CA2048477A1 - Plant chitinase gene and use thereof - Google Patents

Abstract

ABSTRACT
A DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in Sequence 1 or an analogue or subsequence thereof is disclosed. The polypeptide encoded by the DNA sequence, also termed the sugar beet chitanase 4 enzyme, has a high antifungal activity due to a hitherto unreported bifuncitoanl catalytic activity (i.e. a chitianase and a lysozyme activity) which makes the enzyme highly effective in inhibiting the growth of chitin-containing fungi. An even improved antifungal effect is obtained when the sugar beet sugar beet chitinase 4 enzyme is used in combination with other pathogenesis related proteins, especially in combination with a second different chitinase and a .beta.-1,3-glucanase. A preferred use of the DNA sequence disclosed hererin, optionally in combination with DNA
construction of genetically transformed plants, especially genetically transformed sugar beet plants, having an increased resistance to chitin-contiaining fungi as compared to untransformed plants.

829540ab.001/LS/JKM/A12/1991 07 29

Description

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8~r~!bi f`-`~i? !~E ~c~cMi!A !~ 7 ~f 2 20~477 in~ recl befor~ it~i def~-nci~ ~CC~ rii5~ i3Ct~ a-~ u~ Clt';'.
Al30, t~l~ defer ~ mech~ni~ ~f ~hc pli~nt: ~y n~ in I ~.Ci~ b~ Li C~ ng tO ~f~ el~ b~t C~l~ in~c.l.~u~
f~,r~?, t~ n~ cl ~nd nece~ci~r; proc~clu~ t;~ ~r~A~ .ted p - ~}n~.s o~
5 pl~nt~.$ ~u2cepti~1e ~o in~ec~ior~ s~it~: 2i ~em~c~i, e.g a ~ gici~e, eithe. ~s ~ pr~ hy ri~ ~reat~cnt af t Hc~ t~ie ,~ 4f 3 ~}le~ct~l ~re~en~ h~r d~irf~ a ec~log~1 n~r ~o~ ~n *cci~lc p~int cf ~ nd i~ ~oul~l be de.~
~.bl.~ co be abl~ to ~i~hance the defen~ f t~he ho~e pi~nt ~ ~st~ by îO i~ d~c ~ ~g n~ r i~prc~e~ ~e~ by ~e~ c~ ;L~ S~, . A
furth~r hd~.~nt~geou~ ff~ct vf ihis ~;~rf~,:cg;~ w;~ul~l b~ ~ht' :Ir~,~diat-.
inhibit;on o ~h~ fung~l att~ck ~:h~ch jr~ ob~./irit ~ 'ing ro r~t~rd.~d ~pi~lerbie ~;tfiihlshrlicn~ f Lh~ ~.nt~ing ~ ,. in plf~t ~ .p~
L~nd thlls f~n r~v.~fl11 reduct.~orl in rh7.~ .f fec~. ~)1 Lh~ iz~ ir~n 15 Th~ of ~;"in topr~ ogen f~ } ~ r~
gt,~l~`fii~, t~ h~ t ~ c~r~Li~uti~l~ ht~ j C~ c~ t-~r. ~ ` C-~S ~i ~:h~ ~yp~nt~ nz~ in<~c~ ~s~d ,~ ~, 3 -gl~v~ s rc, be e:~pabl~ Q r~n~ym~ticAIly di~,e~;tk~, the f~in~ .e~ll w.~lls !;~ ~C
to r~ tt ~ainlv in colul)le ~litz~erA. n~ ol~e~ of ~ 1 P ~,Lu~:os-2 0 ~rn i n~ ~nd D - f~ r: os c .

(;llitin~ nd ~ gl-~cnt ~ cti~it~ h~ he~ b.~lV~.~I .,1 pl.ll-lt 5pt~ t-~ .C.~,IC~ ~; ;t.~LIr~ O, bnl 1*~ ;'ot,'~ 'irl`Z, ~ JIII.Ir.-~
cumbt~ h~.4t y,t~rnl ~nd p~ rlnd i t h,~:~ he~ .h~ h~,r . ~ rin ~:tl~ n~r~ r~;p?n~ f~,f 2 5 ~ , f Pl~s~. rhitinAse~ b~ n ~ rifit~ ~inA ~ r,.ct~ici.7~ci lLnr~l ~t~p p~ t~ lCI~ t~.s ~Lr.~t~ o~, tt~m;~ ,e~tl .~r~ IJN~3 ~nd g~llom~c clon~s h~ een ol~L,Iin~ ~1 Lh~refr~ P1~ in.~r~ r~.
rc~ w~d b y ~ol ~nd Linthor~r, l~Y~ qnd Rollcr, 1988 3~) Sev~r.l1 pu~-llc~ltior~s h~v~ di~;cusse-l brc~rl~l ~rid pl~n1; chiLi~
t~ ~t~e ther~f in ~ n~}~cr~ ,f rr3llst,et-~, pl~ v~g an Incre.~se~l r~is~n~e to ~riou~ croc,r~; inis~ tlgi.
~, ' ' ~.~

3 2~8477 ~P (~ ?97. ~-~5 ~ .9~ ~; b~is~ y t~ T ivr} L,~ 2t'~
.~Ys ~t;~ t i~ t i n ~, th;~ 2~ a5~ gt~lle ~y ~ ~nt3~hd~lcet~ ~rl t.~ pl;~ e~ re~ nT; Tv p~ o~ ns . Ch~ irlar;e g~r~ h~ sv~r ~e~ ~ild ~tner plAJ~t~: T~ LO
m~s ~e n~ 2t~nt~oned P O :~90 123 ~ W~ 88~t~()97~ ~nd l1S 4 ~40 34Q di~ ::lv~ the u~ ~f chl~i -n~ bact~f,~rial c~rig. n :la~ tnt~ ~t.~ns~nrctioll c~ n,g~s,i.; p~e~, r~h1~r~a~e ~.f plant orl.~ rlcTt 2r,er:tic~n~d t~r ~1rt~ rlati.;~ tZrliy m~nt1One~ in generril tt~rms.

10 W0 '~'0/07011 dl~Cl~ DI~A cftnZ~ ; co~npri~irZg a hi ~ It ~ t~.l prcr~,ot~-r vper;~Zb!y l~n~:etl tt~ a DNA ~,eq~l~n~ ~-nccZdir)& .~ p~;Znt e~ltit.a.~t~ tArhi~h ~ st:r~lf.~t-. flrfZ ~t-;~d ~r~ .h~ t~ns~olrrZtit.n ~f p.'.. lr~ C~:
ov~rf~p~ .sior~ of c~),i.t~n.1~ {n the F~1 iZtl~ ~Zhd ;:~., c 'v~ ~vni`~rC;ng ~esi..~;t~ncr~ to i~litrnt p;l~hc.genic f~-ny,l. l~lr~ t~,nl; p~ n~ ~t;~lrZ..~s~! tZX
1~ t~mpZlif '~?d is q b~n chi~ina~t-.

~P O ~Z'~.Z ~ .P O 307 8~ ZII<j Et' O '3? tO~; d~ ri,t~.~t.l~Zc~
c~f t~ar~ f~t1iC~ pl..~r~ h~rbou-~ ~r~y, DrZA ~qut ~ ;Z~ tl~ "l~ o ~t~ne~i~; r~la,~d pt:ott in~. (PP~}'), t~ tll~Sn~-~. na~ lucnl-;Zs~
Pat~ho~t~"~ rt~t;-r cd prc,;~ ror~ r ~ n~ i r ~<ln .~ Z;~i~
?0 ~l6r~r ~"~ ~ p!~'Zr~t!; nt -- ncZr rrlt-r~t.l)t~ù

In ~oncl~ri;-tn, ncZn~o rZf rl,~ olb~ t~ cl t~J p~rht lt ;~L .~ y ht~r chl ti~ rZt vr t~ u~- tt~:r~ ~L i~ ( t',~C~ CZt' triqn~igt~n ic pt *nts ~

At th~.~ Phy~.c!cht~lTic~1 !;o~-L-~..y ~ rc-p-.~ tr~rn.q;-L.,r.l' v~u ;'S No~ ~it~h tTnit~ i.n~;t~om A~ril i1~ 19-~.9 Bit~ ht ;;~ y .~ lo1~
lflr Rlol.o,ry of P~:~nt~ t-'~o&~ nr.er t~:eior~ ,t pr~ v~ orS
c~iscl~.~.c~ d tht~ ixo7.9~:ion ~sf~l p~tr:i~ic.~tk~n ;>f 6 c~i;in,~ o~rl yt~.s, in~t~ldln~ ch~ln~t~ ~, frt~m ~h~ len~t~.~; of ~ ;.aL h~ t pi.~ ; Inf-c~d by Cl!rno.~pol ~.f bt?f .lc ~\.t~f, ~; ~hyto~ tho,st~r~ic ~ co.qt.~ining l:urtgi .
.~0 lh-~ fa`liti~Ase ~iot~rl~.yr~e~ ~ere char~ctt~ri_nd b~ r~ ~tol;~cul~r weig.~t:
~r.cl k~s~etic.~ o~ ~hi~.in }~ydro1ysi.~. ~tit;'n.ise p-r~p.~raf~ nr ~
c~t.e~ t- b~ ~p~ of hj~d~o1yz l~ na~1~ sy~-~e;,izt~& c hi ~ir~ {i~t-.~
~50bi f~l/lS~ A12/l~t 0'7 29 4 20~8477 ~11 w~ th~ g,r~w~n~ f~ir~h~r c!~cLi`L~ W.~'`
~ep~ rted ~Ind th~ ~h~ tin~cr er~ 2PS w~re .~o~- s~p~ ; àl sc~ ;ed .

PiY th~ pre~nt ir2~Jent;~rl .~2 2~ve3 ~1~12.~t chitina~e h~s t>*en cluci~l~e~d ~hich, either ~l~ne c,~ ~n c~ Ln~,tion w~ ~h oth~ p~ ogene.~is~ 2tf~d S p~te~.... s, ~l~ows p~O~12iSln~ U1l3 in th~ /ibl~ n t3~ c:hi~ c17n tainir~g f~tng,i.

BP.IEF DISCL~SUP~E OF ~HE~ ~NVENTION

In one .SI~pcct the pr~.~ent ir~ention relaEe~ t~ ~ 2~r~ seq~el~cs CC-~2-1(~ p;~i~1ny, the sl~gar bP~t chicirlAse h D~IA ~equ2nce sho~ in S~uenc~ I
or an ~r2~2logui~- rh,:ri~c f, the anaio&t3~ bcinB t D~`iA s~quenc~ el-c~ ng a pol~f!pt~-lf~ hftv~nfc~ th~ ~ntifung~l n~c~.vi~ of ~h~ SU~S~lr b~ hiri-nAse 4 ~ r clelni~il her~?in and ?~in,~ a ~hnra;~;erlstlc p.~tr~ ~if r.h~? DN.~ C.~ ui~nrf~ sn~wn in ~,f~qu~
15 1, lt~ hybri~lizinfc~ with the DN~ *~ui~nc~ ~hot,n in ~;rql~i'ïl(:C' I .lC jS'~.
r~f~ r ~Itl~ cf~ndit.ion~; spi~cl~ d i-1 th~ ~M~ ri .~t' ~i f~r~
nrld~r che hf~ tLn~; "ldentifi~ "~ Or D.~ bcl~r~;in~, t.~ rilc ~-hit;i ~0 ~,enc fa~

îii~ e--cl~-ting a pol~po-pri~ h.~ r; ~h.~ ;~rni~ id ~ u-~n~ vi ~.
sur,nt' l?~ r~ n.~t~ O cl~(>wn i r~ S~qu~ , or ~v) ~nco~lln,~ p~Iyp~pttrl~ bi~in~, ~f~cc~ cd ~y ~r. .. ~ d~ .e~l 7~ rlr.~in~ ~t~ r hf,et ch1tinnc~ 4.

? r.~icin~q~e 4 r~NA ~ ncf,~, St~ lcP 1, S~)wr~ "~ .~;fi.'C~Uf rlC~!
l.lf~t.inF~ b~lo~ wnC; det~ tinf!d c~n ct,t~ of ~ rDN/~ Ci~>n2 isi?iA~f~l from ~ sui~;a~- bc- t cDNA l i~r~ry ;Ir;~pared ~ .crib~l ir~ the M~L{:r;~1 and M~chods .c~ctit~n 'oelos~ i~n the ~15iS of i~bri~iz~ .rl wic~ !53' 30 ~pecitic olion~lcl~ot.~ probe. ~ vli~,ol-ue1eo~ide- p: . r~ a p~e?ar -ec~ on ~he ba~~ vf A tr~pt~c p~p~lcl~ prod~--t~ f.o~ oit~n~ia~.l~
puJ:e su~ar b~et ~hît.in~se ~ obtained ~s d~'`.t i~c; in Pld~ 13 -C; and 82!~4Sab.. ~l/l,S/Jl~l,/A12~ 1 C7 29 s 2~477 .~eeil,od.e ~Ar~.i l n ~ rpl~ I belo~ pr~ ?d~re ~ f ~": ~. .i~). i~ L.i~ h~
~hitir~¢s~ 4 D~ ser~le.lce l~i o~lt lirled in Ex~ple ~ lo.. .

P~ior t~ t~ pr~ent ln~r~ n, i1h~ ~in~ ~ld ~j~`eS~YAIO~ ~ ~r.~ ~U~
~>~ i na~ ~, e~lzylQe ~>.. tl~ e~ d-~ r~ S~lg~
S ehfell)a!iJ~ 4 hnd n~t ~een t*pnr~ n~ r~ n~;c~.on i~rl~ n that it coul~1 bP i.n~er~tlng ~o i~Qk for ~e~:Y ~ eiiC~.i. In fi~
tkP ~nieic?l ar~lys{:g of -iug~r b~~. chitin~i~ie. ~ hich ~e~ lr d ~ir en~ a .r,m.~ t,~ncti~,t~al ~IOI'Ziitlin, ~iu~,ge.~ d ~h2~ the chicin~
~.n.i;~t~f ~Ad ~ lfJW chl;in ~?~fir?iiy ~;d ~:ht~ v~ r'~-.rJtai_~c: ~.t~Vi~j'.
1() Thus the en~ ne dl d not see~ t~> ~e of ~ny p~rticuldr in~:er~sr. .

Th~ lcLclation of che .~ino ~c~id seq~encc: .~f i~hc ~ r beet: cl-~ti n.l'ie ~ t~A.C al? impQrt.~nt .~;tep in the analysi.~ of r.h* ~In ytl?e. I~ s, fr~,m ehe us~ino ~ 1 5eq'1CnC~ it W.~3 c]e~lr thrt~ th~ ?a~r~ t b~ C~ L`
n~s~: ~]~n~,s rO ~he plMnt chit.Ln.~e.~ of tl.e hev~L~I c.l.~ t.h~ i r IS ~Jnt~ s .~ 'rt- tieq?~ hev~ do~ q~n .~ld a ~U?lCt ti.C ~ omr~ io ~.s t? l~C~ h~ bi~ ;o ~:h ~ r.i. ~
dn~na i of th~. ~ n~.ymc hr p rt of .ho ~ n~m~ liCt~ i ex bind t.o c~l'L~in stn~l c~itin cotlr.aitlin~ tr-,~Cllt-t~ h~;c.,.y.l~llc, gc2nic fungi.

20 By hy~ oph~,,h1c cl-.~;trri~ ;In~l2";i': U';i'~7i C7rlt' .n~.hocl ~lr~u~ li2~y, t~
~ hori~ r. ~ 8~, ~}1C pri n~r~ t:~r~ ~,f ~ !..i i2~
heen fou~2d t~-~ b~ mor~ con~pti~ t ~t~an 7.'.hf' ~tt';lCt.~ll'r'.~: )' O~'L ~ r~r chlt'2~ sl~.. b~ r,~,~t1~, t~ th~- c~ t 7~ 't ~'~l;t'~ t ~ ib *ri in fl~r~ r d~t~i1 hel~7w). It ;.~ Lci.p;-r.ort cl,.,. ;',i- CG~lP.~ r.
~5 st~.~c~ r-~ c2f ~litin.~ 4 i~ n s~t'lv"l1t"t~"~ i7- f~r-~ l'o~
r.c;, ~,r~ C~ . chlt:is~ ~Jttuctur-~, P.~. in ~h-- e.l~ i v~ phycc~
p/ltho&enic f F1~1-r.her~ r~ itl (`On~fi';t CO o{r~ kno~n h.2sic c'r,itir2..-... '_~ .L.'.- /~hn~c be~.~n oun~', to l~,ck f~ ~` ceru~irlal f~txre~ or~ a~ ~ ha~ th~' 30 ~ ~2~ ~r;; tr~7.-,~;~r~ tei~ to ~he il~.. er7~ r ~:p;2~ hu~ .2 rh~ ~r."~uole~ ~21c pre.cen~:e ~7 Lhe erL~ , the incr~rcr lula~ ~"~acl.
ha~; ~7~-~n experiu,~21ta1Iy verifi~d.

82SI450t7i.0c~ 2/lr7~

2 ~ 7 7 Th~ r ~t ch~t~n~ r~ Lrià r~ iL .~ULi~rS ~ Ly ~ t~ g.~ ~isy ~n~ 3V~' sh~ p~r~.iculnr~ Lni,~7~ n~
~ec~ c?l~? ?,rowt~i ~f l~}\~o~ r\jt~ r~ n ~di~ le us~
of ~ corib-nAtIor~ of ~hP ~ r be,P~ chItirlaise ~l ei-~y~e, .~ ~con~
S d~ffL~r~ chl~ln~se ~in~ 1,3 ~>.uc~n?~ h~ L~ p~Jt~3-p~3rh~y,enl ~ fungl h~ becre. found ~.o rc~uY r. Ir~ ~n ~c~r. L~ ~pr~d ~n~if~ r.e~ ity as CO p~r~à to ~h~ h~ su~,ar bL-~ ch na~t? 4 alon~ . Thls SiJ~t`le:r~;lsr~ arl~:i'tur?~21 eff~cc I~ ~ep~r~ ~tl Eol' ~h~
f~rst ~ e in connecto~ with chi.s ~ppI~cari~on.
Accord'1n~ly. ~n ~inoth~r l~port~ , .hæ pr~sen~ ~n~r.~ion rel~es t~ a ~enecic: construcc cotnprisine one ~ ~nbre ~opi~?s of a Dt~A s~ n~ on~t~
~?q~oCc ~thowrl in s~q~ence I or on qr~ u ~her~o ~ric ~l~fint~ )V~
lS or l sub~.~quenc~ t:hf xeoi (furthor ~I~incd h~lo~) .

on~ or ~ore ~opies of A 31~A s~qu~ncc~ ~nco~lin~ u ~;-.ic~
dl ff~r~nr. frocl th~ su~cr b~t chi t1n~se 4, an~l one or ~ore copi~ 3 of ~ DN~ ;equcnce er~codin~ L,`) ~ ".. nu~

~?.~ l 0~ r.~ A ~ t~t?llCnCcC.C t~ , f~lrl~ ci on,~l l Sr cJ~rl r~ 4 .I p I IJ~
2C~ ~nd ~ trar~;c~ip~ n ter~ln.ltor c-~p.~ u~` i.':ri~;.rl,~ t~
qu-!nl PS int:o f~ t.ion;?l pol~pep~id~

c~ ;ti~u~!nc~ of ~.ht g~ t~ o~ r~ 5f~ ;r,~ ;t ~r.D urth~r ~x~ in-~d b~low.

'rh~ m~.~n u.~ ` th~ g~n~-t;c ~os~.t~ .s.~. Gf tr,~ illvcs ti~ ir, t;ll~:
~5 producr ion of t- ~,cn~tlc;~ tr,~nsform~ nt h.--vincJ, ~ .c~i resi.~it.~nc~ ~o ~:hitil~-cont ;llnin& plan~ ?.~tho~,or-.t. s~tl iJ~ t~,pnt:h(, ~,~?nj,C fungl a!; co~s~.trcd r.o ylnnt.c which do no~ cv~r.~ h~ ~ ol~c~ c~.
~uch ~:?~ ntr~s~s~onned c~r n2~t:~r~l1 pl~nts . ~ n~ ef:' ] y ~ rla~ u~u p].~nts ar~? a~vnntac$eo~l~;1y p~eparc~ e o~ t pIanc ~ n-.r;)l-ln.1;ior 3V ~rc~or h.~rbv~s~ , th~ g~ot!tic con~ o~ the -~n~ cltlvn.

R29~ob~ ~s/JKM/Au~

7 2~5477 tno~e 4 DN~ q~7c~ or ~ g~ e;, ~ r.~(.u-lnr ~ ~pPC~C ~;u1~quer,~ r~oF (~ wl~ f~ r lo~ ~y ~ls~ be used in tl~e is~l~ti.cn ~,f 5~ q:~$!n~e~ longIr~y~
to e~le ~h~tinar;e 4. ~e.~ f~ily ,~ defin~d ~bo~ ;c, ehe ehit~rla e 5 4 D~IA .~qu~nc~ or ~n ~n~ ,ue th~ t) vr ~ gfr~ri~ .^orlci-ru~ ~f ~he ?nt~on ;:~AV ~* ~ d in ~ me~:h~d c,f p~epxrin~, ~ polyp~pr de, e g. x r~c~mbin~n~ sug~r ~eee chitina3e en~ne, or h ~o' jpep~ d~ ~L~u~e hf~ pot~nt ~ne~u~ga7 activi~v The po~r~p~df~ ~r P~`L~?P~P~id~
tur~ ~y by pr~p~ireci bJ ~ of ~ ornbj.nr. h D~f~ L~hr1iq-l~s ~nd }u~y 10 ~s~ r~d In t~e antif~lngal tre~t~ent of v~rf~?~1.s ~:r~d~1~r;s, ~.~p~ci~
f ood prod~l~ e~ .

DETAI ~D DIS~ OSURE O~ 7~ 'NTION

Th~ it irm.s~ 4 DNA .se~ enc-~ Seq~cr:c~ ~ er1co-!t ~ ~h~ ri~./c ~ect ch.itlnA.~-~ 4 cni~yme, ~h~ amino f1c:id ~C~ o~ ~hicn nl.~:o 3~pca1a 1 S f~on~ S1~yucrlce l . In ~hc preser~ >nt.~ he ~ D.i ~~ inf~ " nnci "st.ly,;~r t~t chi rinas~ r~ u.~ 7 intercharlgcfl~iy .

One c~ Dc ter i ~ f e d ~ i t i n,~e ~ 7~ ,f inv~nt i~ nr~ ~n nn.~l.o,~ c the~ ec~ . tf~ r11ej ~ r.~ c.~ ~ pol ~,po~
h.~vir~, t.~ n~f~ s~t i~ic,~ ~) L~ .t ~ r nti~ r~,nl ;~c.tl~ y ~f ~ r ~ c~lt~ L~ `tl'i~
tl ~hss~ i t i ~i A ~ i ~un(~ r. ;< ~ I A~ y ~ t ~ "; ~ t .i v .q~ ly.~ yr~ ctLvi~ . f~r A~ ir~ L~,L~
~wnr~ hl~ sA~ C~ it~ h.~ t~, r~ -, f~p.. r.~ ~l lor ~:lln~r ot.h~ t l!U~ C pl;~nt. chi~ina~ oI the h~vt~in cl.~s~;.

~5 .~n ;sc~ rclAtllc/~ h~r~ ieh, c~ m "~h~ ri~-~nli~7s,~ r.y .,~ ~r .t~ r h~t c~ic~n.~s~ 4~ d~ >t(~ the ch,~ t~ri~ ~ln.~t~
ct1vity of ch~ ~?n7.y~e, I ,e . ~lle co~bin~t1on ~f chi ~ e nceivity nnc~ c>~ e iSCt.i~rity fo~lnd in t~e sugar l~eet chi ~ as~

m~ t~I~ "o}si~:~nase ~ctl~rity" denoteti el~le en~y~4e'~ a~iti~ ~0 ~ec~
30 pG5~ ch1t 1n ~n~ chitin-cc>ntaining st~uc ture, c~rld ~e Chl e~Z~4~c' vi.t~ y b~ de~t!rmine~ by 1) a btolo&ics~ SL~y a~ 3 C'~eLiiCd 8~4S~bi.~Ol/lAS~/A12/~9~1 07 29 ~;a~ ~n ~h~ lc~gi~ y, ~ Q~ 2 O ~ 7~7vwir~
hyphr~ of p~thog~nic fu:~lgi, i . e ~ ~.he ~ .y vf ch' ~ d~ ! 4 ~o d~-~:.rc~ hyph~ tl~t t~ r r~ d th~ vwLh ~ h~ e, 15 di~`e~ l~/ ob:~ervott. 1~ th~ ~.hemier~ saJ, ~he ci~ p~,sf ci~n of 3~,-5 c~hitin ~y chitin~Je tc. res~lt ~n ~lin}~ e.s ~ hi~i~ i.s ~or~-tored .

~he biologi~ say mQy ~ eal~ried ou~ Sin6 ~lly o~ .h~ .i dif~ent:
rDethot~s dr!~;eri~ed ~.n R~y~ 5~1s ~nd M~thod.-' ~lel~eL~ In le~ ~ht 7s~ading "Anti.fe~ngfitJ. Rct i~ ity" . ~c:n ta pc,siti~e result is ;~bt~il)t~d i~l rlny oÇ
10 the.~if ~th~ 5~ . the o~s~tzvi~rtc~? of rlescruc cion o!` -i~ rph~e ~aLl~
.,tnd re~.~rd~elorl of th~ gro~eh of t:h~ ~1ng<l1 hypht~t, i r i~ cdk~n s?vidr~r!;:e o~ hi- loK50~1 chf't{nc~s~ f~ ao~ ity.

rhr~ ctt~ al at;.~y If~y h~ car.~led c,ut ~I.C ;If~.c~ibr~l io ~;'Z~lt~t'i;115 fn~d M~thorl~ md~?r ~ hen-ling '~lhe r.~cllochæwi.-~l Ohit:in.l';' :-.S'.~.I)r.
~'~ Chtcin~fce ~ cr;L~rlty i.ei .chr,w~l ~y hyrlruly~l.c cf 3ti-chirin ~nd t:he~ res-.lltin~ f~,~na~iort of ~,~inl~ rlin~ i of chi!in in ~ ; A~:S~:y.

lhe t-~nr~ "l$~.~;o~yme ~ctl~ty" ~;OrlOt.?'i l'llf- ,?ll~y~r- ' c fur~ ] c~ nl IIy.~lrtg nh~ ly~cioz~lf.~ oct;~ I, cl:;~,tiT~f z! jJ~ c~lrlyi~ out.
t:h~ o~ y ~r~sc~ ibr~d ;r~ flodt." ~ ; che ''O h~ `fiYt~ .y~ ynt~ y~.

~r wil1. h.o tlndr!r~.rooel th;~r ttlo nt-.r ;'~ g~ ti~ hr .~iU~ r bt!f`l:'.
chi~n~ c f~ ~s ;~ qu.~.-f't.~t iv-~ q~ ti~ t~ St -fl~cr~ng ~ho nh;.lit~ of chc poly~)o~itid~; to d~.c~.r~-~y L oL3pOi~
~t~it~ ) tl'~ h) p~ : W.~tll!; ot .7 ~ c~ ni ~ r,~ )y 2~ .'7it~ L r~t.. ,xdin~, f.ll~` F',ft~Wt-~l oL l-i-li' ~

t~;~lOf~ of thf ,^hitir.~ D~.~ .s~ t nc~ c~
t. V~ t ~ p ro p~ r L i ~ l i s t t~ d ~ t~,v~ t t e L ~ .r. ~: d ~ ~, d~!~irlC tl~t' l~n~lJLO~It'S of tht~ in~enri~ fll-i' *.~ ne~i 1t~ L Lrci'-~
cail~ e~Lo~.

30 Tl~e t,-~rm ~c~ar~o~eri~ c par~ Ised~ in conn~e~i~n t.7i~ h ~:he al~ri-lo,~le d~lned ~1 {~ abo~e dt-~nD~t-s a ncicl~o~-~de ~ e...~e ~hic h ~s s~bt~n~d frQm the n~leocide seq~ .ce of: ~he c ~ in~ !j D~A s~ q 9 204~77 vr ~h~.c~ rt~lcle~-.;r~ q~te~3~e ~-:c~3~ o~ s~
i,t;n.q~ Dtt~ quea~ .IEid ~h.ic~ O;t~ p;tlyi~i~'p~ t` '!~ L~
~t~it,~d ~.h~? .3~ f~ &~ta~ ity ~ a~ l~t~ r~ ase f~ pi~
r~e ~ t3~acte.~istl~. p9"~ c~,l:Qpr~ ;e~ S~q~i~r!~t~ c 4 I~NA s~ u~ e, ti~ tlb~,q~le2~i~';e 7)~ L ~ i"ol~St-'`~ih`i~r ~ Y Ia ~h '~
nu~ e~7-:id~ .. f tht~ chitinasL~ NA j~:tg~ teiLI,~, C;ir.il?~ 01'` ~a or ~,or~ sepAl~a~? 3~lcleotide ~eq-~er,~ ctf t~ cni~ .s~ t~A St~-tce . In oFd{~- to h~ W rhe pO t ~p~ Ge ~n d bJ r h~ ~h~ te~
ti~ p.~rt~ of th~ ehi~inase 4 ~NA ~.q~-~nee ti~ re,~in ~ ts ehr~c~eris~i~
10 at~if~i~g~ ei~it~, the p~re ~i~l nortn~ be only ~ ~nAll rt~h~r sf l~Cle~tit~ r.'~ *t' ~hr-trl th~ c~t;.n~ e~ .c~.~, e.~ Q, s~eh ~ 1 25 n~lcleotiL'tes ~shc>rt.~r.

A typ;e~l e.~ tlplr,~ of A eh.qr,~ c~list:ic ~J~," sf ~he e~ .ir~ e 4 ~iA
lie~Ul~nCi' in~ln<lel, th~ nu~lt~ot:id~ x ellco(lit~, t~e ~e~ r. -~ite r~f .`l IS nas~e 4.

n~ ,g~le def;tleci in ii) .~r~.~ve i~i ~ DNA s~!t~ler.c.~ ~hiel~ rfdi~e~;
wi~ t~ ehlrirl.se ~ DNA ~erlrle~ t:n~ler the eondi~ c~e~-.;fltd il~
thll? hM~cr?x~nl.~ .~nd ~lethods" ~ction h~lc,w ~nd~ e..~ nt, ' Iclenct -f`.ic~ion ~f l)~A b~:!longin~, t.~ rh~ ct~i~inls- . ~,rne r.~,t,tl~. r'r~ e~-ncli ~0 t:;nrls d~fiA r~d fe~ eh~ hybIidi~.~ci~in ro t.;~ f' ~ .lC~ d orl r~hr t d~ e~A~ n~ Arr~ .tril}~ .uDb~ r ~!r k~ ;;r~ t ~
r~5er; ~t~d f~ ')t'~Pt l`ti~ s' ~ r J .~ ib~ F~Y;I~nIf~1, l~ b~lcw 11~ t~ t.~llL ~v~t~ , Ar~y ~ . ! "~
?S t~ t.~ s~ ]frlCt~ ~.tnd~r tht~ I~yl,~ t i~ v~ r~ rl r.h -~ ~t~,t,~t~ t.~ l t.t.~ d pnr ~ f " M~ r i ;~ .nd ~ hG~ r i ~ llSl~ -it~ th~ c~.h~ ;t~:t- L~ ~r~ ~1mlly .~-tl 1~ r"~ J~ r~cc~
p~t.y~ it~ in~, t~ r~ ;,d ~Ir~t. l F~nf~, LI .~ .f. ~
b(?eL t~ . F~ rtn~rt~, wh~, t~t p~ly~p~ic;~ JIlU~ Ih 30 .~ c~l ~N~ t~ e~ t t wit~h nntl~ r31set1 A~ , t~,a~
~hiS~ flsl ~. lr is ~ strOn~;. in~ nr.;on rh.;~ cht- yol.~r~)e~cidc e.~ct/S.ie~lb~ 't~e ~)NA set~uen.c in q~te~tio~ elon~ c ~e ~;~Agn~; t~ c~;.t~in.' st~rt~lo~ic~l cl~ss. Stlch DNA 5e~ t nct~s cons~': s ll~ t.ng p.~r~ of pr~se~t l'r~e~le{on ma; ei~her t~on~pr~ t q";'~ .5 isol~tefl fVfJLI~ ''La~.'..lr~l 35 s~u-.ce~, e.g. pl~nrs, synths~tle~1ly pr~dl~leet~ rS4q~\Pn~:es or mcy be ~Obi.OOt/lS/Jl~l~A12/1991 07 29 20~477 ~o 5 ~r~ ~ f . r.~ ~ i t. r~ l l V f~ D3~A ~; ~ t~ . g . ~. ~ {~ L i . ~ -: C~ r . ~ ?
f~Jllr~r~ ;eq~le~ es l~lc,rly,irly, ~b S,7'~ h;t~ 3is~ S~l~t-! ~
al.~;a t~rL~e~ "ehLtLn~s~ 4 re1~ eà 3)1YA .seq~iinCt~sn.

~r~ t~lC~g~ e~iyled ;I~ i 3 ~ e ~ c ~ ~N.~ 5~ }~r~ c~
~, p~ p~ It~e cc,n!p~ 3ln~ am;~ id ~ 3,n~e ~;h ~`r~ q~l~t~C~ i~
i . e~ . tlle .q~nin~ ae~ eqr,len~e of ~h~ ,re ~`:3i~ir~.r~se ', ei~}~ e ~ lc ~11 kni~wll ~h~r .he ~uRe n~lnv r~c: Ld h~ ~n ec; ~y ~r3~ i r"~ dt>~
th~ O(iC>n llS~f~ beIng reIa~-ed, intt?2` ~t2;r2~ J ~71~ e~ o rh~
or~Y~ 31 in ~Itl~-:c~tnn expres~;nt; the nucl~oei-lo .~equ~ f. ~ , 01~` c7.`
10 mnr~ nllr.~ ot~ lesi or codoni~; oF th~. ~hlt.in.~.i* 4 ~NA Si~yu~l.o~ ol ~n~?
iT~f~r~t~ /r~ r~.ly ~ rxchnngeci by ~ e~ ;cSI, 1.Ji~ n ~?~ L::s i~ s~ r;
po~ `r)t'i.~ t~ic~al to ~r ~ bi-.rfir~ y li}~ t`~ .C~ pe~
C1~t~ 0(1r~t~ b~ t~lt? chltlnAs~ ~; r)NA .~ C~ U~ n.

The .lr~f,l~g~ d~t'~ o~ ; c~ -c~ t"~^i"''~;'~irlg ~
lS p~ t.i~lr ~hi(~h f c r~cogni2t:ct by .ln ~"ti'i",~ d ~ st S~ b~et C}~itill~ . T~ th~ t~ lt. ~ t~ ;t, ;,~ t~.~rrb ;~ c~,~,o'~ (l h~" i.;
u~,c~l i nt:t~r~h;.~ hly wir.h ~ir~di~ th" A.~ i t: i c ~ , Ex-lmple 3 1 0w, 1 r, h~ en ~ot~nd thnr. t.hl~ .C~ r ~ t C}li; in.l';C` !~ , ym~
or~ tt.~ ~ rlt~ s~rolo~ic~ ' hn~ r~ Y~ t(> Il ~(~ r~r!or~ cl .i.~ thc l;tt~ t\~r/ . A rec~llt ~er.~Ic>,~ nrl;~i. o~ n rhp~
1 ;h 1 e in~ls* h.l:; rr~r~t~ t r~ f~ s~ k!Li.- .~ bet~
~t.l~C~ .ti~ ;t! ;~:J ~ Lr,r~ t. ~h~rtr~ e !~, .ir~ - l. r~ IC ~Li~il' p~ -3 c~l~ i t I r~ h~ l r.~n~,: . t ~ ; u f ~

Tllt~ ~r~til~od~ to ~ t i~ hir~ir~"~ r~~ r~ r~ir h~.WP~ I) tl~ pol~vp~p~ ~d; ~ r,r.~.. >~ y r~ it~ .' 4 .)t~ lC IIC~ c,r t.h~: ~n~ri~ntl~ lv~ F~id~ r.~ i h~ .q r~ t~" ~ ,,r ~ c}
o~ y b~ rn.~ lfic ~ rl~ c,r ~ lr~
.~nri~ par~ic~ rly clllr.~.l)ie .~I)ti~c~d~y i~; ~ rr.~"~r~:1c~r~ r ~ic,Iy-3 ~ ~ c l o ~ i b Q ~ r ~ p ~ ; g ,~ e o t l r~ .) t' ~! c ~ z ! ~; r . i c r: p i t~n;~b~ y cl-e ~ ;ti~ia~ 4 ;)~;A .cf ~ r~ .sllc~ ~, ;. r~pr~s .~ ,c~ .. r~r.
ln ~urther detal l below.

eq~ ; of ~e ~n~ ;pl2~ *;' ~ n sr~y- Cvr.~p_ n~t~r~ s wr~.~l a.s ~nt~le~ n~R ~ ~ -i, t~ U~ ~L ~qu~

2~8~77 C ~t 1 1 J i.~ 5~ c3~ r i -Y~ ~ d ~ i r~ L ~ l y 3, ~ 3: 3 ~.~3,L5 l,! ~ ~,t: 3 3C~
Qf ~ 2~ rl~ g jF~cj c3~sc ~ ib*~ ,r~ 3~1Q ~ 12 pr~pi~-"t-s ~y ~ J~r3~ 1 r~,rA~ ds r~ L`~ '3~`_3~ir~ A
e'~ , . .5riing tl'3~: p~'l3~C~p~,'e'j s~~ r~ p~
~:~rrlth~ . 0~ C~5l'~ 3!~1S~ ~ hie ~ i se~ 3.~` b~L ~3~ c9~A .
e*~.cis5~ic, ~i~e~; cDt~A ~ d .~i3n3s~e~ n~ d ~*r.~ol33ii~ ~rad _~ra~L~hQti~
orlgln .

Ir, rh&~ wing, ~hc c~mposi~iosl ~f the ~:hS.ris~ , i)'.~ s~eq~iaen~
tnd each of th~. c om~:iras rJî s he ch~ ~ n.^3se ~ e..~ ,~9- -32~ by ~i5 ~U .~equ~.~r~ r,d wlth the ~ 3inO al`k5 .';'aqUi'`YlCf.? SltOrr`ii it~ Sf~y1ltt~cf~ 1 ar~
ft.lrth"~ .?s- r ~b~. d hnd ~ r~ t '~t uth~!~ pli~nt chi~ s~: .

chitl~n~sf: 4 DNA s-.-tcS~ .o~.pri~ t l~ r ~ ? (r 1 /0~ c~rl~oding ~ alnil;t~t aci;t r(~ld~ s, .e pc~r~ ucl(.~id~s 7i~
~h(~ g ~ eirl ¢iii?m~ c, 35 a~tin~? R~ r~`~;;d~le~: ~f~ FaLt. ;lIU`
ti~ ', 7??.,~ e-t~v~ F, .~ f;~rl-~r;~ iJ ./-f.~tlir' c~tlt~:. 'frt~flD ~ t~ r.n.ll p.~ .f tl~ r~
~ ~C~ t.~ ; t i .'i i ~ to ~,t.~ f lll i T~ t.~ f.~ f~ ,y .~ ,t~. i ,lf.~. i cl ~.-r ~A .~"~q~ nc.iln~; n~ t~ ft~i ~ iSo~( v- r, hilC. ~ e ~l~` t ;'~ rfi t l~! t~
~N/;~ ne~ r.c-~ i of i-si whc~ 5n~ gll~tL~lirl (~JGA f'~! U~ n ~crO ~ tL
20 rlai~e? .~r,~ thri~d o~,~ at~ ~In.~ h~ c tr~;"~ri~y Qtr~ n~i t l:y (~ idt~
lUpl~! 4~ s~ ~r,i~trt (~>f th¢~ iCi~ r~ .e- ~"~
ledtl~ c~l .

t. c i~l i t; tl l l .r, ~ .C n~ `t ~ , l i v i ~ d ~. rl t ~i, f ! r S t - l l i: e~ t ~ e;
cl;~ , th¢~ n~t-l-h-~v~!in cl-ec i ;~n¢l t;h~ Cu~ r~ s` ~1A :C

2~ U~ ?t~t ~it;nn.cl~ t ~. .1 b~ ln~ t~ ". 1~ V
t` 1 f\ .c: c ~ l~tw~t~ L, i è. i .c I j s t i tlC t ly ~t i f f (~ r ~ r ~ t ! 1~ ~! t ~ L SJ ;I ~
¢:hitlni~ ,f` thi.c ~ s~. Wh~ c ¢`hir~n.~ i(tsl! ~V~ a:c~"
tor~ t~, p~ o~ p~et, popt;~t~ t-¢~ tiei~it~
tl;~.~;t 2) he~ t~ L w~ ight.c ox ~ 3~ Ex iel~e~Li~ r.i~
11~ t~ C ~ttli~ r ~lt~ QOI~C~ J~ C/l ~ D~ 3C; _~ ti~ ¢l f~,r t:h~ ~h~lAI`i' en7.~e) . In a~ t'~:lh, .Cih~ odi~:., Ltl1.3c~ ist.
t~ in~e 4 c~ nGt rt~c~gt;~ c~to~ vt?~ b ~ r~ tlf i~ ¢~
~h¢~ r~x~m~ie ~ 0), it ls ~ t ~h.~ '-'h~ t t~ie ~ r.l ir ~r,~

' '' ;

'`' :

1~ 20~8477 t ~ f ie ~ i t ~ e r o l cl g ~ ? ~ 1 W-L !~ 5 t .h cL tl -~ 3.1 l ~ t ~ ~ h C~ l i T"
rl~ses from ttl~ heve~n ctasw~.

`rh-^ pr~ma~ titr~ r~ c~ ~:~e ~L~i'.U~e ~chl-Flil~s~ c~ .rl~:d -~
; c~f ~ t .~ o i~C I f~ f~ I It~ r~ `i 2 t.i '; . f,~ L~hi~L I . ~5: ht--h~ve~T~ dc)rn;~ln ~r:id `he ~inf~iv~ r,rf~L~ r ~ t~sL~lt~ ,Y~ r.. .,f ~he p~ p~e c~.h~ o~ r~ a}~d *;~ Lt~ L.~ ~ j*f`".
.s~nlpa~ecl ~o the h~teilL ~iitr L~:ture . ~t~, f~lYle~ .î3 ~iiCim~ ill C~irt~ 06 ~;~.{r~ d ~fc~d~les. It~ ~h* ~A~ic f~hitl~?~.2 f~ .e ~
~t . ~ LnTI.!t ) ( S~l irlsh i ~ ) ?, ~ h?- ~ ~ r. ~ L CC~rl~ s 4 3 ~2~ o f~ t t e.~ S du~ . r~r~t ~h,-~ `lF t ;~rL~ L. i.i"i~ C ~.~rlt~ s '> '.) ~ s. i At i d t f .~ ~ rli l e; . ~ ot t~ t ~ e ~e i n d~2M.~ e ~ ? i ?'L~`.t f ~
CiC)~ li.t~! OTe thi.t.intis~! li lc ~;hrJrti'r' thf~tri i-bi:?r Ci' ;.lf` rOI_`LtCC~ i'`hiit:tir)~
it;na~t~t? 4 h3s ;~ ~f3int'~i.Til~ ffinit~ whict~ l~ t^~ '; t.~ ,r~;t~tC1~ i5 th.,t. ~: the t)ther bt1si~ chitil~nxt~, be1~?r~in~, tc. r.h~ ht ';'t' ' ~ t',i~;_. . For l5 COTipf?it i !~ht1 ve?~y pot?r O?' Il<) bi~ o~?~ r~ 2.t';t~ f ri?rn t:h~ hel,~ i?,~ t`1.'tt.~; 3re !x~ inf~d~ is '~ 1 tss ~f C';li l~ .t~ OA`; not C~.>~ r.Tltf' l~t~vcin dO?~tsiiTl, ~ C?l'l~ i'` Flltlt..'iiCIl ~ Olit~li.!~ ~.ii` ~C~ilV -'i.Oj~y b~!t:W-'r'lt ~ ' f~inctiG~ l d~ritaios o th~ hc :- lrl cl.t`. ' ~t~1 ;hr rlt~n ht;~if. irl t~lU!i.'; i ~i vl~t y l~i?~l . In sit~3 ~ ..J OII ~t~ 'C l~?n~t I ftr'; I Ihcli t~ i!;t l.t ~0 ~I~"~ ;t~ ch~ c'~ r,~ fY~ L¦l~ h~V~ . s~.; rt~ t~l2'- t~ t' .~
~ Oltt thtr` n~ `Vt` in cI ft.~C

Ir~ 7,rrlt~r"l, 2-h.- ~.p~.~. if iA ~ct't itj~ ?f th~ n~>n ht ~t-it~ I L.~n; ~h~ ~n i~lit:
c~ .,S~ ~r~m ~t~,b l(~t- ,-sl~l f~t' bil.'`~ l t t~t'li~ iTI.~'.'.' ` fr ~ y (I~ ,' f lh*si~ )90! ~tr-- .tpprt~sximr~:t 1J h rC~ t~'A'C' _'~ rh.~,r c,f ~'tt~
2 S ~tt~ Vtl! i t) t` 2 f`t '~ S C } l i t i tl;l .2. t' C .

t f~ t h ~ r, c r I . > l ~ l n t ~ , ' t~ l ~ t . ~ ;, ~ ? ~ L~
~;Ci~ ~'-?Si~ " r!'; t`.~lnr.trt~ tht' ?F,3 ~ LA~ it'~2 rt'~ Jl~ t~lf.~
~ltr~C; iOnfll dhm.-~ i rl t.~f tl~ b.qc, C tol! ~ O Ct~ t ll.'lC J~ ; 2 tl tltt:
spc~ '.flt~ t~'ti~it.y wr~,c; t'Xp*t~C`t~t~, Chitlf:.~ t~ v~, p~ fn." ~
30 t.rt~l;'tt?lv ~ A~'ttt W~lfi by f f~ prt ~ f~1lt ' I~V~ L~ hi>~Jt~ to ~ iU~ L
t~ t'`h'; t",i,l'~St~ Tl ~, .qtt~ C ~rorn b~ f:y (~es~ nt5t shLs~ r~n ;~rt;~
Iy~t~ t`f~ t~ )cht~mg- ~-l en.Y.,~ .; s~.~" L~ J ri~ ir. ~ f~t'~ I C
Me thods " be low .

3~45(biool/ls/JKM~Al2/l997 tr7 ~f .
~' 13 2~ 77 ,h t . ~ V ~ I ~ p ~ i o n, i ~ ~ 1 f i3 ~ 3 ~ E ~ t.
p ~ o~ rt ~ e~ h ~ 7N~ P ~
p~lXt ~ c~ s ~ he~ ; d~m~t~n S-~ ''pi'¢ j.~ y ~h~ pa~ n ~Q~n~ P
f~ cti~n~ &ln c~ he ~ s~ e ~ 3. 1~ 5 SS~ U~ f~ ie's i~ prer~q~ fc7~ ~L1~7~ ¢)ly~
~xpr~s ~ X ~ eq~ler~¢ ~ t~ r.~ipù~ e 0 ~ ~s~ r~
w~ri~h it is p~oduc~àt che r~ t~ t, origl.i C!~ hc -.;¢~ilaL lc~leL~
s~c~u~ n~e ~ ts~ y ~ ncli rl*~¢~ e ~he l ;`C~i~2 7.' ~t~q~t~ nc:~
n~turhll~t FtSSC~Cl~t'ed with the s~hir~n.lse ~;~ en2 ac ~)rf~ ç~ 7i, eh, ~ p~ t r ~ n ~ ~ re ~ in,~ ;A .~ ?q~ nc~
~C~.c~t'(~ to th~ pr~?S:enr ~r~t~c.io~ N~ q~ t-~n~ e~-t~ , 7~ g3 clf ;h~ ~hit~rt.c~ t~ 4 ~ ;
r"l e~o~linEs th~ h~v~in domi3i.n ,3nd thc~ fur:ctic,r~l clçlrr.~Sl~n o~ r.ht?
r l~et chit:ina~ 4 eltz~me, or an analo~le of c5~id D~A
t~ The rt-rt~ ,uc" i.~ Se~r~l tc) LIS ,~ 5~!q~el1C~ t~ ti~

~i) ts a c~t~r~ct~ri~cir: p~ t of ~uicl Dt;.t -~qucnce, Ai~ ybridi:~r ~ wi el~ h.i ~.cc~ plepe.r~d frc.n~ . ~;d 2 nce, A~ t~nt~o~ fJly~p-~lr ~ . ~ IL.i~:'i .)(.-ir~ Uf' 2û ~ h~ p~)ly~ C~C~ i by ~ t ~ A ~

n~ l y~ -)t t tl~ Krli 7.~d ~y il~
r~ilse~cl ;~ in.~t ~1 prly~ -.r;d~ odid by ~ Cf.~ "~

A ~ti 1 i ~ r:~ in~-~r-~crt~ <il~q~ c f ~.h~ N.i S ~ tl , I onlt) I .( ~: i. It~ Uc 1~ o t: i d~ . I 7 1'! 7 ~ ,. rt i ~ ~ r~
;`5 q~ c~ r~ in ~ Ct~ 1 cr~c~ t~i~ f'~ 3~
!;u(~,nr ~ et: (.-t~oi.t inu~i~ 4 orlzy~ , or ,~ Oj'r~Ur .lr ri.~ !i,'iA X~ t`~if:'r .
The t~ "'nn.~lo~ue' r~ rc to ~ Dt.A ~eq~,.cnc~ ~Jhich ~i~ ix ~ c~ r~ct~ristii~ part of .~i~.id DNA 3cq:~f~

~i $ ) ~Iy~ridi~e~ w~ b~ pal~r~ r ~ }~ .~iii r)~.A
quence ~sabi.oollLs/J~ Al2/~ 07 1 2~s~77 encocles ~a poly,l:ier~ lc~ h.4~ri~ !r.' .'i~le i`~ Clcid iCq~.~e.nC's~
i~S ehs~ ps~ypeptici~ enc~ cd b~ .s3~d l'~Sr~ Cs.~:2rl¢`¢-, or Bi~' 2n~d¢-~s ~ poly,ss3p. i~ ~hich i~ r-s s~gr~ s~ ~y ~r~ S ibDd,-' ~aJs~ed ~g~ins, ~ p~l~ps p~i~le s~n~o~ slsl i~P~sein~¢ .

S Th~ ~na~ 0~ S ds~:tns d b~- ths~ p~operr.~ Ai? -Ai~3 ~nd ~
,~s?.~ ve cSr~ d*fir~cl in ~ ~{lrsilar r~armer ~o ~he ian~log~es s~ he chiti-n~e 4 r~l~A ~que~lce defined by ehe prope~ti;~;. î)-i.r) .lbc.v-In a urther asps~c.t, the presene tr,~etl;iol-, .elPtes ~.~s~ ~- DN~ nc~
~ ;npri.;l.n5~ s~l~ar b~c!et chitina~i~ 4 eetl~ . It~ rhe p~ t~ cc~nt.exr, ~e 1.9 ter~n~ "~e~s:" is ws~si ts~ in¢~icaCe -.~ A :i~r~ e ~kt h ~ vol~ed in proSt~- in&; ~ polyp~prids~ c~l~.in ~n¢l wh;C.~ c~r~r~¢~s r~ ;o ns pIs~cer~irlg an~ f~ OWii1~, ths~ ¢~s~¢'s~r'l~, res~ic~ 5' ~.1E)C t.r¢ ;~ 1 3' -¢.~:~W~l~.t~
q~ler;¢~s?s~ wsitl1 .1!-. interven is~r4Us?nC~ h-~ $C 3110~1 ist~ ol-.~;,wth,~ch ...re plnced 7;~t~tw~-~n indiv7dn.tl c~i~ii;!r, ~.Cf,lr~nt~ (~r~ ~,iilc~; c..on;, 1'5 or ln t:h~ r?' I.l7,~ti~rcstm nr 3' clown!;~re.tri l~t~l<~l, Ihs~ 5' ~.~t:i~:re~m ~ s~ior 5:0111~ i S ~ 7~ f l gl l t, ~1 t s r. y s c~ n~ wl7 ich CC'h tl' ? . ~ fs~ X~ l l 0 ~
~s ~t is~, r y ~ y ~ ~ p r om~t ~ 3 ' - ~ o ~ r r~ zi c)~; c utt~ ~ ~ i t i tl~lf ri( t ~; wl~,', c~ t~ ?lv-~d ' n C~ ; Cf7~ t l~ ' ipi' iS.`;~ IC
~ n.~ .tll,l opLi~ eq~ t~ t-~ c.p( ;1si~>l~ L~tr jt~ .t~ t i~ v~
'20 t riitnCe7 ipt attd th~ n-r;~ cs~id r~g,iS~n An ~V~ ],t` c-f ~ IA sc~qucr~ci t?f S ~ tiO!~ COII~ C; ~ .it~.i.r~
4 ~,t. n(~ ; c~ r~t mi c '~ r ~ . L),'~ t~ f~ t.i~, ~,t iiS,' m~e t~hit;n~ 4 clc~rlc ~I?It t.)~ th~ ;c~lrttio.- of ~ c;~sii~, in E~ s~ . T~ti pht~ tf id~ ,s~ L ;~ "~ r~
'1tt ~l~tt~idllt~ t7-c7. is !;hOWT~ X~qt~tt~ r,it'~ d ~; c~ n l~f r.h~
p;3r~ .~ se~ <~ D~iA !~t~ r~;~ of ~,s~ c~ ir~ 7'"~
~ 3t.~wr~ t~ rtt~t~ ~ nr~t~S~ fllr~ L ~ ;c~ ;ct~l h~lc;~, t"~ rl~ 1L~i ~i?qnsij?:lCt~ of ~-h~ itirlase ~ D~P~ S~c~ Ln ~querls 1 (~ c~ p~ .
sc~n~. ~re~ h~)wn i.n S~luen~P 6) it i~c c~nte~pl~t~ h~3~ clf~o~.~de~
:~() 3'~-35'? t~t~ She t:`oitinZ~s~ ~. gerte s~.~quencæ c-~n~ tP tht~ s~clr~ d~n of the c:hitina~e 4 g~:ne.

R~lSlb~ StJKMfAI'~I~l Q'7 ~9 , lS ~8477 ç d ori .I S~ C~I1(Trr ~ r i ~ o~ } t :l l f r~ ~A :i ~ s ~ s~ f ~ c i2r ~ i T. lf; .
~T.~ f~fl,T Ci~ D~,~ Xs~ cf* r~": ç~ TItt~r~ 5 ~ JWli ir! .~s~q~t~ l;< ~-~eS~ p~ T~ rl~ror~ r. i~ r~vt~J Ç.~~lrl . ~ Ç~ .r~Çl~e ~rA
~:O~tip ~ '; riT~ly ~ s~ ;t~r- A~ f3 C f ~ I t~ Ç~l. t ~ S~r~ r e~ s~i s~,e~ s~ do~ s po.t;i~iorl of th~ ~ntro~ iie~.~d T,,~ e~--~fi spor~ t~ f.~ ~O~iitio~ be~ ~7~r~rÇ~il hJ~T~:1s'~Ot ide~s 2~T~' e~hrl 3C~6 i~7 ~hr r~
n~se 4 CDNT.~ ~?que~SCe sh~-w~ in SequPn~e 1.

~c . s ~ S ~ gt~ A~q~i t~ t ~ s~ s ;- ,. Ç,l~ ~ ct ~ e ~ T. * .~ h~ 3-i Ex~rple~ 17 ~nd 18 below.

10 A~i nils~rlti~tted nho~Je, ~;he kn eà~,e Olr ~ in n . t~
suP"?r ~eet ~:hitiYI~se 4 t!i.?'~S i~ pos~ib1e tc~ ç ~ r~ J?r ~n-i ~ ~ ~IC ~ r~iit ~. t ~ ~e ~ a~?,~ ~ t~t?r. E~;t t r ~ ~: t s he e r?~ nlf~, t~ c~;~r~
Otl the ~a.sS.s of ~ ~omp~rL.con ~itln th~ n~nir,o fi~ld ~e;iu~ Lh~:t knowr ~:hit~ !;t"i. t~n ~ ipe~ kls:- r~ rt ~c r.ht ~
IS f~r ;tlr,t,qnne, .~ t. v~npr~ ?~ th~' C~Ct.i~'~! '.it:~` (~. t?,~ c~, I p.nt.ç;.
c~rn~ti:;lrt~ f'pit.(~Çp('Ç; i,î rl~! f~tlr~y~ç~ ;~nd ~ part r~ n;i6`L-.? fnr t.he er~yrno'.c. snl~ ,r~t.~ `C; ~icit~, ~n-3j~r bin~riin& "co~

l`hæ Ct nt~;lpl.lt~ ~1 p~ç~ n ~f ~ht' A~ t Vt~ slre ~,f i~ç ~ ~ h- ~r. nl~
ns~ .t~ G en, .yn~ ~", ~ h-.~*n ~ d by ( o~n~ t i s-;~n :n- ti, ~ -n ~. ~, r - n~
~(t c~th~r ~ J~mf !i f .~ I'L~ . tr~, t~t ~ 3~A<~l j .s i s ~ f ~},i~ c ~I- c. ~ h r~ !X~ l E~cm~lrl~ b I?l~l~;, 1 r i tl~ t;~ , of rh~ c~ t ~ hi~1n.~.~.. '. .. ~.r~ i",~
r r! ~ i du~? ~. l 84 ( A~ nd 1 ~ . lu ~ .

C~r~ thr l'\.tCi .~.' of t~lr :~D- s ,r.~ t~ r: ~}.ic h r ~ t ~ 3 ~ u ~ u ~ ~ v .~ l v ~ . L l ~.~ y, ~ i; y ~ n n rin~ if lO ~.tc i(i c~ t ~ or Lflt? t?n }~m., i r ~ 7t ~? tr~
p~'f~ti~ f t~ rt~ ~i tjf Lhi~ ~?n~ rl~e ~ if~ r t~ r~ ~t -~t ~ t",~ t~rl,:y~nt ' -i C p ~ >~? ~ ~ t ~ t l f~ ~ ~ i o ~ ~ r ~ ;. c ~ t ~, j c. ~ 1 CJ ~
P~hf.~ thf' c~ tirlo ll~lt~!~ oF t'1'1f- ~ t~ t'~ >~' t'~ t r.'l~'f? il>~ if ~f~!.Ly ntt~ t`~tt~ r~ , Ll~ tf'~r~ V~' f~lt1ri~ tf~d ~I!;tt, t~t~ tltinO tlCii`l tesidllcli tol~ir~, ch~ irs,p~ s oi tll~
~n~ tf~ mri,y ht' ~ lC~ tr~f~t~ t`~ t~vtsi~; t~f ~LI~ e ~ t~i sp~?c.1ti~ ~3nin; acids ~t ;s pt,ssi~,lP t.~ .s~tc~.f,.n.-.~'.1y ~o~l~'y ~,hf? ~ y;!~
` ~so ~it~; to ohf~ 3 ~llodifii?d mod~ c,l: ;~tt't-'o~m of ~.h~ e~,~yrnt~ & ~ith .:
:
8294~fx!iool/!ts~ MtAl2~ fJ7 251 '~
' 20~,~477 r f~ r~ `e A ~ ,y ~ l f~: a~C t i'\.~ l P~ t. '~
~rof~lf~l~f~ ;p~ if lf~it.:y, .in .Imp~ f~fl ~ L,;i~ .f', '"~
~),;?i'~ f~ o~i~ f;~d ~ pe. S~h s~lc~ c.~ . 'h~y ~ p ~ '.
o~ we~l-k;~ow1~ prlnc~pf~g o pro?ei~ e~:gi-:eer~,, C Icl~ ~s s;~ -d~ i:e~ ~:e~ r.~g~lesi~, e . g. f~'i descl~1bed in~ '~pif~ 16 ~. elow .

A s ~ p l f~, t?~ e p l ~l c ¢ ~e ll t h ' i r ~ ` L~ C; .- ~ 0 5~ ~ T r p r ~ s ~ d~ ~ f ~
pf..';i'; i~'~7) ;7~3, 2~ ~nd 207 w~ ~h Ty~ fesid~;f ~ is *.~.p~ 'i t-~ cl~ r~,P ~he t~f~ f~ S~bfi?~ t~ r~}ti~j ~.o e}~ .J~y~ ail~ pech.~F~
tl~e sut~s'~ate ~ipeclfici~:y Li~cewi~e, ch~h~,t~ e~ ;h~ -3~in~ 3cid ~c-~sî-O Cil.l~'s ~;~n!~ti~utl2~ .hi- .~cti.~e si:~t.~- o~ ~M:~rlo AC~ s~dlles ~ ich fo~m th~ SttuC~ re of t:he fotdPcl ,?l~;yr~e ;~re {~x~e~:eecl Ci~ lrlF3~lellc.r~, e. jg., t ll~? c.~ lyt:'~c ~ec~ ?st rflt~ ~pe~ - i fi~ t:;,~o.~ s~lbs~:f ~e ~ind1ng? ~ y l~i! f,~un~l e.'! ri~ ~u1i i~ t~rvv~d pr~p~ rtl. ~ v~' t-l~t' C`~ t i~l~J~?
rr?~.?~ii;~ r):~me, Oc ~:t?~.~r!;~, tht~ rll~ttltr;~ i~f' th~ ID~ t~ ~:V b~
r ~ oll t w; t l ~ r~-t C?n t:he ~ t ~?. pt?~ l F i ~:
lel~iir.~ fur~ctl~n r~f t~ resul~inf?, mod;'le~ n~yl~

rJ ~ ,?l~lir\j~ t~ ; t ~ r~c~ f ~ , (J b; ,~ "-,r tll r? ; t~V c~ t l 1>~ ?A 5~! ~4u C! ~ j?~ '' [~ ; t~ . iI i 1. i?, I .. I'~i ~",~ m;~y ~ lt}~t~- 3lo~ or ;i~ c~?lr,l.i~A~ . D?;~ ;lGt)~:~
, (~ j 7 ~ ~ C? t . !, ~ p ~ o t--~ t " c, ~? . ~ ~ ? Ll: ~ I V ~, ~ rl ~ t~ ? ~ . ' . I' i I I ' ., ;. ~.~ ~ ~ I ~1-~h~ r,::: rl~ .t.,.~ L~nct,~"- ,~ r i ~ 5, 19?1, l~ h.- r ~ n. ~l~?r~.
.S~ it i~ A ~ 1 - 3~ ur~ f~ r, r~ ('U(:ti~!t~
n ~ r ~ t r ~ln~ -m~ d ji l, o~ i-, r r l ~ r ~- .t~ 7" ~ f~ ~ t p l ~l n t, r~ p~ t^i ~ y ~ tl ;Irl~? ~ r~t ~ ~- if ~l~-"~ , i Vi t y, '?5 ~ , L~r: Ir~C~ m~ rr,,~, r~ t ,.~.~L
.r t illf` ~r?,nr-~-tir l~t: ~,f ~ 1' i rllr~ c~rr j~ . f~ ,?~
?f~ OW.

I g~rlc ~!lr~ .4 ~ tI`L?;:_'I' Q~ '~oir.. i.~",; 1"-.
~; L ~? ~ f t-~ e~ t, 1 ~i e~y~ c~ J 1 ' t~ . , . s ~ t?; ~
~t~ ~f~r~i r~ CC~ ,inrl~ rr~- r~ >f ~ r~i?, L~ c~,y i ~ pcc frc~l ~.f.~ ~f~ e~: t i ' l ~ tl ~ ~lrc~g~ t ~ t~ a ~ ~ e l ~ h .
t~ .e~ y,y~ scd her~ c.~;e ~ r~st.~ of c'.~ d~ r~c~ ut c~mpl~n)en~ 3~t)~ be~ n ~he ~ ti~ a.~ f'~.lUr.~l~C~ f)(~i~~p-r~de ~ iC~ t.~ r~lr~ c ~ f.1yr!rpr~ , t-,~
3~ t ef~ r~ , u~ th~ cot~p~r;~ y ~t~ r~ C3 .~ f-, 8~94Sabi.lY.)l/lS/.lY.M/AI~/1~91 07 ~Q

':

1~ 2~ 77 e O ~ ~ ~t ~ C~ f, ~ U ~ .. r, . ` ~ . t n ~: .
le~, 19~ô, The ,~ nt~ acid ~J~L~t~ , b~s, ~ 7~osd frc~
r~ y ';:~ c~b~ ed b~ ~ c~ J~n~lo~ l. ~ibl~, 3.:`; ~ 'q'.~5~C'i.n' m~ ls. ~t~ t~ Z~ !O~ y L~ xe~a2:~iy d~ti~5~i~ t~ a~
~" cs ~ i r ~ p ~ t ~ . t~ . ~ i *: ~1 b U ~ g ~Z~ Z C~ 5 ~ u ~ 5~f- ~ ~ r t '_ acct)unt .

ln ~ r~cL~e her~s~ , t~le y~es~n' in~erZi~-icn elai~er to fL 7~ri s5 q~Ze~ t~.t~di r~;, a ~ h~i t iT~é?e`.e i3~ierLL.;~ Ll~ s t ~,OY h~mc! -c~r~ g~ t: ~.hit~n~ , rn-."~ s ~ :
.i;et,ltLel~,c~.s ~;ecJt,*r~ :nd ~: t~? Ui~ 2C~~no~ uc~ P~ .`t~
bt~c~t c~ clr?~ r~c~od;-d ~oy t:he DN.`~ r~t~ri~ ~? -;2:(;'41t ;1~ s~ t?n~t~ 7 .
T~e r~ini~ r~:c of 2~o~olt~ of .~ a~it. ~,0~. h.1~ C?~II d~ (.)n t~l~? b~ f nt~ is O~ a ~apf~ s~ d chi~ :sc ~ f.";l ~rl tlil2 ~ 5 n~r ~ir~ ~rV~ I) wl~icn hfl!~ b~ ~?n ~hown ~.c~ b~ nc$ `.n t n~5 X~5~ r C~ Ar;~C:~ 4 s~r-.lo~,lcal ''lfl-`,': (~r~ C.xnn~ ? It ) Th~ cl~,;r~ ~f hc~ n-~c~r~y 1~ 0 ' ~ C~ )J~;t! ~ ic!l do~. n~>~. t~ClC~?~ t~. C~ f~
4 ~ 7 ~ t C ~.S tl'lf? mi~lx i m.~l Cl~,r~? ~ ; e ,~ ( 5~ ?p ( ' :i b l ~ tJ~r. ~ p~ t? hr? l t:>np~i r~ f~ ?. ~. t rl~ .~ ~ ~ c 1 ,~ .c .c;

;C~ r ~ t~r hot2lc~ y ~irh ~ C;l?? t~ 7~
f~r .~ .Io~r ~If~f~ hf~mr~lc?~ tr~l t2~r C~tLI).'s~.q 1 ~'rl~:Vf~
r,~ r2 e~ r !;imil~rir.y h~ Sh ,1~`2 .;~ ti~S~
s ~ t ;~ tl ~Ibr~v(~ prf-ft~: R~-l y ~ 1~:
W~ l. 65Z, f f~, ~Slt`. 1 f~ 7n~. 11C)~ "~ r~
`r~ S~1Y R~ c~ ;r~ri~l~ L~ ,rl~ b~ .t:;~ f.

"(' ~ C)Si ~,r~', Stl~t~ olr~O~ t`~l tt~' t;~ t\.~
e~ y~u,.~ ~t~ t~r d ~iy t~ s~ t-f l~ 2t~s~ S-~q~2~

,~,y~ r ~ f ri nN,~ \It'l~ -' s.'r~ t~
~30 ~lL~illVIO~ r~; stO t.t-ie cU~ be~c t~ ti~ ! 4 ~i:r~/u~ d 1~ Cs~ OI.
s~tp~ ;Vtl~ t~L~ ,;;lt bt ;, t. t'hS r.i~ ;r'~2~ S~2r.
5 e q ~ c ~ h i t 1 l ! cl ~; t ~ 2 ~ ~ e c~ ~ r~ ~^, c: ~ t ~; ~ rS; " ~ i O .~ r2 ~ J
~orlt,~inPt~ IY~ ttlr2 ~"~n~SIn~t~ t`,lOYli? /~`hi iir)tlL~ .'}ir;~P~! (J!, c~ c~.ibl.~tl i, Ex~pl t~. 5 t~5a~ ~c~ Rl2~

lg 2 ~ 7 7 ~ ro~ E~iLI~pl~ 10 ;. ~ i c~ ent ~.h~ , 2-' ~;' .'~.'~ ' ' '1 1'1- '' ' ~. ' '. ~' ' .i '' .'` i !

.~U~,~3 . ~ r~ h i t i ~ !'C,~ C"~7~ f ~L~, thL~ }~r~? .~ ? ~t i~as ~? /^ ~ r~ 4 ~.h ~ e~ t:~:an ~ h-~ s~ ch~ ç 2 ~ n~ i~ r~?~it:
r~l~s~;e~ o~ h~ it~ s ~ L-~ ~-)n r?7n~rl 3~r.~ ~h~ ~t~e -pol~p~ptitle.~i b~lor:g~ng to th~ hi.-.-irl~si~ S Ea3~ ho~
r e rl _ ~ 'L C~ t`c 4a t !_ ~? ';' 71 ~ lC ~: O r d i i lC~ ^ Ci ~ t- n i O ~ r ~ he ~
0 s::OrQpr l ~je3 h ~NA ser?t~r~ce which cn~rootes .;i tlo'L~ d. ~-h~. ~`., i s re;~c,~, 7ii":ed b~ a~ it.~to~ly l.~iser.~ lit~ ;rt~ -t~,~.' ' '`i,~'_t~ ~t, ~'. i.- n~t b} ari ~;t; ib~y ~ ~ i !; cct ~l~d i t.'lC t !.~ ll 7 b~ It~ { ~ - t~ ~J
urt~lr~ ~,y~t:, r~ ?c~nL i'j~V~ ?.~ ii~ r;
,C;~7`~Ut!t'~Ct' COm;,~ C1~flU~nCi~ Cl'.'fi.~ ' t '1~0~'? ~ir\f\;' i.~ :in~; t:t r 15 ~.ti~ 'r~ NA !;e~ rtl~c,~ ~,r g~rlf ~ r~L~ ,;T:,~ r~ liC~
]~ !;' t~T~ t)~ r~c ~f;~:l d~t. ~ t~l-f~ if ~f}\~ r ir~
~,r~ t~ 3~ ~;r ~n,.~ 12f~ ll tr~r,t ~f t~ "~t~rl ~"~ rt.i~3 ~;c~ ~s r~
~7~CO~ yp~pt' ~ h~ r~ n~ ` I " i~ 'tl~ n~ 't. ~ ty ~>f t.t~
.~,t~lg.~r hr~r. c:h~l'in~lr~C 4 ~ irr~ r~ i!lr~ t ;Ir~ti~ MI rf~cti~ y r~r., ,4 ~ t t o t ~l r~ yJ ~l L` ~ 6~ T 1: ! 1 i \ i r l ~ L~ j~L! ~ ) r r~ 3 r ~.~ TI ~ L ;l ~, f T-~ r T't ~-r.~ c( ~lu~ r ~j~4 ;~ n.~ -L~ti ~ t'.l t~;.lr .'1 mf~ 'ir.'(.l j~?Y/~ f' ,~
t ~ f'~r'~ o L i. Ult~ ., t h( r~

t~ b~ >~ ; r~T fCt. nl~f3 . ,; .~ rh~
- r l rl~- c ~ r~c ~ L t ~ " , ~ L L; ~ t Tl~
t. ~ C j ., ~i l L~ i t i r ~ r ~ u" ~ r ! ~ t ~
t;~t ~ hr~ r ~l rt ~f ~ t,~ ". A ~ ",- ~ , t~ i ti ~ t t ,,~ ,t-nt~ o t: ~ c.~ r ~ l e l i~ t ~ ol-,l .: h c ~ ; ;t . .~ l s ~ t ~ `, ~ ~'j'.i :' ', ~; ` ' W~
t~?~ e~ o ~ ,;l~nc~ ~r a~ J ~ t; t~ ' t: .

A ~ d;~led ~3~.4 s~ t~e (:~.,t~ le.:; l, jjt ''~ '.lf.
35 by ll~;e c)f s:Lte direeted ~ c~ n~

t.101/~S/nC~/A~2~ t 29 ~9 20~77 ~ ! l c S ~ vs l t ~ t~ r ~q !~; p C C ~ ?~ r s~ r ~ r ~ r~
r~ ~ c~ 7~ 7~iA s*~iu~A~ .. S~;f~ls ~ r~ Ji.. ~ pf~l vpf~ s?
t.~?~7C7-; r~t~ti r~k, bi.~. w~,it ~ r~ 7`'f~!''~'.': r~ ~
~;~.lg~L ltjS`f~ 3 if~ S. E'~ '`.f~f~l~,?~ 7s.~?ri~ f~ `f~S~ h.
S c hi~ a~;f~ ;i fr,`fi`~ tr~lU~7`1~:~` t~r f,~ t ~ g~ ri;~ 'f` -q~ . t~ ~ C~ .r: ~ ~ 1 J ~ t~ f.~ i rVh : j~4~i f -r 1~ t i A~ f, i tlr6~ .5Uj~af`' l~t,':~. c~ ;e b ,-~Y,~I~t ~ ,',,",t~ ' ," 7~
q~ss?f~t't~ 1 ~; f~ ttt.~,f~ t~equ~:fiAe Cr~r.~ 1 i'' ;T~ h~ ~c ~ t~ if~ `~
b~e~ ~7~ in~S:t~ i~ e~z~ s g~ t~r~ aA ~ ?,~-~2~, t'7St~ `.g p~pt~ e (~;h,~ 'r~ e f~f S~-'flt~. s~.b71~,renS~?~ " ;t t~C~ 7ir7.~ ~i's cbde ):

S l~^F^D ~-L~N-A-P E T V A ~3 N ~ ~J-T A F F'~

T'h~ ;f .~nc~ is~ r ~ ~ t ~ 7 7 t ;~ rt;lll L l l r~ p~ i f i r> t~ t ~ ~ ~ p ~ r ~: 7 l~ t V ~ ~ } ~ 7- 7~ t~ S ~ t-t i n ;~ Ex.~7~ t~ 7~,7M~ ~ t?qUf~ "~ rn.7,~ ) .4 .~lgr~rl~rlnt .7t7r~ riiç~ t~:~C ~ lr~ ;rlf, ~ t: l-7;t~'i~ .
.t:r~ s t~ f impro~ },~ ?~ r,~ f i.r~y, 7~.01y7r~e~t~i-t~ t'~ r~lt~ mt?rr~, t~ r7~?~ ':'"1''' ~" f n ~ 1 r~ r~
o f L~ t ~ ?~ Dl~ f ' t l ~ rlc c ~ y, i~ J :.~ f r ~ r ~ t. , I . ~
r~ ~ t ~ t ~ ?~? C ~ W l 1,~ t i ~ t ~- u~ r ~ tr t~ v~ t: t~ Ll~r. ;~ , r ~ f7 r"~", ~ t i 7 ~ t ~ , t l l s i f ~ . t.~ ~ f v i. ~ C ~ ' t l . ,~
sh~ pt' l y~ ] ~ ~- i l t t~ f rh~ 1? ~ hl -. . I .~` . t ~ (,' i i! ,t, t~ f. I f~t.~ nlt~ ,t ~ : L~? ;)~ .. i C ~ '.'' r ~ t ;
2'~ i~tcr'~v~ ir.

-r~ r ~n~ ?i~tA ~:Pq~ t~ r~r,lr,-!i;t~ , . F 'h~ ~ t~:i .~;.1' .~ '~rt vr~ t~?,~ t~ ; r t-t~,t1 1 llf, rh~ 1.> ~ t irlr~ lirlo ;~i(l ,C~ nr~

S-I-G-F-D-G~L-N-~ P F.'l'-~ r? A-~7-T A ~'-lt :jO ~.~ ,c~t~ rr.~id.~ ti.?~?t~c~d f~ r?~.t ~.~qll~r.c~ t.?t~ ~t~ t~ lt.-i'`l~; I.J~lll~;t'.~ 7~? ClC?7~ lC7t~1 irl S~q'lt~f~ ?? ~'tt~ 1 C~l fe5pr~ I t~? tll Q,7:~

s~ tftn~ oE t~r~ pept~de ~-?2 gi-'it 8.?r?'t'.-, jrs t.i~ pt fc~ th,~ t t~l~ t.

82W50t?iO(`?l/~StJ~CM/A12/1~?1 t~.7 ~

2 ~ 7 7 . ir~ p,o~ t ;.~* ~ p~ p~ h - 2'' .ti fr~ l o~lng prpt ~ ~e C: P-~- Q I T W

w~.ich i~ ~:h~ f-r~J, -~ pept:J ~2 4 .1'.~ L' ._~ f)~i ~;?
q~ e~ o~ , t ~ y'~ p~r~ tid~ ~ - 2~

T-A-F W-F-W M ~ N v 11 S tt~T-~^M-G-~-G ~ G ./~ S I

?I r~ f; I~ f.~ :C Y i L ~ E~ L~ . T?~
~c~rlF~ >r~ r~rl l~w~ re~ t~ T~ :it.~le:

~ -`¢`.; f ;~ ' t,f ~ t"~"~ 4 ell~y~n~
Exnmp 1 f` 16 be l o~ .

,t.~r~l, t s~ )?t?l~ ~r~ r~ ir~ 0'~ -JI ':,;, t~ r~
t i.~jr; i '; f~ .tL~ vl ltf~ t~ C~ r '~ f .~ f ~f;.
c~f~ofJit~ iti~ Ulr-7.'i~;i'1,6 rl~ c~on~ t.~ t~l''~' t II i t; ~ e.1~ r~ n~ tt~ c~ r .~ i f ~
;~c~ t.~ t~ Dt~A '.~ f-ll- f ,tr. r'~;';` Il'-i~'.?-'.C ~

~;o~l ~ 1. r,'~A l~lf.,rl~;ir~, t~.. rl~ "~ t ~ i f=~ "n .?i ii , U ?~ r~ ? t .tclf- f . t,' t~ t ~ J t ~ , C C~ f` i f~ ~ 1f` i l 1 ; t ~; t l .i r ~ ~ ` f~ tt~ t l ~ l l r ~ ~mi l t c~ t ,~ h ~ ~ f~ L . l t ~; ~ . `: i t t ~ ? i ~ T ~

;;~S c. f n ~hitin col~urm".

,l~,"~ -f ~ ~ r~ t ;-~?~- h;~. tll ;3-?i~ `t '~ fA~ .rn.~ i , .~rn~ ,r~. 't~ bf.~ ry ~ ~! C i il~ i f~ hl ~ t, r~ `?l'J~
i r~ rl i~lt~ r.t: ~i i t~ i.o ~ r ~ t~ :-; ;J~ .t.~ f~
~ .; f.` ~ C ~?t? rb~ C .t--r~ C) ~ ~: " ~: t . 1,~ L
.?0 ~ it~ r~ tnfr~ r ~1 bC~tl~ ~r? r:f) ~ ) . t ~/It~ 1` .. i t~ Q~
`ff~n:t~ ~Ji~t ~ r~-ty~ .it~-bi~)itlf ~-~t!:bi.i'~y Cl? i.~?~ t~
O~J l *.' .~f. ~ r~ i h ~ tl!~ 1r~ f~3 i ~ i 8..~i t)QI/~ tl~3~ t'~ '-f .?~

F F ~ F l ~ E L-: E T~ P . l ~ L

21 20~77 irsert:ion of the PNP~ ~eq~ence eneoding the h,e~ein do~2in of s~r beet ehitinase 4 are chitin~ses o ~h~ n-he~ein class or cucu~h~r clct~s (e.~. the sugar beet chitinase ~E disclosed here~n).

A fur~her interestin~ subse4uence of the pres~nt inYent~on s a ~ubs~quence of the chi~ina~e ~ ~NA seq~ence Sequence 1 encodin~ th*
le~der peptide of chitln~se 4 o~ an analo~ue ther~oF ~hlch ~ybridi-zes to ehe chitin~s~ 6 DNA ~equence at S5~C un~er ~he condition3 specified in ~Materials and Methodsn under the headin~ ~Ideneif~ct~
tion of DNA belongin~ to the sugar beet c~ietnase 4 ~ene f~m~ly" and which is capable of di.recting ~ passenger polypeptide eo w~ich it i~
fused o~t of the endopla matic retlc~lum of the cell in which rh~
f~lsed l.eader and passenger pblypeptide is prod-lced.

As ~xplaln~d nbo~e, epitopes of ehe sueRr bee~ chicina~e b enzy~e ~.~y be ~sed to raise l~onospecific polyclonal and ll~onoclonal ~ntibodi~s which are u~eful in ldent~fyin~ chitinase 4 isoenzy~e3 belonglng to the chltinase 4 ~erolo~i~al class and for epitope mappinf~ ~t~icable epitopes ftre expec~cd to be fotmd ~mong the hydroph11 tc peptides of the chitlnese 4 amino acld segl~ence Sequence 2, b~c~uce these pep-tides see~ to be sl~b.stantially dif~e~ent rom pept~d~ p~r~'s of other chitin~s ehan su~ar bee~ chitinase 4 Anr.ihodie~ (either monoe.lo nal, monoxp~c;f:ic Ot- polycpe~ific) m~y bc pr~p.~re~l by u~ of CO~l~'eTl tional ~e~hcd~ .g. .~.~ de.~cr{bed in ~he ~teri~ls .~nd Me~hod!; ~ec-tion helow on the bn~s of sysl~hctic~lly pro~luced peptlcle parts o~
th~ .~u~ar b~e~. c.hitin~se ~ rn~.y~e. B3se~i on a convcntional computer 25 AnJllysl~s of ~he chitln;~e 4 ~NA a-ld Amino ~ci.l seq~lence, th~ follow~
ing possiblc epi~opes of th~ ~e~uence h~tc beetl i~ nt.l f~ed Peptlde 1: AGKRFYTRA
Peptide 2: NPSKQ
Peptide 3: GG~S
P~ptide 4: TARVGYYTQYCQ

The~ ~pitopes are belleved to be particul~rly sultable for the produetion of monospecific antibod~e~ ts suga~ beet chitinase 4.
Peptide 1 and Peptide 4 ~e believed ~o be the most suit~ole peptide .
,, , .~

~ Fr~ EEE r~ F. ! ~-' 22 ~ 7 7 ~equ~nces to be u~ed in ~he p~od~lct~on o~ morlQspeciic ~f,~lbodi~ ~o chitin~se 4.

A DNA seq~ence compris~ng ~ quence o the present i~lventio~ in wh;ch one or more nucleotides have b~en ~odified, e.g. ~s ~xpla~n~d S abo~e, an.i having suh~t~ne~ ret~lned the function ~nd/or charac-teristics of the subseq~ence sho~ld be understood as be~g ~lthn ~he scope of the pre~ent ~nvention As ~entioned nbov~, b~cterial as w~ll a-. plant chi.i~ses exis~. In the ~resent context in which arl importane ~se of the ~NA sequence of iO the invention is in the const~uction of g~netic~lly transfo~e~
plants, che mose interesting types of chieinases are belIe~ed to bP
plant ch~tinases, And flccord~n~ly ~t ~s preferred that the D~A se quetice of the in~ention or ~n ~nalo~,ue or a subseq~tence thereof i5 o plant origin. ~.pecially interesCing ylant chi~inase DNA seqtlences Are der~ved from a member of th~ f~mil5~ ~enopod~aceae, SolAnaceae, ~piace~e, Br~ssic~ce.~e, Cucurbit~ce~e or F~bnceae. Ex~mples of 5~ch pl~nts ~re corn, ~lfAlfA, oat, wheat, rye r~ce, h~r~ey, .cor~,hum, tobacco, eo~ton, sug~r beet, sunflower, c3rrot, canola, to~to, pot~t~, .soybean, o~l ~eed rape, cabb~ge, p~pper, lett~ce, be~n and 20 pea.

Th~ terms ~sequence~, "s~hseq~l~nc.~" ~nd ~nalogu~ u~d h*l-ein with rCspcc~ to ~equence.c, .cuh.qequence.c And a~ o,ues ~ccordln~, to r.he lnveltion should of cour!;e be ùn~ler.tood ~s no~ c~mprîsin~ these phenom~nn in th~ir n~turAl envi ronm~nt, h~lt rtth~r, ~ n ~.so 25 l~red, pu~iie~, in vi~ro or rccombinant form.

e chitin~!ie 4 DNA sequenee of the Snvention or .~n ~na~ogue or ~ubsequen-;e t~reof ~s deined nbove nnd e3pe~inlly ~l ;in~le !;~rnnded DNA or ~NA sequenc~ which is <;ubstnllti~lly comple~entary to either strand of sueh a D~A sequence ~ay be ~Ised to lsol~e cor~espondin~
sequences ~rom other pl~nts, whereupon ~hey, if desirAble, m~y be modifîed ~s desc~i~ed he~ein.

From the abo~e expl~nat~on ;t uill be cle~r thst the chitin~se 4 D~A
seq~ence of the in~ention or an anologue or s~bse~en~e ehereof may 8~S~i.~OI/LS/JK~/A~2~1991 m 29 .

23 %~48~77 be f~sed to one c~ ~o~e seoon-l nucleoti~;e s~ n~ ~c; ~nc.o~ne ~ s~e.ond polypeptide o~ p~qrt ther~of unde3: eonditloFIs whieh ~nsure th~r i~r leac~t p~rc of the l)~P~ sequence of 1;he 1nvent.ton ~s~ ~xpre~:sed in c:on~lnctlon with the o~her nucleo~dr. sequence(s) j e. ~,. in ~he for~
of a usion protein. For instQnce, ç~ I)NA seqttence of the inventior encocllr,&, ~ polypepti~ havlng the ~nt~f~nga1 ~etlviey of ~he ~ug~r beet chitin~e L en~y2ue IDay ~dvanta~,~ou~ly bP fus2d to ~ C-~rmin~l sequence erlcoding ~ ~ignAl pep~l~e which gives rlse t:o t~ sporL of ~l~e fl~sion protein express~d tkerefxom to specific org;~nell~s ~f ehe 1(~ orgRnis~ expressing the polypeptlde . Signal pepti~es involvf n& trans^
po~t will be di.scussed in further detail below. incerestirlg subse-erlcf~: o~ the ~.h~tina~;~ 4 ~)NA seq~l~nce, .~lch a~ tho.ce (le~eribed élbove, ~.g. a ~lbceq~lence encoding the hevein domain an:l~or an epi-~ope, m~y likewise be fused t;o DNA sequences encoding othe~ proteins, tS .cuch iq.s en7.yme.~:, e.g. c~itin.tses, in ordcr to coner to the p~oteins thc desir,lbl~ prop~rti~s of the polyp~pt df~ ncod~tl hy tha ~ub.~-ncex of t~l~ chitin.,tsc 4 DI~A sequencr~.

Also withi~l th~ invention is. ~t polyp~pt;d~ ~ncodod hy th~ chit:inrlse DNA e;equence or ~n atna~oy,-lc Or sub ;r~q~lrnc r~ ~hcrc~f rl~, clt~in~c~ abo~re, 20 pr~f~rtthly in .n non-n~t~r.llly occurrin~P, or rec-o~ irl.lo~ fc-cl}l. A~
comp~rcd ~o î~hc ~ tturnll~ occ~lrr n, ch~r.lnAc~ n~~rl~ hr.~ 1~lyp~p tld~ f th~ ~v~n~.ion h.~s th~ ~clv,~rlt3~ llrtt it ut;ly ',~r~ ~a~.;ly pl'o-CIUCCd Ln 1nL~C~ c~ n~iLic-~ by u.~c~ of w~!t7 kn~ n f~onv~nr~{on~tl rf~cont~i-nnnt produ~.ttone. t.~chniq~ s, e.~ i cl~sc~i~erl in ~;u~ Lrjolt ~t ;l., ?S 1990. ~rlcl thi~ it ntr.ty b~ obtninod In n form which 1.~1 fro(~ f~orn im-pu~lr1~ n~-m~lly ~4sso~ te~l ~ith tlle n~ltur.llly oc~ r ;r~ r be~
<~hitlr~ 4. Th~ polypeptid-! oL t~e inv~ tion mar l.~ cort-.~titll~t~t ~ ;t)~ifun~,.l Ct.)ll~C~ iOI~, C.~,. ll'i C~ ;c~i~ol.`r~ ~.)c~ W.
As ~t ~ xt~ n~ bove ~,cl frl t~ yle~, ~o ~o~ w, t~ gi;
30 bcct: chi~inn.~ t~ cnzy~ hrt.c hr-(~n s~lown t~, h~v~ n~un~f r c~f ;Idt~n-t~g~O~IS p~oper~ s lnc lucling, a s~rprlsin~ly ~ligh an~lfw~ '. act;ivi~y ~s, co~ltp~re~d to oth~?r known chltfrlas~ uch ~ other known ~ugar bee~
chitinac,es, probably due eO iC5 d~l~l chltina~e~lyso~y~ne ac~.lvity and 1 t.~ cotnp~ct str~ ture . Al~,o, th~ s~:rc,ng h~v~in dv~ain l~f ~ ? r.~gar 35 beet chltina~e 4 en2yines ~dds to ~ts ach~an~a~eo~s propertle~g. Thu~, the ~lse of a DNA S~,qt~en~e ~nco~lng the $~gar be~t chi tina~e 4 or ~n ~45tlbi.001/l~e./ll~M/A12/199~

. .

''`'L- FFi-l~l Fl[~1 jlir ~ E:~-E Tl: lt..~ I F.:-~.s 24 ~4~77 logtle ehertiO$ f~n¢:odinK ~ polypeptic3e h~l~in,g he ~nt:f~ L~ c--tivlty as dein~d abo~re i~ expect~d eo be very int er~?~tin~ in the constr.lctiotl o genæt~cally Dlodiffed pl~n~s htlvin~; ~n Incr~ d re~ nce eo ph~tnpslt:hogeni~ f~ .gi tl~ co~p~e~l to l~n~ nsfo~m~d 5 pi~nt; c ~

Accordingly, in ar~other i~por~Arlt ~spe~t, thæ F~esen~ inve~t~ on ræl~tes to a ~enetic ~onstruct co;:lpri~ing 1) a promot~ funceion~lly conn~ct~d to 2) s~ DliA tiequence co~pri~iing a chitinilse ~ DtiA sequen-æ or ~n ~n~^
10 logue or A ~bseq~.~ence thcreof Q!~ defined sJbove and ~) s~ L~s~n~;ript ion t~rnin.ltor f~nctionally conneotecl to t.he ~NA
scq~ence .

enetlc con.ctr~ct ~qy ~e ~ ed ~n ehe c~n.tr-uc~ion c>f t~ eenccio.ll^
ly modific~l plsnt in order to prod~lce .~ plAnt ~iho~inp, ~In in~ c;ed 15 ;~rlti~n~ DCti~rity R!i d~tcr~ d ~y ~'t~e p~oc~ Le gi~ r~ .'x;~in~
nnd thu~ ~n 'Incr~ ed re.s~ct~nce towsirdc phstop,l~ho~,en;c funy,i.
~lcthcr1tlor~, it is ontc~pl~ d th~t ~he p,L'n~'ti~ ~OrlSt~r~lCt m~y b~
n,ed ill incre~ th~ c hi~in~ ing cap~bil j rV f .~ p1.~ r. An ~ mpk- "f .~ gen~tic .Onst'r~let t.S def{ned ~bove 1s ~v~n in E:~a~ple 18 below.

F~rthrrmctrr:, expel-{meI~es h~ve reve~-led (vi~fe ~ .Dp~e 2) tttl;~t wh~r phyt:op~th~,en{c fun~ c~ n~ T. vir~t'e~ nre Lre;I~-d ~
compo.~itior~ co~ ?xi~-:în& ;t polY~ e ~ ving the ;torIf~ et~ 1y of thr ~t~ r bcet chitin~se 4 in ,~d~ixture ~ith ~n ~cidit: chitilla~
~S a~ 5i.c a-1,3-Rl~lc~/n~se the ~row~ rd~e o the fun~1 hyphnf is dr~.s~c.~lly recl~c.e.l ;.tt~d rl~- n~ber of ~errn;~.~tlng ~pore.~ ~rc: dccre.
ed. 1n this connection, ~t ~s conte1~plnted ehnt the syne~is,~ic effec~ ~ill be obse~ved in gene~Al ~,rhen ~ he ~-lg~r be--t ch~ r~lnhs~
~scd in comb;na~ tn wl~h o~her chltin,ases aT1d ,,3-l,3-gl~can~
preferably ~f plane ori~ln.

' . 8~t~i.~i~s/~cM/Al~t~n ~'~t r~ f~ l-r ~ EF E T~ -J~ 3.~ F'. ~

.s ~h~s, in another i~portant ~spe~ e pr~ent lmt*nt~ori rel~a~es ~ a ~en~5c construce co~p.ising on~ or mo~e ~opi~ of A DNA sequence a5 deined ~bov~ co~pri~.;ng ~he chitinase 4 D~A ~equence sh~wn in Sequence 1 or ~n analogue o~ ~ubse-quence thereof, one or ~ore copies ~f ~ D~A sequen~e enco~ing a polypeptide havingthe actlv~ty of a second chitinhse diff~rent fro~ ~he su~a~ beet chitinase 6, an~for one or ~ore copies of ~ DNA ae4uence encodiny, a polypeytid~ h~v5.n~ ~-1,3~glu~,an~ ¢ti~5ty, ~;lCh of the ~NA scquences being func~ionall~ connected ~o ;I prolnoter antl ~ eran~eliptiOn t~r~in~tor capable of ~ressLng ~he nN~ se-quences into functlonal polypeptides.

The polypeptid~ with ehitinA~e or ~ 3-elucc~n-lse acL~ r is pr~-1~ fYr.qbly of platl~ s)ri~in, The chit.in.ls~ ar~(l ,B~ ,1uc~n~.se t~ce1~
may b~ doc~r~Ined ~s cxpla~nrd 1n t.he .~:cc~:~on "M, tl~ri~ls ;Incl M~hods"
be low .

n~ l~..rt:icn~ r int-~r~xt i~ n~ti~ coll~truct ~o~l~pri~ir-g l~)t~ 0~l? copi~.~ of ;I DNA ~ q~lel~c~ i~ ,v~ ri~:;r,~, r.h-'>O ~h~ t.i l)~ t I~l~iA .~ t~ .c11~,w~ t~-~n~ n nn~ 7,\l~ or ~b.~c ~cnce thoreof, ~ne c~r more. eopi~.C ~ DNA sc~q~l~n~e el,cod~n~ n s~1dl~ rhit.in~
ha~in& ~ pl cqu2~l to hr lec.s th~n 4.0, rlnd one <~r mor~ ~op~.e~ of a I)NA s~tlenee encoding a ~)a.s~c ,B- l, 3-gluc~nasc ha~i~g a pl of at le~st 9.0, :
cach of ~;he DNA sequences being fttnctional!y Cl,~nn~ct~d to a Frrolttoe~r ~d ~ ~rAnscrlption ~er~in~eor ~p~ble of ~.~pressSn~ NA ~-q~lences ine~ ftmction~l polypeptides.
., , :-:

26~ 2~8~77 In rh~ E)r~aene c:onte~st, ~ cidIc ehi~ln~.~'` is ~l~fined ~ A ~hiti~
n~e hA~ing a pI of less ch~n 4Ø Pref~rably, ;hr ~cl~ chitinase i.~ a chitfn~e which hydroly~es ehitîn irlto ~hi~ooligo~acch~rid~s of the h~x~Der ty~e The ~cidic chitna~e s pr~fPrAbly of pIant vrigin.
Exa~ptea ~f s~ch ch~tin~ are .uc~b~r lysoz,v~efchicina~ and Arabidopsi~. as ~ell as the acidiro sugar be~. chitn~se SE having the ~mlnQ ac~d seq~ence sho~2 ~n S~quence S ~r an an~logue Qf sa~d D~A
s~q~ence encoding an acidic chit~n~se h~vlng a pT of at the ~o~t 4.0 and prefer~bly capable of hydroly21n~ 3H-chiein into ~ainly hc~i~m~s In the prr-~sel)t conre~t, the term ~b~sic ~-1,3-gluc~nRs~ ans i~ ~
I ,3-,1~1canase hav~ng A pI of Is~ore th~n ~Ø Prr.~ferably. the ~asic ~-1, 3~ ca~ase is o~e ~r1~lich ls c.3p.lblr~ ~,f hy~lroly?.ing elucAn ~nto mainly dimers, e.g. n~ deer2n~;ned by thc 3H~ arin ~ssay described in thr: M,3tcrials ar1d Methorl~ seccirJ~ bL~lvw. lrl~ bac~c ,B-t~3-gluc~n~
15 is preferab~y of pl~nt origir~ mpk:s of ~ .~ult.~ht~ h.~c.ic ~l-1,3-gluc.a~ r~ b~';;c B- 1 ,3-glucan~ d~or1~1 fro~D tobacco ~Shinshi et ~1., 19$?()), b~rley (Finchcr 1~t nl, 1985) or su~eitr b~ct, e.g,. the b.~ ga~ b~et B l, ~-glnc~-tn.~ h.~ n~ of ~hlch i5 ~,hown in S~ cn~-~ 9 or ~n ~nttlo~,uc th~rc-~f cncod1nf~ n b.~-.ic p-1,3-~ 1a.~
20 h~rin~ a p o ~t~ lcast ~.0 ~ln~ pr~~rably b~ , capab1~ of hydtoly?:-~n~, 3~1 1Atnin.~r1n tnt.o mAtnly ~l~m~r. of ,~ -p,luc~m. Th~ ba.~.ic ~:u~,ar bcc~ p-~,3-~,lucnn.l~;c 3 ia ~lifcrcn~ ro~l otlltr ~l."r ,~ 1nc.qr1.
ir~ ~h;lt it do~ not con~in .~ C-r~rminal ~xt~nsi~n. ï`hr a~vnntn~ uc~
~f f~ ~ ot ~ )g t1~ 3';i~ r h~ ctlnt. ~ y in p~r~
?'; du~ t~ thic IAc:kine, C-termln~ll C~tt~n~ on Th~! e~;pe~ nt ~i repor~e d in E~;1u~ 2 hek)w show th~r. t~he con1hin<l~ivn of th~ sug.~r b~-~t chitin.4.~e rJ ~n<.yme, rnl Ictdic ch~ ~ inQ~c an~l a li.~; je p-1~3~ c.~2.se resull:s il~ ~YI inc~e~s~a 3ntlfun~,AI Acl:lv~ty B'; coin p~re~i to the ~nt1fun~,Al actlvity of enc~ of the cc~n~.tit~ r.s. 'I'hf`
30 incre~se~ ant1fungal ac' ivity obser~ en using rh~c sp~cIfic combinati~n is p~t:ly belie~ed to b~ e ~o th~ fer~:nt mode of acti~>n of the acidic chitlnas~, b~sic ~-~,3-~ e~r~se ~nd ~iugar ~ee~
chitinase 4, re~pectively. When th~ ac~dic chltina~ ls one which hydroly ~ c~.in 'n~3se pr'l~aril~ ineo hexa;~ers (~s co~p~red ~o ch:Lf 1 2~ 20~77 n~se 4 wn;c~ p~ima.t~ily hydrolyxe~: ch:Ltin i~ltO di~*r i? iL~ lc ~-1,3-&,lucna~e ~s one which hy~o1~s~ glUC.`a2'. r~riil~.~r~ly ~ P di~r~
i.~. is ~eli~ed eh~t ~h~se different ~e~ g D~c~d~s IDay be ~n~c,lved in t:he re~lt.ing A~v~nta~eou~ tc~tnl effe~t.

Furthenav~e, ~h~ ~ynergi~tic ef~e~t ob~ined when using R e~bin~tion of S~he ~ghr ~ee~ ~hitin.lse 4, ~ polypep~ide ha~in~ the ~qeti-~fity s~
~;ec~nd chi ~lnasP diferent fro6 chitina~e 4, e . g. an ~idic cht:i -n~se, arld a polypep~ide ha~in~ the act~vity ~f ~ 1, 3-~.luc~na~e, e.g. a ha~c ,~-t,3-~ canase, is believed tc> be due r~ the f.,ct thi3t 1() such combination will ~ttt~ck both th~ ch~tin ctnd ~lstcalt c~nstituents of the &~11 wctll o phytopatho,genic fungi And al~sQ ~rt~ o the ~ell w~ll in which the chitin and glucan constituen~s ~re intim~tely &ro~.s-l.lnke-! to one another. Th~ ,B-1,3-&,lucana~ie fttrt~e~ serv~s to remov~ the outcr ~ can l~yer cov*r~ng the c.h~t~n ~r.ructure of :hitin lS cont~(ining plctnt p~thogerls, e.g. phytop~tthogen{c fun;, rt!sultin~; .in ~tn e~ o.s~lre of the ch~in st rttct~re ~o the en~,ylrt~ic .~c~ i.on of the chlt ina~e .

D~iA ~ uences f~r.&o~in~g tl-e .secon-l ch; tln~t.~e reF~rrcd to nbove and th~ ,~-t,3-gluc~nase mAy be obtalnccl, c.r,. Trom .llr~:~tly knoh~ ollrce.s, ~) or Tnsly h~ ldcntif~t!d snd isot..lt.~d frotn nn;ur,~l ~.ourct.~s, c.~" Iny t~se of t}~e techniq-les disclos~d hereill.

tt: w~ll h~ lln~er.~itt>oti rh;lc n ~r~,~ ntLmhl?r of di ~fcrell~ ~CTIC~it Co~l-.~tr~lct.~ leflnod ~>ove mAy he de.~i~.n~tl nnd prep;.~et~. ~itnout ~ei nn ~X~t~ Liv~ L, el~Tn~TtL~, of t.~c y,~:T~CtiC coll~.t~ crs wl~ h ~;t~,~ b~
25 v~ c~ c ~ r o c~ o~ > t~l~ nNA ~ J~nt~,t~ of ~h ~,~n~t;t~. Ctln~tr~l`r., t.h~ CiflC ntlCl-Or.~de .~equcnct- of e;lC~I ~, t~e I)NA f-qllene~*~;~ r.he typ~ of ~)rom~r.er ~qntT tetm~nntQr con .l t.o DNA sequrnce, nn-l thc ~ypc o uny ot~eL llssoci~t~d S~ llene~ ~:, e . ~
C-teTmiTlill vr ~-termin.~ qu~nce ~de~,crLbed b-low). l~lu;, p,enetic 30 const:nlc~.s of the pr~sent invent:~t>n m~y vary withln widc lir~its.
Normsllly, thc con~b~n~tion of e~ch o~ the ubovc ~ell~iortecl v~rls~bte el~me~ of ehe genc:t~.c eon~tt~lct to be cho3cn wi'1 depend, e.g on the desiTed stren~th ~f the cll~ifungctl ef.C~ct to be obtalned which may be ~eter~Incd as a unction of gene clc~.ag~ ~tnd ~p~cific ~scleo-:`:

8~50bi.00t/LS/n~/A12/1991 07 2~

:

't~ r F F~C-l l r l L ~ J ' ~ E~EE '-'-I t~ r . .~

28 2~8~7~
tid~ sec~uenc.e c~:~ e.~c.h ~f the DN.^; ~e~1encc~l e~nd th~ p;. ~ nd ,i ~ ,th o the pr~v~er ~nd ~er~n~t~or ~ for each DN~ sequence.
Hbwe~er, in designng ~ genetic corlstruct of the in~enrion w~,ich ix to ~e expressed in ~ given orgar~is~ such ~ a pl~nt, ~n~ ~ust ~e 5 ~w~re ~ f ~che po~ible tox~c effect of ~ ~oo hizh expres~ibr~ ~f ~7ne c~r more of ~he proteins encoded ~y ~h~ ger~ ic cons~n~ce ~hch, e.g.r may lead to a lc)wer y~cld ~ f the t.rar~for~ed o~~nls;c, e. ~. plallt, ~:~
c.bmpare~ to an ~ntran~ormed ~rg~n;sm or ari organism not ~.on..aininb the genetic construct . Also, ~heT- th~ genec9 e chnseruct ~ he inven 0 tibn i5 too large, it may be ~ifficul~ to ob~(lin ~ ~table is~rro~uç-tior, thereof ir~o the genome c,f ~he planc wh~ch may le~d to exc~sicn o ~ part of or the entire genee lc con~trucr. from the genome of the pl ant . Thus, the genet~c construc~ should be ttd tpted so ~.h;it ehe expre~;~;ion products thererom ~re gerler~tlly ~cccptshle tc~ the ho~t i5 or~,anis~.

It is bcllevt:d ~ha~ ~t ntun?~e~ o copie~i o 3 4 of so~e ~)r ~11 of th~
D~ t7,uence.~ of th-- eenet~.c con~truc~ o~ th~ in~el-tion will ple.~et~c ;~ tt.~o l~rge t~ eri.~l to be int r(>cll~c~<l i n r.ht z~ on~t ;~f t!te pl~nt .Expre.c;.~1On of ~rot.- ~n~ from ~h n ~enetic conxt- uct mlly .llso prove 20 ~o bl~ too he~vy .I burden or ~he ~ n~ ~t.~sultin~, in ~t.ar~l~tl ~roweh ~tnt~vr re-luced yiel-l. 'tlowever, witl~ t.h~ f.t~r ;ncr(-R~n~, kno~lc~d~,t.~
wi,thitl th~ fi~l<l of pl tnr gen~t.i en~,irl~l rln2" it~prov~d ~r;lnst>rl~lt-ti.on nnll hl~lo~,icnl contn~nm~nt t~-chniqut~s n~ c cl.~vclvp;~l le..~lin~, to ~le possi~)ility cf itltI'O~lUl.~ tr.~t l. rOte;~n ~ rl~tlc ftS~7Jment.~i 25 in,.t~ it pl~tnt wlthout c.lUCtn~, t<~tarttcd ~,to~?l, yit~lt3 t~ s~co~lllJination~
~1 evcn~6.

Ar ~re.~t-n~., R ~en~t.Si~ tctn~trurt l.s prt~f~rred whit-h con~ail):; otlly ,1 l~w t~opi~ t~f tht~ Dt~A :~?qU~nt~t~ t~' t~t! l~lvct~Loll. At ~o~ t i~.
pl~crred ch tc e.tc'tl o chc D~A !;et~uellct~s of the g,ellt t~tc con.~truc~ of 30 the ~nvenc.lon ~s E~resen~ in only on~ c.opy. ~hc~ con/;~ruc~ion o a &enet(s: ct>n~truct cont~tlnin~, Ont? copy of ci~th of th~ D~A seqnence.~ ir illustI~tted in the e~amples l>elo~

.As lltenr.ior~t~d above, a signifit^ant antifung,a} effect ~ obt~ine~ fro ~ prvt~in encoded ~y the o~hitinase b D~A ~e~uer.ce of ~ne inven~ion o-~
3~ ~n ~nalogue ~hereof. Acc~rd;ngly, ~t i~ eont~pi~ed tr,at a ~netio .~

:
.~

29 ~ 8~7 conr,truct of ~.he in~n~ion, in whieh ~wo c~t?ies of th~ chi~ ase 4 D~A seq~sen~.e ~f ehe ~nventi~n or ~n Rn~lo~ue thereof/ ~nd 0?1~ ~vpy of eAch of the DNA ~equen~nes eneoding ~n ~cidi- chitin~se ~nd a ba~tc ~-l,3-~lu ~n~se are ~r~ent ~y sh~w very poeent ~n~ifung~l effects ~hen pr~ent in ~ eene~icaliy ~r~ns~or~ed plant of the in~ent.ion. It is bel ieved that s~eh ~ genetic eon truct w~ll noe pos~ ~ ~oo h~y burden on the plant in w~ich i~ ls harboured. Of cours~, ~150 the ehoice of e.g. promoter used for eaeh nN~ seq~_nc~ will ' n~ ence t~e ~molmt of protein expresse~ th~refrom. Thi~ will be further explaine~
below.

The genetic construct of ~he invention ~s dæ~cribed above may be pre.~ent on vne o~ sever~l ~N~ fr~ments. Dependin& o~ the si-e of the genetic construct to be incrod~ced in an org~nis~ such ~s a plant, i ehe CJSe Gf U plant ts~pica11y by mcAns oF a p1ant rransformation ~ector, {t m~y bf: a(~v~tntngeo~s t~ ~ntr~duce the con~e~uct by tscie of two or more pl~tnt tran.r~fv~rnation v~ctorx, And accordill~ly it ~y be ~d~tnt~geous that the ~scnetic~. t,onstn~ct. l~; present Otl t'./o or ~or~ DNA
fr~-gment~. ~he use of more ehan ont~ ~ec~or 1~: discus~it~d b~ ,w ~en the ~se of only one pl~nt tr nsfor,~ntl~n ~ccec~r is desir.lt~1c, it i5 adva~t~g~us that th~ ~enctic co~-~tru~ i, prcr~nt on on- D~A fr~-rnent, When a pG1yp~pcltlc ellc;o~led by ~he D~ lerlce Or Lhc invcr.~1On t~
~o hc cx~re~s~d in nn or~tn;~n-, e.~. in ~ plnnt, 5r. l.~ d~ ie t~
Lhc DN/~ .~eq~nce fu~hcr csompri ~t~ n~clc~oLi(lc .~ cnc~ o n~.otlin~; a 25 le~lcSer s~qu~n.o . The leader scqurn(-c rn~ he th~ n~turul lcud- r .~c -qut slc~, oX ;~ r :;t ~ n~ tle~ 'ro~i, DN,~ r ~ t n~ :In~)r~ pr~
in. In ~ny e~e~lt, tho l~nAer ~oqt~e/lc-~ is to ~c f~nc.tion.ql l~; ~on~er~d to thc DN~ ;u~nce so tllLt~ tht~ polyr- pt iAe ~xprc~ cd ~rom ~.ho rn~ ~
.~lltin~ nuclt!oe~dt .-re~lence serv~s ~o d~rccr tlt~ p~1y~pti~ cncodcd 30 by ~ht~ DNA ~què~e ou~ of r.hc ~nAopl,i~sll~ic L~:~ iculu~ of the c:cll itl wh~ch it i.~: prhduc~?d nepen~in~ the nat:ure of the le~der se~lue~lce emplc)ye~l, the polypeptide Inay be dlrected t.o spec~f~c l~.~ations of the orgP~nis~ irl ~hlch lt i~; pro~luced, e . g. to Iy.~:o.somc s or vacuoles, or the passen~,er polypept}~e u~y be e~cre~ed ~nto the int~ce1lular 35 room. ~he l~t~r sequence may be elth~r 'l-~ermin~slly o~ C-~er~1nally po~ltioned.
~'~

5~nq~/

, . .

FFCII I F' I [~ I Irl I t ;F.~ F, ~

2~118~277 l~e l~ture nf th~ el-mln~l sequenc~ ~o be ~l5~ ill e . g. d~pe~rld vn th~G p~ticul~ ~r,g~nis~ he p~ here~f, e . g. ~lte speciic cell or tiscue, Ln ~l1LCh the p~lypepcide e2~c~ded by ~he ~A sequ~nce of the ~rencio~ o be pr~uced Anc~ ~ which part ~f th~ s~3e c~l l o~
~Ylother l~c~t.ion ~n the c~g~nis~ th~ poly~.pt4cle i~ t~ b~ tr~n~pc~t~
ed. ~ t~p~cal l~a~ peptide h~ or~ ~f hydroFh~bic ~iI7.0 ~cids and th~, a su~tablc le~der seque;~ce r.o bG u.ed n c~nneceon wth ~he l)NA sequenoe of ~che ln~ren~i~n 1~ ct nucleotlde s~uence o~risin~, a stretch of cod~ns ~rtroti~n~e hy~lropho~ic .~i3~t~ -7~ . Suit~bl~ no~t-10 l'm'.e~ng ~xtt~npl~s ~ ~ le~de~ sefiuence to bc use~ in the pr~sentCOtttext ~tre the N-terLttinftl leader ~i~q~nc~ of ehe ~-lg.~t beet chiti-nt~.s~ I ~nzytne, ehe nucleotide itnd Amino ~tcld eir~ nce o~ whI~h i~
shown in Sequencc 7, the N- ternti.n~l leatl~r ~eql~nce of th~ ~enoDtic chl tin~ci~ ~6 ~.lone ~ thc tllti.no ~cid sequencc of ~hich i~; showtl ~n 15 Sequertce 3, ~.tnd th~ N-t~rn7;nal le~der se~tence of tt-e aci~lic ~o~gar beet chitinase ~E, the ~no ~c~d .~nd nuclet>ti~le ~er~ nce t)f which is shown isl Seq tcnce 8.

F~lrthenttor~, It; IDay b~ ;tdv; nt. get)tts t'~ t~ ;lt lea -i~ on~ o~ ~he D~A
S~ slceS of the ~,~n~tie con~r.rucr of tll~ inveiltio-: u7:ttl~I coutpri4es 20 ~It C-tenttina1 .~i~qll~onee ~no~lisl~; .I s1en~l pc~ idc capabl~ o dlr~ctin th~ polyp~pt i~le cnco~cd by tl~e DNA .~iC~ r r,c~ ro ~ p.qrt r>f t~n ~r~,an i isl ~hich it lti r.o he ~ r~sse-l, e.g. th~ vacuol~ Irhe C-teillliri~l ~e~ lc~ lo;~y b~ t~ C~c-~rm~n;t~ ;r~nsi(~ r~orl~tà~ ;t~.s~ci~.~te~l ~it DNA ~;equ~llc~ ~ ir r~n~ ~ or rn~ty ~- d.~r~re-t f ~ n ch-~ hO~;t irl wl~ t~
2S p,enetlc enn! tl~lc~t is to be cxprc~iied or mny bo of nnot~ er or~,in i ls ~u;p~c1~11ly relev~t- in co"l~ecrio~ ~ritll tl~ hi~in.t~;~ 4 D~A
~q~le~lC~ 1 tll~ DN~ qu~nc~ of rh- h~<~ g~ t B l,l ~
3 both o~ whlch li~ck ~ C-tertuin~1 e~ rl In D~i~ rie~uence~; ~hlh nor~t,~lly ~otn~rises C-~ermlnnl ex~n~lon, ~he r~iltllral C-rel-m~r,ill ; 30 s~qu~llce ~an be re~ e~ with anothcr ~.equesl~e Non lll~itis~ e.~a~ple5 C-~erminal sequerlce~ to he ~nc1uded lr( ~ g~ne-~ic cOIlCit~ct o~ the invent~on are C-ter~in~l ~equenee~i setec~ed from the ~ollô~in~ quenceci:

8~50bi.001/~;tJlCM/A12/1991 0~ 29 31 2~477 th~ nm~n~ q~n<e o.f ~ r be~t ~ t ~1L:~iz~ . r~l-lowin~ polypept~de L ~ C Y ~ T P ~ D W G L K K 1, t~ ~ A R S ~ S S S ~

rhe C-ter~in~l ~n~ o the ~ug~r heet ehir.inase 4 ~rc~ding the fol-lowing polypeptide L ~ C *

the C-terminal ~equence of a be~n e~itinase (P~A) enco-ij.ng ~he follow~ng polypeptide ~ C Y S Q T P F G N S L L L S D L V T S Q *

1~ t~ C-terminal r.e(tuon~e of A ~S~C eo~c~o Chi~indSe encoiling the following sequcnce N 1.1) C G N Q R S F C N ~ L I. V D ~ M *
the C ter~ ;cquence of ~n ~eid~c t~ cc,o chlti~ nco~ing the followin~ ~equence t~c C~ rm~nl~t .~q~ o~ ~f~ y C~hif~innr.~ CH~f; ~n~ iirl~, r.h~
followi~g seq~onoo N 1. D C Y S Q R P ~ , or the C-ter~in~l ~e~n~nc~ of a ~t~iC p~l,3-Gluc;~ r.r fr~m ;oh~cco enco~ing the follow~n~ sequence :
C V S G G V W D S S V E T N A T A S L V S E

~ Th~ cholc~ of ~hether ~ C- ~r~in~l ~fequen~e i~ to ~,~ acl~e~ tv oi~e o~
~Or2 of the D~A sequences of ~he genetic oonstruct t~ e d~t-r~n-e~ e.g. on the b~s~s of to ~hich pl~nt comp~r;~el1t the polypep ~de ., ~,O~ CMJAl2/199l 07 2~

'.'~
:, expressed from the sequence is to be directed. Thus, when it is desirable to control a phytopathogenic fungus mainly present in the intercellular space of the plant, it may be desirable to avoid the use of a C-terminal sequence. When a phytopathogenic fungus mainly present intracellularly is to be controlled it mayu be desirable that most of or all of the DNA sequences of the genetic construct are provided with a C-terminal sequence capable to transport the polypep-tides expressed form the DNA sequences to the vacuole.
As it will be apparent form the above explanation it is important to obtain a sufficient expression of the polypeptides encoded by the genetic construct in plants containing said construct in order to allow the polypeptides to exert their intended function, i.e. to exert their antifungal activity. One essential element in obtaining a sufficient expression is to provide a satisfactory regulation of the transcription and expression of the DNA sequence or gene form which the polypeptide is expressed.
The expression of each of the DNA sequences of the genetic construct of the invention or of a gene comprising such DNA sequences are accomplished by means of a regulatory sequence functionally connected to the DNA sequence or gene so as to obtain expression of said se-quence or gene under the control of the inserted regulatory sequence.
Typically, the regulatory sequence is a promoter which may be consti-tutive or regulatable.
The term "promoter" is intended to mean a short DNA sequence to which RNA polymerase and/or other transcription initiation factors bind prior to transcription of the DNA to which the promoter is function-ally connected, allowing transcription to take place. The promoter is usually situated upstream (5') of the coding sequence. In its broader scope, the term "promoter" includes the RNA polymerase binding site as well as regulatory sequence elements located within several hundreds of base pairs, occasionally even further away, from the transcription start site. Such regulatory sequences are, e.g. se-quences which are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological conditions.
829450bi.001/LS/JKM/A12/1991 07 29 2~8~77 ~ oon~tltutl~e pro,l~o~er~ is ~ p~o~o'r:er which is ~ubJect~d t~o 5U~-stantially no regul~tion s~tch ~ Induction ol repr~ssion, ~u~ which ws for ~ steady ~nd substanti~lly unch~ngi~d transcr:~ption ~f ~he D~3A sequence co ~Yhi~h it is f~ tion~lîy ~ound in all ~ce~e cells of S the ~rg~nl~ pr~ided th~ other re~ulr~ments ror ~he tx~n~r~ip~ion t~ t~tke place is fulfilled.

A ~'regulat~ble pron~oe~l" is n pror~o~er the funccio;l o which i~
regula~ed by one or l~Q~:~ f~otors . ll~ese factors ~ay e~ ther be SUCh which by their pre.cence ensure e~pr*ssion of the ~elevant DNA ~e-quence or ma~, alternatively, be strch which supp~ess thc e:;pre~siorh th~ A sequence so th~t their ~bsence cause~ r.h~ ~1A sequenc.e tc~
b~ ~xpressed. T~tus, the pro~oter and optionatly i~s as.so.lar~d re-gul~ttory seqlle~lce ~ny be ~cti~ated by the prcsence or .lbsencc of one or mor~ f,tctors co ~tect tr8tlSeript10;1 of e~cl- of the ~A ~equence~
1.5 of th~ ~,cnetlc construct of the irvent~on.

Other types of r~eul~tory sequ~nc.es ~re up~ rearQ .~n~l do~ns~reant sequer)ctri invol~red in con~rol of t.ermin~tion of tran~.c~iption (~r;~ls e~ipt;ion terrtti~tor~) nnd remo~rtl of ~t~trons, n. wt~l~ a~; 3~qucnc~:s rcspbn.sil~,le or pot~adenylntion, .~n~l init iflttoll c,f ;~an~ tion. W~lcn 20 r.he r~&ulntory .sequct~cc i.~ r.o ~nc~ion in . pl2,nc, ir is p~:cLcrt~bl.y of pl~ne oriF,in.

-~ F~ctnr.s rep,ulnt.in&, promt)Ler ~ICt~Vi~y m~y ~nr;~ depcndirl,$, JnC~r uli~, on th~ kln~l of promorer c~luplt~-~cd ~s well rn~. nn r.he orf~ ni.r.m Ln which it i!; to function. ~ s~Je sp~cific rey,ul,l-ion n~ y ~ rcr,ul,tr.e~l l?y ~5 certaitl /rlt~insic ~r~ctors which en.cure thaL r,cnr~. et)~.vdirly, proteills ~pccilelc to n ~,ivr~n ti.ssuc ~re tixprcr.~.cd ~.x;~mples oL ~is.sue spt~cilic pr~m~)t~:,r, ~re l~ .~p~c~fit~ p~OLtOt~ c~c~ ht~ ~'t~ o~lyll a/~
pr~mor.t?r and the A~S promotcr, an~! furth~r root ~:pecific, st~em .~peeific, ~iee:l spccific 2~rt(l pe-~l ripec.ific promoter.C, Also f,~ctor~;
~o ~;t.~ch ~ p~ttho~,enic ~ttack or cer~.~.;n biological factor~ h~e b~erl shc\wn to r~ul~e prot~ote~s. Furth~nnor~, hear-resp~ns2 promoters ~nd pro~otcrs invol~d in the developmental r~ulat~on of plants ~y ~e found to be of interes~.
~.
829~iool~Qs/~KMfAl2J~ 07 2g . . .
.
;' `

t i~ L ,'~ FP~ '1[~ F,EE T,-l J~: t ~ 8~ J"'J F'.1-' 2 ~ 7 7 In ~he present cone~x~ u~able ~onsti.utive pro~
fr~m the group ~tn.~1~ting of plan~ promo~e~, f~ngal pr~ ot~r;, b~c~e1:ial pro~oters, Dr plant vlru~ pro~oters.

A pre~red ~roup ~f pla11~ vtrus promoter~ ~re pro~r~ers ~hi~h ~ay be S derl~ed fr~m e eau1~flow~r mo~aic ~r~s (C,~Mv). Such pro~ot~rs nre norm~ stx~ng consti~utive pr~moter~. Ex~mpte~ of ~ p~ef~red C~V
pro~ote~ Is ~t C~Mt' l9S promoter and a GaMV 3S5 p~omoter tOdell e~
~1 ., 1 g85 ) .

Ot~eL promoters may be deri~ed fro~ e Ti-plasmid s~lch as the oc~o~
pine ~neh~se p~omoter, the nopaline synth~se promorAer (H~rrer~
Estrell~ et ~., 1983), the ~annopin~ $ynchase promo~er, ~n~ pro~o-te~As from other ~pen re~din~, frames in the T-D~A ~t~ch a:; ~R~7.
Furt~er ex~mplcs of suttable promo~ers are MA~J35S (J~lnsser~ and Gardner, l~9)~ MAS dual Tr 1,2 (Velten et al., ~984) an~l a T-2 ~NA
lS ~erl~ 5 promoter (~on~ and Schell, 1~8~).

Tlle re~ulatory se~tlence may be ét chitinase pro~oter, i.e. a yromoter whi(:ll i5 nat~t~r,tlly found it: conn~e~inn wlr.h ~hitinJ~ ene~ ~t~1d lnvo1~cd in th~ t~.~ns~rip~ion ch~r~of. A ch;tinn:;~? promoter ~Rjr b~
oht~ine~ fro~ Rn isolae~d chitinf~.~e g~n~ n ..1lre~1dy knowr chittn.~n~ n~ ~r a ~en~ which mlly b~ id~nti~i~d ~tnd i~ol~r.~
by the n~t:hod~ .lisclo3cd her~in 1-ypicnl1y, t~ hlti~ c~ prnmot~t ~sho~lld be otit,~{ned Frot~ t t~hicll h;~ bc~tl ~;llo~n o tl~r~ ;I f~;t r~pon~,e to pa~!lo~ n ch.~11rn~ n r.h-t~ n.~nni-n~;nn, f;~it- r~-~.po~
have been ot~;erv~ in p~n s:nd hArl~y ~nd ~t i~ conteu~lated th~t ~5 ~.h~lt:1oa~ r~ o~rs from th~ pl~lnl:~ m.~y b~ n.~ -1 for ~ r~s~lt p~rpo.~c. An ~x.~m~lP o such plou~ r~ ; th~ cl~ieinr~ c promot~r ~,f p~ . Vlld, 1992 ) . An ~x~ f ~nother pro~oter ~hictl i~; c<~n~u~-pl~r.~ to h~ ~s~ful in ~hc pr~ n~ context ~s the ,nr h~r, c.hir~i-na~ ,xo~o-el (S~quence 7) ~nd the .CU~Ar b~t ;~e~t.otl;~lro~yae;(l synthAcc pr~înot.t~r (AIIAS) ~P. Sto~lK'~rd an~ ~. Bojs~n, ~ani!;co A~S, Den~ark, pcrson~l co~nt~nicRt.~on)~

Optionally, nnd lf desired, ehe n~c~lr~1 promocer may ~ J~odi~ied for th~ p~rpose, e.~,. by ~odiflc~tion~ of t.h~ pro~oter nuole-otid~ se-8X450bi.~01/15/JKM/A12/1991 ~
.

!t. ; ~ F ~ !! I r ~ EE T~ I F'. L--2 ~ 7 7 quencr ~ a~ to oot~in Q pro~c~*r Eunc~ion~ng itl anO~ler l~ eL ~h~nthe s~atural promoter, prefe~ably b~ing st~nger.

A~ staeed abo~e, each Df th~ co~in~ ~NA ~equenoe~ of the gene~ic cGnstruot of che invention 1~ f~rAc~ionally connected to ~ ~r~n~crip-tion ter~ir,ator. rhe er~n~c~iption term~na~r ~rves to ~r~in~te th~tr~nscription ~f the ~NA ~nto RNA and is p~efer~bly xelec~ed from t~e eroup eonsi5~in~ of plant t~`ar.sc~iption ~ermina~or se~uences. b&c^
terial sr~ns~rip~ion ter~inator seq~J~nces ~n~ plan~ ~iru~ ter~i~tor sequences.

10 Sp~ific exR~ples of suitnble tran.~ripeion termin~t~rs ~re a NOS ~nd OCS tr.~nscriytion term~n~or ~e~uence of the optne synt~s~ ~ene~- of A~ro~ct~rf~m ~He~rera-Estrella et ~l , 1983), ~ 35S trdnscr~ption terrnir~aeor se~ence of the c.~uliflo~er m-~t~ic v~rus (Pac7kowski et al ., l 98~), A PADG4 tri~nscription tennil?a~or tv tho D?.`JA ~ne 4 (~ing lS ct ~l , 1989), lnd . PADG7 trAnscr~,rion terMinator ~v t:he T-D~A gPn-me or ~ore of th~ D~A sequences of the ~erlettc con~ra~ct o the inv~ntion may ;ldvi~nt~,eou;lv bt fllncriol~ conr~c tcd to ~n ~ahancer seq~lenoe whi~h resl~lts In ~n incle~:td tr~ erip~io;~ d ~ rcssion ~0 of rh~ ~MA s~qu,~ncc(x~ Sl~1tt~1y enhilnccr ~le~qu~nc~. nnd ~-"~ns or obt;~t~n~n~ i~n irlcre~ d trln~:crlptio~ ?lld e~y~r~!;r~i~n ~re kt,owr? ln rhe ~r~

Th-! ~.peciftc prom~-te~-s ~Ind cho ~.prc~f;~ rPrlri~ tOr~ esyecti~ly, ~o he c~or~n~cr~d with ench of th~ ~NA secl~letlc~ of ~he r,-n~t~c <orl~rllcr m;lv bc thc snme ~r ~ifierent 1L M~y h- ~n ;-clvant-~e ~o ~I!;e di1f~`rC
prolnorers ~nd terminAtor~, r~.pecti~cly, bec~ - thr~n the xi~ of roct)~l?n~qtio~ vcnts, whlch m~y l~-td to ~Y.cision or prlrt~. of or the entire eon(?-~c c~nstnlot, ;Ir~ voided In a f~lrthcr ~spect, the pr*serlt invention rel~t~ ~o ;~ ~ctor whieh ~s capable o~ replicatln~ irl a host organ~sm and whlch c.~rrie: a D~A
seq~enc~ of the invention COmp~isi~l~ a chi~ln.~se 4 DNA sequence ~bs~,3ntially ~s sh~w~ Ln Seqllen~ I or an analo~le br ~bs~q~ence ~h~re~f, or A genee~c cons~r~ct o~ the ~nventfbn. The vec~or m~

/nc~/Al2/l~l ~ 29 ~ r~'C!li F~ L~ E,E!~ r.!_1 -~ ,'3i'ii -; ~J, ~ ~

36 ~8~77 e~ther be or~e wh~ch is c~pable of ~u~ono~o~ replica~ i . . J
plasmid, or one w~lich is repl~c~te~ ~ith the ho~ chro~o~or~e, ~uch as a bacterioph~ge or i~tegra~ed into ~ pl~nt genome vi~ the b~rder ~equences of TI veceors. Fo1 produc~hn pu~poses, che ~ector is an exp~ession vector c~p~ble ~f exp~essing the DNA ~equenc~s ~n the org~n~sm chosen for the prod~tction. Th~s, the expres~ion ~eccor is a veetor ~Ich earries ~he regula~o-sy 6equences ne~essary ~ or ex-pression s~ch as the promo~er, ~ initiation s~gnal ~nd ~ Cer~ elo s~nal, etc. These regulatory sequences m~ be ~he ones c~rrieA by the ~enetic oon~tr~ct of the in~n~ion. The ~eceor t~y ~lsv be one used for identification And opeionally I~ol~t~on of chitinase genes or ~essenger.s fro~ other organism~, e.g. other pl~nes, for which p~trpose expre6sion îs not re~ired. This ~y be Aone, e.g., Q~
described below.

In fl st.ill further aspece, th~ present In~ene;on relates to ~n or~an-ism which carries .~sld whif~h 1~ eaplble of replic2;ing or ~pressin,~
f..n irlserted DNs~ see,~ nc~ as de~ined e~bove, L.e. ii chitinase ~ DNA
s~quence com~risine ti nucleoti-i~ s~q~ence ~ubstAntially ris Sh4WT~ in Seq~encc ~ or sn ~in~rilogue thereof or A chir.in;~se ,~e~f~ or pseudo~ene compris1ng said D~A sequ~nce.

rhe t~tr~ ~ir~iert~d~ '.ndi~ates ~h;1~ the D~ ~f?~ nce (vr ubscq~ern~c or .~nalo~e, or ,~ne or rseudo geI)a) hrris heen ~nsf~r~e~ o thf~
or~.~r.nl.~m fr .~n ~nc..~stor ehere~f 1>~ l~f~ s 4f y,cne~ic mani~ll.r.i~.rl, in ot~ r w~rds, the or~ nisln,n~;ly b~ onc wh~eh d-id r~o, nu~ lly or ?~ rontly (~nt-uin ~ ch .I DNA ri<q-~nc.f ill L~ r!omc, or It ma~ ~,e ~ne which nat~ur-llly or inherel)tly cont~l1n~. s~tch ., DNA ie~lence, but Sl~ u lower n~mher C;O thilt th-; or~,s~n's~ w:;~h ~ inr.cr~cd DNA ~oquenc-hSI~ hlg)le~ nwllber of ~l1ch seq-~enc~s ihnn its n.~tur~lty vcc~rfin~
cottnt~:rpArts .

~le DNA sequene~? e.arried by thc brgan~t~1 m..~y br part o~ the gcno~e of the or&~n~sn~, or ~fly be eslrried on u vec~or as defined ~o~e whioh is harl~o~leed in th~ organism. The D~A sequence ~ay ~ie prf serlt i~ the ~enome o~ express~on vector ~s defin~d above ln fr~me wi~h one or more second D~A seqtlences encodin~ fl seoQnd polypeptide or p~rt there~f s~ fl~ ~0 encode ~ fu~ion prote~n, e.g. as defir~d ~bo~e.

~50b.. f~ JKh1/A12/15191 ~7 ~

v'~ FF~ !r~ T ~ EEE T~ F. i,'' 37 ~ 7 7 The or~nJ~m n~ e ~ higher or~anlsm ~l~eh ~1S a pl~n~ or a lower org.qni ~m such R5 1~ mioro~r~nl~r~. A l~wer org3hi~ such ~ blc-terium, ~ ,ra;r-n~gat~ e bac~er~ uch as ~ bae~ of the genus Escherichf~ . E. o~>li, or of the g~nus Pseud~ aj~, c.g. P.
S pue~d~ and P. ~iu~.c~s, ~r a gr~-po~itive b~cteri~ ~iuch sl~ ~7f the een~s ~il tus, ~.g. B. su~ilis j or ~ y~t ~uch ~S of the genus Snc~h~ro~es or ~ f~ s, e.g. of ~2 e ~enus Asp~iii~s, i~ u~ful for prodtlcin~ ct recombinant polyp~ptide ~s ~efin~d qbo~Ye. A;, r~an3 or~ganisms inher~ntly produce ~hi~ina~ he ins~rt~on of ~ DNA ~e 10 quenC~ or ~ genet~c constrtlct. ~cco~dng to the p~r-sen~ in~,~nt:ion may leAd to .~ oos~sider~tbly incressed tchitin~se and opeion~ B-1.3-gltlc~n~ ex~ression ~n-l a correspon~ngly incre~sed a~tifung~l n~ rity. ~te r~combin~r.c prodttction may be perfor~ed by U5C 0~
ct~nventional t~chniqt~es, e.~, ct.s described by S~mbro~k ~ tl., Ig40.

15 A.~ lt .ill be di.~.ctlsscd in ft~rrher detrlil below, a micrclc r~,a~i.s~tlprc~ds~ ng chit{na~e may bc used ~n combatin~f~, soll ~l;tn~ p.lcho~-:n~:, . p.,~tho~,ens prcsent in rhe 30il nnd rf sponsibl~ for r~t~trc-lecl growt~l or deA~h of the pl~nt. E~ ple~: of ~cueh pl. ~l~ pa~hoges~ r~
soi 1 f~lngi pr~nr in c . ~. th~ rhizosphcire .

~0 Also, the <)r~ani.sm mJ;ty 1~ t ccll I in~ pli"~r. c~ll llnc . ~ost prefe~;lbly, t.h~ or~;lnism i~ a pluslt, i ~ ;ei)e;ic,llly modifie~l plant .s~ch t~ will h~ ~I;scu~:s~d irl ft~rt~er d~ltil b-low.

A.~ m~ntiorl~d ~bo~, r~e y,crletic co~jtr~lct l.c ~ f~.bl~ to b~ usf:d lrl mod~fy~n~, t pl~tn~. Accor~lir~ , ch~ ~e~esl; in~ent-ton ,lso rel;l~s to 2~i ~t ~oneticftlly tr.,~s1~ fo~--n~d pl.lsl- co~n~rlci~)~, in i~ ,enom~ .q ~ iC
t~o~lstruct A.~ fifl~ ritb->v~ e Kc~nct~ ~ rf.~'.iL'Orlll~,d p~ lt ~1~'; dr`
Increi-~,ed ~ntif~n~ ctlvity comp;~r~d ~o a pll~nt wt.i.~ docs not h.~rbollr a gen- clc constr~lct of ~ho ln~nt:~on, ~ n untrnn~for~
or n~rn~;-l pl~nt or; pl;ln~ which h;~s been genec~c.~ r;nsfor~ed, 30 but. not w~ th ~ gelletic constr~-ct o the ~nve~tlosl. Norr~ally a con~;ti tn~ xpr~s.;lon o~ the polypeptides encod~d ~y the gene~ con-S~ JCt is desirable, b~c in certain c~ses it ~1; '02 int~r~in~, ~o ha~e the expre~sion of the polypeptides es~coded by the ~enetic cor-`:

., . .

t~ FF~ r~ r ~ FEF Tl-~ -;rr~ F'.

str~c~ r~gulated ~v variotls factor~s, for ~:~a~tple bj- ac~ols -,ue~
tet~pet~atur~, pa~hogen~, cnd biolo~ic~ ctor~.

Chitinase genes ha~e be~n fo~nd in ~20nctcotyAedonous ns ~ell a~ di-co~ donous pl~n~s and haYe theTe bcen found tQ ~e e~pre~sed into S çhit lnase ~.tetive in destroying ~he c~ll w~lls of phytop~hoger2ic fun~, Accordin~ly, ehe plant to be transformed by the genYtic cQn;truct of the in~tention ~y ke A tt20110COtyledono~ls ~5 well ~ts ~ ~cot~ledonous plant, s~nce the ~enetic construct is expecte~ to be ~c~ e in s~tch tO classe; of pl3nts. Non-l~t22itin& cx~mples of monocotyle-~ono~ls plants which may be tran~i02~ed a~e corsl, oat, ~theat, r~e, rice, b.trlcy and sorF,ht~n .

Non-limiting ~a~,ples of dicocylecdonou.s pla.~s wl,ich ~ay bæ gen~ti-cally tr..~2n~afor~ed ~re ulfalfa, tob~cco, cotton, st~gar be~t, sun-flower, c;arrot~ cAnolt, tott2aLo, potato, so$bean, oil s-~ed ~ap~, c.2bbag,~t pepper, lettuce, bean and pea.

It wiJl be ~parent from tht~ above disc1~:a~2., thae ~hc ~,ene.tlcatly trnn~fl)rtn~d plrltnt accc)2din~t to the l~lVetl~iO~ !lS a?? inCte'~lStd re-~;istance eo chicin~contaf niny~ ~l;mt pilt.~o~,e~ .U~I as phy~.or?t~ho~,ellic ~0 fnngi ~nd netnar.o~les ;~:; compare~l r.& pl~.n~, wh{rh h~?~-e n(,t beerl p,eneei call.y r~r~rl~;ormed .~ceor~lioK eO the ~f~Uerlti~n o~ as c.om~ :~t~ to planes ~rhitch t~o nht ~l~trbt)ur tho geliet;it con.ctrlsct ~ (le~incd nbov~.

11~ ost: imp~rt~ t ~hitin-corlt.,~ini~lK plant parh~,g*r~ o be col~crolled nce.or~ ; to th~ ln~elltiorl are repr~ nLtd by p~yto~choe-:ni~ funF~f-25 Phytopacho~enlc fun,~,i dif~r in thc w~y ~ ieh t,~ey ir~t.t rat r withthe~ir host ~:?lt~nt tll-rin~e, infecti~ll. Sonc .~ eclt?.~ ~n~ he pl~nt vi;~
n~tl.lrnt t>peninF,s or wo~nd~ Lissur? and grOw ir~ Lc~wet?n the pl~nt cells, in the ~ntercell~ r sp~ce, ~luring ~he elleire in~ection cycle, The f~n~t hyph~e excIete to~ins or en~y~*s th~?t ~e~keY~ or d~st~oy ~0 the plant cells ~nd thereby p~o~ ide the f~n~ s wl th ~ell const~it~ents le~kin~, o~e of the plant cells. Other f~ngal p.~tl~ogens i~nmediately destroy the hos~ cells by penetrating th~ cell wall of heal~hy hc~t cells ~nd di~inte~r~e eheir protopl~sts.

`:
8294SObi.0~11/LS/J2~/AI2/t!~l 07 29 ~' .

F, l ll, F l [lLIl l r . , !~1, E ~ r .~ i F . 1 -~

39 ~ 7 7 ~el~w ~re elYen ~o~e ~x~ples of ehlti~ .d ~l~can con~i~inir~g phyto-patho~n~c furlgi wlt~ different hos~ ~n~er~ctln~ ~trategie~, ~11 of which ~e cont~mplated eo b~ ~ertsiti~e t~ ~he ;ritns~nic plants of the lnv~ntior~t.

5 Ce~o~p~r~ spp. i~ ~ fungu~ th~ growth of which i~ res,ricte~ eO ~he intercell~l~r ~p~ce. Conidia (i.e. ~pores) rom th~ fur.t~ts ~errtin~tte on the leaf surfa~c and penetrate through ehe srOmatA of the leaves.
Inside the leaf the pl~nt cells close tu t~e h,~hae gf vWi.ng ~n the intercelltllar space are se~érely affected by the to~ins e~creced ~ro~
LO the ~ungt~s. ~te toxin~ c~usc the plas~a mc~br~ne t~ de~rade. ~hereby the cell. content leaks out ~nt.~ the ~nt~rcellula~- ~p.~e. Later in ti~e infection cycle the plant cells collap.~P anù ~crotic ilreas con~ining dead pl~lnt cells and f~tngal ~yceli;.t e-~erg~.

Vertioll?i~lm~,tlbo~enl~ is a root pathu~en which propsgates .in the .5 ineerccllul~r space, btlt: which pene.rates throltgh the upcn~n~ t~tde by ehe emer~ence of laeeral ruo~s, throu~h ~.c.h~n~c~lly inj~led i~e~5 or b~ dlr~ct penetrat.ion of hyph:.1e chrough thP tend~ root tissue in the r~ions ~f cell clon~,a~;~T1 or m~r~stemi~ ~c~lvic~. r~,~ Lun~u.~ d~
stroys the p~r~n~hy~nto~ lls and t1re ;rac~t~y ~lemer1ts are mec}~ar~i-c~lly pluK~,ed.

Oth~r ~ e pathoEenl~ ~np,i wit.h ~ int.-r~ctl~ in.rc~ion ~y~
lnc]~utle~ rc~fnl.t sclero-iorllm, R~ octrfn)-~ so~ 'h~ tc>r~
me~3~ errl~.4 ~r~d f~<!;min~spcJrium .~pp.

C~ c~rlcllm lind~mu~lliRt)ut~ CAUSe.S R~ nthr~cn(ij~". Conidl,l f~
thi~; f~ng~s y,ermln.lte in n f~l~ o wa~r in rhe inftc~iol- co~r~ and the pro~l1c~d ~ be p~n~tr~qt~ th~ c~clc~ and ero~s in~o .he epid~rmnl c~lls of b~n Lcuve.s flnd po~s. D~r~r1g the ollowing inf~--~ion, the ~mg~ ~ct.c as ~ p~r~s~tiC pachogtn, pen~tr~cin~ living cell~: Rnd C~Sirl~ disintegration of ch~ protopl~ts.

30 Fusa~ium Spp. is a typ~cal svilborne fun~us l~fe~tln~ the plane~
thr~gh the roots, where the ~yphae penetrat~ the epi~en~l ce o youn~ roots an~ invades the xylem of rbot~ ~nd ste~s. Ihe ~ssels 82g4.50~.0~1/LS/Jla~JA12J1~1 ~7 2g F~ T ~ F;E[- r!~ F~ o 20~8477 b~come plugged wi;h gr~nu~ n~t~rial ~nd ~ro~ndin~ h~
outer phloertt and corcex are de~royed.

P~coini~ gram~nis c~uses ~Stem r~r" ~f wheat. The ~.poridia genminQte on a fil~ ~f war.er on the surfac~ of the plant ~nd tlle ~erm t~b~s S penetr~re the ctttIc~ . The growing myc~ia produce h~u~toria that penet~ate the wall~ of the host cells and Inv~gin~te th~ir p~oeo-pl~st~.

Ustil~go ma~df~ ~s ~ f~n~us with mainly i~terceliular growth, ~ut occasionall~ p2netraees the c~ all of hose cells.

10 In a ~ur.-her a.spect, the pres~nt invention rel~tes to seeds, seed-lings or pl~nt parr.s obtaiIled by grow;n~ the ~enerically transformed pl~nt .~. described above. lt will be tm~*rstood thaL any pl~nr. yarC
or cc11 ~erivable from ~he geneticAlly transformed plant: or the invent;on Is to be consl-ler~d within ~he scope o~ the presenc in~eII-tion.

In rccent ye~r.~ rca. cfforr. tl;~s bcesl foc~lse~ on ~levt~1Opilig usefulmcrhod~. for constructlng no~l pl~nts or plant cells ha~in~ .specific .lnd de.~rablè propertie.~. by tr~nset^rin~ n~ g~n~tic i~forll;ation enco~ing the dcslr~bl~ propert~e.~ r.o eh~ pl~nc, ~n~ n nt~ber of such 20 m~:thods basc-l on recornhln~nt D~A echn~ y .~n(l sui~al~lc plant erAn~
ormtlr.ion syst~ms aro noh .~v;~ilable U.su;~ , r.h~ ne~ic in~o~ Cion i~: int;rodueed in~o the plnnt hy u~e o ;. v~c~r ~ysr~r,l or hy dir~ct 1ntro(luctior~ ,. by us~ o f the mctho~s ~,iv-n hy 2ie~!la Es~rell.~ er:
~l., 1988, Ro~,~r.~ et al., 1988, ~:~ul et al, 19~8, An pt nl., 1988, ~'i Hooyk;~ 88 , Hor~ch t. ~1 ., 1988 , P.eyn~rr ~ et .. 1 ., 1988 , f~nd Tomns et ~t., 1990.

Ih~t~, In rtnoeh~r ~.tspect, t:he pre~cnL Inver~cion rf~late~. to a ~rans.or-m~tion ~yste~ colllprisln~, At l~ast one ~ctor ~h~ch carries a genecic con~truc~. as ~efin~d above ~nd whic2~ is c~pable o~ inet odueing the 30 ger~eeie construct into ~he ger-ome of ~ p1ant such as 3 p] ant o~ th~
famlly Chienopodi~-ceae, in parecul~r of thP ~enus ~e~, especia].l~
Bet~ vulg~r~s.

41 2~8~77 N~rma' ly, pla~ r~ncfc~rm~ n sy-.r.ei~s ~e ~ased on r.~:e ~ Jf pla~ s o~ pl~ riv~ives of the b~t~ri~ A,gl--o~cr~riL~n2.
t~iJQ be,C knvw~ ~grc~b~c~eri~ are Agrob~ct~f~t~ e~c~ien~ ~nd Agro~
h~cteril~ ~hL~ogent?~ (pl~smi~s the~es:~f ~re irt the f~llowing t~med S p~i ar~d pR~, respec iv@ly). 'rhe u~e o ~uc}~ plant tr~rlsfs~ tion syste;ns is b~e~ on t~e ~bil;ty of the bRCterlh A~l~o~e~teril~m to tran~fer ~ ~;pecific pieee bf D~A ~T-DrlA) to ~ plan. cell ~n ~ w~unded ~reR. In nature, the I~DNA ~ loc~ted betw~erl ~peoific b~rder DNA
sequence~ on the pTi or pP~1 whieh fll~ther earriDs virulence genes 10 necessa~ for the ~ n~;er of the T-D~ to ~he p1an~. Thf? A~r~b~c~
t:e~ cr~nsforr~ati.-,.n ~ys~em ~:efiiates eh~ ~ran~er of any DNA se-~uerlce 10cA~ed b~tweer, the "borders" and thuç, it is possible to exreh~nge the wild ~yp~ A,~r()baf ~e~ T-DNA with nny desir~b1e piece of DNA to be it~trod~ ced ineo 2 pl ant, 15 Prefera~.,l)~, thc p1ant trAn.c~onnatio~l s~rsten~ ~f the ~nventio~ is base~ion disar~ned A~rob~ eri~ h~bourlng d~riv.~tiv~s of ~h~ pTi or pRi f~o~n ~qhic~ th~ wi1al type T-DNA h~ ecn removed.

~ormally, the v~ctor ~iystelh with wh1ch t~l~e p1~nt i~ ~ranriEo~Ded comprise~ orlc or two pt;~smids, In Lhe or\e-pl~.sm;cl sySrtw (alao ~ermed 20 ~ co :Ln~.egr~t~? vecLor ~y.scf m~, thf- T^DNA of pTi or pR~ h~.s b.~e~
removed and repl~ced l y ~he DNA r.o h~ tr;~ns~rrf d in~o ~he plarlt ce11 by u~;e of homologo~-s rccombin~cion. In rl ~ o ~ slc;i.l sy~ n (~ c, t~:rm~ binary ve~t.or cy.~r~m) i~ot~ Lll~' T-t)NA rinfl rl f l-,<.r(l~r~ h~vc been ~wo~r~d rom th~ pTi o~- p~i. Inrrodller.lon in r.i~ di~ rmod Aæro-25 ~c~erlllm of .~r ~ 311 pl~smid fn nr.;iir-~irl~ th~ D~J~\ LO be r.r.~ncfer.rf~<l becween pT;. or pRi Ldentic~l borde--~ anfl ;~ s~lit.l'al-- u~ i~,in of r~
c~qrior-, resu1-s in n v~cror sys~em whcre ~h-~ vlr~-l*~ i(>rl~; nrc ` loc~t.-ml on thl` disArmed pRi o~ yTi uncl th~ T-DNA ~n-l hord~ -e 10cat:ed on a~other p1rlsmId.
' 30 An ~ mlpIe o~ n .quit~ble pl~nt tr~nsforma~ion vect:or i.~, p~121 ~r,d .~ dexivat:ives thereof, e g. us descrll?e~ h~ Jeffersorl 1987~
:
Suit~bly, the ~ectc~ to bc use~ ls provided ~ith suitab~e marker~
eucaryot1c ~s well as procar~otic, e . g~ genes encodirl~ antibiotic resistance or h~rbicide resist~nce or g~u~orc~nidase (~tS), e . g.
., Z32~.50bi.001,~ ~1/A12~19~1 01 29 ~, ~`

FF'I:II ! F I ~ICILlT ~ ! E,EE T~ F~

` - 42 2~ 77 hygromyein cr o~her ~no~n msrker~, e~&. th~ ~rke~
sey, ~990 and R~ aerr.s et ~1., 1988 rhe ~arker is to ~e present so es ~o ~e ~ble to determine ~hether the D~A ir.sert has been insert-ed in ~he desired position in the p~a~T~id and co be ~ble eO select 5 plant cells tr~tncfor~ed with ch~ ctor.

The use of ~ore than one vector in one transformatiorl event wlll according to the pre~se~cly kno~n pl~nt tra~sfo~m~e~on techniques requ~e that different selective ge~es ~re present on e~t~h ~c~or in order t~ be able ~o follo~ the success of the pla~t e~nsform~tion.

In thc çonstr~ction o a transgeTllc pla~t using ~ plas~id ~uch as A
pTl or pRi or deriva~ive thereof i~ is preferr~d ehae the genetic constr~ct to be in~*rted in the plant is first constructed in a mlcroorg~ni.sm in which the plasmid c~n repl~c~te and which i~s easy ~o maniptll~t~. An exa~ple of a useful ~icroorg~nis~ is E. co~i. b~tt other microorgaT~is~s h~ving the Rbove prope~ties ~dy he used. When a plasmid o a veC~r sy~tem ~s defined above has been const.ructed in E. ~oli, it i~s transferred, iC nccessary, ~nto a su~t~blc Arob~c~
teri~lm ~train~ e.g. Agrob~lcccri~ tu~ef~e~en.c.

The pln~;mid ?larborin~, ~h~ etie eon..truct of tII. in-~rltion 1.c eh~
20 pref-?r;~h~y tr~.n~:ferr~ n~o ~ suit~b'~ A~yr~h~rerill~r str~in, c.~,. A.
L~lm~f.lcicn3, s~ .s ~o obt.~n s~n Aro~ac~riu~: cell h.~rhot-5n,c the ~,enet::i.c eonstrucc o~ c lnventlon, the D11i~ of which is s-Ib~:eq~lently trnn.çferrcù in~o ~hc p1;nt cell to he ~o~?ifi~d. Thi~: ~r~nsfor~nat5On may ~e pcrfor~,e~l in ~ .r of wny.~, o . y,. a~ crib~? In (An e~
2~ ~1., 1988).

Dircct infeotion o pl.~nt~ t.5.c.cue.~ b~ Af~rc~b~ct~ri~lln ic ~ s;n7pt~: tccl~nklut? wll;ch ha~ .n w~dely cmploye~ n-l wh5<.?- ~ drs~:ribod i~ (Bnt-chcr ~ l., lq~O). Typically, a pl~lnr. to b illfecr~l 5~ wolmded, ~.g. by out.t~n~ the plant. with ~ razor hl~cl~ or p~moturin~, the plant 30 ~.;th a needle or r~bbing the plant ~ith ~n abra~ive or brushing t.h~
pl~nt with A steel bnlsh ~e.g. AS describecl i l Example 15). ~I~ wc~und i5 theI~ inoculated o~ith ehe A~rob~teri~, e.~. in ~ c~l~pen~iGn.
~: Alternatively, the infection of ~ plane ~nay be done on a eet-eAin part or ti~:~iue of th~ plant, i . e . on a p~rt of a leaf, a rooe, ~ stem or :

., .

. .
',` .
~' L:: ~~ F~ t~ -r ~ ,EEE r~ rlJ~r,'~ F -:

~'3 2~8~77 ~nother part of tht- planr.. The ~ncc~a~es~ pl~ne or pl~rll p:~ c i$ ~hen sub~eceed t:o ~el~ctl~n ilnd regener~t~hn Rnd gro~n Vll a suit~ble c~le~lre medium and Al10~7ed to develop ints~ ture plant:s. ~hLs ~cco~plir.hed by use of m~thods knv~n in the art~

S Other ve~ su~Eable ~eehods for ttansform~ng the pl~nt ~ by use of sonic~ n, electropor~io~ ~Jo~sbo, 1990) or p~rtlcle gun meehods, e.g. .~5 d~sc~ibed by Klein ~ ~1., 1989.

~'hen ~enetically ~ransfor~ed plant ce~ls Are produce~i these cells ~y be grown and m~intained in accordan~P wieh well-kno~n tissue cult~rin~ methods .~lch as by culturing the cells in a suitable cul-ture mcd~ upplieci with the nece~sary growth f~oti rs such AS aTnlnO
~cids, plant hormone~ it~mins, etc Regenerfltlon of ~h~ tra~lsfo~e~
c?:lls ~nto genetically ~odified plan~s m.~ be accomi>lished us;n~, known m~thods for the re~,ene~ion of E~lanc~ f~om c~ r tissue culture.¢, for example by selecting trAn.cformed shoot~ ~sin~ ~n anti-hio~ic and by subcultur~ng ~ shvots on ~ medium contain{ng the rtppropriAte nutrients, plnnt hossnones, etc.

tn accordanc~ with ~oll-knot~n pl~nc br~in~ te~hr!iques i~ will bo understood thnt th~ prodt~ction of n eenor.icAlly tr;.tn.¢form~-~l p1a~lt ~.~y be perfo~me~ ~5 a doubl~ trqn.~fol~tt.ion even~ ~introduclll~ che gene-tic eonxtrtlc~ in two tran~;Lormnt~on cyclec) or may ~e ~soc-a-~wlth ~ of con~entlon;tl b~ee~iT)~, ceehl-itll~e~. Th~l~, two ~enetical1y ~odif;.o~ plnnt.~ nee~,r<~ing to ~l~e yrc~;~n~ invention m~.ty he eros<;
hree~d in ord~ ~o obtr,tin q pl.qne which CO~ iT~S the ~,on~-ic con-25 sLrucr. o~ eaf.h it~ paront plnnt~.

~s wlLl be underctoo<l fro~ hc introd~ctory pnrt of ~.h~ pt-f`X~llt.
; spee1.ficat.ion, ~h* cllitinnce ~ DNA .ce~tlenn~ of t.he ~re~ t inv~rltior o~ ,~t~ analo~tlo theroof may be U~t't3 or di~no.c~ic purpo~es, t,rhl.ch will be further explA~ned in the ~ollowin~,.
, .
:`~

44 ~0~77 VariOu~ ~ypes o~ diAgrlos1~ m.~ be p~rfc,r~ed b~ ~se o~ .hi~ ~;eii~s~ 4 D~A sequence of ehe ~v~ntion~ In a gl~en example, rhitin~se m~5-3ezlger RN~s ~ranscri~ed From a gene be1vriging ~o the chitin~e 6 gcne fa~ily may be qu~li tati~ely AS well ~ q~n{i~,ively d~termined by hybridi2~eion tQ the D~A sequence o the in~ncion co~pri~ing ~e chi~na3e ~ DNA sequence or ~r. ~na'Logue or sub~e~u~nc~ th~r~of uncle~
condleions suitable for said hybridi~ati~n. Further~ore, genes b~-long~n& ~ ehe chitinase 4 gene f~mily and prerent in ~n or~niszr, such as a plant m~y be l~ntified ~nd ~solAted by use of the DNA
seg~ence of the invention, e.g. by sereet Ln& a gene l~brary of such an or~anism.

Whe~ the DN~ .ceq~lence co~prsing the ch1tin~se ~i D~A scquence or an analo~le or subsequence thereof is to be e~ployed ~cr d:L~nosric purposes, ~t w~ll of~en bc usef~7 to provic~e it ~ith a Inbel which may be used for de~ecticn. Use~ul labels .~re known in the ~rt and is, e.g. ~ fluorophore, n r~d~o~ct;~e isotope, an isotope or cl eomplexing agent such AS biotin.

Also, the ~A sequence of thc in~enrj.t>n ~o~yrisin~ ~hc chit~nase ~
~NA sequ~n~e or an .~n~].t?~6ue or subst!quence ~hcreof l~ay he~ u~i~d in a ~0 met~od of iso1a~ing n ~,ene or Itless~ cr belon~inr, to or iori~(l f~o~
the ehir.inise 4 gone fnmily from ,?11 Org3~113m, t`.g~ ;I plnnt, in p~rti cul~r a d~oor~y~ orl~ the metho-l e.~prisislg hyhr idi~ in~7 ~ nuc-e{e ~oid con~nin~n~ x.qmple ob~intd from n ~,ene lihr,ry Ol c~YA 1ibrary ~ronl rh~ ~rganism with the ~NA sP~uence o ~he invent.~on eomprlsin~
t~ chitin.~t~ ~ r?NA s~u~r,c~ ~r nn nn.~10~1t~ t~ u~ of, opt.ion.11ly in ~l lnbelletl ~tr~, in a t~cnaturt~ fD-~ o-- .ql~ r~A cvpy tbcreof undcr cotldlC;ons fa~orn~?le to hybrAdl~a~ wect~ ~hc D`iJA
set~uerlcc or RN~ co~y ~?ncl ~h~ nuclcic ncld of th- ~am~le, al~C] reeov~r-ine ttle hybridi,:e~ clDne so as to obtnin n ~ne ~1t' ~rl~'A ~elon~ing to th~ chltin,~se 4 gonc f~?~ily of eh~ vrganis~.

The ~d~?tific.aeion and isolat~on of a gene or cD~A ~lone in a sa~ple be10ngin~ t~ ~he chitinase 4 gene fa~ by use of the chi~in~se 4 DNA s*quence of the invenrion or an an~1ogue thereof, in pareic~1ftr a subs~q\lence th~r~vf, ~ay be ba~d on standard pr~ced-tres, e ~. as dcscribed by Sambrook et al., 19~0. ~or in~e~nce, ~o char~ctefize ,~
P294SObi.~ S/JKM/A12/1991 m ~9 ,:
~' %~477 ch~tin~ 4 rel.at.ed ger~e~ in ot.hE~r pl~nts. lt i~ pl~e~er~d ~ pl st~ndard Svuthern r~chrliqus~s.

The chitina~e 4 D~A C~quer~ce ~f the inven~ion or an ~logue or subseq~e~ee ehereo ~ay also be used in a r~ethod of ~u~nciiy~rl~ the ~mount Or a chitinA~e ~ r~la~ed ~e~s~-nger presen~. ~n diffe~ei~t tissues in ~n or~anis~, e.g. a ~l~nt, the ~etnod co~prl~ hyb~idi~-ing A n~clPio ~ containir~ s~mple ~btained fro~ ehe ~,r~anis~ ~ith the chitinsse 4 DNA sequence o~ ~he inv~ntior~ eoaipri~in~ ~ r.l~cle~eid~
sequence s~st~ntlally ~ ~houn $eqence I or ~n ~nalogue ~'nereo, especlally a subseque~ce the~eof, ~p~ional1y in labelied For~, ln d~natured form ~r ~r~ ~N~ copy thereof under condi~ions favorable to hybriti~.ation bet~een .he dende~re~1 D~ sequence or ~A copy and ~he RNA of t.he sa"iple and de~eri~ining the ~niount o~ hybrid~ze~t nuclelc acid (Barkardottir et al., 1987).

IS The hybridi7.ae~0n shv~ld be caLrfed c,ut. ~n ~COoI~ance ~L~h conven ei.~nal hybridization ~ethods under suitable condit.fons w;ch lespeot to e ~ ~ri~gerlcy, il~cubfltIon tirl~e, tt~ pC'~a~U'e, ehc rAtlo ber.weer~
the D~A ~equen~e of t.hc im~ention c~mpl icing r.he r.~hi,~ D~A
sequr?nce c~r att .llaloeue or suS~r~eqll~nce thefcot ~o hc ~Ist2Ci $or the idcntific~rion ;~nd ~he s~.t~p~e t:r,~ h-? ~n.ly~e~d, b~fc; nnd ~alt concc~n-cr~tiorl or othc~ e~n~litio~ of import~nc~ for r1~ h;~ridi.a~iorl. Thc ~hoice of cc)n~litior~ will, l~rc~ , cle~erl~ on Lhc d~,r(e of co~
p~men~arity hc~w-~n c~ Ifa~netlL~ to hc hyhrt<li~it, i.~ hi~h ~e~re-? ot co~p~ell~ent.qriry r~q~ire~. more ~;~rin~,ent .o~ r!o~ h 2~ low .~lt cont.:t~l~tx;ltions, lot~ lo~ltC ccr~rl~h o ~h~ ~uFfcr ~nd h~ehi~r temp~r~ltures, whor~.~s ~1 lo~cte~,rce of COmpl~?~n~nt.~ri~y rcr~uiL~n; lc5r, ~tringen~ orltitions, c.~,. hi~ilhl' ~ t ~ c~n~ iQn, ht~her l~nlc s.tl.~n~th oE th~ l~ufr~r or lnwer ~emp~ra~ur~ or t:hr hyhridi~.ittion to ~ke pl~tce~

The suppor~ t~ whi~lt DNA or R~`A fr~m~nts of ~he ~i~unpi~ t.o bc Qn~ty~-ed ~re ~ouTlcl in d~n.ltured form is prefer~'r>ly ~ ~oli.l ;~ppOr. ~d ~ay be .Iny o ~he supporc5 convYntionally ~tsed in D~A and ~NA .l~a].ysis.

The D~A sequence u~d for dete~tin~ the pre.,ence of tr5e onitinacie 4 rel3ted Rene ~s prefer~tbly la~e~led, e.g. ae expi~ined ~bo~e, ~nd the 825i~5~i001/15/lKM,tA12/19~

~6 2~8477 presence of hybfidl~ed DNA is dete~;n~l by ~torac~io~ pL.~" -~cin~
tillatior2 countin~, lwnînesceno~, or he~ic~l r~àcCibn~

Anothe~ app~oach for d~tecti~ ~he pre~enoe of a specific chitln~s~ 4 related gete, e.g introduced by the ~enetlc ~ethods descr~bed pr~?-vio~sly, o~ a part ther~of ;~ an or~aniS~, e.g. ~ plant, ~n p~rtic~lar ~ diootyledon, is to e~ploy the prinsiples of ~h~ Il-kno~
polyn?er~s~ chain reAction, e.~. ~s descri~?d ir. the ~Material~ ~nd Methods" section belo~.

~he sample to be ~nalyzed for the presence ;,f A chi~.in~se ~ rel~ted ~ene or part therco~ in accordance with the methods o~line~ ~bove ~ay be taken from cl~e g~oup of plant pa~ts consis;ing o~ lea~ec, st~s, t:ubcrs, flowcrs, roots, spro~ts, shoots ~nd ~eeds.

~he ~ e principl.er. flS de.~cr~bed ~bove mAy be used in the isolation of D~A sequences to be used in the prepAr~tlon cl ~ ~erletic onstruct i5 Or the inverltion, e.y,. D~A scquences eslcodirl~; a polypept;.cl~ havin~
~h~.t~n~s~ or ~ g~uc~nàse activity.

Restric~ion fra&mcn~ lClleth po1ymorphisn~s (RFLP~ .-r~ cre.~c.inply usl?d eo follow sT)ee~f~c nll~ c of ~,rne.r~ ~n vnl lou~ or~ n{~;m!:~ Thf:
~ s ~ th~,s~1v~ -~11c.w~ . r.l~ r~ ~ c~ ; m ~ r.~
~m]ink~d or link~ 1n croei~oc 1nvolving ~th~r ch~ acteri~;ti(~;, e.~.
pa~ho~cn rer,k~-~nce nnd ~orpholc.r,ic-lI chnru~erir.Lic~ .-Ctl .-~i tuhet : colour. So ;~r, ~1 ~tho~ tl;-; pri,~ e~ nl)loyi~l in htut"~tl~., but it h.~.~; Al.CO beetl employ~ n pl.~nr..<;. It ~s coneer"plh~(~d ~'laL ~he ch;tin~ D~ sr4uen~.~ of the invcnrion or n ~In.~lo~,ue thereof ln.iy b~ u~:e~ll in ~FLP~nfl1~ ol chiLinar~c ~'~ relu~c~d ~,t'l~C''~, e;p~ t ~n ~ue~r beet.

In n further a.~pe~c thc pre.cent ~nv~nt~on reI;~tes Lo ;~n ~r~iun~
composition co~prîsing n polypeptide encodcd by 3 D~A se(~en~e cvm-prisin~ che chititl~se ~ D~A sequence sllo~n in Seq~2nce 1 or an ana-lo~e or ~t~bseq~ence thereof ~c ~e~ined ~bo-~, or ~y a genetic con-~: ~truct ~f the invention as defined above ~nd ~ ~uitab1e vehiole. In anot~r embodi~ent, the present inventi~n ~elfltes to an antift~ngal col~pc.sition ~o~prisi~g a ~ioro~rgan~sm ~ap~ble of expresslng a poly-.
":

:
:

1 .L -' ~ FrCII I F l l ICII !T . ~ El~ CI l r,C~ J~ p p~ptide sncoded by the DNA ~e~uen~c~ compri~ing the ch ~ 77 sequence shown in Seq~ence I or hn an~tog~¢ or sl~se~lence ~hereof ~.
de'~netl above, or by ~t ~enetic eonstr~c~ of the i~e.nt'~n defin~
c~b4ve ~nd a suitabl~ vehicle. Microo~g~nis~s s~it~bie ~x ~onst-ltuents in an ~ntifun~al co~posi~lon ~re mentioned ~bo~.

rhe antifungal composit~on ~eco~din~ to the presehL ~nvention ~ay be prepared by ~ ~eehod co~prising c~tcurin~ a mi~roo~gsni~ h~Tho~rinx and ~eing capable of ~xpr~ssin~ a D~'A ~equ~nce of the in~re~tion comprisin~ th~ chitin~se ~ DNA sequence ~ho~ in Sequence 1 or ~n analog~ or subsequence thereof o~ Q gene~ie con~trucc o the in~en-~on in ~n ~ppropric~te ~edium .tnd ~ndel conttitions whIch rt~s~lt in th~ expression of one or more antiungc~l polypeptide~ ~nc~ded by the D~IA seq~enccs, optionally rupturin~, the microor~niCi~s so as to rele~se tllceir content of exprecsed antifungal pol~peptid~.s) in~o the medi~m, removil~ cel1 debri~ from the m~di~ ar-d optionally .~ubject~
ing the metl~llrn eont~inirlg the polypepcide(s) t~ free~e-d~yin~ or spray-~r~in~ th~reby obtaining An rtntif~ln~l conlpo~i~ion comprising the ~ntif~,tngQl polypeptide(.~).

l~e ctntifungnl compocltion accvr~inf, ~o the inv~ntiotl m;~y be u~ed in comhat~n~ or inhibitine the ~,ermination arld/or Kro~ of ll phyt.op.q-thoKenic fun~llc i n or Otl .I pl.lnt or in ~ny o~her m3terial ir- which etle pre~;cnce tf ftln~l is un~ sirn~le. Th~ 11 he fllrt~ler ~iscus!;cd b~low.

'l~e antif~lngtt~ compoc5eior- o ~he invcn~ion ~h;~ll, of ;o~lr.~e, b~
2S ~dnpr~e~l t~ its Inteslded purpo~`, bot.~, with re ;p_~CL ~o the veh~cle r.o h~ tlse~l und uith recp~ct t~) the orm, ln whlch ~h~ .~nt.if~ln~ get~t:
i~. pre¢ ~y t~e tero ~antifun~ enr" is Ine~rlt th~! active eorl-stituent of the c~l~tiungal compo.~itton re.,pGnsible or or involv~
in prcvidinr, the Rntifung~l ~cti~i~y. Ry th~ certn ~antilln~1 p~ly peptide~ eant a polypepeide en~octe(l ~y thc chit-inAse 4 ~A se-quenc~ o~ the ~nvention or Qn Qn~log~te thereo~ or 3 g~netic conseru-e of eh~ ~nv~ntion havln~ Qn~if~n~ cti~ity, i.~. chltinase activity ~nd optiona1ly ~-1,3-gl~c~na~e ~ctl~ie~ ~s àefined ~b~e.

8294~ tLStntM1~12/1991 0~ 29 .~

48 ~ 7 7 Not-mall~, ~he ~rltif~n&al ~en~ is in t~.~elf a mic~oo~.~
be prepared b~ ~ ~icroox~anis~l. In ~oa~ cRse~ the m~ct t~a~y ~d ~ne~perlsive w~y of prep~r~ng rhe i~n~.if~lrlgal compvsitlon will be to ~l~e the nic~oor~ani~m as such or the ~ediu~ in ~hich i~ is grown A~
the nntIiun~al agent. ~le ~ntifung~l polypeptide(s) expre~ed fr~
the ~ic~oor~ani~ms may be s~cr~ted into the ~edium, e.g. QS a co~se^
qu~nce of the ~ction of ~ 5Ui t~ble si~nal pe~tide capa~le of direct-i.ng the polypeptidr out into the m~diwm, or ~ay be rc;e~sed from the ~icroo~ganlsm by well kno~tn mech~nical or chemical means. ~efore USQ, ~r ~ e ~dvant~geous to remov~ the ~licroorgan;s~.~ or ~n~ cell debris from thc medi~.

~e ~edlwn ~ay, ln pr~nciple, .serve as the vehicle fox the ~ncifung~l ~ge~t but it is pIeferred to ndd a further vehicle cu;te~ for the part.~¢ul~r intended use.

1~ A ~ulture of th~ microorganis~s expressin~ the nnt~fun~,al polypep t:ide(~) rnay he obtaSned a~ described ~bove using rnethods kno~n in t:he ar~. As mcntion~d abov~, ~t m~y h~ n~c~ssa~ ~r .~v.tt.r.~e~o~ls to suhj~t the microorg~nlsm c:~llt~re to a Lulrtl~er tr~tm~n~ so a~; tv rc~lca~c the content of the ~nt1fun~ o1ypel~r;~k~ Inrc~ th~ m~
20 or ~o incre~e ~he ~mo~nt relenced by .c~cr~tion Th~ m(di~ compr;.~Sng a ~lb~,t.~nti.~ ount of th.~ antiLu:~;ll poly~p-titc(s) m~y be d;r~ctly ~pp1~ed t,O che .~o;l tn whi~ t:he ~ nt~ re present o~ irn ~hich the plnrlts nre to bc ero~n, or to th~ pl~nL~ or pl~nt part~ or to t~ irri~;~ti~ wat~ it.~ tiv~ly, ~ y b~
2S ~r~at~t witl~ th~r~c~ , op~ion~ irl c~imbin.ltion wi;~, ~ cvnvctltivn~
~l s~ed concin~, compo.~ttton.

~-e ~-icroorgas1is~ e~.p~cs~in~ c ~ntiun~,nl pvlypr~ptidc~) clr1 ~c ~ppliet io vario~ls ormu1ations contai~ gron~lui~lly ~c~p~;ib1e vchicles, ~.~. At~uv.~nes or citrrt~rs, In ~lhsages ~tnd COIl~PntratiOn5 chosen to m~ximi~.e the beneficial effecr of the m~croor~nis~.. How-~v~r, ch~ mioroorg~nis~s ~y also be dis~ribut~d as s~oh un~ r cir-c~stances ~lto~in~ e microorgAnisms ~o egtsbl~sh th~mselves ln the materlal to be tre~ted. When the mic~-oor~ni~m i~ ~ m~croorg~nism ; conventionally fo~nd in the soil, e.g. a rhi~ob~cterium, it wiLl 82~50b~ LS/JKMJA12/199t 07 2~

49 ~ 18477 r,f~n~r.~lly be de~ ble ;:h~L Ch~l ~rf~rlsformed micro-rg~d ~ ~J
it~tf in ~:he so11 so ~har it continucus~y ~y secrP;9 ~he ~n~ifung~l polyp~p~lde~ 011t into the soil ~rrounding ~-he plant.

~t may be advant~eous to add the icroor~ani~ms or ~.he m~di~ ~om-p~ising the anrifun~l p~lypep~ide(s) eo pre-~i~es, e.g. nr~ifi~ial growth media o~ othe~ ~oll mixe5 u~ed lr) ~h~ ct.l~ivation of ;he pl~nt in question. Fc~ ~u~h p~r~oses it is conveniene th~t the microorga~-isr~s or the medi~m is in ~ solid forrn, e.g. in a po~d~ry f~rm or ~n the ~or~ of a ~ranule. rhe powder~ fo~ ~ay be obt~ined b~ con~en~
10 tionRl meAns ~ e ~ g. by ~pp1ying the microorg~nicm on a p~rti~ula~e carrier by spray dry~ng or an equivalent rnethod ~hen the microorgarlism expre.~ing th~ ar~tifun2al polypeptide(s) is ~o be ~-sed i~ a hurTlid st~re i t ~ay be Ln the form of ~ ~u.cpen~ ion o:~
d{spersi on, e ~. as an aqueous SuÆpensio~

In ord~r rAo induce the chieln~se AC~.iVit:y of the t~ansfo~ned microor-g~nis~ it mAy b~ a~v~nr.i1g~0~ to ~dd fl s~ TTIounc of ohtl~ tv ~he medi.n1n i~ which the tlt~nsfor~ned mlc.roor~nis~ îs presenC.

In accold~nce wiCh th~ ~bove, thc presen~. ~n~enr.Son f~rrh*r reI~tes eh il m~th~d of inh;bltine, or tht~ germil1ati~r1ilsm~or ~ow~h of fi chît~n cont.aini~ 1an~ p~hor,~n, ~nch ilS ;,hyto~tho~et~ie un~,u~, in ~r on a pl~nt, whi-:h n~et:hod coulpriscs 1) tr;1nsonnin~ th.~ pl~nr. or ;~ p~r~ tt1ereof ~ith ~ r,~n.~ ic evnstn-~1er.
~.~. d~fi~ed abo~c ~md re~ener.~tin~ the resultlT1e tr~ln;ormed ~lant or planr. parr 1nt~ enetic~lly tr~n~form~ ,t, ~lnd~oL

2~ ~) tr~at.in~ th plallL or n pare ther~of, il se~dlin~ ~r ~ d L~ om which ~hc plnnr is t.o be proy;~;ated, or ehe mt~ m on w111ch lt ~.
,rown wir.h al1 ~1ntifun~1 compos1t~on a.s defined ~bovc.

Uhll~ gen~tlc tr~n~fo~mation of plants i5 for most pur~os~ r~ t.he preferred mechod, it may be an ~d~ane~ge to co~bine ~ransform~ti~n with treatment of the plarlt with an antifun~a~ ~omposiri.on o~ the inventlt)n. S~nce the gene~ic trRnsform~tlon is ~ e-~on~u~in~; ~nd /nKM/A12/1~1 ~ ~

.`
'~

so ~4~77 in certAin ~spect~ diSfieulc proce~, it ~av be ~n biologicall~ b~secl composit.iotl inste~d of ~r in addLtion t4 ~h~
conventionally ~l~ed An~ f~om an envir~nelltal p~nt of ~i~w undesir-eble chemical fungicides 5 in ~ost c~se~ the ~aeeriAl t~ be tr~sAted wit~ eh~ ~heifungal eomposi-tion of the inYe~tion is ~ pl~t. Howeve~, ~ n~her of ~hitin con^
t~ining ungi exist which infect other material~ than plants, e.~.
food prod~cts su~h ~s hre~d or bread productsi ~ilk product~ chee~e, meat, vegetables, cereals, in whi~h th~ presence anc growth t~f fungi are unde~lrable. ~t i5 contemplated ~hat an ~ntif~mga'. composition ~c~ord~ng to the present invention m~y ~e used ~o control or eornb~t such ~un~i. In ~his re~pec~, lt is contemplated eh.~ atso beverA~e~
and (:ont~iners ~any part chereof) u~ed for food pro~uc~s or be~er~ges mny be ~-re~t-ed with an arltiflln&al compositlon of the inventlon eithe~
lS ~5 ~I prophylRctic treatment or ~ comba~in~ treAtment.

rhe present inverltiorl ;s urther illuct.rAted in the fol~owin~ se-eneis, examples and actorl~p~ ing drawlngs, b~t no~ ited hereto.

The drawing:

Fi~. 1 desc~ 5 th.~ rifi~:fltiOll bF suK.3r l.~eet ch;r.inas~ 2, ~ nnd 4 20 hy ttono^~ cation cxcmlnE~,c ~llron,atogr.~l.hy ~ t pH 4 . 5. El~io~ f t:h~
p~ eir!4 was ~elformed wlt:h ~ 1 in~ p,l.~dicrl-. of ~ 1. Thr nhsolb.~n-:e w:~.q recor~cd ~t 280 11~-~iy . ~ <IPx~:r;l>~ t.h~ l>olyp~ ptide pattcrrl o~ ~u~;a~ h. ct: chitir~as~ 2, 3~n~ ter puri.ficll~ion on ;I M~.no-S ~'P~ lumn. T~nes contai~ 50 J~, 25 of thc f~ wLny, proteins. ~an-~x n nnd h, chitina~.c 4; l.~r-e.s ~ nnd o.
chlr.l~ 3; l~nos f cnd E~" chitir~. s~ ,nd ~nt .c c ,Ind h, n~. lecul.~r wei~h~ ~rker~ e I-rot~ns were stalrlcd with ~ r.

Fig. 3 shows che ~na~ysis of the wate~-sol~lble pro~lucts rel~sed fro~n 3H-chitin hy chitinase 4. 3H ch~tln ~as incu~ated wleh 4 ~ug 30 chitinase 4 at 37~ for 0.25, 0.5, 3 a~ ~4 hours. A~ a ~o~rol 3H-.~

~ s/~cu/~2/~

. .

,;
:' 51 ~8477 chiti~ w~5 incu~e~d ~tholl~ el~yme ~ 37C. for 2b hour~.. ,", ch~co~
ol;go.~cch.~rldes releil~ed were sep~rat~d by T~C and 'de2ltlfied by co~pa~in~ thei~ migration ~ith th~t of N flce~ylz~ucos~minY (monomer~, chitobio~e (di,~er), chitotrio~e ~tr.lm~r~ ~nd chitotetr~ e ~tetr~mer) standards. ~he ~adioacti~itv repre~el-ting the ~hitooligo~ac~h~rides w~s d~ermined by ~c~nelll~t~orl ~ountin~ afte~ cueting the 'rLC pl8te into pieces.

Fig. 4 shows the lysozy~e activi~y of chi~in~se ~ of the enz~te w~s incubated with cell wallc from ~icrococcus l~rscdei~ s and ehe decrea.se in ab~orbanc~ at 450 r~ was recorded at specif_ed tim~
inter~als. 1 ~l~ of S~ (~SIlre ~:llen~) was used as ~ control, ~nd (50 ~g ~nd 5~g) ly~ozy~e (ly.s) was used as stan~ards.

. 5 show.s the lnhibition o~ Lhe ~ro~th o Cercospor~ b~ ~ co~bîna-tlon of chit.inase 4, SE arld ~lucanase ~ using the mict-o~cope slide bio~ss~y. Ater 48 hou~s o~ incubation the cultur~c wer~ s~a;rle~l wSrh alcofl~lor White an~ investi~atcd ~n~cr fluor~ePn~ ,ht.

Fi&. 5~ .~hows thc growth o th~ fuA~ 5 wh~ 20 ~r, ot oach of th~:
~y~ s ehit. ~., XF: ~nd elucnna.~e ~ wer~ ~Idde~.l t.-> r.h- c~llture ~t time 0.

l`lg. 5R ~how.~ the y,row~l~ o~ . ~o~ltro~ culr.tlre where nv allti-L`un~l proCeins h~ve h~rn ~dd~d.

~i~. t~ çhows ~he lnhibi~ion o growth ~f Cerc~spora by chi~inil5~'~
u~ing hc ~icrv~it~r ~lat~ bina~:.cay. The time COUfSC` .~urve~: Sah~or b<~rl~c ~t G20 s~) descxlbe r.he ~rowth of ~he fu.leus duri~lg che fir~t 25 g2 hvur~ of ineuh.~r.fhn. The ~bs~rbance (a~ d~.cation of the growth) wa~ ~*;~ured ae ~ to 16 hours ti~e int~rv~1s and ~ach ~easurernerlt is ~;1 AVerage o~ 5 replic~tes. Curve A i~ a cor,trol curve showing ehe gro~th of Cercos~or~ when no gro~th inhl~ltors ~ere ~ded to the culture. Curve ~ shows the grow~h Of the fung~ç whe~ 20 ~l o ~
chiti~se oontaining fractlon ro~ the c~ n-colu~n was added ~t . .

52 2~8~77 ti~e 0~ In ~urve C 2Q ~g of purified ch~tin~e 4 culture ~t time 0.

Fig. 7. .is an autorndiography sho~ng ehe effect o~ chitin~e 4 o~
chi~i n in ehe apex of Cerco~poc~ hyphae. Incorporat~on of 3H-~belled ~-acetylglu~osamin~ Into the hyphae of C~cosporA beeioot~ w~s per-for~ed ~y gro~in& the fung~s for 2Q minutes ~n growth m~ium csn-tn~n~n~ rnd~o~cei~e mono~er of chi~in lncorporation o N-~cetylgl~l-eoseamine i~to the cell wall in th~ ~pex of che fungal hyphae is seen as blark dots.

~ig. 7A 3hows th~ hyphae befor~ treatmenr. wS th purifie~ chiti-n~Se 6 Fig, 7~ sho~s the hyphae after th~ radioacti~e inco~poration follow~d hy t~eae~ent wi~h p~rified chi~in~s~ 4 for 2~ hours.

Fig 8 S~IOWS ~he separ~tion o~ ~ryptic peptide~ of chi~innse t~ by 1.5 rever~e pha.~e liPLC on a Vyd~c RP-18 colu~ e pey~i~es w~re eluted with ~ linear ~rndient from lOX to ~l5X Acctonitr{le frotQ 2S to 75 min~tcs, Buf~r A wa5 wutcrl whcre~r, B wnr, ncetorlitrile Both sol-~ents contait~ecl O lX tri1uoro.~c~ic nci~ flow rn~e wa~;
~7 ~l/minuee.

~0 ~ 5how~ the sep.lI~tion o~ rce ncidic SE chi~cln~ io~yme!i on nn anion exeh.qn~e coIumn (Mc~no P) by ~he FPLC syst~m Th~! yroteins w~r~ t~1 w;th a lin~nr sod;um chloride ~rndient in n ~5 m~t B;.s-Tris buff~r at pH 7,0.

FLg. 10 describes the two d~fferent ~erolog1cal classes of .sugAr beet, the ch1t1n~se ~ nnd chLtinase 4 olass. 5 ~g ~f both ch1tir~se ~3? kD) ~nd 4 ~27 kD? were blotted on to the ni~rocel~ulose membrane before reaction with antihody to ~ugar be~t chie;n~se 2 (left) or Antibo~y to ~ug~r beet chitin~se 4 (right~

g ;

k. ~ t', FF'-I I F ~ . E~E Ts~ '?1~

53 ~8~77 Fi~. 11. Hybri~i~a~iorl of ~iff~nt ehitins.~e ~nes ~ith ~ c~cin~e 6 e~NA probn under .~pecifir hybridiza~ion sor~di~lo~i~. The different chitir.a~e ~ene~ ~ere spvt~2d on Hybvnd N nylon ~br~nf~s as 1 ~l pro~es o~ ~ pl~s~1d preparatlon ~n~ining the chitina~e .~quence~.

1 a chitinase 1 clone from sugar b~Pt 2 e chitinase 4 ~lone form sugar beet 3 a ~hitinase 76 clone form su~r beet 4 a chitinase clone from pea 5 a rSE" clone fro~ ~ugar beee 6 a chitinAse clone 1 frorn t~bacco 7 a chiti~ase clône 2 f~oTn tobacco 8 ~t chitin~ e clone 3 from tobacco 9 A chitinase clone from bean a chitin~.~e 4 1~ko clone from rape seed.

t5 ~e b~brldi~sttfon w~ e~trried o~lt nt. ~5~C 5n ehe ~ollowin~ h~.~hridi7,a-tion b~lffer: 2 x SSC, ().IX SDS, lO x Denhar~t's, 50 ~y,~ll Salmos~
~perrn ~NA ~nd A chitLnnse ~ cDNA se4uerlce ~ts prob~.

Fir,. 12 de~.crih~.~ t.ho ~nducr.~on ~f chft.fn~.~r ~nd ~-l.,3-~lucnna.,e in ~ r h~r l ~' ;t~ f~.~r itlf~r;-T- wir~ r~ ri/~l.q. P~ t.
w~rP ;r~o~ul;lr~d with ~t suspension o ~ung~ pores. Iff~avec. were harvnstocl aftor cpnc~fied t:lme Intorvnl~ nnd crudo ~tract- we,e prepa~e~l. En~ nc ;IC~ iC!; oL Chi~i.nll.sC arld ~-1, 3~ atl-:~se wet~e me;~ s:itl~, th~ r~dictrac~l a~:says witl) 311-~h~ jrl ~rld 3}i l~lnarin tlle s~ tr.~te, re.specelvely.

Fig. L3 de~cr:ibes the Lmm~nodetectlon of c~lg~r bee~ chItin~se 2 and b and p~ canase ~ in pr~t~in ext~acts ~ ~erçospora infected su~r beet leAves. L~nes I An~ c contain proteln extracts fro~ in-fecte~ and oontrol pl~nts, r~spect~vely. Antibod~es r~lsed a~ nst chitinase 2 (left). chitinase 4 (centse) and ~-1,3-g~ucan~se 3 (right) were employed.
.~

R25~lSObi.OOlJL~/~M/At2~1991 fD7 29 '-., f~ r 1,~1 l EFE T,~ :,-" '~ f, - j 54 ~84~

Fig. 1l~ irec~ed ~utag~n~sl~ of ~l~irlo ~cl~ onte~pl.~ted to f~,rm p~r~ of th~ ~cti~e ~it:e of the ~hl tirtase 4 ehZ~me by th~ us~ vf the Pt,~ r:*~:hni~ue described ~n "M~terial~ and Methods~. SD0 i~ t~ed ~ ~' primer~ ~or ~tll ehe su~gested PCR-re~etions. The seq~tence is 5 incii~:~ted by the arrc~ nd i~ cho~en 5' to ~:he ~ique ~ ir.e. The sequences for the SDl, SD2, SI)3, 504 and SDS primer~ ~re indi&~ted by arro~s. ~or the~e 3' primer~ the coinple~erlta~y ~eqtlence ~ith the indic~ted subs~itu~ions ~re u~ed. The primers can be u~ed for ehe following substitutions:

Sl)~: Trpl70-Tyr TGG ~T~C
S1)2: Gl~ l90~Gln CAA~CM
SD~: A.~pl84-~Asn GAT ~MT
SD4: l'rp207-~Tyr TGG~TAC
Sl)5: 'rrp205~Tyr Tt,C~TAC

15 The ~'t,~ produ~ts ~rc diz~sted with the relev tnt re . ~rLction enzym~s and exc:hanged witt. tlle corr~.cipo;ld;n~; ~,cc;ue~lce .in ehe ~h;t.inA~ie 4 g~ne .

FLg. IS. Conscr~lctior~ o ;~ hyhrid a-1,3-p,lucJn.~s-~ C,rine ~on~tr~lct with ~ t, t.~rm~n~l cxtensiorl fro~ ~ob.~cco 20 Fig. 15A. A ~l&qr beet cDNA 1'3-1 ,3-c,l~l(nr~nRse cls~ne wirh .~n ~und~r-lit1l?<1 tobac.co C-t~rmln~l ex~et)~ n Fi~. lS~. P(:l~ prlmers ~hich c;ln 1~ tl~:P~ O ;hrltl?,c the ':t<-p codon t~ ;,nt.r~d~lc~ A pnt-t of t~ -t~r~ tl~ sl, 3 1~rs~
~s creMt;c~ t ~h~ 3' en~1. Thc nrrc,w!; is~3ic;1tf~ thc PCR pri1"ers;
25 <)r t.h~ ~' primcr the seq~en~P 1mdernc!neh ~hc :~rl~>w ~s ~scd, o~
the 3' pfi~cr the ~omple~entar~r s~quence ~./it~1 th~ 1ndicntcd .c~lbstitutic>ns is usecl.

Fig. lSC. Fo~r anne~led synthcti-: oligon-lcleotldes containin~
the l~st part o the C-term~nnl extensio~ ~ stop codon, ~ S~I
. 30 site ~nd an BglII cite.
,~

.~

,J~ FF;~ F i ~ IJT , ~ri E,t:.t Tl- J ~ t~ 1 F,--' 2 ~ L 7 7 The fuse~t gen~ product can be ~ e l:ry c~igec~.~ng the glu;:.ir~, gerle wlth ~b~I Qnd EcvRI ~nd lig~;n~, it ~ h the P5R pro~lc~ digest2d Wit~l Xb~I ~nd ~r~I ~nd the anrle~led s~r~t.het Ic oligo~ncl~oeides dS -~sted with ~aI ~nd BglII.

5 F~g. 16. Construction of a hybrid chi t~n~ie ~, gene constn~. w~th a C- termlnsl extension ~ig. 16A. Chi~inase 4 with an tmderl~Lned tobacco C:-t~ntl ex tetls ion ~i~,. 16B. YCR prlmer~ ~./hic~l can I c ~ced ro ~nrroduce A SmaI site near ~he stop codon in tt~e ¢~i t~ ,en~? . 'rhe arrc~ s inclic~t~
th~ PCR pri~ner~; for the S' p~ Itner the sequ~r:ce ~lf!~d~rrleaeh the arrow îs u!;ed, for the 3' p~ n~r t~ht: ~omplement.~ry seq~l~n.e wi th t.he ind~cat~d ~ubscieutlons is ~Ised.

Eig. 16C. ~our annenled syn~he~ic ~ligon~lcleot.i-ln~ cGnt.~inin~
the sequence for the C- c~ ir~ ;ren.~1 on a .h.1n;~ed stop codon, ~ Smal ~ite and an EcoRI ~ice.

T~ f~ n~ product c~n 1~ mi~ hy llgc~i~.in~ e ( llitin.1s~ 4 gen~
wit~, Biltn~ nd F.~:oRI ~tnd ll~ r.lnE it with Chc P~R p ~ (t di~,~.cted ~sith htlmH1 nnd Smnl nnd ~hc llnne~ d sylltlletif o11g(!rlllc1eocide.r, ~ s~ed wiC~I Sm~tI ;~lrld Ecol~l.

~'1g. 17. ~onscr\lcr.i.on ol ~he p1.ln- ~ral~formdtiot~ , rol- p~LJ,r~4 CVnt;l illitlg th~ chitin.~ 4 ~rlA ~:~quencc~ <:hown in Sec;uence 1 . 1~
boxecl seq~n~ s ~.ndi~ te ~.he hl .S r.hitinn~.c 4 cDN~ the enll;~r,cr ~l 35S
prombt.er An~l the 35S terminntor seq.l~rlces ~Is~d or th~ ceorl.~tr~cC.
p~15t;~.1 is p~lue3c~1pt c~rryln~ ~he g66 hp EeoRI fr~ enc encodirl&, the chitinase 4. T~le h;~cchecl hoxes ind~cflte t:he co~1rlg regions con-t~ined in the final pr~duct. Kb3 (~K~3) ~nd Kb4 (-1~J) ;~e synt:h~tie oli,gom1cleotides acting as primers in the pol~er~se cha1n reac~on (PC~ using pB15~ A ~s templace. The DNA cecjuence~ of ~B3 and KB4, respectively, are given in Example 18. Pl~sm~ pP~i48 carrie~
., .

56 2 ~ 7 7 c~n~en~ionRt 35S enhanced pro~o~er 8lld a Conver.tiol~al 3J~ .. n::Le~.~2`
sey~fated hy a polylinker containing ~ tle c'ol~lng sltes The pl.~nt tran~for~ation vector p~K~, carr~es ~ right and ~ left T-~NA hor~er seqne~ce from the Agr~b~c~eri~ T~ pla~mi~ pBIl21 (Bev~n et al, S 1984), ~ ~US ~en~ w1th a 35S p~oter and a cv~entio~ NOs c~r~-~ator, a conventîonal ~PTII ~ene with a 3SS pro~ter and A con~en tion~l oCs ter~irator. A polylinker con~lning sev~r~l uni~ue 51 oning sltes is sit~ated between th~ GUS ~nd ~he NPTII genes Fi~. 18. C~ns~ruction ~f the plant ~r~nsfonnaeion vector pB~L4~4~SE1 containing the DNA seq~lcnces encoding chitinase 4 and SE, r~spectiv~-ly sho~l in Se-~uerlce 1 and Seguenc~ 8. 'rhe boxe~ se~l~ences ~ndicate the ~S~n cD~A, the e~hanced 35S promo~Pr and t~e 35S ter~inator sequences also used in connection w~th the conctruct shown in Fi~.
17. yS~rl is p~luesript carryirle th~ 5' end of the ~SE~ gen~, pSE22 :is l;k~w;se pRt~escr~pt c~rry;n~ ~lmost the ~n~.~Le ~E~ C~NA~ The hatched boxes indicate the coctin~ re~ions con~ d ~n th~ final pro~et.. S A~TGTAC3 ;~ ~n ad~ptor u~l for th- Kpi~1 HAndlll llga-tion. pPS48 i~ mention~d in connoction w~th Fi~ '~. The constr~ction : o~ t.he planr. r~ran~for~t~on ~ctor h~rboring ~.he chlr.~n~sf.~ ~- sequence ~0 (p~L4K4) ls ~ cribe~l in Fig. 1/.

Fi~,. I9 (:orlstr~lctlon of the f~ nr. t.r.~r).~forl~n~.;on ~ctor ?~Kl~.K76 cont:~inin~, the y,cnol~ic chi~inQ.ce 76 Ecnc, thc seq~ence oL ~hich is showll in .~q~*~ 3. The boxed s~qu~nc.~ dic~itv rhf- ~;ht~.fn.~ 7h y,ene, the cnh~nced 35S promoter and the .~5S ter~in~or .~equcnc~s.
2S pK76.1 ~s pUC1~ rryisle th.` HiT~ ficoRI fr.~ellt ~n.odi~K chiti ;C 7G isl thc HindII~/Ec~RI ~sitc of thc p~tC19 poly1ink~r. ~le ha~c~ed ~)o~t~ indlr;ce th~ coding ~e~ions c~nr~l~n~ the fln.~l pro~cL. KB3 ~n~ 340 ~c s~-nthetic olL~onucleo~ides :.lct:ing as p~mers in ~,e pol~ner~e ch~;n re.~ctiorl ~PCR) usi~g pK76.l ~; templnte. The ~0 ~NA sequences of KB3 and 34~, respecti~ly, Qre sho~n in Ex~ple 18.
P1A5~id pPS48 WR5 us~d ~n cor~n~ction wA~th ~ALg. 1~. The pl~lnt transfo~at10n ~ector pBKL4 {s descri~ed in Fi~. 17.

`:

`
... .
' .

!-'i FF'I~ lr ~ hlEiEE ~1_! 'r~ r,'~ ~r`_l r ..~

57 2 ~ 4 g ~ 7 7 Fig. 20. P~ amplific~tion of ~ part of the ~SE~ c~NA u~ 2S a templat~ N~ W'?~ r~?verse tr~nscr~bed t~sin~ ~ prir~?~r co~Si.~;ting o~
oligo-dT linked to t-~o r~s~rio~ioll siees (770). Ampliic~io.. w~s ca.rr~d out ~sing a 8en~ spec1 ic ~1~ed oligonucleotide linked to re~tric~ion sit~ (XbaI-KB7) ~s ~h~ 5' pril~er a~d 210 ~ th~ 3' pr~-mer. ~ seoond ro~n~ of .~plific~tion w~s then c~rried cut uSing arlother gene specific ~lxed ~ o~eleoei~e l~nk~d to a restrlct~on site (B ~Ir-KB9) a~ th~ 5' pri~er and 2?Q a~ ehe 3' prir.e~.

Fig, 21 describe~: the separation of .stlgAr hcec ,8 1, 3-eluc~nases 1, 10 2, 3 <~nd 4 by Mono-S cation exch~nge chromato~sphy ~t pH 4 5. Elu-tion was per~ot~ed with ~ line~r gradient of NaCl. ~le ~bsorb.nce wa~ m~asured Ar. 280 n~.

t`ig. 22 describes ~e const~-~ction of ~.he pl.~nt tra~ form~t~n ~eceor ~BKI~.K~.KSElCl corlta~nin~ the ON~ sequences encodin~ chitin.~se t~, ~E
and ~-1,3-~lucanas~, respec~ively, ~nd showrl in Sequellce 1, Sequ~nce ~ ~n~l Sequence 9. ~he ~oxed ~quenc~s ~ndic~ee the ~-1,3-gluc,~nAse cDNA, the enhanced 35S promor.~.r and the 35S tet~inAtor. pGluc 1 is pBluescript carryin~. thc 1249 bp EcoRI fr~t~;ltCnt cncodin,~ the p- 1, 3-~ucan.~.~e. The h~q~che(l b~x i ndi C,'tt~'~ ehf' codt n~. re~t~n Pt .~mid 20 pPS~8M i~ the samc .~ PS48 dc~ic~ibcd ln Cotlslc~:~io~l witll th~ c:on struc~ .~h<~wn ~n ~`t~, 17. ~xc~ c th.o.~ t.h~ plA.cr~lrl S~ lem-~nt~d ~rith two ;~ ionnl r~.tricti~!n ~ite~. (F.c~RT ,~ncl KpnT~ ;tch ';it.C
~f t.h~ ~35S-3',~t b-~x. Th~ ConStructi~.~n o ~he ~ nt tf~ f~,rn?~r:if-~VCCI:~)r hnrb~r{rl~, the chir~tnAct~ 4 r~n~i x~ uencc~ dcr.cribcd in 2~ Fi~ nd Fi~,. 18.

:"
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S Msi~ral~ J -P. ~r. ~., lg89, Proc. Na~l. Ac~d. S~i. VSA, Vol. ~6, pp.
~96-900 Mornon J. P. et ~1., Hydrophobi- clus~er an~slysic: an efflci~nt nt~
w~y to colpare and at)~lyze ~lno acid sequences, 1987, Else~ier Science Publishers B.V. ~Biomedical De~islon~, Vol. 22t, No. I

Mur~shi~e ~. et ~l., A ~ ed medium for ~spid ~,rowrh ~d hio~ssay Wi~h eOb~SCCO CS1ItUreS~I 1t3SJ2~ Physiol ~ nt, Vol. 15, pp. t~;~3-497 Myerc et al., 1988, CA~IOS, Vol, t~, pp. '1-17 ~t~s~ us J, M. f~t ~l., 1990, 5th Jnt. SY~T~?. of The Mol. ~enetic of Plnnt-.~icrob~ Interaction, Ir~ ld'~t~ 'iw~ r~f~rl and, p . 218 OdYll J. T. et al , Idfnri~ifat.~on o '~NA ~:~q~l~nc~ rs~ e-l for acti~ y o ~hc caS~ 1Sw~r ~O~ if~ '.S~ pronots~r, 198r, N~r1~r~, Vol. 313 Ooms G. ~t ~1., 1982, Plas~itl no. 7, pp. 15-29 T'.~.c~kowsls; J. s~S: a1., Di~c~ ~,s.~no t~a~l~Jf~r ro rlr.sr~., t9"~t Tl~ M~
J~nal, ~ol. ~ No. l~, pp. ~717 ~722 Rt~yn;lcrLs A. T.~t ~ 198~ ele~rahlf~ And a~crt~f~n.3blr: ~u;trk~r~i, Pl~t.
Mf l~cul.tr Rlolo~y ~nual A9, 1-16 Rogers S.G. et al., Use of cointegras.in~ Ti p~.~smi~ vectors~ 19q8, PlAnt Mol~cul~r Bic-los~y Manual A2, 1-12 S~ac et al., 1990, Plant Phys~ol., Vol. 93, PF~ 907-914 329~sSobi.~O~/LS/JlCM~A12/199~

Ij F . ~ r ~ r,. . iJ_, ~ F~ "~

62 ~ 85~77 Sarttbrook J., Mo1eeu1nr Clonin~" A l~bor~t~:ory :nanual, 1~ .. t.~i-tion, Cold Spr~ng Harbor l ftboraeory Press Saul M.W. et al ., Direet DNA tr~ fel t:o pro~oplast.q f ith ~nd witho~lt eleetroporation, 1~88, P~.~nt Mole~ulht~ B~ol~y lla~t~lal A1, 1 16 Schap,~,er ~r ~l., Ani~l. Bioehe~., 198t~ Vol. 166, pp. 368-37~

Selstes M. E. et al., 1980 Anal. Bit~ehen7, Vol. 10'3. pp. 67-'0 Shin~:hl 1~. et ~1., A~rie. Eiol. Chent. 4~, 145S-1460, 1983 Shin.r~h{ H . et al ., E~ivence for t~ ntl C - rermin~tl pr~e~-;s i~tg o a pla~r~ defens~-r~lat~d enzynte: Primary str~-et-lre of tob~eeo preptio~
1, 3 -~,lue~nnr.e, 1988, prGc ~ ~a~ cacl. ~;n~ ~ USA, Vol. 85, pp .

Sh;.nshi H . ~ 1., Structure G~ ll tO~Ieco ell~k chirina~ ,ene:
eviclen~ ~ha~ diff~r~nr el~t;rl,,tç~ genen~ e<~n ~ricie by t:r.ln~po.~.it~on ofsequences enevding a cyst~ine-ric.h d~ , 199~), Plnnt Molec~llar ~iology, Vol. 1, pp. 3'>7-36B

Sierk!; M. ~ al., Cittillyt;e mreh,tnl~m of ~mp,nl Kluec)aJnyl.tse a~
~lefit,ed by ntur.i~ n<~ of Acpl76~ (`lul79 i~nd ~ 18Q in tl,e. rnzym~
from ~.sp~r~,i..lllls f~wnmori, 19qO, Pro~eitl Eoy,illt~{ting, Vol 1, No. 3, pp. 1~33-198 .~
Stoug,~trd J~ ~t ~tl., 198fi, Nnt~re, Vol. 321, pp.6G~-~7~.

Tom~.~ r),T ~r. ~tl ., 1)1 r~:t t?NA trnn~;fer lll~o ltt~ct pli1nr e~ snd roeo~r~ o~ tra~!s~;enic plan~.. ç ~ m~err~prot~etile bomb~ In~ 19~0.
Pli;1tn~ Mol~c.~ tr R~ology Mr~nunl t~l3, 1-22 tl~d K. ct tl., 1991, P1~,trlta, 184, In Pre~s .
2S Velten J. e~. nl., Isolntion of a d~lal pl~nt promOter fra~ntent from ~h~ Ti plt~mid of AgrobRc~erium etlmef~ci~ns, l~t84, The F~BO Journ~1, Vol 3, 2~o. i~, pp. 2723-2~30 ':

~45a~i.QO~t~ tA12~ 7 2g ., ':
. .

~ . .

63 ~8477 Vi~ers, A. J. et; ~l., lg91, H~1. Plant Microb~ l~terc~:ti~n, vol 4, pp . 315- 323, ~ldrosl C. ~t al., Resîst~r~ce to hy~romycin B - A new m~rker for p~ant t~nsfor~ation s~udies, 1985, Plant ~olecl;lar Rlol~sgy, Vol S, 5 pp, 103 - ~V8 ~lng D. et. ~1., Conservcd ~un~ic>n ln ~ic~ti~n~ ~b~ .~ of ~ $lngle ~osophL7a hsp 70 I,ro~oter heAt shc>ck el~r~ent when fused to a s~inimal ~-DNA promot~r, 1989, Mol (;en Genet, Vol. 219, pp. 9-t5 Woocl 1~. I. et al., 1985, PNAS, Vol. R2, 1> l585 ':

:
:;

6~ 2~8477 SEQU~N~ lISTIMG

BRIEF EX~I~NATION

Setluence I ~he ch~tina~ b cDNA sequenc~e ~nd thf~ chiein~t~;~ 4 ~r.ttino ~tcid sequ~nce (harboured in the cr~N4 ~ugtr beet chitin~e 6 clone ~15) Sf~qu~nce 2 the partial D~lA seq tence ar~d a~ttino 3cici sequence of the ~enontic chitinase 4 clone Sequence 3 the D~A seq~tence ~tnd deduced nmino acid sequence vf the ~enomic clone chitinase ~6 Scqucnce 4 A comp~ri~on between thP nNA sequ~n~ of the c~hicina$e 4 cDNA ~t~qu~ncf~ shown in ~cquence 1 ancS the geno~ic clone r~hitinase 76 shown in Sequence 3 Sequ~nce 5 A contperi~on betweren thr~ ~trt~rto .~cld seq~t~nce o~ the cl2itinase 4 cDNA sequence .~hown in Sequencc 1 and chitinase 16 shown in Scqucncc 3 ~;equer,ce ~ ~ c orl~parisvll betwccn thc non- coding 5 seq~l~ncc of the chitina.ce ~ An~S e.h~rin~ e t6 gent~m;c ~ttl~enct~.
.shown in Sequence 2 al~tl Sc~u~nco 3, rc!;pec~iv~

St~quence 7 The DNA scq~lencc o~ Che cntire ~u~ar bt~ct ch1 tinrlsc I
~ene includin" intron!; proulvter ~Ind le.ld~ ~ sf!queTIf e nd the ~mino ~c.id sequence ciecluc~cl frtJm t~-e cvdin"
re,rion of che ch~einA~e I gent~

Sequence 8 the cl)NA Ci-~trin~lltce <lnd ~.he Se(lu- t~cl amino .-tcl-l s~cluence vf the Acidic su,~ar beet chitinns~

Seq~ence ~ ~he cDMA st~qllence anci the deduced ~uDino ucid sequ- nce of the basic sugar bee~ ~-1 3-gluc~an~se 82~450bi.001,~S/ntM/A12/1991 ~ 29 .
';

~J I ~_lb i ~ / l r-F~I I! I F~ EIEE T~ t 1-1 jr~

2~8~77 DErAILED EXPL~NhTI~N

S~qtlence 1 The ~NA and de~ucPd ~mino ~c~d sequence of the Bl5 chiei~ 4 cD~A
clone isol~ted from ct ~-lgar beet ~ZAp cDNA ~ihrary lhe ~eq~ence is 966 bp long and er.tcodes ~ protein h~ ing 264 nmino ncid ~esidues isl the polypeptide cha~n. She lead~ sequer1oe e~on5lse5 of 23 asLtino ~cid re~tidues followed ~y a hevein dom~tin of 35 amino ncid re~i~ues nnd e. unctional domail~ of 206 am~no acid r~sidues.
~tcr the st~>p co~lon, the cD~A hA~ a 158 bp 3' f lanking r~ion with a put~ive poly~del1ylation si~n~l ac ~ it~on ~47 etnd a pol~ A tail.

For ~omparison, the chitinase ~ te, the part;.~tl nt-c1e~ri.de ~eqt(~ncf.
of ~hi-h is showr) in Sequ~n~.~ ?, es~co~e.~t A pr~teln ha~in~ 265 a~tino ~ci.d re.cidue.~. The ~i~nal .~eq~ence encodcd bv th~ ~el-e eons~.ctts of 2 nmino ncid re~idues.

Sequerlce 2 The part.i~,tl DNA nnd deduccd nmino ncid sequ~rl~ o a ~lt~nli~. clon~
es~cvdin~ the c~1iri~ se 4 ~ t' i~ t:e-l from a S~ b~e~ EM~L3 y,es~o-mic ltbr~ry Thc scquence i~; 691 b~ los~y~ ar~tl enC.o~ ; rhf fsr.cr. 11? of CS~tc ?.~5 ~ssnino acitls of tl1-` chitin.~ pol~p~tide ch.~in ~h~ dcr ~cqucncc co~ ,S.~r.~ of ?~ ~0tno Ac~s.d r~sldusc.s followcd by 1l h-~cirs~lc-ls~;.si.rl o~ 35 ~1tn;l~o ac.i~l~. T~lf' ~rr.i.slty ~ f~nc~1 clon- t-.~s.~ .~ S' r.on--->csir1~ re~ion ol 355 bp with ~ TA~A bo~ sequcncc (TATAAA) loc.s~ Sr~ ~)o~s eion ~87, which is 70 t~p ~Ipstre.~ss o~ t.~e AT~; stalt codon.

R29~s~0bi.001/15/~/A12/19~1 0~ 29 ' :

Sequence 3 2 ~ ~ 8 4 7 7 Thc D~A ænd ~duct~ R~ino ~cid seq~e~ce v~ ~ $~nom~c c~on~ encodi~
the chi~inase ,6 ger~e içol~ced ~ro~ a suga~ beet F.MBL3 genomic libra-ry ~hP se~uence ~s 1838 bp lon& and encod~ a pro~in h~vLn~ 268 ~ino acid ~es~d~es in the polypepe~de ch~in. ~Ie~ Le~d~r ~et.;uenc~ con~
of 24 amino acid re~idue~ follo~ed b~ a ~e~e1n do~ o~ 35 and ct funccîon~l do~nain of 20g ami.no ~cid rt~sidues. The ge~e cv~t~ins one introTl whlch i.~ located ;n posit~on 87S to 1262. Th~ ~x~ct locfteion of thi5 int-L-t!n is bas~d on ~n ~ gnment wieh the E15 ch.i~ L cDNA
(~quenee ~). The intron ~ordexs conr..ain che consensu~ GT/AG se-q-~ences. A TATA-box ~eqtI~rl~e (TATAA~) is lvea.ed at posit;on 378 wh5.o~h ).s 90 bp upstrc~m o~ the ATG star~ codon A p\~tar.~ve~ pol~-A
sigrIlll (AATAAA~ is loc~ted at position 1725.

Seq~ence 4 compaliso~ between tl:~ ~NA ~t~q~Ienc~ o~ the ~IS ch~ t cr~ clone and the geno~lic chitinase 7C clone Th~ ~o.s~ton of the chi;inA.~t- 76 in~ron i.c cnsily ~ccn dt position 87~ tv 1262. Th~ ho~olo~y o~ ~he ;eclucnces is abolJr 73~..

20 . indicat~s iclt~ntic~l n~lcle(,tiAes.

S~CS~UellC~ S

A comp;~ri-ion of thc ~ o 3ci~ quesIceci ~f th~ ~ennmlc chit.~n~s.e :~ ~.lone .~s~d the ~l5 chit.inA~ cDNR clon~
, .
A hon)hlogy of a~o"t 80% is ~n Th.~ ~xtr~ ~ amino acid~ in chit;.na.~*
2S 76 ~re the a~ino ~ciAs (~er Thr Pro) in poiition 6~-64.
':
: indicates iden~ical ~mino ~cids.

.,:
.
82~5~ ool/Ls/~M/Al2~ Ot Z9 ~.'`
. .

Seq~enot~ 6 ~ 8 4 ~ 7 A co~parison c~f eh~ norL cod~ng S' ~cqueilc~c t f ~he _hl~ln~t~ 4 and 5' seq~lt~nce~ of ~he chi tinar.~ nd çhitlnase 76 ~enomic sequenoes 8 boxes of stron~ homolo~y ~s obscr~ed. It i~ con~emplate~ ~ha~ s~mt~
5 of ehesf boxes ~y be of r~gulatory importance.

: lndacat~s identical nucl~otides.

The ATG ~tart codons hre underlined.

~equence ~

I)NA ~equence of the CH1 gene 0 The a~ino f~cids of tllt~ t',HI pro~ein ~r~ sho~n below thc corrt~sponding codons Thf, follo~in~ .~re undf~rllned: TATA-box (T~T.~A), methionine s~rt codosl (A~'G), ~ntron borders (AG ~nd ~), r,top coclol- for trans-latiosl ~TA~ nd ~oly~denylation .~gn,~l (M TAAA).

Seq~lence 8 15 Thc DNA ~nd d~tlc~d Is~ino acid ~ ncc o~ S~" cr~NA c~onc iso l~et~l from A s~p,.lr boo~ ~ZAP c~DII/\ 11bsllry 'l~e soqu~nce Is 1106 bt~ lon~, And cncod~s a proe~ ~n hnving 293 ~m1no tcid ~osi~ucs in eh~ polypeF~ei~o c}lai~ endor s~ t-en~ con.~i!its o 2~ O ~cid residuc~i ;m~l the unt ~on~l clo~airl n 2kR Amino acitl 20 r~itiut~; Tht~ cDNA c lone h~s ~ S' non-~o~in~, ~e~,ior~ of 11 bp and a :~' f l~nking ~egiorl of 202 bp Set~uerlce 9 ~e VNA ~nd t h~ <leduoetl al~ino ac ~ d seql~ence of A p- 1, 3 ~ canaae cD~A
clone isol~eed fro~u a ~uga. beet ~AP ~DNA library ~94SObi.~l/~ SJA12~1941 0'7 29 -IJ!-~ t 1 ~ , F-'l-ll 1 F l rlCII lT ~ l E'~:E Tl l i ~ 7.~ F~

~8 ~ 77 s~qtl~nee 1~ 1249 bp long ~nd encQdes a prot~_n ha~.rl~.,., . . ~.c, acid resi~ues in ~h~ p~lypept~ct~ chain. T~le cbNA clone ha~ a 5' n~n-coding region ~f 33 bp and a 205 bp 3i fl~nkln~7 region con~ainlng a putative poly~denylat;~r~ s~n11 ~t position 115~ anct R poly A

8294sob~ /LslJKMlAl2/l99t 07 29 I; f F~ ! F I -:li-ll .iT ~ E~EE r ~ F J -~

69 ~8~77 tL~T~!:RIAIS AND t~l~ODS

Biolo5~1cal m~e~

Plants Seæ.d~ of Be~R vuig~ris, c~r. ~ono~a~, were ~own In cl~y mix~d peat (nCycasr) and placed in ~owth ~hRnbe~ wich ll~13 hours day~ni~ht cycle~ 25/18C {day/night) ~nd ~o% ~-h. Li~ht in~en~ity w ~ ~ppro~i-r~ately 25000 l~x ~Osra~ HQi-T~, 400 ~/DH). Three week~ ~fte~ sowing the seedlings were r.?pl~nted sin~1y ~n 12 c~ pl.~stic pots contai~;n~
the same growth ~edi~m. T~iee c~ day the plants were s~.lpplied with w~ter cont~inin~ O.lX Ferr.ilizer: ~t~erne~ universQl fercilizer~
4:~:4 (N:P:K). Si~ we~kc ~fter s~Wi~l~ eh~ planes were ~e;~(~y fo~
infectio~ experiments with Cercospor~ b~Licola.

Ncori~n.~ e.~h.~c~,M ~r~d ~r ben~ia3i,-na plarlts were obtaln~l as cle~cri~-ed above.

t .5 Ftm,~ i ~n isol~te of the E~rneus ccreo~ r?rn ~CtiColA W.i` used ~or inF-?(~.tion ~:xpor1mcnts . 17)~ ol t~, "F5?3`', ~r,~, .>bt; ined rt~m Uni~ ~c~ St.l~s ~ep~lrCtnen~ of A~riettltur~, Agrl(t~lr.ur;~ r.~.qreh l)ivi~ion, I!orr.
Coll in~, Colc-r~clo, USA .

20 ~11 is~ c~ ~' ~h~! ~'unr,t~ rif~ lR'',~ ) w~ hr.~ l fr eh~ Alneric.~ll Type G. Lture Colloe~ion.

~rcl~h of Cerco3por~ .~p~c I ec Th~ un~u~i wa:; ~,rown on .col~d ~,r~wl:h m.?~ n 11~ P~rrl ~lishP.s. ~.t.~r~lf "Pc-t;~tc) Dextro~e A~,nr" ("Difeo", ',9 ~,/1) w~s ~ ,ro~ch ~e<l~llm.
25 A pl.~lg o~ ~Iye~ WLt~; plne~d ~n ~hrn e~nt~ r of ;:h~ P~?t r~ d~.sh f~nd the et,tLtùrc waS ineubated ~at r~o~ e~p~r;~.t~lr~ for 4 w~Pks. ~Iye~ for sp~ ~e Indttetlon wa.ei "ha~ve:~eed~ by ~tletir.g of th~ ol~ myce~ at"
incl-ldin~ ~oihe ctg~tr.

82~5oe~oD~ Al2/t~ 7 29 IS:55 20~8477 Sporulati~ o~ Cercospora ~peoie~

Mycelî~ was mix~d wth distill~d ~ter (l.2) irl ~ S0 ml st~rile ~l~ss tube ~nd ~o~ogeni~e~ using a ~Ultro T~rax ~25~ ~ixer oper~ted at 8000 rpm for 2 min~tes.

5 1 ml of th~ ho~ogen~e w~s tr~nsferr~d to ~ Pet~i di~h ~on~ining solid sporula~ion ~ediu~. ~V-8" was used as ~e~ium. I~ cont~ined 200 ml ~V-~ Juice (Cambells, I~aly~, ~00 ~ a~er, 3 ~ CaC03 and 20 g ~gQ r.

~ suspension was ~llowed to set~le or 1 hour~ Af~er airdrying the iO cult~re (~pproxi~stely 1 ho~r~ the Peer~ ~ish W1~ clo~:e~, s~nl~d --nd pl~eed In en incuba~ion ch.~m~er ~ 13~C and 24 hourc l1~t ~cool whito).

After 7 d~ys of inc~b~tion the spores w~re harvested bv po~lring 10 ml dis~ille-l ~at~r onto the Petri dirih ~ 1 flrmly brur;hing thP .surf~ce lS oE th~ cul~uro ~i~h a $~rile brush.

The rcsu~ing spor~ s~spcns~on con~incd ~Ipproxi~n,~t21y ioo,ooo ~ pores/~l .

Infec~ion ~ C~rc~.cpor~ pocios Fox inoc~ tion, 1~.500 .sporcs w~r~ s~-~:p~n~ 1 ln I ml o~ ~;.tr.l~r cnn ~0 tninin~ 0 ~u~, o Tween-2~ in~ rl chromA~oe~lp1~ t-,m1~ r th~
r~ pe~lsion w~ Applied t~ ~hc uppi.~s lei~ .surF.~cc of .~ix~ k ol~1 SU~J~r b~et or Nic~t~ n~ Rllt~ un~ run off . Imm~?dintcty nftcI
ino~ulation ~he pl~nt.~ wcrc p1neccl ill a "~lsr ch~mh~1~ kep~ at 30(:, .: lOOX r}1 anc1 ~4 hours li~ht (cool whi~e!. Af~er S ~ys of inc-lbatior-25 the plants wer~ mc->ved to n ~;row~h ~h.~ber kep~. .r~t 30~C, ~(1% ~h ~nd ?~
hours li~ht. Approxi~ately 10 ~y~ nfecr ino~ ti~r~ necroeic spots developed on 1na~ure ~e~ves showin~ that ~ nfect{on with Ce~cosporn h~1d been est~bli~he~. After inoetllaticr., the ~tthr bc~t pl ~ts were harve~.~ed ~t specific time int~rvals or ' 82~ lSlJlCM/~l2~ S55 .

,': .
:`

J ~ F~r~ r~ T ~ T~ -'` i F'.--:L

2~1g477 '71 ij sm~ ics,tl~ pu~ifl~s~tt~on o~ ch1t:in~se ~, th~ cld~e et ini~.~.e ~SE" and ,9-1, 3 A gl~tcana.~e, and ~;i) a titne co~r~;e ~t~lC3y to deter7nir,e the expression level o` total ~nzy~e activity usin~ rs~di~chf~icsitl 6tsss~tys ~.tnd ~t.-~oblo~ting.
S ii~) determinat~n of the ~xpres~ion level of e~ h of ~ en2yme~ ir.t tr~n~enic plsitn~s u~ng the abo~ i) techn~que~.
i~olation of ~RNA or use in th~ c.onstruction of ~ cD~A library.

Inf~ctic>rl of l~ic~t~ t ?~ncs wist~ the rooL p~ Og~?rl 2~iro~ tonia ~olani tO An isola~e of R. sol~ni W$35 Obeained ~rom Dr . K . T-avellfi -Kl c>n.qvi (SalonSk~, Creece).

An inoeulu~ of R~ sol ~s?i was pre~i~recl on ~n~ley gr.lin~ soake(l twict-~in 1 X : f pvtato dextrose ~roth .~nd slutoctaved. The gra~n~ wt~re inoc:u Ii~ted w~ts~ agar disks osE t growin~, Clll tnr e of eh~ fu~ t~ nd in~ bntt lS ~d for two w~ek~:, after which they wer~ air~trie~.

Alr.ern.~ tely, clisks o~ R. 5or~lni p,rouin~, on pO~ftCO de-~cto~.e ~;Ar cfln be used dir~c:tly f~t.C ln~)c~l~,um.

The ;~noe~ ts;~s mixecl ir,r.o po~t~n~ o11 In diFferen~. (o~ .ri~ior~
~r)cl chc trnnr.~,enie ~ nr.ler.s ,rhich h~ e~n rc)o~-~d f:o~ 3ay~;, w~re 20 eri~nsp~ te~ in~o ~he in'ected coil. l'he p-rcenr.;.~i~ of -nlr~ lng pl~rl~!i mny be re~.f,rd~d ~ft~r 1 2 ~nd 3 wc~k5, r~`!ip~ ively, .In~
ate~ ~ w~eks the !inrvi~r~n~ pl.lnt.~ r~ A~:sesr~e~l f-,~ r~l~t ~
A1terr,.~t.~v.-~y, .~ d~ f~o~tl trlln!;p,enic pIrl~s were~ ~o~ direct1y in the incc ~e~i ~o ~ I .

25 Æx~:r;tt~ n of f~r~cin frc0 1 ~ of~ .c~ r h.`~ .tf m~r~ri.q~

More specific~Ily, ~h~ small .licAIe pur5f~c~t.1On U.~5 carried OL~ ac follow~ ,g of Ie;~f m~eeri~l w;ts hottlo~ni:~ed by -~ Ukr3-Tul ~a~ ho:~o-genize~ in citrate buffe- ~û I M, pH 5, 2 ml/g tis~le), ~:~n~inirlg 1 mM uf b~th benz.A~idine~ diehioth~eitol ~nd phe~ylL~Iethylsuphonyl fluo-30 r~de. ~rticul~ee ~Qattprc were ~emoved by ce~trifu.g~ei~n ~ l 5,000 ^.~

~S29450tx.001/lS/Jl~MIA12119~1 0~ 29 lS:SS

' ., r,,. ! ~ ~ FF~ F lrll.~ r ~ L~Er~ lr~ r..l F '.--2 ~ 7 7 8 f~ n~t~ e ~-pern~ C~ isin~, ehe ~n y~ w ferred t~ anoeher test tube bt~fore the ~en~rl~lgAt~on wn~i repeated~

L~xge sc~le ex~r~?~tlon of p~o~en~ rrof~ s~r beer le~tf mA_erRI

To ~ete~mine t~e aneifu~tal pot~ttial and th~ amino ~cid sequence ~f the enzynes, l~rge quantities of p~tre enzy~es are required To obtain s~tffjoient quan~ities, i e m~, quanti~les, a lar~,e sc~le purification of C~i~inAse 4, the aei~ic cllitin~se "SE" and ~-1,3-gl~lc~nase ~as cnrried out from 2 k~, of leaf mater1al frbm nat~ral~y ~tfectt~d s~gar beet plarlts, cv ~Monova'` Nnt~ra1ly infeceed le~ es carry~ng 50 or more necrot~c le~ions were picked in tl~t.~ field at a br~e~lin~ station it~ I~aly (Maribo-l~aly, ~oto~na~ ~tnd s~ored at 6C ulttil the ex~rac ~ion of chltlna~e 4 wa~ c.~rried out Prepnratlbr of a chit~n coltJmn 30 g of chito~an (fro~ Protnn; Sea C~re P, No 70, ~orwty~ ~a~ ~~-sc>l~ed ~n 600 ml of 10% acetic acicl ~ft~r 30 ~lnuter., G00 ml of ~cthnnol wns slowly a~lt~d while mixing The rloudy ~ coo; s->l~t~on was filtered twice to rt~move p~rtict~lit,e matexials; fix~j~ with ~lass wool ~lnd th~n with ~ sinter-~ gl.~is funnt!l Thc filLrn~c war. ~r~n~-f~recl ~.o ,1 ~e~ker orl ~ m~,n~Lic stirrt~r, n~d ~0 nl of ~c~ nhy-dr~dc wa~ ~owly ~dde~ Wlth exterl!:ivc ~;tirrin~ AfLct-~ppl^o~i~n~c!ly ?
~inutes, thc ~;ol~ttion turn~d 1nto ~ l Th~ ~elotlon w;~ ~11Owel to proc~ for 1~ minute~ bcforc hc ~,cl w~ c~ int.o picccr~ wi th n ~p~tul~ rhe ~el pieces were tr~n~ rr~d ~o ~ W;~rr~r)~, blclder, cc>-vcred ~ith m~rhano1, ~d ho~cni~ed ~ot ~ rrlinu~er, ~L full po~cr Mcth~nol, ~ct~c~c nc~ ,d ~t)r~.~oted ~c~ nhydrid~ ~erc relrJ~cd by filLration ~n .~ B~tchner Lurl~lel usin~ Whe~mnn No 1 fil~cr p~pcr l~e f~ltr~t~ w~s t~nsfcrre~ to ~ b~nkcr, t l of t M N~t~C~3 wns atlcl~cl ~n~
thc pH was ad~usted to ~ ~ith ~ ~ ~aOH 50 ~l of acetic ~r~lydride was ~lowly ~dd~d and the pH ad~u~ced to 9 Tht~ r~tior, ~s all~wed to t~ke place for l hour before the flnal pro~ct wa~ collecrt?d by f;1-tering on a ~u~er runr.el After extensi~e wa~hing ~ith water, the prod~ct was equilibrated i~ a 10 ~M Tri~ ~ffer ~t p~ 8 C before storing at 4~C The yield W-~5 700 ~1 of re8enerated chleln A ch~n 8~9450t~001tLSt~KM/A12/1991 0'7 ~9 15 'S

~ J~ l' FF~ Fl[lC~ ,EFE T~ F~.--73 ~4~77 colu~n ~a~ prepared rom the regellerit~d ~h~n b~ us~ oL ~p~L~ conv~rL~onal prooedure according to Pharma~is.

PrepQr~ttior.t of ra~iOA~ir~e <~ol lo~ h~ti~2 2 g of chitosal~ wis ~c~tylAted wi~h 3H-labelled ~c~tic ar~tydride as des~rib~d ~or the synthesis of unlabellYd chit;n (see above). After ex~ensi~e w~shing vf the 3H-labelled chlein on a B~tchne~ funnel, lt waq trunsferred to a beaker. 5~ ~1 of concentrated ~ce-cvld HCl w~s added, ~nd the chitin was dissol~ed by stixr~ng for ; minutes at 0C.
The syrupy liquld was filtered t~ro~l~h a ~inteted ~lass fu~mel and slowly poured into vigorously stirred 50X uq~eo~t~ ~th~nol to precipi-tRte the chi~in in a highly d~spersed state. I~te residue was sedi-ment~d by c~ntr~fugation snd resuspendec~ in t~i~e~r severnl ~ imes to r~o~e excess acid and ethanol. Finally the eollo~da1 chitin t~as suspende~ in ~00 ml of wa~er and sonlcated for 5 x 1 minuCr a~ f~
powc~. The 3H-Iabell~d cihie~rt was stored ~t ~C ~efore ~.ce Prepurution of a ~mlnar~n cotw~n : ~vinytsulfone activueed ~arose (Mini-1~ak h;&h, ~FM ~:N ~F.C, D~n-mark) wn~s employed ~o immobiliz~ l~minar5n ~B-1,3-F,lucan~ (from l.2minilri.l di~itatc- Si~;m; ) . 50 4 l~ini leak High wns di:;pes~sed in ~00 20 ~1 1 M por~ s~sltlrn phociphitr~ (K P) .-~t pH 11 ~ 0 n~ mlnarln d~ ~.solved in 5 ml H~20 ~ax .ld~]Cd . The re;lc~t i~ n WA~ owed to yr~ c~!t.~nlLor 16 ho~r3 at 2SC on . ~ch.~kin~ r.ahle. IJnre.~c~t d divi~yls~ lfon~
O~ ; W~t'l~` hlocked hy ~ncuba~.ion wl~l~. a x.olu~ior- of )X ;n- rc..lptot- -thnncl in 1 M K-P-~ufcr ..~t p~ . T~l~ re~l~e~on eime ~ > Hou~; at ~S 25~ R~slcl~lal m~?r~ pt-~t?th~r~)t wa~ r-rno~:t~d by t.~et~ ;ivt wa~htng of ehe ,~ n ~ R~lchr,e r funn~?l . Il~e Lnrninarin-A~; rO-.:e w;~ p~nd~?d in 70 mM Tr~-huffer ~t pH 8 C nnd xtor~ t 4C A l~rn;nflri2-l eol~lmn S pre~ ~red rc,~ rht~ ~min~ l t~ro~e ~ir~g thtA co2~rention31 ~oct d~lrt~ accortling to Ph~r~aci~.

30 S~nthesfs of 3~ Rbelled l~mins2rln Lamir~arirl ~as labelled wirh r~dioacti~ity by reduc~ io;~ uith 3H-lal~el-led ~I~B3H6. 500 IDg la~ninarin (~rom ~minari~ dig~tat~ Si8~a) ~as 82q~ 1S/~/A12/1')92 a7 29 15:55 l' F-' -!1 ! F l [.~ E'E~ T~ l ' t._~ L '--J

dis~olve~ 3n 2 ml H20~ ~nd pur~fied ~y precipj.~3~ion ~ty ~ iirjon 800 ~I N~Cl (0.2 ~'~1) f~tllt~twed by 8 ml abs~l~te e~h~nol. ~h~ pr~c{
p~t~te w~ ollecte~ by cer~rif~gation for 5 ~In. ~ 15.000 g. The ~upe~n~ t w~ d~s~r~e~l Snd the p~lle~ ~on~ning rh~ in.~rin ~s dissolved in 4 ml of 0.1 N NaOH rhis so,lutlon WAS ~ansferred to ~eactiorl wess~l containing 5 mCi ~f N~B3~4. After s'-irrin~ for 90 min. at 25^C, 600 ~1 of 1 M HCl wa~ ~dded to de~itroy ~nre~ct~d N~P,3~ib. The reaçtion ~i~ture waS divided into 500 ~1 ~lîquo~s and 200 ~1 of ~aCl and 2 nl sf ~b~oluce eth~nol wa.~ added ~o e~h te~t ts~b~
10 After srorage for 10 min. at 0C, the precipitate wa~ coll*cted by centriu~.~,ation or 5 min. at 15.000 x g. The 3H^lab~ d lamin~rin w~s di~olved in 500 ~1 of H20 and the pr~cipitatiOn was reptated unti 1 che ba~kgronnd level in the supernatant was less than 100 cpm per ?O ~ Iabelled solution of lamin~in w~s 5r0red ~ 20~C.
Before use in ~.he fl-1,3-glucnn~.çb-as.sfly, the sols~tio~ wa~ d{l~st~d 20 fold w~th w~ter.

Rev~ r .s ~ s e - HRLC

A ICor~tron AC ~"uric~, Swit~Llari~l) isls~ eo~ consis.ing c~f 2 model 4 ~0 pump.~ nnd ~ s~lv~nt miX~r ~ s u~iecl GL~ ent cosl~ro~ tta 20 ncqui~sîtion ~,n.~ porfot-med by ;3 Kont.r~n mo~le~ 4$0-M~ D;~ sysCe;r Aceordirlg ~o tlle ~alluf~ct~ s ins~r~ionr.. Protoint c~u~ed from tho Mono S cbl~t-nn ~e~ h~low) ~r~ s~bj~ct~cl ~o RP-HPLC o~ hor ~iDA~
~P4 (Q.~6 x lS cm; 10 ll~ pnr~ic1e c~-; Th~ r.~t;t7l);; (:ro~
.cp~ri~ otr~ ol~mrl OI 1 Poly E (Du Pc~nl de l~mou~ r~) cOlUlllrl.
~5 T~ t~m ~15~<l I`c,r RP~HPL~ b-lff~r .~. Q. IZ rFA in ~n~r ~n~l b~l~`for S: 0 .1~ TF`~ ~n nc~roni r.r~

SDS-P~CE

SDS-P~CE of cr~de pl~nr..~ extr~ct~ or p~rtly p~rif;~ Ch~ Sf". Wf' perfc-~ed on an E~sy~4 eppArAtu.s (Kem-~n Tec, l~enm~rk) ~ ng ~he Tricine SDS-~A~E ~te~ ~lescribcd by Sch~cr and ~on J~gow (19~7). A
: total of 25 ~g o~ prote~n was applied to each 1.~ . P~re chi~n~se : iso~nzy~es were an~ly~d on the Phast Sy~te~ armaeia) in a~cor-d~nce with the ~anuf~cturer~ ~nst2~ctions.

8~s4soe~ool~s~n~M/A22llss} 07 29 IS:SS

-i; F l [ i-,l !T ~ r r~ ,i-,1 5 " ~ F' ~':

t5 ~ 77 ~nzyne ~s6a~

Th~ r~io~hemf c~l chitir2~qse ~ssay ~h;tin~e ~ceivi~y WAS de~r~ined r~dioche~ic~lly ~i~h ~H-chiti~
substrate.

S The specific ~ctivit~ of the 3H-chit~n w~ 46~ cpm,,'n~ol ~-a~ecyl-gl~os~m~ne (Clc~Ac~ equl~lcnt (or 2,3 x 106 ~pm~mg 3~-chitin~. It w~s deeermined by scintilla~ion counting and colorime~ric ~.en~ina-tivn of ~lc~Ac a~.er toLAl hydrolysis of 3H-chitin ~y crud~ chitit~ase preparat~ons froni s~r bee~ le~e~ and exoch~t~nase frotn ~e~ra~i~
10 m~rcescens or St~eptomyces ,gri~

The ~ssay ~i~ture concair.~d ~n a totnl vol~e o 200 ~ f ~n~yme solution, 50 ~t of 3~1-ch;tin suspension ~cont~inin6 I00.0~0 cpm? ~n~
10 /~mol of sod;~ ci~r~e (p1~ 5,0). hf~er mixin,~, rhe en~.ymatic hydroly.~is of 3H-ch~t~n ~a~ nllowed to t~ke pl~ce ~r 4t)~ or IS min.
15 be~oxe ~c1ditlo1~ of ~O0 ~l of t.0 % (w~. ) TCA. In t~rdf~7~ t-~ decre$lse the ~ackg,r-nnd re~d~sng, 100 ~-l of bovine r~s~lirl al~ n ~lO ~n~,jml ~ wt~re rldded baf~rc rhc isrol-1ble 3H-thltis- W~ r~movf~d hv c~ncrifugation l5.000 x ~, for 5 r~in Th~. raA~oAtct~ it:y in ~00 ~ rs~r:~nr. w.
deter~incd by st in~ orl cv~lnting.

20 'rhe rA~lo~h~m~c~ll p-1,3~ 2;s~ ..c;~

t~.. qr~ nt t~ y wa~ 3r~tiol~.h~ y Wi ',~l 3}~-l~qbelled l~qminqrin n~ ;tlbstratt.~.

Tht' ~t~C.~ny m~xttirt~ ct~n~i!;ted o` 50 ~ l oL t~n2yme eYrract~ SO ~ll of 0,1M Na-t:itr~te p~ri 5,0 ~ntl 10 ~tl of ~ d Inmin.l~in (l92~000 c~n1) ~5 Inc1lh.tr~0n w.~.~ c.qrt1t~d ont for 15 min. a~ 40-C~ To ~rrni~ t.e the rcac~ion, lO00 ~ f ~b~ . Et:hAnol ~ncl S0 ~-l of ~ att1s at~d ~ Cl-soltstio~ was ~dded~ After lO isin. at O~C, ~mxec~c~.~r1 IfLrulnarin wa~
rer~oved b)t cener~.fugntlon nt lO . OO0 x ~ or 5 rnir n An ~1 Lqr~ot of 4g~
,ul of ~up~natant ~as tr~nserxed eo ~ ~cntill~tSon v{nl. 5 m' of 30 PI~O~ UOX-40 were added and the ~not~n~ o.~ r;-dioactrvi~ s dete~-mined by ~ liq~id ~.~intillation counting.

8294soeJ~.~olflLs/Jlc~d/Al2fl9~t 07 29 IS:S5 F~ l [ l c~ E, ~ r r ~ ' J F~ ~ r 76 ~ 3 4 7 7 ~,~Sozr~e .Y556ty Th.-~ ly~ozyme ~ct~ ty of ~h;eir,.l~s~ 4 w~ d~t~r~ini~ e~t mt.~bho descrlbed hy S~lsees et ~1. (1980). Mor~ specifi ally, ly~ y~e a~eiv.{cy was measured in ~icrotit~r pl~s. E~ch well eonct~lns c~ll 5 wall~ fror., ~fcroct~rcu~ ly~odeik~icus suspended in Lq 20 ~ e odiura phosphate buffer, pH 7.4, ~ontaining 1 mg/tnl o~ BS~. Th~ inieial ~bsorbency at 4S0 nm was adjusted ~o 0.6 before Ltddition ~f egg-whît~
lysozyme or plant ch~tîn~tse 4. The reaction wcts followet~ ~y measurin2 the decrease ir- a~st~rb~tnce At 5 min. ~nee~vals f~r 50 min.

10 ,~-glucu~o~lldAs~ (GUS)-~cs.~,~

When G~S is employcd ~ç ~ rt~port.er ~ent..~ in col~neciion ~ith the con-.~tntt.~.t;brl of the zetletic~lly tr/ln~Formed ~-lant~i acct)t:cli-lg co the prescnt invnnti~n, the cilrcess of Che tr~rl~orm.-t ion ~nhy ~ dc~.nr-mln~ by usc c~ the following ~I~S-assay de~cribed by Jcffcrson, ~9b~7.
l.S l~eaf e~s were ~lied intt> t.hin .~ectlons (c0,5 ~ ntJ il2cubL~ted in .
2 mM ~olut5tln cf ~:-gl~c. (5-.~>ro~o-tl-chloro-3-lndolvl ~ tet;ronide) d;ssolved in 0.1 M sod~rm ~ho.~p1~ac~ bufer pH 7.0 corta;ning 0.5 mM
pota.~ fcrri .~y.nidc ancl 10 ~ ED~A. The 't.~f ~.ectionS w-~e tre~-te~l fo 2 4 hour.~ t ~7~ ~ rirnied ~ith t~a~cr ,qnd ~h.~ afrlin~ k~t~
2t) ~fr.~ ~eeorclecl bv vi,~rtl ~n.~Pctio~ by mic~o~-copy.

8294tSO~2.0~lJI5/lKM/AI2/l991 b7 ~ 1~:55 J~ ~',~'-.!1 ;,-,: ]r Fr~ P Ir~ci~ -r ~ El=E T~ f~
2 ~ 7 7 Purîf Icatior3 of ~hitinA~e 2, ~ ~nd 4, nc tllc chlcln~s~ '~sE~ 13fld &
t~an~se l~oen2~mes Acidic and l~asic chi~in~;e isoenZym~5 ~ere p~lr-~ied tu~,etheE -~ith ~B-1,3~ canct~es fro~ su~ar be~ leaY~s a~i 5hown in the following flow S diagrai;~

2 kg of sugsr beet leaves 0 1 M l~a-citrate 1 mM DT~ Homogeni2~tion 1 mM BAM, pH 5 0 Centrifug~tion ~eac t~eat~nel It ~ SOc ~ 20 ~in 90X (NH4)2SO~, Dialysi~; 1~ mM Tris, ~ pH 8.0 FF Sepharose Q
lS Chit~n colu~n R~ff FPLC, Mono S FF S~ ~ph~ro~e Q
FF-Seph~rose S RP HPLC ~ro~ato-f~cusir~
L.~inarin-Aenro~ chlt~n~ 2,3, and 4 FPI.C; Mono P
ncidlc chltlnase FPLC, Mono S
RP-HPI~C
~-1,3-gl~canAse 3 and 4 Th~ su~ar o~ct l~a~es wcrc obtnine~ in I~tl~ rge se~tlc, s~e ~Rio logical Mat~ri~l~). In the followin~ each o~ thc purL~ication sceps outl;ned b~lo~ will bæ expl~incd. The equip~ent ~nd procedur~ ~lscd for ~ch stcp nre c~r~ied out ns ~e~cribed belo~.

82~50~1~L5/JKM/A1~1991 07 ~9 15~5,c i ';' F F ~ [II-i lT ~ ; !r, l E.L- E -r~ J ~ - _ 78 ~ 77 ~xtr~c~os~J ~ protel~ S ro~ Cercospo~ ricol~ nfc~t~ suy, c I~:A~e ~

All steps w~r~ perfvrmecl ~t 4C. C~ntrifug~t~on ~s car~i~d o~c at 20000 x g for 20 min~te~ in ~ ~entrikon ~v~el H-401B cenerif~e, f~Ughf~U~ ~he pur~fic~tion procedure.

Pr~p~3ratSon of cellf~ee-ex~acts 2 kg of Gercorspor~ infected leave~ ~er~ ho~ogcni~ed in 4 1 Na~
tr~te buffer pH 5.0 corltainin~ 1 mM DTT ~Dithiothreitol~, 1 mM ~S
t~en~midine) (startir)g buff~r) and 200 g ~owex lx2 (lC~ fmesh si~e. ~le homo~en~te ~as squeezed through a rlo~lhle la~er of 31 ~w me~h nylon gauze, before centrif~tion.

Preclpt~ ior2 ~h he~t and ~ onlu~sulfsc~?

p~rr~ nt fr.~ctLon obtained ater t1 e c~ ntr~ fr~ .ion w.~ he~reA
~t 50~C for 20 min~tes nnd sft.cr cooling to ~C, th-- pr~cipi.aCe ~.s lS co~ cted b~ centrifu~tion. Solid :~mm~ni~m~lfn~ W.l~ dct~l .o the s~pe~l-rl~t~nt until ~ 9~Z s~tur~tîon w~s .~cl~ ed. Af~er ccntri~
~ion, the precipi~n~d proteins were li~.~olvod in ~ rtin~ bufffr; t ~1 of buffer/10 8 o~ st~r~ing m~terlnl.

P~rif1c~tlon of ~hltlnR~ ~, 3 and ~, ncldlc ehltl~ F." nnd 1,3-gl~tc~ se by column chromntogr~p2ly Chitin.~ce ar,~ r~lucAn~so i~;octlzyme~ e r~r~ f~c~ fr~rn ~h..~ R
moni~un ~llf~ p~ccipi~;itcd pr~tein fr.~.r.ion. f~er r;v1~lbil;~rlt~ol-, the ~rotei~l *oluti~n w.~s (lia1y~.~d ~r,~1n~.t 10 ~ Tri~ cont.3in-in~ M l)T~ nt~d ~ mM h~M. D~ntl~ur~d pr~in~ were relov~d hy e~ntLi-f~g;1tioll ~n-l t.l,e .s~lpern.~t.an~ ~as loaded on t~ne llbOVe o~tli.ned t~o colu~ns ~.g i) a S0 ml FA5t F10~ S~m~05e Q (~h~rmAci~) ~n~
100 ml ~hicln .olumn (prepare~ ns clescribed aboYe~, the col~n~ heing connect~d in series. The colu~ns w~re eq~ilil~r2~ed ~ith the Tris buffer, be~ore 281 ml of the s~mple we~e 1oaded IJilb~ d proeeir~
cl~lding ~-1,3-glucana~e ~ere remov~d ~y e~tensi~ hi~& with the starti~g b~ffer. After ~is~onn~cting rh~ F~st Flnw S~ph~rose Q co-U/~12~1~1 ~ 2~ lSSS

r `,~ FF~ r~ r ~ El~ it.~ F.~

79 2~8~77 l~n, ~he chle~n*se w~s el~ted f~o~l t.he ¢h~ti~ cc~l~n ~;it.~l 2acetic ac~id. pl~ 3.2 oont~ining 1 mM ~T. rhe a~i~io ehitin~e ~SE~
w~s elue~d fr~,~ the Fas~ Fl~w Se~har~se Q ~oll~n with the T~is~ ffe~
~ont~inil~ 0,5 M ~aCl.
S Pu~ific~tios~ of ~-1,3-gluc~nsse Sep~Rt~t~n of ~ 3-~1u~:anase on C~ti~n ~x~h2-2g~ Chl-o~t~gl-~ph;~
Prot~ins whie~ were not adso~ed on either the ~ast Flow Seph~rose Q
nor the c~hitin ~olumn were colleete~, and c~.oneentrated t o 60 ml b~
press~re clialy~i~ wi~h an ~nicon PM-10 fike~ (D~nve~, ~, U.S.A.~.
10 After dialy~;is overni~ht AgAinst 20 ~M Xa-acetate buffer ~t p~i ~.2 contalnin~ 1 mM DTT ~nd 1 mM BAM, the prote~n sblut~on was k~a~lecl ~n tc n S0 ~nl Fa~t Flow Sephar(>se ~ c;ol~ (Phan~a~ ) e~uilibL~ted in the dialysi~ buffer. U-~ads~rbed protel.rl~ werP removed by w~.~hi~g wi~h the e~lilibra~iorl buf~ ound proteins were el.ute~ with ~ 600 ~1 1.~ linear ~radient from 0 ~o 0.5 M ~aCI ~n ehe s~.nrtin~ hnf~er.

Three ~a~or p~ak~ A. ~ and C o~ B-1,3-eIucnn,~se activ~tj were ob ~ .rved. P~ak ~ w/i~ furthar fraction~t.~d ~y ~ffirtity cvlu~m chrom-.tcog-r~hy on ~ minarin-Agnrose. Penk~ A ~n~l ~ w~r~ n<>t utth~r p-lLificd.

Pl~t i~.ic~clon o.f p- t .3~ U~:An.~QCe Or~ m.~n~ri~ n~-C~ C

A ?~ ml colu~n f l~tminnrin~A~nroc~e w~s ~q~lilibr-~tt~d wi~h .1 10 mMT~i~.; bu'~r p~ ~.0 aont;ining 1 ~S o both D~T and P.,~M. rh~ proreirl frn~r.lon.s from peak ~ ~as combina~l, aoncer,t~1~te~l b~ ~r~ u~e ~inlysil;
t.o l.S ml ~m~ dinI~ ng.tinst t~l~ Tri:; butf-r. Afear lo:~irl~ of t~
.~ample on the L~ntinnritl-Agsro~ colwnn, ~ha flow through the colum ~5 WBS stoppe~l or 20 ~inuta.~ to :~llow th~ ~-1,3-~;lu~nrulse t~> b1nd to t~le ~ffit~icv llgtr~. Unnbsorbad prot~i~ wa.~. r~o~ d ~y wn.hing Jit.h T~i~ buffer. ~-1,3-~lucartnse wn.ç elu~:ed witl, 1 ~ ~aC1 in Tris buffer.

Purifi~t~on of 4 ,B-~,3~ ea~2ase i~oenzymes by FPLC

~rA~eions fro~l the Lamin~rin-AgRrose coI~mn ~irh ~ 3-~lucAnR~e ~cti~ity were co~bined, concentr~ted and dialy~ed o~e~ ht ~gain~t ~S~x~ noM~A~ S~SS

~ ^F~ rll-lLI r ~ .EE r~ J ~ ~r~ Fl . r~

. 8Q ~8~77 20 ~M ac~t~te buffer pH 4.5. ~he pro~ei~ were sepc~rated ~ c~ r e~teh~llc~e col~mn (Mono S~ (Pharma~5~ on the ~PLC 5y5te~ usin2 ~
lln~r Ma~l gr~dient. Four ~ajor proteirl pe~c wer~ ob~et~ed ~ee Fi~. 21). ~ley all four hy~rolyzed ~he 3H-labelled l~min~ sub~tr~-S t~ in the rcdiochemical ~ss~y for ~-1,3-gi~canase (se~ ~bo~

Pu~f icRefon of the ~-1 .3-gluc~s~ on Rever~e Ph~e RPLG

Tlle p~rific~eion w~ achie~ed by injecting t~e ~P~C-purified ~-1,3-~lucan~se in~o t~e a~o~e des~ ed Poly F ~e~er~e p~ e HP~C col Non-adsorbed ma~erials ~buf~er~, salt et~.) were re~no~ed ~y wc~sh.in~
with lOX acetonitrile in O.lX TFA (trifluoro acetic acid). Proeein~
wer~ elutPd by e~ploying a line.~r gracli.ent of Rce~c,nitril.e from 10 to 70~.

Af-er Lhis de~lt;ng/purific~tion ~tep, pe~k 3 ~n~ ~ were ready for i? N-~c,rminal a~ino aci~ ~equenc;n~, ii) ~n~ine ac.id compo~le;on .~ lyciis (see Exa~pl~ ~), iii) ~ryp~ic ~ es~ion to ~cllie~ pe~.itl~
and iv) injecting into r~bbits to produce polycton.~l ~nt;boclies.

Purlf~.Atlon o~ c~t~ln8se 2, 3 ~nd 4 ~lut.ion of the ~.hitin eolumn with ?0 mM i~ceri~ a~d, p~l l.?, yielded ~0 fri3-:tion~ (10 ~ r;~cLion) with clli~inase ~ctivi~y. Ihe fractions w~r~ colnbin-d, AdJtlieed co ~ , con(~ tr~t.i~l ~o 1rJ ~nl ~n~l <lialy..ed ~3n~t i~ 20 m~ N.t i~c~t~te bu~er ~t; pH 4.~.

2 ~l aliqll~ts wcr~ lo~ded ont.o tho .~bove m~nt~on-d ent.~on cxchan~,e col~ Mono S) ~y rl-,e FYLC syst~m (P~ m.ci~ ;oll-ads~rt>~ Inat~
ri;~ ; w~re rc~ovr~ hy ~ h~n~ w~th che .q~ti~te h~ffer. ~ t~-,n ~c r.he chitin~l.se i~ocnz~e.s wiqs ~ch~ev~d w5t.h .~ l~ne;lr ~r.~dienr. rro~ 0 r.o l M NaCl in thP AeetAte h~t~er. ~l~ el~tion proflle is ~;h~wn i.n Fig.
1. Fo~ fllrther p~riflca~ton, the revor.se phase V~AC RP~ HPLC col~mn w~s cmpl~yed. The condir~ion~ ~ere ~si~ r t~o ~h~ deser3~ed ~)o~ve in~
connectlorl with ~e purif~catlon of ~-1,3-~lucanase.

- ~w~xkx~l~5/nKM~Al2/l~l ~ ~ lSss IJ', ~ c ; ~ ' F~ l F ~ E~EF rl-l t ~ ~t_l î l F~
~8~77 Purificae~on of a~idlc ~h~tin~e S~

.P~ O~qtfhlî of th~ scldic c~lir. 'n~e "SE" ~n ~nLo~ h~&~e ch~orr~-cography The ~cîd.ic ~hit;n~so ~SE" ~as eluted fro~ thc abo~e de~rJb~d Fast 5 Flow Seph~rose Q col~n wi~h t.he ~ri~ b~fer con~alnin~ 0.5 shown in the p~riflcation ~scheme The proteins we~e ~i~lyzed agaln~
10 mM T~is-HCl, pH 8.0, an~ loaded ~n~ a 40 ml ~Fas~ Flow Sep~arose Q~ column equilibra~ed ~th the sa~e ~ffær. The protein~ were el~d wieh a 800 ~1 linear ~odi.wm chlori.te ~radiene from 0 to 0,5 mM NaC'..
Fr~c~ions containitlg c.hitinase activity as de.er~ined ~ e radio-chemical chitlnase ~s.say t~.scribed above were poo~e~.

Pllr.Sfic~ion of ~ci~ic ch~inase "S~" on ~lr~r.~r~f~co.~in~

The prot.e~n fractions were dialy7ed a~ainst 25 mM Bis-~Lis, rdiu.ted to pH 7.0 with i~in~di~cer.ic acid. A lS ~ polybuffer ~xc.llan~er~
is co~tunn (Phflrn2~c;rl; ppJ~ 74) w~ ~q~llllhrated with r.h~ c~lrt~ h~lf~r Qnd 50 ml of the sample was lo;lded t~nAbsorb~d prorein~ lrer~? rer~o~ ed hy wa~;hing w~th ~he ~ Tris buffrL!r.

Applicca~ n Or Pol~buffcr 74~ nd~usted to pH ~.fi crc-.lted .2 I tne.~tr pH ~,radi~nt from 7 to ~.6 .2nd g~v~ d-~.orl rir~n ~ r ~l pr~le.ein- .
~0 ~he a~icli~ Chitina!:c SF.' ~.; r.ti~l r~ain~r3 on thr~ t:o11lmn slt r.his pH but. i r. Wfl~ de ~ r~ed by ~t~lr~ition of n 3 M ~;~Cl t.-, t.lle Polybuf ferJ4 ' .

Put ~flc;2r~ior~ c~ acidic ~l~icin~se "SE" l~y FPLC

E'rbte;n ractiort ~iL~I hlzh ch~tin~cc ~c:ti-~r1t;~ ac; ~l~-r~r~ y t~i~
~5 radiocl~c~ic~ll chit~nAs~ ASS~ty d~sr.rih~rl ~bove wer~ p~lol~d ~lcl r~ialy-z~d a~ai.nst 25 ~i Bis-Tris at pH 7Ø l~e proteins w--re resol~e d on A Mono-P F~PI.C colum~ ~Yharl~tacia) eyuilibr~ d with ~h-e ~is ~r is buffer. After an :~nitial wash with the s~rtln~ b tffer three t~oen-zyllte~s of ~cidic chiClnase SE~ was separ~ted using ~ lirle~r s~
30 ~rAd~ènt from O tr~ O . 3 M NaCI (FiE . 3) .

~50e~001/~5/JK~/A12/1~91 07 29 ~S,SS

,'~
~.

Ff~ F~ r:t~r ~ ~ ] r.,~i~i~',:,`:,~. l-'~ 1 F . ,--, 82 ~ L 7 7 an~l~sls ~f the en~.y~Ac~c ~ ge pR~t~l.n ~ su~Ar b~r ~ .r~ e 4 4V ~1 ~f 3H-l~b~lled ~hitln (50,000 cpm! w~s in-ct~ar.~d with 7 ~g of chi~inase 4 (puriied as described Rbov~) in ~ O.1 M citra~e buffes ~ pH 6.S. The to~al ~olt~ w~s 300 ~tl Afeer a specified t~m~ (~5 ~in., 30 min., 1 hour and 2~ hour~) ~he re~tc~on -~ts stopped ~y ~he additlon of 300 ~ll of 10~ (w~v) TG~. The unr~acted poly~er of 3H-l~bell~d chitin t~t~ re~oved, and an ~tllquo~ ~00 ~tl) of the super~
natant was applied to a thin layer chro~tato~raphy (~) plate (Ssli~a g~l 60 H, Merck~. The ~obi.le ph~se ~as n-propa~ H20,~H3 (70~30fl;
v~v/v ) .

A stand~rd of ~ ac~tyl~ cos.lmine--derived oligosacehar;des was pro-dtlced by flcid hydrol;~is of chitin (Rupl~y, 196~). This stand~rd was tw~d ~o lo~al;.7.~ the products ~rom tht ~nz,vmatic clea~g~ on ~he TL~
plt~ .on~s of in~eres~: on the Tt.C plate t~ere r~ov~:d by scr~pin~
with ct razor blade ~ an~ t~e .~ cn g~l con~aining ~he 3H- labelled oligosaccharide~.i w~ trRnsf~rrcd t~ ~ scint.lllsttion vial 10 ml of seintillrttion ti~uid DimIl~.lnie ~P.tck~trd ln~r~rlts) w~ro ~ddod ~n~
th~ dioactivity w~i (leterm;ned by r~ liq~ l cie~nr~ ion ~ye~tro-photometer.

Antifu~g~ t~.v-lty lbtt.ory eff~?ct ol -.u~,lr b~er cl~1r;n,~.~- ', h;~ q~ll o~;erv~d on Lh~: ~,r~w~ L l?l~t}~ C~?-t -?.~p~)r.~ b--~t~c~ TL ~f ~ l r~?~?.~;Ci t!it~
ulo~ t~mh i l~At ~ on wi t)~ c i~ ); r. ~ ct~
25 ~ 1 ~ 3 ~lucana!;~ 3. Germirl~tion of ~o-l-'. nn~ er~,w~l of hy~"~e rorn ph~top~lhogenic f~ln~i~ e S C.?r~o-~o~ , in ~lle l~res~nc~ of ~n~if~.~r1g;~1 protetncilm~t)~ nAIy~ with ~.hree ~ffert~ne m~hod~;.

~et~t~d ~ c~rrte~ t on micrv~cv~e ~ le~i cv~fr-~d w;th ~ thLn f11m ~ 2~ m ~nd l~uh~ted with either ~f*r (contro1) o~ r.~ti~.
of` th~ ~ntiL~Iog;1 proe~Sns. ~rmInation o ~;pOres or ~ro~th of th~
2yc~1iu~ is fol1Owed b~ ~t~t.~l~ng ~ith Cal~vfl~lor '~h;ee ~efore ~rl~ly ~is by R fl~OleSCent rlliCroS¢ol~e.

k~ 5/n~11A12/t~~ 5 ~3 `~ 0 ~ethod II ~s ~rti~d out In miero~it~r y~al:es oon~:Qinin~
medi~, 10 oc 100 spores fro~ reo~por~ ffer (contrc.l~ or th~
~n~:if~ng~1 ~ro~ins. '1~.¢ ptates are irls~lb~eed at ~5~ ~fore the optcal der~sity ~t 620 ~) is de~e nnined at speoiif~d ti~e inter-5 ~I.s.

In mcthod 111, r~diotra~er techniques in co~biFIa~ion wlth ~tox~-diogr~phy ~e u.sed ~o demonstr~te tha-~ ~hitin ~nd ,B-1,3-,~ c~n are impox~an~ ~ell ~all componenes ln C~ c(~por~ and that: fhitin~e 4 ~n r~mo~e radioactlvi~.y d~posited In the hyph~e ~ip of Ceroo~por~.

0 Me~hod I: Microsc~py Sl~de Bio~ss~y 'rhe ~nlcros~opy lides were eovered with ~, thin layer of poti:lto d~x trose ;~gar (PDAj end stored fo~ 6 ht~urs on moistenecl f~lter p~per in petri dishes. 10 ~1 o a spore susp~nsion (lO.000 spores~l) w,ts pl.~tked ~n th~ ct:n~t!r of t~te .~lide. tO yl G~ a 1() ~I Tri.c~buffer, pH
8.0 or îO ~1 o ~t preparzttIon containill~, 20 /-g of -he nnt.Lftn~l protein to he t~ted wa.s nppli~ Co t~le .~pore cuspension. ~he ~n-ti~ungtl prot~i~ wrls dissolve~ th~ 'rr't~-huFfc~ anc' il~?r~d r.hrou~,h R t),22 ~m filrer be~ore mix;n~, wieh ~ht spOre c~l~,pension Th~
p~tri~ish wts s~tlt-l wlth t~pr nnd incub~t~e~l ft~r ~ hollrs .~t 'tOC
20 ~nd f~-ll lighr.. ,~r. the ti~e or ~v~ tatio~ rh~ c~t~ture was st~in~
w~.th tht2 fl.uor~sc~!o~ dy~ (~al~ofl~or Uhlr.e (O.OSX (w~ wart~r) for 10 rttjt~, (,;11cofl~lt~r ~h~cc binds priLna~ilJ tt- erlt wsltl-. contni~
n~.ol!n~ str~-~ttlr~s o ch;r.in, rtnd the fl.uorcst,en~ ~ly-~ /mty ~.hercfGr~
~ier~t~; s ;~ m.~rker for diff~r~rltia~ior~ l .srowr.l~ of t.he hyphae c~
2~ w.~tl.l.

Mec~c~d II: Mio.rotirer ~'IAtt' nfo~ sl~y 100 ~1 o pot tto tlt~xtro~t? hrt)th (PD~? Ilqt~id p,rowth w~diun~ W.~i plAc~ed in e~ch ~e~.l of n mi~rotit~r plat~ . ~ spt rf: ~iuspersion of CerooSpo~
(lOO,C~00/Lnl) was filt:ered r.wicc thro~lgh ~ layeLs of stertle gn2e to 30 remo~e sm~ll amount~ of myoioli~ fr~gmen~s. The ~pore suspension w:~s ced 1:100 And 1:îO00 with ster.ile w~t2r, before ~1~iqt~o~5 o 100 s tr~nsfL.~rred to the microeiee~ ~ells. The ~ntifun~l proteins were dI~sol~e~ in the same b~ffer ~n~ ~ræatæd dS de.~eribed ~ho~e f~r 829450e~001tlStJKMtA12~1991 07 29 1S~

'; ' . L~ F~ fJ!rl~ r ~ E,~t- rl~ ."- ;-,-l-..., F ,,J

84 2~
~ethod I. i~,e bioAss~ys wer~ arr;ed ~t with 5 repe.~t s ~
dilutivn of the ungal spor~. The ~ic~r~iter pl~te w~ 3~led with cape co ~oid e~.~apOrAtiOn ~ d con~am~na~ion. Aft~r Incu~io~n ~t. room ee~pera~ure on ~n a~itator oper~ted wi~h S0 rpm, the t~pe waS r~move~
5 ~d t.wice a d~y, the absorban~e w~s m~ured at 620 I?~ The ~ermina-tlon and gro~h o~ the f~n&~s was follow~d f~r 4 d~s by n~e~r~ring ~:he absorbance. For e~h co~nbinatic)n of ant.fung~l protein ~nd spore dil~ltion rh~ ~bsor~ance v5 ~im~ WA~: plo~ted.

~le tllod III - Au rorad~ ogrsphy 0 Cf~rcospo~a cultu~es wf~re grown c>n a micro~ie.o~e r~lide ~ describe~3 in method I. i.~quid ,~,rowth ntediurn (PDa~ contfllning 31~ hel~d N-ace-tyl~7,lucos.~ille ~7c~s d;~.itrlhuted unifonslly OVf~ a onfn day old (:~-lr~r~.
A~e~ onlhatlon for 2V min. ~p~1lcie l:th~11ing~, th~;- re (:tion ~,rowth-/in~.orpor~1tion~ ,ts sr.opp2d b~ ippirlg thc ~ ro~ .ope r~lidc in 6X
15 (w~vj vf '~CA. The prepRrQt:ior~ i cl~.i,y(lr.~r~d in ln c~lt;.1r~oI gt ~di~nt ( 70-100%~ uncl dried.

Aftf.~r th~ puls~! 1tbe~1ir-g. S0 ~0() ,ul of ~ fi~cri-,-- cnntflinin~ ch.itl-I~.t~;f~ ~ in tO mM ~ri~i bu~ s .~t ~li 8.0 w~ di.r.trib~tf:cl ,ver ~n~ hi~lf o ~ fl%e-l and d~hydt~tr.~d cuit~t:~:. Tt!e mi-roC.~ p~ ~ idf! w~.s pl-~ce-l ~!0 on ~2oi~t~n~d iltcr p;~per in ;i ~f~t:rldi ~h At~t ~n~ , thf~ p~tr~ -clish, thf-~ prepatr~ri.-n w;~ri inc~h-lt~d .1~ 3~)'`C fo~ 2n h~ r~ C~SI~yL~l~
~r-~.t~:m~n~. w~ r.opped by dippinp, ~he s.li-i-. in 6X ~ t~nd ;ho yr~yar.~--t.io~ w.~s clehy~ki~tf d In ~r.h~tn~l flr; clcs(:~;bed ~bovo.

~ microscop~ s1ide t~.3~i (o.~.r.,-d wSth nn n~l~o~.,td;u~ ,,hic f~rrul~ion 2'i ~llford K S~ Aft~r dr~yinf, Lh~ emt~lsion ~xr-~nclv.~l; wi~h ..1 ";~11 dryer`' t~ slide W;~ n~d ~n ths d.~rk fot 7 d.~y~. ~r. 7~(` rnd low ret~tive h-l~tidi ty fc.r cxpos~rf~. Th~ el~t~ll<iS<~n ~:~s d~ve!op~.d l~y pl.ae;r,~, tlle slide in Ko k D-t9 d~velop-~r fc>s 10 L~inute~ follo~ed l,y ixation for ~ m~nutes ftnd WtlSh~l in r~mniltg w;it~er for 1(~ m~nuc~ rer 30 drying the prep~rlttion wa:i ready fc r cl microricope an;~lysis of the h~ph;,te of the f~mgus.

g~ ;/JRM/A~2~1~91 07 29 IS:SS

t~ .; . 1 F f 'l-l' ~ G~; r ~ E.r E r~ F', ~ ' - 85 ~ 7~
~roduc~on ~f ~nCItx)d~es for ~e ~rn ~erolc~lc~l arl~

~rod~ctii>n o~ pol,~clo~I an~ihod~s ~o c:h.r~inR~e ~, 3, ~nc~ 4 Free~d~i¢d purified chitilt~se 2, 3 ,md 4 ~obt~ned a5 c~scr~bed abo~e) were di~sol~ed in Tris buffer ~10 ~, p~f ~,0) ~nd di'.~ted 1:1 5 wlth ~reunds incom~leee ~djuva~t P~lyclonctl 2nt-~otlI2~ wer~ ~~lsed in r~-bbit s accordir.~ ~o con~entional method~ by Dakop.~tt3 (Den~rk~.

Produc~ic~n ~f monospecific poIyclvn~l r!r~ti~od~e~ ~ chi ina~e 4 pepti des ~e procedure w~s carrie~l Ol.tt hS described in de~ail fc r the prodttc^
0 tios~ of ~ono~.p~cific nntibodies to AH/~S pop;~des (;~I.t~( U~SC:l ~r-d Poulsen, subMittf~d). Basccl ort con~put:er ~nLtljsi~ of rhe ~tmino tcid S~ql1erlCe fOI' Chitirl.lSe 4, four pcptides ~ere ~ete~t~d o~ the criteri.
of hydrophili(:ity ;~n~l vnr{nbil ie; bccwe~l ct,~rin"~,e ~ ;~;ld other h~qsic chieirl.t~e~. Pcpeidcs werc CUst~>ll~ c~t~ehc~ d ~y C~ol~hricl~7e ~.e~eé~rch ~iochemic~tls (UK) ~te~ ~ruc~ur~s w r~ ~erlflcd b~ ~ass ~ eerQ~copy ~nd ;~no ~cid nn~lysis to estim.-t- pur1ty.

Bcfore i~ ni~;~riol~ th~ pt:ptlde~ ;e ~onJ~.Ie,lced to d;phth~rl~ toxo id. '~e c~rri~r, diphth~rla to~oid ~ s e,~ r~od to th~ r.(~xoid-.~.ul o.c~cc~ny~ st~L ~ri~.tci~ hy rene~.lon ~itll c~r~o-15~n~ld~ (E~
fc~llo~ocl by ~ lr~xy ~ 1fo.~uc(:inis~id~ ~er tlle e.o-.lnlinp" th~ f~ur ~liff~rf~nt: p~pr~i<l~-diphchrti~ oi-l e~n~ ntc~ w~ ifif~ by r, fil~ tion ~n ~ ph:~cryl Si-~0~ columtl. ~le ~eh mo1ee1l1nr ~ ht r;~tion.~ wer~ colle~tr~d, ~se~ dri~(~.-:n~ di~ol~ is1 w~rer. Irn muni2..ltl.Qn in rtlbbit~ ~ere p*rf-,-m~ n~ d~ ribtml ~Ibov~ for tho p~o~uctl~n ~I p~lyc~on~ ntibodies t~ c`lltir-lA~t~ ? nn,~ 4.

SPS .PAr~ .~nd fmmu~oblotting For i1~unoblotei--~, protein~ were trails~tretl b) sc~-dry blo.~ng onto a 0.45 ~m nicrocelltlos~ ~em~rdne ~hleiche~- ~md ~ch~ell, ~GR~
aft:er separh~io~ by SDS PAG~. The anti~en~ were p~b;~ with prim:~y polyclon~l rabbit antibodi~ ;sed ~gain~t ehitlnAse ~nd 4 ~$e~
abGve) and stlbseqt~en~ly v~uali~ed us~ng ~lc~llne phosp~t~se ~on-s/JxM~AJ?.~ 9 IS~iC

86 ~ 4~7 ju~,~ted s~condar~ an~ibodies (Dakopa~ts, De~a~k? ~col-d i ., - '~ i Andersen (198~

Anal~sis of the amin~ e~id cornp~sitlon o~ ell8 p~.~r~r~e~ chl~.;In~3~ iso-e~zymes 2,3 ~n~ 4 and ,8-~,3-glucRn~e 3 ~nd $
,'.`~
5 Asfter freeze-~ryin~ ~he purlf~ed c~litina~e leoen~,~es 2 1 3 and 4 ~nd 1,3-gluc~n~e 3 and ~ wer~ subjeoted to auDin~ ac~d an31y~is ~s described by Bafkh~It and Jensan (~89~ . An aliquot (4. 2 ~g~ of each .~ of .he chitinase isoenzyme3 ~rd the ,~-1,3-gl~lcanase respec.i~ely were incub~ted w~eh 3,3-dithiopropion~c acid to leri~rati;!e ~he cys~elne 10 Leslclues befoze acid hydrot~si.s. The detenninaeion ~as repe~eed twice .
;
PrePAraC~On and a~l~nc~ ACld sequenc~ un~lysi~ of ~r~pl:ic peptlde~ of sug~r be-~t chltlnnse 3 arld 4, "SE~ J~nd ~ . 3-g1UCUnA~I?. 3 And 4 Trypt~ fges~ion 15 Arter RP-HPLC ns described ~bov~ .~nrl frc~,.e dryin~ of p~o-teint.: were r~di~.solve~l in ~00 11 o~ 0.2 M Tris-ll6'1 ~pH 8.4) oont~
in~ u.~ni-line hjdrochlori~le. 2~ c~1 <~i thi~!hr~ Sto; w,~ klf~ rd he prot~!in w~ ro-lu~.~d ot 37"C for /I h;~ur~. ~lo~l~. r n;rros~ n. 30 rLM
i o~ . e r. . lln i d~ wr. ~ dd.~ d l~ n~l ~ h ~ r. i o, l wn ~ n l l o~, ~ d l o l ~ r ~
?0 the ~lark or 40 ll~inut~ qt 2',~C un;~c~ T~i t~'o~ . U:~r~ tC~.; iC)~lO;l(:t t ~)~id-~ w.~ n~cti~ tl~ by ;Id~litlo~ of S ~Ll of ~-m~:rc;l}~oei:h.3no1 fo1-lvwed by in<:uhAe;on or lS IllinU~eS ~It ~5~C in Lhe d;lrk. '~~ r. tein ut~on w~ irlly.:e;l a~Dill~.r 0.2 )i .-mrnonl~n r i~rl~oî~t~ (f)ll 8 ()~ fbr '>4 ~lours ~ ;: 4~r, i.n rhe d~rk u.r.inK Eyyt~ndorf tec ~. tllh~ ]y~
25 t~lbi~ 10 kl)a C~lt o~f; Scr~ or~:, 8~r~ ) in~er~1 u~ ru-.~th .
p~mct.ured lid. I'hQIc;lfter, l~reCipit.~t~ rot~ a~ tf(l ~y cent~ g.~t.ion I'or S ~ninutes a~ 10,000 x ,~ .~n~l t?le .~u}!ern~ r~ ~a.~:
trAn.sf~lre~l to ~nother tes~ ~:u?~e. I'h~ ~ro~ in ~. llet W~5 p~rtS~.lly solut~ilized by ~dctition of ~ few phrt~cle.~i of vuaniclin~ hydroohlor;de 30 ~nd in~ubated wit~h 4 ,t~8 lPCK-tre~ted trypsir: in 2C J~ f ~nmoni~
c~rbonate ~p~ 8 . 0) ~I~. 40DC fo~ 30 mi~ut~ . F~nA11Y th~ supe~n~t~.nt an~l 6 ~ f 'rPClC- tre~ted tryp~in we~e added. ~e d~gestion w~s al -lowed to take pl~ce ~t: 40C for 4 hou~s and stopps~d ~ dd~ ~io~ o~ 2V

.
8~450~1s.0~ A~2/1991 ~7 29 15~
'' ..'1 F- F~ r ~ r ~: l lh l E'EE ~ F' . -,'' ~7 ~ 7 ~
~1 of TFA. The peptide sbl~eion w~s ~ub~ected to RP-Ht'L.C o~
CL~ column (0.46 ~ 15 cm; 10 ~n p~r~cl~ 5ize; The ~epar6t~ions ~roup, California} u5ing the mv~ile sysee~ describ~d abo~e for ~P-HPLC ~f protelns ~See Fig 4). Collected pepei~es wer~ ~iluted 3 ci~es with b~ffer A ~tnd rech~oma~ographed on a ~v~lo~il C18 c~lu~n ~0.~/ ~ 10 cm; 5 ~m particle size; ~r. 0 Schot~, No~o-~ordisk, Den~Lt~k) u~ln~ rh~
mobile syse~m described above. S~lected peptldcs ~re ~ub~ected to ~mino acid ~equence a~alysis.

~nti~o ~cld sequencing 1~ Amino acld sequencing of ehe pept~des was done with a P~lsed Li~uid Phase Prote;.nJPeptide Seqùencer model 477 and a HPLC 0ll-line PTH^
Amino Acid ~nalyzer modei 120 A ~rom Applied Biosysee~5 (CA, USA~, accordin~ to the manufacturers instructions.

Bacterlal ~tralns and cnzymes 1.; Res;riction en~.ymes, Klenow polymcrtse ~rml T4 ~A I i~,fi.~C w~re .supplle~ by Bo~hringor ~annheim .~nd us~d in accor~lr.tnce w.~h t~e mantlf~.tc^
turers instruccions.

p~lu~cripc was suppli~d by Sr.rata~n~ (US~).

p~l w..~g .~ur,~ d by Bo~hriny,~r M~nn~Ieim.

20 Fot subclor~ in E. eoli, ransfer of DNA W~! carri~d ,ut ~sing DHS~
. o~ lls (from BRL) nccordine to tiI~ m:~nuf.~eturrrs inr,~r~lc.~i~)nc.

I~O1A~10n of RN~ fro~ s~gar be~t. l~v~

tsolation ~f RN~ w~s c~rried O-lt as d~.erIbe~ by Stou~,a~rd e~ ~.tl.
(1986).

25 Only sterile solutions ~d gla~w~re bRked o~ern~h~ at 180~C ~ere u~ed ~n order to avo~d eont~ination wlth RNaçe. 2 g of Cerco~por~
inec:t~d ~ r bee~ leaves (c~. Monova) obté-ined ~ de~erbed abt~e were ground in liq~id N2 and transf~rIec; to a 50 ~ aleon t~be. 20 8294~x.00~/LS/~/Al2J~ 9 15 SS

FF" ~ EFF. , 1 1 ~ r . h' -, R8 ~8477 ~i o~ extr~cti~n huffer ~lOP ~M Tr;.~ pH 7. S, 50 ~ F.DTt^. . . ~ G.`) and 2 m1 Of 10X S~S, 0.7 g ~f ~-1RV~1S~rO~S~ e, O. C5 g oE ~lrintri c~r~oxyli~ açid and 1 ~1 of (14 ~ ereapco~eh~nsl ~ere added ~nd the result.ing mixture w~s inçub~ted f~r ~5 ~inute~ at 65C. T~e 5 lne~lb~ted mlxture wa~ spun at 10,000 ~ ~ for 20 ~inutes ~t room temperat~re in a Corex tube. The s~lpe~-n~tent was transferred to a F~lcon tube ~nd ~.7 ~ of C~Cl w~ d~s~olved the~-ein. The mi~Cure w~s ~pun for 5 ~inutes ~t 10,000 x ~ ~n~ room te~perat~re i.n a Corex ~ub~, ~nd the supernata~lt wa~ ~r~nsferred tv 6 S~' 55 tub~s e~eh tO containing 1.4 ~l of CsCl cushion. (The CsCl cu~hio~ prep~red ~y dis~olvin~ g.6 g of CsCl in 5 ml of 0.2 ~ EDT~ and ~dding H~O
tot~l vol~e of 1û ~1).

The R~A of the ~upe~latane was pellete~ chro~lgh the ~ushion ~ een-trifu~atio~ for ~8 hours nt 37,000 x g and t8~ in ~ Beck~an SW 5S
roCor. ~h~ gr~en supernatant ~as remo~e~. and the CsGl c~ h~on was caref~llly washed twiCe wieh ~2 The C.~Cl cushion wat~ re~oved ~nd the ~A s~ellets wer~ dis~olv~d in a ~ot~1 os ? ml o~ H20. T~ .NA w.~.c prec~ipitated with 7 ~1 of 96% e.~hanol for 30 minutec- a~ -20C, sui)se-q~lently ~pun ~nd redi~solved in ~ ~1 o H20~ i~cl~ted to 100 ~M ~aCl ~ncl preclpit~ted wi~h 2.5 volumes of 96X ethanol. The r~s~ltlng RNA
pellet WQ~ spun as~d di~solved i~ 1 mt of H20. The R~iA C.onr~nt w~.c me~!;ured by <.Ictcrminat;on of t.h~ OD2~U (OD~60 - 1.O - ~0 Jl~ of ~A/~ n(l th~ RNA w~ tored n~ -2~C. Yiel~ bo~r. t ~g oL' ~otal RNA.

25 Purif~cntiosl of poly-A RNA

Puriication w~. ci;rrl~ out. nccordilly, ~o Chir~wi~ 7 (1~18).
T~e RNI~ wit.h ~ pol~-~ t~i1 Wh!; purified ~y .~fflni~.y ch~omJt~rap~ly through ~sl oli~o-clT c~Ll~lo~ c.otl~n O.S ~, of ol;~,o-dT ..ell~lo~e W~5 u~i~ed ~n 5 ml of o S M N~Ot3 for 5 mlnu~c~ (1 g of o~ieo-dT cellmlo binds 1.2 mg of pol~-A R~A). Th~ r~sult~n~ ~ixtu~e W~5 ~e~ltri~ ed with 10 ~ T~ pH 7.S un~l pH reached 7.5. An I cm co1u~n with a di~eter o 1 c~ de ~nd equilibr~ted with 20 ~1 of ~olw~r buffer ~500 mM NaCl, 10 m~ Tris pH 7.5, 1 mM ~D~A). The ~A W~5 denatured ~t 6SC f~r S ~inutes, asld S vo1u~es sf column buffer wer~
added to the R~A before ehro~ato~raphy thr~gh rhe c~lu~r~. Th~ "r~lr.-~ /nCMrA1211~1 ~ ~ ~s5 ~-!'' ~Jt, I4'~ FFCIII Fl[lll,lr ~ ElEE -r~l It~ .t:i ~9 `~ 7 7 th~ough~ ~AS coll~cted and su~ e~c:l c~ chro~aeQ~ ph~ r,2~
coluxnn ~ilS ~a~;he~ wi~h column buffe,. un^ill OD2~,0 reached 0 ~t or less. The poly-A RNA w~s eltlted with TE buffer i~ 1 m] fra~tlons, anæ
the ~NA con~:enc~ar.~on for each of the rac~ions ~as deeeI~nin~d at S ~2~i0 rfhe polv-A R~A-os~neair,~n~ fraot~on:i we~e pooled F~nd adjusted to 100 D~ N~Cl ~nd the RNA was preeipitated o~rernight wlth 2 . 5 ~o-l~mes of ~6X erhar~ol at: ^2QG. ~he pol~^A ~A ~as spl~n and d~ss~ d in H2G at ~ conceneration of 1 ~ 1 and s~ored a~ -20~C. Th~ y~eld w~s about l-2X of total R~iA applied to the col~n.

10 Isolaton of geno;~lc DN.~ ron stugar beet l~vrs Genom;c I~I~tA was ~solRtr!cl ~orr~ st~ty~r l~ t l~ (fiO.159.83& 131-4) (~ellnport~ et 4~ 83).

2 g of C~rco.~por ~nfec~d su~tlr beet l~es obrai~ed ~ cribc~
Ibov~ wcAr~ ~round irt liquid ~2 r~-1 fro,.f?n, .~nd the fr-ozc~ materi31 lS w~lc tr~nsferred to a 40 m1 poly~tlyl~ne c~ntr~.fuge tub~. 15 mi of : extr~ction b~tffcr ~100 D~ TI'i.'. pll 8.0, S0 tn~ ~bTA ~nd S00 ~ N~C1~
wc-c .~dded toget~hèr wi th 1 ml >f ?OZ ~l)S .Irld rt~r t~ g, clle rn~x ture was {ncttbAt~d r.tt C5C fo~ 20 ttlin. 'j Irrl of ~ M ~ tflcs~lm .~ tate w~re ~dd~d, thc ~iolutioll w,~; mixe(l (Vort.cx) itr,d ~n~ubrt~cd Lor 20 mi~m 2~ n lc~. Sub.~q~t~nt~y, L-h.~ m~xcurc ~r~l~, vcfi.ri~ f~r ?~ m1n. .~
~C, t "000 x ~ c ~up~rrl.t3rtt was fitterf~ rhrc~ ,h 1-? 1;1yt~r5 Vf mirilclv~ to d rle'~t C~ntrif~ t.llt ~ 5 m~ ico-pr~,punol w . ~i add~-l ~ncl ttl* ~nixture w~ cllhrr.-~d for 30 min. .~ -20C. A~f~r at ot.~r c~ntrlf~l~t,t~on nc 15,0~(~ Y~ y, lor 15 lDin .-t ~ h~ r~ wn.~
2~ w~t~;hc~l wit~ -;o~ er.~ nol :~ncl thf~r~ r (iri~r~ brictly brt~rc beir1g r~.~t~ nde~t 1rl 0.7 ml ~-f X-T~. t uff~r ~5() m~ Tri~ li 8.0 ~-r~l lO m~l h(~ .ct~tcp~n~; ion wn~i ~r~n.ri`~rr~Cl ~o ~ pi-erldo~ f tul f~ s~n~2 t!n~
t~.;n~;.3tetl o~ ~ Inilm ~ c~ ern~r~ftnt wi~.c extrrtctcd ~ r wi!:h ph~-nnl~chl~roform . The DNA Wcl.~i pr(!cipi ~ted by ;Iddi~l~, `7$ ,ul of 3 M N,~^
30 acetclte and SOO ~1 of is~-p~op~n~l, mixing and spinr,~ng ~r 30 ~;e-con~l~. Afte~wards, th~ pelles: ~a~ di~s<3lved in 400 ~1 of H20~ ~nd ~he s~sp~-2sion waS adj~l~;ed ~o 100 mM N~Cl an~ pr~cipi~ed wi th 1 of ~6% ethanol. The s~spen~:ion was een~rifug~ for 5 ~ih. ~d ~he s~ern~t~nt r~rnoved. The pellet was dried ~ fly, and ~he DNA dl~
35 solved ~n ~00 J~l of TE b~lff~r. 'rhe DNA concen~r~tiGn waS ~ete~i~at~

829450eJ~.~)I/LS/lKM/A12/1~91 Q7 2~ IS:SS

.

~ 7 7 using the absorbance a~ 01)2~, where OD260-1-50 ~g T~t~A~.. . ...
wns storecl a~ -20~ nti1 ~Ise.

Constn~ctlcrn of e suga~ ~e~t cDNA llbr~ry ~n the b~sis 3f su~r beet t~RNA ~ol~ted as described ~b~Y~ a A~AP
S cDNA library w3s con~tructed by Str~&ene Clonlng Sy~tems.

Constrt~ctlon of B sug~r beet ~enG~1.C I)NA l~brary C~n the bssis of geno~ie sugar bect DNA obtn:~ned as describ~d above, whieh had been p3rciRIly digested with SAU 3A, a genomi s~ r ~eet libr~y ~s con~tru~ed by clorlinK the geno~nic DN~ ln the Ba~llI ~ite 10 of the vector E~BL3~ libr~ry w.ss construc~ed by Clontech.

Plfl~n~ br~ri~.~ for ~crcening for relevan~ DNA ~equen~es The ti.ter ~f the libr. ry (eith~r ~>f the cDN~'~ or the ~,en~rn;e 1 ihrary~
w~s dete~mlned ~ccording to S/~mbroo~; e.' al. (l990), nnd ahout 106 phP.ges wer~ used for e~ch s.reen~ng. 1`ot e~h 24 . 5 x 2~ . 5 ~m pl~te.~, 15 2 . 5 ~ 105 phage~ tere mixed with 3 31 o the E. co;i ~tr~in XL L-Blue (In cnse of a suy,~r beer ~.ZAP c~NA l~l-r~ry) or LE392 (in e~s~ o the 3u~ r be~ec genon~ic l~,r~ry (~`MBI 3~) nnd ~rown in LB ~liu~l with 10 mM
MgS04 ~nd O . 2X m;~I~os~ ~o ~n 01~60~ . Thr mi~;tu~e W,ls ~lIow~ r.o s t~n~l at 3 7 C f or 20~ 30 ~1 nur.ec .

Sub~qi~q~lently, 30 ml ~f to~ ;8~r ~O~Z ~&.~rose in LB m~:di~ wit~ ~M
M~S~4 ~n~l 0,2X mnlr.~ "C) were lldded ;Ind the ~es-.lr~n~ ~ixture pl;~te~ on~ 24.5 x ~4.5 1~ pl.-t.es ~ontain~nr, 200 ml of L~ ~-y,~x ~n~l allowed to ~,row ovcrnight nt 37UC.

TrAtl~er ~f plaq~es to nltrocellulos~ filter ~n sltu 25 'rhe s~reening of ~ZAP reco~nbinant clone~; by hyh~ id;zsl~ion to sin~
pI ~que~ ln 5 i tU WRS done n~ follo~s .

Af~er ~,ro~th c>vernight A'C 31~C, the plates ~er~ cooleA for ~bo~lt 15 ~in~tes ~t 5C. Ph~g~s ~n~ DNA ~ere transferred to a hybo~,d-N nrlon F l r~ r ~ , F,~-E Tl l lt~ l F . 7:1 91 ~877 ~e~brsne (A~ersh~.) by plRc.n~ the dry fil~e~ On the IL
Pha~,~s ~ere flllowed to ;~-Ssor's ~ e filter for 1 so ~ Ijolnu;es~
Duri~g ~dsorptiv~ it w~s ~onven~nt e~ mark the filter Rnd plRte ~tîth a needle for ~rienta~cn. l replic.ate fil ter~ w~re mad~ the m~rks S on the pl~te ~ere filled w~th ink and It was then po~ le t~ ~ark the rep]ic~te f~lter~ ~ith si~ilAr marks.

rne fCl~ers were then ~l~ced ~i~h r.he ~pl~que side~ upw~rds on Wh~c^
ma~ 3~M fil~er pap~r sheets ~o~lced with 0.5 M NaOH 1.5 M ~iaCI for 30 sec.onds. rhey ~ere then ~sht-~d for 30 seccnds in each of the follow-in~ ~olutions. 1) O.S M ~aOH 1.5 M ~aCl 2? 0.5 M Tris, pH 7 5 1.5 M NaCl 3~ 2 x SSC (modlfied Bentos~ ~.9?7). The fllters were air dried and ill~in~l~ecl with W for ~ minutes with ~he pha~e ~ide upward.s.

PrepRrat~n of rndloflctive probonc ror use ~n screenln~ for s~sgnr b~et chlt1nase 4 in s~g~r beet ~DNA librf~ries Rele~nt oligonutleoti~es ~ert! labelled by pho-:phorylation with bac-~trio~hAge ~4 pclyn~cleoridt~ k~n~.se ~ccortling ro rhe ~et.hod ~esc~lbed ir~ Sambrook ~t 11. (1990). More specificnlly the oll~onucltotldes w~re s~lthe-.i~.od wiLhou~ a pho;pha rou~ ~t ~hei~ 5 termini and ~ere labellcd with ~ 32p from ~ ]~Tr u~ine th. en7.y~r bnct~rio~
pha~c Tt polynu~.teotitl~ kin~e.

PurificAtlon o~ rat~ih~A~)e.llhd ol.l~onuc.l~ot~des by pr~ciplc~clon wlt.h eth~nol Af~.~r in~cti~nt~o~ ~ ch~ bactericp?l~e T ~olynucl~o~ kis2a..~ ~y 25 he~ ., 40 ,ul of H2(~ WA~ adrled t o the t~ . rl,e contellt of wl)iel~ ,t~
subjec.~ed to thorou~h m5xing. ~rhen 2~0 J~l of a S M ~ol.Jtion of a~-moni~m nc~cate .Ind 1 ~g of herr;n~, sper~ DN~ rere rldded. rr~c ren~ult:-ing mi.xture WRS m{xe~l well, and ?S0 ~1 o ice-cold ethanol we~e added. t~g~in, thorou~ ing wrl~ perfor~ned, nnd the resll~ting tQiX~
30 t~re was stored ~c)r 30 ~Inutes At O~C.

The r~diolabelled ol.igontlcleotide wa~ rec~vered by c~ntri~ug~c~i~n a~:
12,00Q x g for ~Q minute~ at 4~C in a microuge. ~ ing an au~omatic stncM/AI~J~ m 29 L555 t, ; ' ~ F~ l l F ~ T ~ E r t 1~

9;~ 4L 7 7 plpette device ~uipped wi~Lh A disposable tip a] l ~ I;e ;.~ .ttQnt fluid (which contQined mo~t of ~:he tlnln~o~por~e~ [7-3~R~ p! and f~ny free 32p 8enerat~d d tring the pho!iphory~Ation retction w~r~ c~ref~tlly reuto~ed. ll~e re3ule~ng res~du~ ~as redissolved in 100 ~1 of ~2 and 5 10 ~1 of 3M ~odi~m atc~ta~e ctnd ther~after 25C ~i of Y6% ethQnol were added. ~te ~ixt~tre ~as .s tbjected to cenrrifugation ~or 20 minttte~ at 4Q~;, dri~d and redi.~s~l~red in 200 ~1 of H20.

Olig~n~cleotid~ hybrldiz~tion ~f chitinn~e 4 DNA by f~lter h~b~id~
tion tO T~-~ oliK~ ucleot:i~e hybrtdtzfttion procedure ~Ised ~limin~es ~he preerential meltin~ of A- r V~r~ -C h~3se l.>~iI`'.~, tIlowin~ ~he strin~el~cy ~f the hybridi.:~.ttion to be con~.rolle-1 ac a fuoction o prob~ len~th vnly ~tc hybr~d~z~tiol- W;l'; carrie~l out ~ss~n-ially IIS
de~cr~b~l t)y Wood c~ ~1. (158S). The nitroc~llulo.~o filter~ obt:~ine<l lS es ~escr1bed above were tlet~e~l on the ~urf~ce w.5 ~h 2xSSC: ~ncl sul~s~-q~ nt.ly prehybr~(liz~cl ln hybLidizsltion buffer (6x~C lX B~;A lX
Fie~o:LI 4~()0 1.% IltJP 50 ~g/ml o~ heaL lenttc~rated sat7rn~n ;perm l:)~lA
S0 rnM so~Iiwn phosph~r.e, IH 6 8). Thc hybsidi~.ALion w~s l~erforlned at 37~C or 4 hvu~r. in ~1 p1A.<:ric b;~t; with shnkinf~,. The fileer w.ls hybri-diz~d overni~ht in ~-c same ~;olucion ;~ r;~clio;~ctive oli,~,on~Ic1e-otide ~r~"~e ~thc 23 m~r ch~r. ' pr~ t 3~C with ~hak~n~ lxl()~
c.pm/rul solut;on of th~ hybri(li;~ ion b~lff~r w~.s usecI. The fil~er wr~s rln.ce~l t~re~ tin,es ~n fix~iS~: a~ 4~C nnd ~:hereJ~f~.er w.lshtd twi~:e ~OL 30min. at: 4C in GxSSC. ~rrhi?r, ~he filt.-?r ~ wf~sh~ ree tir~cs in TMAC-bnffer (3 M Te~rtlmethy~nnnoni~un etllorid-, S() ~M 1ris ~H 8,0, EDT~, 0,IX ~ S) ~or ~ min. .lt 37~C. (The tetr~m~ thyl~lmnvniu~ll ~hloricle is ma~le in il S M sto~k ~ivlu~ion. Slnc- TM~C i~ y~ros-:or)lc, the act~ml mol;lr concentr~ltion (c) rmlC.t ~le~err~ined frorn r.he refr~-ct~t~r~ in~Iex ~n~ by t:he ~orm~ c-tn-~, 331)/0,018) . l~le fiLLcr was ~0 ~hen w.q.~hed ~wice ft:r 20 IDin. ~n TMAC-buffer at 55C.

l`he ~ilters were dried in the air a~ room t~eulperature. Inkmark3 on the fllters ser~in~ to fllign the autorad~ographs with ~he file~rs ~d the a~ar pl~ees were marked wit:h an autoradiogrdphy marker ~Ultcr~it, Du Po~t de Nemours). The fil~ers were covered wi~ll Saran Wrap and an X ~ay ilm lAGFA CURIX RP2~ were e3~posed ro the il~:~rs for l~- 70 8~94s(k~03~/LS/JK~JA~2/1s~

~ r ~ FEE ~ '' F.,--~3 ~ 7 hottrs a~ -70nC with ~n in~.ensifyin~ sereen. The ~ . ..L~ped and p~cIcive pla~ue~ we~ id-?ntified by alignin~ the clors vr~ the film wî th those on the agar plates.

P~cklng plaques Agar fr~gments c~nt~InIng pos;ti~e plagues were pick~l from the ag~r plaee using mild sucton and pl~ced ~n 500 ~ f S~ pha~ebuffer (Sa~brook e~ al., I9gO) and t drop of ch1Orcfor~ ~ont&lned in ~n ~?ppendorf tube. The eppendorf ~ubes ~re 611OW~d to s~and for }-2 hours at room te~pera~ure so ~s to allow the pl-~ge p~rticlPs to diffuse out o the ag~r. About 106-I07 pha~es per pl~que were ob-t~ined, The ph~ges were t~en d~l~ted in S~ ph~ bll~fer and mixed w;~h 200 ~.
of Xl.l. ~ e ce11.~ (OD600 ~ 1). rhe ~ixtur~ wa~ ~111Owed ;o stand for 1S 20 minutes ~t 37C and 2.5 ml of ~op ~garos. (48~C) was ~dded. ~e ~ixture w~s poured onto 9 cm ~etri cli~hc5 ~nd fil rer prints werc m~d~
f-r ~escreenin~.

A singl.e ~ell isolaced pos;tive plngue usefu1 for ~aking ~ phage ~tock to be used in ~he i~ vivo exci.sion w~c pic~ed from th~ ~g~r ~0 plates .~ccc~rding to th~ ~c~ho~l ~cscrihed by ~ brook e~ ~1 (1990) uçln~ scver;~l ster,c of rPplating ~nd r~sor~enin~.

A ph~gc .~t.ock w~r~ prcp~r~ ncc~c~rdin~, tv thc m~Ltlod c~ tm~rook ~1990).

~n ~Ivo ~xc~ion In vfvo ax~lsion of ~laq~as wns p~rform~cl ~.c ~eçaIib~.d in "tn ~ivo ~xci~ion Proe~o1~ in th~ Ins~ruation M;.nu~tl ~CAT~ 2362~ u~ust 30, 1988) f`or nndig~ad La~bd;~ 7~P II Clvnin6 Kit, S~r~tc~one Clon-~ng SyStemc, 8~x~s~l~s/n~/~î2/l~l ~ 29 ~55 _ _ , _ _ 9~ 2 ~ 7 7 Pr~parat~c,n of plttsnid Dt1A
~-Preparatiorl ~f pl~tsnlid ~NA ~s ~odified a~cordin~, to e~,e ~e~i;od of Sa~brook e~ 1990), ~2nd ua~; perfor~ecl a~ follows :
~act~r~tl stra~ns (DH5~ nnd XLl~Rl:l~) h~s~boring th~ ~lasmids were ~,so~n Gverr~igllt Ls-. S ~1 ~f LB al~clit~2 c~l~plié~ wich th~ rcl~ f3nt an~i~iutlcs stnd 5 ~l of the oværn~ght cultur~ ~as ~Arve~ted by cen^
tr~fu~;~tion fcr 10 m~nutes at 3000 x g The pelle. ~tA!; r~ç t~p~nded 1 700 s~l of ~olution I ~50 }L~2 gluco~e, ~5 ~t Tris pH 8.0, 10 ~1 EDTA) in 1.5 ml t~l~es 400 /~1 of SO1~ n II (0 2 ~ OH, 1% CDS) was ~0 added, the rnixt~2-r~ ~ubject~d to gentle ~ixing stn~ placed ~n ice for 5 ~; t~inurec ~ 300 ~l of 5 M K(~Ac pH 4. 8 was added and subjececd t:o eho-rou~,h mixing. The sesulln~ mi;~turQ Wft.~ plac~d on ice for ~0 mln~ltes flnd cllbse~u~!rltly ~;~Jected t~ centr1fu~,ari;n .2t iS,ûOQ x g ror 10 ~is~utes At 4~C~

The sup~rnrlr~n~ (900 ,ul~ w~ tran~;ferred to new ~ubc~ nd 0 6 ~ol~e (540 ~-1) of ieecold isopropRnol w;is ;Idàe~ resul ~ing mi~ture was ~11GWed ~ O stdnd for 15 minute~ t~ rooll~ temper~ re l~le mi~t~re w;~s ~g~in su~ jecr~ct to cenerlfugation ht 1~,000 ~C g nnd /~h(l fvr 10 minu-te~ and the ~upern~t~nt w.~s realovc~3.

~he ~ llc~L w~ dissolv~(l in 100 ~l of T~ ;1tl(~ f, M LiC1 w.
ndtiecl The mix~ure w.~s ~tlowed to ~.e~n~ ic~ f~f 5 ~ ute~i an~t ~;ubje~re~l to ce~rt~ifu~ ion .~tt l'~,OOO x g ;~t~ "h~, for- lV ,llinuc~c :~ Th~ rt~uLlqn~. t~ t.rJ~ ferr~!~ t~ U~ tl-~l SO(~ c f ~6X ~h~l-nol ~ s rld~:led. Th-~ tubes w~r~ cet1tr~f~ ttcd nr l.S,GG(I ~. e .~nd 4bC for 2' 31) nlinutex :~nd th-~ .c~pe~n,3tnnr ~;1S remove~l. The r~elle~ wflS "a~lhedw~.h 70X rr.han~ \bout 100 ~ md dri~d. Th~ dried pellet w~s rcd~x~~lvcd ln ~0 ,1 of TE.

D~ sequeDclng The pl~smid DNA (do-tb~e-str~lnde~l t~p}ate? ta be sequer:~ed ~Ar~s puri-fi~d bv the ~bove des~rlbe~ ~ethod S~quen.Ing i~raS perfonn~ LX fol-l<~ws:

;:

.

FF~ i I F I t~ T ~ F'EE r~ r- . l ' ~8477 A a~iV.ture ~:omprisln~ at~ouc ~ of the relev~2nt pl~smid, i ~.i.i. GL Z M
N~OH, 2 mM E~ITA, 1 r~l of primer (100 ,u~,~ml) ~nd H20 up Co 1~ ,ul wa~
incubated ~t 85C fo~ 5 ~nutes ~nd sub~eque~ p~l~ on ic:e.

The mi-xture ~dQ5 neutr~l~ 4ed b~ ~dding 1 ~;l t~ 5 t: N~ c .qn~l then5 p~ecip~ eed by adding 20 ~l of ~fiX eth~nol. ~2e resulting ~xt~lre was spun for 30 ~uinnt~s a~ 4"C ~nd resuspended i~ 5 ~l of H2~
of 5 x conc. seq~en~e buffer ~s added. The ~2ixture .~as placec~ at 37C for 5 minute~

4 ,ul of seq-Aetide ~Ri~,technolo~y 5~stem~ t~~ Resectrch Products, Du 10 Pont de ~Jemours) and 2 ~-l of seyuennse (tlnited St.~ce~ Bi~ hemical) were ~d~led, res-llring in a total volu~ne of the mlx~u~e .~f 13 . 5 ~l l'tle tnixture was ~1lRced ~t room temperature for 5 minu~es.

3 1 ,~1 of thc labellfng reaction t~as ~rf.tn~fe~red t.~ ~aci~ ter~ination tul~e (t~, A, T ;nd C) con~aining 2~5 ~1 of the dl`,'TP tertl~-n. tin~ 122i~C-15 tnlre. Tht~ mixtures in each of the ~ubes ~ere ~lo~ed ~o r~ac:t for Sminutes at 37C tnc3 ~ ,ul of .~ttop solu~ ion was rldd~d Tht mixtures wcrc t:hen he.~t~d to 85~C nnd ? /~ f the hcnted n?ix~ure was applied on~o ~t 6X sequencin~ gel (Gel^~ix 6 from BPd,) The ~ el wn.~. vAe~lum dr~ed ~r~ xposcd to ~n X-~a~ ilm.

~sbellinp, of c~ r b~cC ~SE~ DNA ~r~beri ~A probe~ r.n IJ~ u~:ed in t.h~ isola~ion of r.h~ su~3r b~cL .qcid~c ch~rin.~se ~SF.~ wa~ labe~lod h~ u~e of t~le StrntA~ene oli~olabcll;n~
kit prime IT, (R~ndom Prime~r Kit) ~c~ot~ g to ~hc m.~ f~l~tuccrr, {n~tr~e~ion.~ Mnre spceSfic~lly, the ollowil-~ pr~c~dur-~ W;lS ~

2' A s~mple colllpri~ g 2'~ n~ (l 23 ~l~ o thc bN.~ te~p1;~;c ~o ho labe-lc~, 0 2~ ~-1 of H20 ~ncl 10 ~l o~ rando~ oligonuc1eotid~ ~rimers ~con-~eitu~in~ ~ roe~l volume of 33 ~l~ were ~dded to the hotl:om of a clean microc~ntrifuge tuoe The reaction tubes were he~ec:r~ ~o 95-100~C in Q bo~ling w~ter ba~h fvr 5 minutes and then .2ntri~u~ed briefly Rt room temperatut-e to co11eet the 1lq~ hioh may have corldensed on the CRp of the ~ube~. The reaction tu~e ~ont~inin~ t~le /nLM~ Q~ ~ ~5~5 ,_FIIJI I F ! rlHI l~ H I EIFE Tl-l l ~ r~r - L-t ?.! f 7r ~

96 ~8~77 DNA sa~ple in LMT fl~A~o~e was placed at 37rc .~nc~ ~h~ ~r a~ents were adtle~3 ~o the reac~lon eubes:

10 ~1 of SX primer buff~r co~t~inis~,g dATP, dGTP ~nd. dT'rP.

5 ~l of l~bel~d nucleotide ~-32PdC~P (3000 Cij~) (A~rsh~m), and 2 ~l of dit~te~ T7 DNA Poly~eras~ T7 D~tA Poly~*,i~s~ w~s di1uted in ice c~l~i En~,v~e Dilution Buf~e~ ediY~ly be~ore ~e to r~ f~nal ConC~ntrQtiOn of 1 U/~l. The reac~Giorl oomposlents we;-e mi~ with the Cip c~f a pipette.

: Ih~ t~he.s were i~ct~bated at 37 40~C for between 2 and 10 mi~ tes and subse~ently, C~le reaction wac stoppe~ by the ~ddition of 2 ~l of S~vp Mix. The probes ~?lth the 32P lAbcl*d ~NA ~ere rurther purified ~sin~ the Elutip~-D colt~n aystem ~Schleicher ~ Sch~

Then, the probe D~A wa.s made ready for hybritli2~tion ~y lhi~in~ the re(luisite nmo~nt of r~dionctive ~robe with 200 ~1 of 1~7 mgJm7 sY~lmon t5 ~perr7. ~NA. The mi~;ture ~as heat~d to ~S-lOQ~C in A boilin~, w~ter b~th for 5 ~7inutes ~nd cooled on ice. The resultin~ probe ~a~ ~rored aC -20 ^C for up to one week ancl heAr.ed co 9S- lOO~C in i7 bollin~ w,l~er b.l~h or 5 mln~lto!i And co~led oll iCt` b~fore ~IS~.

~y~brid~ntion of "SEn-DN/~

20 Fi.ltel rr.i~lts o~Cn~n*d as described abc,ve ur.de~ )l;go~ leo~Lde bri-~;7..~eion" o th~ sug~r bect ~ ~AP ~DNA li~.~r. ry t~ iul) jccte~ ~o prellyt)rid{7.~ .0n for ? ho-lrs .1~ 6?~ nd~r convrntiollal pr-hybridi 7.atios~ cond~ti~)ns u~;ln~ pxchybr~d~n~ sol~l~ion co,nllxi~ ,g 2 x SS~, 1.0 x ~enhardt ~;, O . I X ~'DS ~r~d 50 ~f~l ~.nlsDon sp~r~ D~

25 Hybridi~ t~o~ s ~arried ou~: overnlght using a hybridization 501u tios~ identic~l to the prehybridiz~ on solution ex~ept for the fac:t t.h~t a radioactive DN~ probe prepared ~ s described above h~d been added .

8~50e)~.00l/15/llCMJA12/1991 0~ 29 ]5:55 F F'~ F~ i L~ E Tl_l I r~ t ,~ F . ?'' 97 ~ 77 Afeer hybridizat~on. a wf~sh~at~ p~c~lur~ was carrit~ti dan~ wieh the follow~ng ~che~e.

2 x tS ~in. in 2 x SSC and O.lX SDS, and ~ x 15 min. in I X SSC ~nd 0~1~ SDS.

5 The posîtl~e pl~que~ ~re identifit.~d ~t5 described ~ d~r "Oligonucleo-tide h~bridi~ation of chitinase 4 DNA in filter h~bridi~éteion~.

Identlf~catlon of D1~ long~ng to th~ ch~t~nase 4 g~-le ~ ly To id~ntify DN~ belon~ng to ehe chir.~nase 4 gene famil~, hyb~idiza-tion of ebe DNA in ques~ion ui~h a chitin~lse 4 probe was carried out uc~n~ the hybrid;zatit.~ll procedure dt.~clos~d in ~hybridi2dtivn of nSEn-DN~" except for ~h~ act th~t the hybridization is c~rrit~d oue ; at ;I temp~r~ure of 55C.

Ext lclvn of PNA from agaros~ gels DN~ frAgrneT)ts to be us-~d, e.g. ~-- rhc con.r~.~uct ion o gt~ ti~ con-15 s~r~3ct:s accordir1g to eh~ imt~ntion w~r~ isolat~ s tollows.

'rhe L)IIA was run o~ IMT (1,o~ Mc:lr~ng, Temp. t8tu~t~) ag,lrose (S~ Plllqu~-GTG, FM(~) iTl TAE (0 0~ M Trin~-nt.,~tat~, 0 . 0~2 M Er~TA) buffc~ o I~NA h.~rld wa~; e~cis~d wiLh :~ P,~s~out pipcttt~. 'rO ~ht~ ~xcist~d l)N~, 1 vol 200 mM N~C1, lO ~ VTA wn.s add~d. The gel w~s rù(~lt~ t ~C
:~0 for t~1 min. .~nd r~ quili~ra~!d ts) 3~C. ~llbs~qu~nr.lv, ~U/100 ~1 ot .~t~;DrO~e (~Tee ~)f DNa.ce, ~r--m C;llbioche~ n~ kle~l Th~ ~ix~llre w.~s : n1.1~ d to st~sn~ ou~rni~,h~ ~t ~7C ~snd w~. r~b.cs~q~enLl~ ~x~r.lcce-.l twice w~ phnnol ~n~1 twic~ wi~h chklrDform, sub3ect~ ~o ~tV~: preei pi~.ation ~nd f~nally re~olub~lized ln 1120.

2S PCP~ u.ced for the Ampllflc.Qtion of cD~ ~ncodln,g "SEn, ~-1,3^81~ n4se And ch~t J6 on tkse bAs~s of sug~r be8t mRNA

The prep~rat~on of a p~rtial cDNA molec~le w~s done by use of tn~
~ene Amp~ RNA Amplific~tion Re~sgent Ki~ (Perkin Elmer Cetus, USA).
The PC~ w~s performed ~n accord~s1ce wl th ~he s~nuf~ct~re~ 5 Instruc-. ~:55 ~ '1' F~:H.~ ~!rIC!lJ-r: ~IHIE~FE T~ 2.--li=,~ F.'75, 98 ~ 4 7 7 tion with ~ few modificatlon~ ThQ re~er~e eranscrlp~slo ~ ; ~a~
~110w~d ~in~ the concentrations i~ the scheme below.

~omponent volume F~al c~nc.

S ~gC12 solut~on 4 ~1 20 ~M
10 x PCR b~ffer II 2 ~1 10 m~
d~P 2 ~1 10 mM
dATP 2 ~ mM
dTTP 2 ~1 10 mM
10 ~CTP 2 ~1 lO
RNase Inhibitor 1 ~1 5 ~
Reverse Transcript~se l ~1 S U~l pr~er 270 0.4 ~1 0,25 m~/100 ~1 ~NA 3.6 ~1 1 5 -- -- -- -- -- - ~ . .. . .. . .. .
Tot~l volumc per sa~ple 20 ~1 ; In t~ step cycle the following rrocedure w~s use~.

Segment l: ~2'C for 2 hours Se~ent ~: 99nc or 5 mlnutes Seg~ent 3~ 5'C for S mi~ut~s The PCR protoco1. ~as ollowe~l exc~rt tlla~ Lhe T.~q ~oiymcras~ w;~
~d~d l.~ter tsee PC~ cycles~ ancl th~ ~t"p~r~ture cycling w~s ch~ged : ~o t~l~ followilly,:

IS:SS

99 ~ 7 7 PCR c~c le s:

no. of cycles ~ time (t~in. ) ~: 5 6 5 addit~on of Taq polymerase sind oil : 37 2 : 50 : 37 2 t2 10 lS 42 2 PCR us-~d ln thc ~:onstruction of ~cnctSc ~c>nstruct~ c-f chc in~s~
and in ~q~t~d~rect~d ~:t~cn~ ls on th-~ bnsi~ of clon~.d DN~ tcmpla~s 20 'l~e pr~prlrrleior~ of eh.~ r~lc-nnt ~IA mc~ ulP w;~s donr b~ U'iC of th~
G:cne /~mpr" DNA t~mlllific/it~or~ gcr t: Kit (P~rkin Eltncr C- C~J~., US.~) .~n~l in slc~:ordancc w~th thc mnn~lf~c~ur~.c lllstruc~ion.~ c~p~ for r~
tcmp~r.~tur~ cyc1in~,. H~r-? t~ f`otlowirlf; proc;-~lJr~ W.lS uS~

P~ c~c l<~s 25no. of cycl{~s ~C time ~rnin. ) -~ 35 94 1 ~2 :: 6~ 1 1/2 829~LOOI~ KM/A12/~ 07 29 15:55 t~ FF~ F~ ! r !-il. ,T ' I ~ , ElEL T -i I t ,,!~ -.r'~ F . -:[

l~o 2~ 77 XAMP~E 1 PURI~ICr?ATION AND CHARACT~IZATION OF GHITI~ASE ~,3 AN~ 4 The ~?~thod used ~or the synth~sls of regen~?~ated chitin ha~ be~n specifically de~elop~d in order to ~ake it posslble to ob~ain a high yi~ld of sceive chit~ase 4. A high yield o~ active and pure chi~n-ase is requi~d in order to have s~fficient PLO~in ~aeerial f~r i) de~enmining the antifungal potential, ii) prep~rin& And an~ly~ln~ tryptic p~ptid~ which make~ pos5ibl~
to prepare an oligon-~cleoLide probe suiti~blc ror isolat:~on of ~NA
e~coding R chitinase, iii) producing ~?~onoclontll ~nd polyclonal antihodies .her~o.

The ~sot.~tion and Ch~-r~Ct2ri~atiOn of the DNA ~ncodi~ ~.h* chi~Lnnse is ne~e.~ary when rh~ rNA 1~ to be U.fied ~Or (he ~on.C~r~c~ion of ~neticnlly modificd pl.~nts h~ n~ .~n .increa.ce~ ehi~inase ~crvicy.
15 Also, a high Amc,une of p~re chi~inacc~ is r~q-l;rl~d to l~ake it po.ssible to cl~ ldatc And ch~racreri~ ~h~ ~port;~nt parcc of the ~n7.yl~e .su~h ~s the nctive ~ite.

The re~encr.~te~l ehit~n wa. ob~nlned by acetylatln~ ~he free ~mino ~ o~p~; n~ low ~ wel 1 .~ t high pll ~i de~celhe~l ~b~e (n.s eo~np~rcd : ~O to th~ eon~*tltion.~ e~lod in which thi. syn~ ;is is p~r~o~ed only ;~t low p}l~. The ~ bined met~d W.1~ ';icr, f~r~ nc1 ~-,nve .
m~leh hi.gher yi~ nnd .~ ~ore s~.qhl~ prodtlct rh,~n Ih~ ~oove~l;lon~]
~erho-l in ~hleh ~cetyl~on ls ~rr1f~l ouL onl~ ~t low Th* degree of p~lriey of the enzyme:; w~s ~xamined t.hrou~ o~ h~
~5 ~urif;c~t~on steps hy SDS-P,~ on the Ph~st-Syste~ ~s d~scribe(l ill ~M~.eti~ls and Me~hods~. ~~er s~p.~r~tion on th~ Mor~o S FPLC col-~n ~ig. 1) only a sin~le sil~er s~ined band for earh chitinase iso-; z,V~es 2,3 ~nd 4 ~ould be obserx~ed on the SDS ~el (Fig. 2j. Further ~nalysis bv ~everse pha3e HPLG on a VXDAC RP~ col~mn ~ve c.lly one protein p~ak for e~eh of the i502y~es. ~ is is fur~her ~vidence o~ aho~o~eneous protein prep~r~tion for e~ch ~f the ba~ic chit~n~se isoz~es.

x.~l~t~/nu~/A~ ~ ~ ~5 ` lD1 ~48477 rhe ~ol~otllar weights determ~ned by ~S-PA~E for chlLt.l ~., , "i~ld ~re 3~, 27 ~d 27 kDa, respectlvely (~lg. 2). By is~iecr.t ic ~cu~-in~, eh~ isoeleceric poinec for chtinase 2, 3 ~d 4 were determined to ~.~, 9.0 and 9.1, re.~pecti~ely. Vsing che radloch~mie~l chltinase assay de~cri~ed above, all t.hree i soen2ymes w~t~ fo~nd t~ h~t~e a broad p~ optîmum with ~axi~.t~ a~ti~ity arour.d '.~. The .~p~cifi~ acti~i~y for chitina~e 4 is 4~0 nk~/m~ prv~ein. ~ reas that or chi~inase 3 ~nd 2 are 208 an~ n~.~t/mg proteir~, respecti~ely.

In order to determIne ~hether chitinase 4 ~ an e~.~oc~itlna~e produc-tO ing chitooligosacch,trldes or an exochi.inase lib~r~tirl~ only N-A&e-tylgluco.~.~min~ from the non-redu~ing end of chitir~ or chi~ooli~osac-charides, the pateern of re~ctio;l products lib~rate~ by ~hit{nas 4 from 3H chiein ~as ~n~ 4ed by ~C tFi~. 3). Irrespecri~e o~ dura~
tion of ~ncubation, N ~e~tyl~lu&os~mine w~s only a ~!æry ~inor r~ae~
t5 t~on product, whex~s ch~tobios~, ch~to.riose ar-d chitoterr~osc were the major product. This strongly imp1ies that chlci~ase (~ r endochitit1a9e.

In ad~ition to r.he catfllytic ~c~ ty eY.crted ~n 3H-~hitin, chiti-n~se ~ wa~ also c.~ ble of hydr~lyzing the ce11 w.~lls o~ ~icrc,c~
~0 ~y.~odeictf<~u* u~.in~, ~!e ly~7yme ~ssay desc~ibed 1n ~Mate~ia1s ~ir~d Methods~ (cee Fir,. ~). Thi~ emonstr~et~ tll~- ch~ri~ .c ~; is a bi-funetionnl on~ymc hn~1n~ both chitin~,e nn(l lyso.~ym- a~ivi~y.

.~
EX~MPI.E ?

: A~TIFI;Nt'~l, AG~1VITY GF ~ IFIED CHlTI~ASF.i~ -l,3-(;I.lJC~SE ISOi:N
~Y~ES ~ROM SUC~R B~.ET LFA~E~

Three differ~nt bioa.c.s~ were cor1ducee~l to ~scere.~in ~he ~.n vi~o antifu;-~al act{~ity of chitinn~ and ~-1,3 gl~cana~e i~oenzymes on the ~er~ination ~n~1 growth of Cercospor~ ~eeicola . Irl t~1e same manner the qnt~fun~,Al ac~ivi~y o~ chit~nases and ~-~.,3-gluc~nases from o~her . 3~ so~rces or other iso2ymes ~rom s~gar bee~s mRy ~e d~ter~in~*d using either puriied er~es or extracts çcnt~ining ~he enzyme~. hlso, ehe B~4,~1/~5/J~t/A12/1991 ~7 ~ 15 SS

tJ~ ?1 1~ F~ Fil~ t.~-r :.: llhltFE lI_i l~ F'.,~:-~ 102 ~8~77 as3~ys ~y be u:~ed to ~eterLain~ whe~:her ~ gi`.~t- ~r.
Y~ thin t:he ~cope of the F~re~ent invention .

; ~le~h<~d 1 - I~Lcroscope sl i~e Bio~s~y '`~
Spore cultur~s of Cercospo~ g~!nnir-~tD ~nd grow well on x thin fil~
S of PDA cn ~ m~croscope slide. The growth v3n ~e ollowed by light micros~opic inve~tigations of the num~er of ~,er2in~Lin~, ~pore~ a~d .~ the tot.~1/averct,~e myceli~l growth. Fur~he~;)re, ~t an)~ ~3pe~ific time the ~rowth ~c.tivity can ~e vi~u~ d by ~taining che eult:ure with t~al~l;ofl~or wh~te f~llowed b~ ~icroscopic investig~ion under fluoIec-10 cent I i~he. The number of hyphae with fll~o~escent t.ip~ arlcl the ~xten-sion of the stAin~ng ~t the indivfd~ ip reflec~ the ~,rowth ac tivity in the culture.

t~hen prhteins with stronG antiftlng.al ~ctivity are ~dcled, the number vf ~eltaîntt~inr, ~pore~; are clec~eaced, and the growth rate vf the 1~ hyphae ls drastically reduced. ln Fi~. ~; is sho~l che recule~ when a combinl~ion of t.hi.tinase 4, "SE" nnd ,B t,3 ~luc.ancse 3 is .npplied to the culture. 60 1~1 of protein sol~ltion cont;-ining 20 ~g of each an-t~f~t~ roteins were nppl~.ed to eact, microscop~ slide. When chici-n~e 4 wr~s ~sed ~lon-- or in ~ombisl~Cion ~ieh eith~r ~-L,~ ~lucasl~se 3 or ~SEn alone, the inhibitory f~ff~ct: ~.IS le~;~. pronounceA. ~1either ~
1,3-&luc..~tlase 3 nor "S~" hA~ .~ny ~.ignl~ic.~nL in}libitory ~fff~ct.~lone en combined. ~hW~ en from ~. S whPr~;111 3 ~nz~nes w~rf? ~ fl ~g~f~h~r~ srrOI~; is~ iLory ~ff~r.~ n i~di~
is~, fl ~yl-rgistic effec~ herw~f) <:hitir~ sC ~ "SE" and ~ Z~ICa 25 rl~;o.
.:
~c~hQLt Z 1 I~.f i ~r~e i t er F~ n.~ ,f~

~ f~. germirlrltion of spore~; Mnd erowth oF the ~yceli~ c~n l~e fo110wed :~ isr ~ ~erotiter pl~tc by sfoe.~.~urlng ~he .~sorb~nc~ (6?~ nm) .~t 5pPCi ~ie~ tine inte~al.s. In the ^ontroI experirnefl~s, thc ~,rowth of Cer-c~oi~porA Is initi.~e~d af~er rn appro~. 40 ho~rs la& &cr~od a~d in-cre~es ~Imo~ linearly for ~he next 40-S0 hours ~cur~ A in Fi~
: 6). When p~lr~ chitina~e 4 (5 ~g per ~ell) is include~, ~h~ ini~l lag period is incr~sed to ?5 hours ~nd the growch r~ is dec~ s~d ~s ., jl5/~tAl~99l ~7 29 1S:5S

1~3 20~8~77 compared to the ~ntr~l ~cuIve C in ~Ig. 6). '~t* el~
chitis) colu;~n is sho~7n as a comparison.

Method II~ - AutoradiogrRphy In the ~hird bio~s~y, the chitln In the hyph~e cell ~atl w~s label-S led ~7irh 3H-l~tbelled ~-hcetyl~l~co~ai7,in~. After a ~hort pul~ he radio~ctivity W6tS depo~it~d in the tip ~f the f~gal hyph~te ~see Fi~.
~ hen chitinase 4 ~lone or in combln~tion ~ith "~ and B-1,3-glucanase was ~dded ~fter the pulse l~b~ling, t.he r~ioa~tivity deposited in the hyphae tip was cffccti~ely remove~ The amo~n~s of en2ymes 1~7 ~7imilar to thnt de~crlbed in Meth~d 1 (s~e ~tbove). '~tts stron~ly indiccttes tha~ thc mo~ of act~on of chitina~7e b on th~ cell wall of C~rco~.porct is specific~lly to hvdroly22 ~he chiein fibe~s in the hyphae tip ~tnd thereby ir~tibit cell ~all ~neh~sis.

~le follo~ing conc'~.io-~ ca-~ be ~de on th~ b6tsc~ of ehe above cxper~ments:

It is ~ossible to i~.hible rh~ ~rowth of ccrcv.~)orA in spore culrut~s by ndditiorl of chitinas~ ~r~tct~ons from ~ug~ b~ le.~v~s.

'l~e inhibition is prim~rily s~t .~s a l.ty, Lim~ for e~rm~n~eion~ rh~
I.cngth of which d~p~7llcl~ o~ ~hc~ s~rt!ny,th nrtd con<:enrt-r; rlon ~-f thc growth lnhibitor.

~r;~cLions whl~h non~ ln ~oth chi~inrlse ~nd ,a 1,~ F,tucal-~se h~v~
seron~-?r tr~hi~)itin~ effoct th.~n chitin~s~ alon Chitinnso~,lu~.qn.~.ce fr~tions ~rom C~-~O.~pOt.? inf~c~ l s~ r b~ct pl.qnt.~ ha~e ~ ~tr~n~,~r inhihitillg ~ffcct thnn fr~r~n<~ lrii~cl in 25 tho s~m~: mAt?n~r frou- ~c>ntrol plnnts.

~x~x~?l~s~n~M/~~ 7 ~ ~5s t . i ~ F~ F . ~ ,f`~ .EE r!- ! r l l lr" ~

1;)4 2 ~ 7 7 EXAMPL~. 3 ,~MINO RcID COMF~OSITION A~D P~lIAL AM2N~ ACID SEQUE~E~ OF T~E P~K~-F~ED CHITI~ASE ISOE~ZY~ES ~, 3 ~ 4 Aftt?r freeze-drytng, the a~ino ~cid co~pos~tion of pure sug~r b~ee chitinases 2, 3 and 6 were d~ter~lned (5ee "MR~erials and M~thodsn).

rh~ result~ are sho~l in ~ble I. FQr eom~ar~son, t~e a~ino ~cid co~po~iti.on of chi~inase fro~ barley, wheat and ~,-an ~Leah ec ~l., 198~) a~e included in the Table. The amino acid composieion of chiti-na~e 2 is sImilar to thac of bean chitinase in a nu~ber of aoino acid resdues, e.g. a~partic ~cid, proline, glycine, leucine, tyrosine, ph~nylal~nin~, valine ~nd lysine. l~ t~cntrA~, chici~ase 3 an~ ~ have a .~;ignific~nt different ~mino AC;tl co~posieic,n than any of th~ other chLtinases.

Furthermore, ehe amino acSd ~on,po.sition cle,~ d E~OID ~he cDNA .se-15 guenc~ encoding the su~r bet t chi~ina.se ~ wit~,c)ut siC~,n~t peptide i~
al.so sho~n. 'rh~ cDNA .¢~q~ence ~as c,btail,ecl a~ d~st;:ri~e~ in Exnmplt~ 5 below .

~450c~.00~t/ll~MfAl2~ 07 29 15:55 TAf~LE I 2~48477 A~ino ~cid ~o~positi~n ~f b,~rl~y, wheat ~n ~nd sugar beet c~:Ltlnases 2, 3 and 4 S A;i~ino acid Barle~ Wheat ~e~ S.B 2 S.~.3 S.~.-4 cD~A
~ . ... _. . , Asp~rti~ ~cid 23 28 29 34.4 ~4 7 24 4 22 Th~eonine 13.8 22 22 ~.2 13 0 12.8 12 Serin~ 17.7 24 26 21.0 24.8 24.8 2~-Cl~eamic acid J8 20 22 24.~ ~2.1 21.0 18 Proline 17 15 20 17.1 10.3 10.2 Glycine 30 . ~ 52 3t 3~ . ?30 . 630 . 4 ~7 Alanine 37.3 27 26 28.0 28.2 28.5 26 Cy.~teine 7.2 1? 16 16.9 16.8 16.3 I5 Vnl;ne 12.5 l4 10 8.6 14.4 ~4.3 1 ~thiorlirle1.6 3 2 1.8 1.l l.l Isot~ucinoI0.8 9 lt ll.9 10.9 Il.0 Il ~ucine 11.3 13 17 16.2 ~.0 9.0 8 Tyro.~rle 11.9 l~ 15 11 î 1~ 7 12.7 12 PhenyIalar)1ne 12./ 14 13 11 ~ 13.3 18.1 17 tll.stldine.9 4 ~ 6,6 4.7 5.4 4 l,ysine 6.9 8 8 8.7 4.3 3.1 3 Argininc 15, 2 t4 16 11. 314 . 2lfi .1 15 T~ypt~phan~3.2 7 4 nd nd nd 3 2~
MW (~) 2~ 29 3~ ~0 fi ~7 h 27.7 ~.5.9 S.B.2 - suy,nr b-~t chitln.-~ce 2 ~,A 3 ~ ~ugar b~c- chitinn.~ 3 30 S p~.4 ~ gar b~ct chitinrl3~ ~
~t)NA - .lm;no ~cid co~pcsition d~r~ . fro~ ~.h~ c~N~ .cegu~nc~ ~nco~irlg thc m~turc protein, chir.ina.~ 4 nd - noe ~eter~lned.

~45~c~.001/35JJKM/~I~/l~l (~? 29 L5 55 L ~ , E t-t-: r ~ f: -, r 10~5 2~ 77 Try?tic di~ ior~ o~ SUgl!il beet chi~in~ 3 ~n ~

Analvsi.s ~f tlle pnrD chitin~.s~ 4 en~vlLe h~ r~v7 ~le~ he Il-7~rmin 71 p~rt of the ~n~y~te Ls bl7ic3ce7~ us by ~n~1~J~:S S of the mature ch:ltinasc 4 l~ wa~ not directl~ pos~ib~e ~o det:ermin2 l~s 5 a~tino acid ~t?g7~nCe ~nd in ord~r to ge7~ s7~ffi. ie7nt info~nation abou~.
ehe en~y~te wit~ ehe event7~-7al aim t~f beir7g ~ble ~o i~;ola7~e and char~c-terize the ~7NA by which it i~3 t`nCOd7sd, it ~.S cl~sen to subject the ctat~lre er~2y~te to tr~ptic ~iges~ion i~ rc~er tt~ o~ln ~lot7sin fra~
ments (p~ptides) s~lsceptible to am~no acid seq~encing.

10 The tryptic di~estion of the purified ch~ri~ se e nz~n7ss was carried out ~s cl~scribed in l~aterial.~ ntl 7~ietho{ls~ a~>o~e. Thc t:rypt~c pep-t~dt~ were fieparated by r~ver.se pha~7~-HPLC on rh7s vyc~lc P~P-18 col~l~Ln m~ntit.7t~e~7 ~7l>0Ve tlndt. r the cc~nclitions .~p7; if iec7~ in Materials ;~nd Me-thocl.~7' .~ee (Fip~. 8) . Peptides t eprcsentin~, large pe~ks at ~n absor-15 bance of 21~ nm ~nd displ~yin~; .a Iligh rer.er~tior~ iruF~ (indlcatirl~ long'~ polypeptitJe cha~.ns) Werc .rlecte(l ~or further p~l~iicatio-- on a Develo.~;il RP 18 col~n.

purifled pt~t.ides w~re ~ubjected r.o ~ ino nc.d set~ tlce ana1;sis.
as descrihed .~ove in "M.4reriitls And Mf~hods ~nd rhf~ mino acid ^e-~0 q~l"nct~ of cnch of che pcp~icle.: is shown bf~ltw in Tab~e II.

tJht!n comp.~rinp the .~nlino ~cid Sf q~lenc~; ot i-~tCll of the p~:pcides with th~ nmino ~cid seqt~ences o~ knowtl ~hit inrlses. (110t o~' Su~ ht~et origin) r~ 1 ow ~ie~;rce of ho~olo~ y w;~ v-lnd .

One of the rryp~ic pe~ri~lc-; prov~;l to ~- very a~ t.~ o~s v ~or~tt ~S the b~ for the ~.onstruc~ion of ;Itl oli,~olluclt!o~i~ie probc. ~hus b~
rtn.~t~sis of rhf~ ~tnino .~c.i~ scq~ u-ct~ ol ehe r.ryp~ic pep~ .2~ it WA.q fo~nd that t~ e of t~lis seqllerlce in the c(,n; ~-uc~ on of .~n ol~.eo -n~lcleotide probe ~ould reqltire only ~.''b' codon choices. Thus this peptide was chosen tO form the ~si.s of ~hP const~uct;on of hn vli~,o~
30 n tcleoride prob~ to be used in the ~sol.2tl0n of ~NA encodin~ chiti-na~e 4 (see E~c~mple 4 below).

I~SSO~L~I/lSt~/A~2/î99t 07 29 tS:5S

~ TA~LE II ~0~8~77 Tryptic peptides of ch~tlnar.~ 3 and ~, Chitinase 3:
S 3-10.3 s-T Y-C-Q-S-Y-A-~-F-P-P-N-P-S-K
3-16.1 A-C-~-T-H-E-T-G^H-~-C-Y-I-E-~-I-A-K
3-16.2 V-G-~'-Y-T-Q-Y-C~Q-Q
3-22.3 G-P-~-Q-I-T-~
: 3-23.3 S l-G-~-~-G-L-N-A-P-E-T-~-A-N-N-A-V-T-A-F-R

: 10 Chitinase 4:
4-4.2 V-G-y-Y-T-Q y 4-19.3 G-P-L-~-I-T-W
4-22 S-I-G~F-D-C-l. N-A-P-E-T-V-~-N-N-A-v-T-A F-~
6-2~ F-G-~-C G S T-D-A-Y-C-G E-G-C-R
: 15 4 24 S-P-S-S-(: G-G-S-V-S-S-~-V-T-D-A-~
4-26 T-A-F-W ~-W-M N-N-V H S V~I-v-N-G-Q-~-F-G-A-s-I

EXAMP~E 4 ISOLATTON A~D CHARACTERIZATION Ol~ cDN/~ c..~.ri~ r~R CHIT7N~SE 4 2~ Fro~ the nm1no ~cld .~eq~lencc obt~n~.a ~or p~pr.5~1e 4.2G (.~e- T~llc II
in F.xAmple 3), th~ follow~ng ~cry .~p~cific oll~,on~t.~ o~id~ n~ probe s~thc.sl7ed u3~n~ ~ DN~ .~ynrhe~ cr 381 A ~ppl;~ Rio~.y~cm.r).
.~
Pep~-id~ 4,~6 .~ 2S T-A-~ W-F-W-M-N N-V-H-S-V I-V-~-G'-Q G-F-G-A ~-I
I I

--- F ~ F ~ M N N ---Phe Trp Phe Trp Met A6n Asn : TTT T~GTTT TCG ATGAAT M T
C C C C

82945~k)~.001/lSfJXM/A12/1~ .55 .~

, ~ .

FF~ F~ CII-Ir ~ I~IH'I t,EE r' l 1~ r~ F~

108 ~8k77 UçLn~ thiç ~ene probe, the expre~sion cDNA llbr~ry ~ ed u~is~ the procedure ~i~en in h~ateri~ls An~ ~ethods~ ~bove. g cDNA
clone~ were obtained fr~m ~ZAP, and one of the cloTIes ~s fully sequenced while the oeher~ were only p~rtly seguenced. The sequ~ncin~
w~s performed ~s ~escribe~ ~n ~MAteri~ts And Meehods~ ~bove. An o~t full leng~h cDNA clone~ chit 4-B15, ~as obtain~d ~rom th~ ~ZAP
r~r~ ~nd the DNA ~equence the~eof ~ ~ho~n in Seque~c~ 1.

On ~he b~is of the cDNA sequerlce, ~ deduced a~ino aeid sequence of chlt~n~se 4 waç obeai~e~ S~q~ence 1. '~e deduced amlno acLd se~uel~ce w~s Ali~ned wLth the partial seqt~ence obts~r~ed from ~he ch;t.~ase 4 prote;n ~AS deçcribed ~n E~lmp~e 3 above) and an almoçt IQO~ ~deî~tLey w~s observed. This demon.~tr~teç th~e the içolAted cDNA clone codes for the chitinase 4 pol)~eptide3 puri~ied by the chromatoeraphic proc.ed~xe descri~ed ~o~e. ~le chit 4^B15 cDNA clone i!; ~66 bp lon~
and encotles ~ protein ha~in~ 264 amino acid resid~es in the poly-~)ep-t.i~le chain out of the 2~5 nm{no acids predict?d for ehe chitLna.~e gent>mic ~NA. Th~ te~der ~ uer~c~ con~is~ p~o~a~ly of 23 ~ino a~id te.~i.d~les ~out of 2~ ino aci~ residu~s ~ et~r~ine~l for the genomi~
chitinase 4 ~NA, see h~lo~), f~ltc~ec~ by a h.~cin ~ in of 3~ ~n~ a f-lneciorlal d~m~in of 2n6 nmino ~cid re~dtle~ Afte~ rh~ .crop ~OCIOT
the ~D~A has a 1S8 bp 3' noncoding reglon.

.~ meQtiOnc~ ln Exampl-! 3 Ah~ve, it h;~s no~. b~n yc.s~ibl~ ~o segucnce thc N^t~.~htln.~l ~mir~o ~c.id sl!q~enc- of oh~r.~r.~e 4 dlr~tly, ~ecrlrl:;c ~le N-tet~in~ hloc.k~d. Howcvar comp.~r~.çon ~;.rh wlle~t ~rm ag-2~ ~lutinin (W(`~ rld ptta-o chitinft~ ad ~o th,: y,U~S!; ~f thc first ,~tmino aeid ba;n~ ~ln. I~ere;lter the rec~. of the ~,mino ~citl scgucnc-, of ~h!~ c:llititl~sc /~ N-t~rr~ln.~ s cl~duced fr.>rn ~h~ Dl~ u~!n(~c ~c~ bc Cln-A~n-Cy$-Glv-Cys ......

~he N~ee~inAl seg~2en.~ of chitln~ 4 W,3~ f~Jr~her e~amined by d~-30 termining the mc~ecular w~ight (MW) vf thc mflture ~ hi.tin~se 4 by electro~pr~y m~s.ç speetro~ try ~s descrih~d by G.J. Felstner et al., 1990. A MW of 2S893.6 +~- 10 wsS obser~t~d. On thc basis of the ~mino ~cid seq~enc~, a ~ of 2Sg23 c~n be ca~culated. Gi~ei~ ehae ~he ~ure ch~t~nase 4 oontains 7 S-S-bridges (loss of 14 pror~ns~ and that he fir~e a~ino ac~d ~ésidu~ Gln is conYerte~ to the pyroglue~myl deri~-8~ nKM/A~ s L': -`l FF~ 'lr~ ,E'EF -I~

2 13~ q 7 7 tiYe (105~ o - ~2 ~ lS MW~, the ~.alctll~ted M~' Gf nase 4 ~s ~894. Thls is in ~eement w~h t.h~ ~t~ ob~r.~.d by th~
ele~r.lo~pr.~v ~ss sp@~tro~et~ic asZalysis And confir~.~ the ~e~ced ~-t~r~in~l ~mino ~cid ~equence glven abo~P fbr ct~ mae-lr~ in~se h.

l~Ze N-t~Zinal ~mino ~ZCid seq~Zence c~uld b~ deeerm~ned fQr chi~inase 2 and che follo~ing ter~nal anZino a~id seq~e~Z~ ~as ~o~Zd in c~1ei~
nas~ 2: Giu-Leu-Cys-Gly-A~n Gln-Ala.

T~ble III

G'oZllp~r~lson of th~ N-ter~in~l amin~ ~ci~ seyuence ~tw~en dif~erent chitln bfnding protcins:

WGA-A QRCGEQGSNME(;PNNLCCS~-GYCG~IGGD`l'C(:KG--CQNCACWl`S
H~v~i.n: EQ**Rk~GGK~*******~*W *~*~ST~c**S~ I**S~-~KD
Chit. ~n: EQ*~R*AGGAI.**G(,N**~*F-~W~STT~***P* **SQ *GG
Chit. To~.: EQ**S*AGGAR*ASG**~ *W*-k~r~ *~'P* ~h*~tSQ-*PG
Chit. SB 2 EL~*AGGAL*IkG*~ *-~W*~NTNY~*~
Chit:. SB ~: *~**C-A~LC*SI~FG~GSTDA~**E~tCR~ CRS

* - a~ino a~f~ r~sidues idPr~tic~l to ~(;A A

~:XAMP~E S

ISOLA'rrON AN~ CHA~RCTERT~ 10~ OF ~11~ SU~`AR ~ 1 CEN~ LG~ ; (,IIIT
76 AN~ ~HII' 4 S~r~ening of ~00,~0~ clones from th~ rl~pllfi~d F~BL3 library contAin-l~g ~,enoml~ ~ug~ bee~ lnser~s fro~ p~rci~ 3A di~estion, rcsul ted in the lsolation ~>f t~l~ee clones w~rh the CH/ c~NA ~s probe.

2S The three ~y~ridi~ing cl~es were ch~racterized b~ res~ic~Lon ra~-ment an~lysis and sequencin~. ~hese ~n~lysis showed. th~t c,ne o the ~lones cont~n~d ~ ch~tinase g~ne, no~ c~lled Chi~ 76, the sequence 82~k OOJ/LS/IKM/A22/199l 0? Z9 IS.SS

_ _ L !~ F~ I F ! r~ 'T ~ H ~-.EF TC ~ F . '~r!

of ~hlch is shown in Seqtlence 3 Seq~encing of ti~i 2 ~ ~ 8 ~ 7 7 ei w~th th~ prime~ us~d ~or ~crePnin~ of the ~ZAP li~ c; ~ser x-ample 4?, And corltinu~d ~ith othe~ pI~ ers co~plemes~ear~ tv sequenc~s in~ide the chit 76 gene.

S The chit 76 ~ene eodes for ~. 258 ~ino ~cl~ lor.~ chi-inas~ which h~s 80X hoDol~ t~ the C~ ~mino ~cd ~equen~e fvide SequPn~e 1~ but o~ly 34% ho~olo~y eo the entire ~H1 pro~ein (ViQ'~ Seq~lerlce 7). The ~ene cont~~s Or~e intron ~hic~ is located in posi~ion S7S ~o l26?.
The eXact location ~f this Ineron is based or, an ali~nmenc ~ith the CH4 c~A Sequence 4. The intron borde~s cencain the corlsensus GT/AG
sequences. The chit 76 in~ron i~ locfieed e~ctly at t~ s~me p~ition es the second intron in the C~1 ~,ene, when the amino acld sequence~
of CH1 and chit 76 ~r~ aligned.

A ~AT~ box seq~ence (TAT~A) i~ located at p~ieion 3~8, which is 90 hp ~Ipstre~ for rhe ATG start CC~dOtl ~or tr~n,la~io~ poly-A signal (AATA~A~ ls loeated at po.c1tion 17~5 In Sequenee 3 In a similar ~y 3 ~,enomic clone eneo~lin~, eh~tin.~e ~; ~ac isol~ted.
The DNA has been p~rtiall~ .s~q~enced ~nd ~ou~. ~5~ nucleot.ides o~ the .~' noneodfn~ rcgion h.~s be~n el~lCidQtC~I. Abou~ 3~l~ n~l~Ieotii1es of thc ?.0 coding region h~ beon ~queneed '~e .~eq~ nc~ ~pp~-:.qr fr~m the Soq~ene~ 2.

~igr~enr of th~ ~)' non~.odin~, ?'e~ic`/nC fr~,~ ch~ twu ~,~noll~ie ~n~ s~ow box~.~ of homolo~,y (~.~,. ehitin.~ nucl~otidec 14~ , G0-1~?, 123-13S, 1S~-17~, 1i4 20/ ~nd 217~ 8, (~eq~leneo 6).

2$ ~a~ied on knowloA~e of t)~e ehi~ /~ RlS enN~ ~cq~nce ~n(.l ~hi~ parLi~lly s~quenc~d ~,~nomic Chitin~!ie ~ eene, ~e ~S~ of chc ~ e c.~n ear;i]y be .~equ~ne~d. It i~ e.onte~plat.ed that che ch~inase 4 gene co~pri.ses at lca~t I in~ron, pro~Ably only 1 corrc~pondin~ to th~ ehItln;.~sc~ 76 sequence.

829450e~.001/15/~/AI~/1991 07 79 15 55 ExAMp~E 6 2~477 C~R~C~E~I2ATI~N OF r~E A~I~IC C11ITI~hSE ISOEN7~ME "SF:~' AXD ~ETEP.-MINATI9N 0~ PAR~IAL ~MIN0 ACID SEQ~ENCE

The Acidlo chitin~ise ~SE~ was p~r~fied as der~cri~ed 1n ~M~e~ial~ and Methods~ above.

After th~ final purific~tion bn th~ Mono P FPLc oolu~r} three iso7y~es of "SE~ could be resol~etl ~see Fig. 9). ~y analys~s on SDS-P~GE only 8i ~in~le protein band ~or ~ach o~ the isozy~es co~lld be d~monstr~ted.
The ~me ~olecular weigh~ of 29 kD w~s d~ter~irled by SDS-PAG.E. Analy-1() ~is by .isoelectric focusing an isoelectric poi~t of ap~roxi~ately 3 . OW~i.S derermined for the ~hree ;sc)~ymYs of ~SEn. ~his corresponds w~ll to ehe theorecLcal l.soelectric poin~ which has been e~imated ~o 3.87.

In cot-trast to che basie ch1tinase~ 2, .~ snd ~, Lhe acidic chitinaçe ~SE~ w~s not rctained o;1 the chirin-affinity colu~n either ar the u.~u~l conditlon, ~t pH 8 (~e "M~t~riAts ~nd Mcthocl~) nor nt h~her Or lower ~1l. "SE~ did, ~owever, ~e~dily dc~,rade che 3~ b~lled ch~tin. The ~njo~ prodtlc~ ~f ~h~ ~nzym~tic hydrolys.~ W~5 r.he hc:x.~tners of chi~in or h~gher homoto~ous of chitill oli~,o s.~c~ ri(lcs.
7~ Since thc m~j-r prod~ct f~ chitinAse 4 ~ the ~i~cr, n di~fercr modc ~f RCtic~n for SE" i¢ ~l~ferr~d. N~ Iy~ acti~i~y ~o~ b~
~cter~incd for "SF,~ ~t pH ~ 5.

~h~ p~rif1~d en~y~ w;~s .~uhjec~d to tryptMc di~e~i~ion ~ ~k~:;crlb~d ln ~M.qter~J~I.c .~IJ M~rho~1s~ and lf~ Ex.~mp1e 3 nbo~c or chi~ e /~ and 2S fi ~ep~ldn~ wcrc sel~c~cd. ~lC p~'p~idt`.r~ w~r~ 4 jccr.~d t~ ~urther purlflc~lt.lon in the ~m~ m;~nnc~r A~ thc tryp~:ic prptld~; ot` chltir 4 d~crlbcd in ~x~mple 3 nhove nnd th~ ~m~no ~cid sequence o~ thc 6 peptlde.~.; wcrc c~etcr2n~ned. 'rhc pept~dc.s wcre .,elect~d u~in~, the same CriterlA AS the ones us-?d n connectior~ with chittna~ic (~. The a~ino .~0 ~cid ~:e~uence of t~e~e peptf des ~re 5how~ in ~able IV~ In ~ddit~on, the N ter~in~l ~nino acid seq-lence w~; also deten~ined a.~ shown in the Table IV.

112 2~4~'477 T~BLE IV

N-ter~1nQl. S-Q-I-Y-I^Y-~-G-Q~N-G-D E-C-S-L A-D-T-C-~
: ~ 22.5: V-L-L-S I-r~-G-C^A ~-G-Y
- SE 23.0 A-D-Y-L-W-~-T-Y
S SE 25.1 ~-N P-P-C-Q-Y-D-T S-A-D-N-L-L-S-S
S~ 26.1 Y-G-G-V-M-~-V
SE 30.4 S-L-S-S-T-D-D-H-H-T-~ -Y-L-~-N-T
SE 31.1 T-T-V-~-A-N-Q~ -L-L-G P-A-S-T-D-A-A-~-S-G-F-I

EXA~'?P~E ~

ISOtATION AND CHARACT~P~Ir~ATTO~ OF I~E cD~A FO~ ~HE ACI~IC CHIrl~ASE
ISO~ZYME "SE~

On the b~?sls of the .~mino ac~d ceq~lence ~f the t:rypti~ pt-~pt:iAes listt?d in Table TV two subsequences fro~ hc pr~ptld~5 SE 25.~ ~ncl 5~
31.1 ~Table XV) wert sel~cted for the synther.i; of mix~d oligon~cl~o-tidos as they had the best codons. Th~ PCR p~imers K~ 7 ~SE 25.1) KB-9 ~SE 31.1) antl the oligo-dT primer (270) were pre~.tre~l in the samt~ m~?nnt!r as ~lescribed ?n Ex3~plc ~ Sn rt l~ion to C~li tln;tse 4. me ??l~cletit~idt? .seq~ ce of the Gcne pro1~ IC ;hown i r- ~; blc~ V .

; TABIE V

~0 N P P ~ Q Y ~ T
KB 7. 5'-GACT~;'rAGAGA~C(.XCCgTG~C~ TA~GA~AC-3' A N Q
KB ~. 5'-G~AGGATCCCC~(;CGAA~CA~ATAl~-3' A C
T

2r0. ;'-CCM GCTTGAATl`CTTrrrrrrl~rTTTTTrrl-3' ~204~xtX?lJt5/llCM,rA12/19~1 07 2~ 15$~

~C .~ - Ff~ !.r.~ r . ~ E:EE 1-1 ir.~ f'.~
2~8~77 ~3 A par~i~l cD~iA ~olccule w~ prcp~r~d 11~ ~Wb ~i~.ep~ 11 tec~nique ar~d ~A, ~he flr~ step uslng the ~t~ove m ;l~in.l.~., pri~ers RB7 and ~ . The PCR techniq~le was pe~ormed ~ desc~ ibed ~ove in ~Mater~ls ~nd Methods~. ~he cDNA sy~he~i~.ed was i.col~t~l on Lr~
S ag~rose gel ~nd the ~ros~ ~s re~oved wlth a~rosei For ~he sub~e-qllent PCR re~ct~on the primers KB9 and ~JO wer~ ~ed. The ~ethod is -` ~llustrated ~n Fi~. 20. The produet ~om thP seeon~ P~ re~e~on was ~loned in pUC 19 (Boehr~nge~ Man~eim) and c_quenced.

The DNA ~q~enoe obtai~led fbr the partial cDNA molec~le c.onstituted by ~ucleotdes ~ 962 of the DNA .~eq~ence shown in Seq~enee 8. This cD~A was used to screen the ~-ZAP cDNA 1 ibr~y described in ~Materi-nd Methods~ and 23 cDNA cto~es we~ obtained. The lon~es~. C~A
clrJne ~as scquenced usin~ the ~ethod de.~crib~d in ~Ma~erials and Methods~ above~ and WA~ fO~Ind to be 10~0 ~p lon~. The se~uence is 1.5 const~.c~Itf~d by n~lcleo~id~ 7 1106 o ~he DNA scquerl~e sho~ in Sequencc 8. As not~ally obscr~d In connl~ct;~n ~ith ~he iGc~l~ttion of cDNA, the entire cDNA w~s o-mtl to be difficul~ r.o 1sol~t~. RescrecrI-in~, of the ~ZAP libr~ry with A 122 ~p ~coRI-Y~pnI from thc 5' ~nd of th~ I o~ge.s~ cD~A clonc (S~2), ~ st!(~uenc~ con~.lining eh~ ~ntir 20 S' ell-l. The clonf~ were 1ig.ttedt us~n~ tht~ ~nT ~ir.~ . Tl-~ st:r~ctu~tl p,cnt hn.~ ' noncodin~ rcglon of t7 hp, ~ det set~ rlc~ of 25 : A~ino acid ~esidun~, A ~lnction;~l do~tnin of ,'fiR ~m1t~ id r~sid~es an~ ~ ~' no~Ieodin~ rc~,ion of 202 bp u~ter Lhe .s~op codon. rh~ c~
scquenct~ An~l th~ ;ln~ino ~Icid ~qunnc~ ~r~ sho~n 1n Scclucncc 8.

2S Whèn t~e lImino ac1~ st~q~It~nce ob~.~inc~ from th~ ~-r~n,ill~L ~tl~l ~he crypti~. p~ptidt~s ~1~7 r~sl~ wt~ro compnt~d J~n ~lmo~ 100% ~ree ment to ~hn ~nDl~ rlvetl s~qu~nct wcre nh~:~rved. Tl~i~: d~monstrlttcs th~t the 1.~olatetl oDNA clone ~odes for thc ~SE~ polypept1de.~ pnrif~
hy the ohromIlLt3~r.qph~c procc~durc clercr;hed ~bcv~. The c~A ~ont~in;
th~ ~ terulin~1 n~ welt as ~h~ C tcr-IDir~ end of th~ m.qtu~ protein.
Th~ N t.~ tl o the m~ture ~SE~ ls app~r~ent from TAble IV.

, ' ~ S~ 7/1991 W ~

.

.

114 2~8~77 TABL~. V I

SE2~5A~L inAhK~V~ 'LFL1SLL~ Si~$$HGS~ IYlYitGONG~iEG5LADTCN 46 GUCU~BER ~AH~T--TTLSt~Ll551~RS~4A~--5~AIY~GaN~HEGSLA~TCA 46 A~A~ID9PS ~TN~TLRKHvIy~LFF~5QsLsKp~D~5RG~i~A}y~GQN~iNEGHLs~Tc~ SO

SE~25A~L S~NYGT~LAi~TFG~TPAL~LA~HC4PATN~ L~Sbl~TCaOAG ~S
CUCIt~BER TGNyi-~v~}~FLssFGs~oApvLN~AG~cHp~NNGc~F~spE~NscK~Q~ 96 ~RABIDOP~ TGPY~YVNVRFLYi~GNG~TPÆLNL~HCNP~A~TCTHF~50VXDC8SRG ~0 SE~25~L I~VLLS~GGG~GRYSLSST~DANTF~PYL~NTYLGG~SSTRPLGVAYLD~ 145 CUCU~BER VKvLLsIGGG~5ysLssAD3~KQv~NEIwNsyLGGosDs~pLGAAvLDG 146 ARhB~DOP5 IKV~LSLG~GIGNYSIGSREDAKVtADYL~NNFL~G~SSSRPLGiD~VLDG lSO

SE22$~L IDpD~EsGDGRF~DDLAR~LA~NliGQKTvyLsA~poGpLpD~sLsT~sA 195 i WCUnBER VDFD~ESI~SGCli-~DVL~C1ELK~FGO~ VILSAAPQCP'PDA~iLDA~K 192 AR~B~OPS ID~NIE~.GSPQHWDDL~RTLSKFSHRG~KIYLTGAPaCPF?DRL~GS~LN 2CO

SE22sA~L TGLFDYVW~FYNNPPC~YDT-5~DNLLS~ T~ V~ANOlFLGLPAS 243 CUCU~BER l`GLFbSV~v6:1i` Y~INPPC~IFAD-N~ DNLLSS\~NO~lTt~ - FP ~ SKLYnGLP~J~ 2'~0 ~R~BIDOPS 1'KRR~YVwIQFYNNi~PC5YSSGNTO~LFiDS~NKWTT~I~QKFFLGLPA~ 2$V

SE225~i~L TDA~-GSG~PA~LTSOVLPTIKG5AKY~GV~LWSX~Y~ YSS~IK ~9~
~UCUi~BER RE~PSGGFIP~DVLl~QVLPTI~SSNYGiGV~ S~FD--t~GY5DSIK ~8 ~RABIDOPS PEA~-PSGYIPPDVLTSOILPTLi<.K~KY~oV~L~-SK~WD~5YSSSlL 299 SE.22S~L S~V- 293 Con~r.~u~ n~th: ~04 CUCU~ER GS~G 292 Id~ntity ~ 1~7 ~ ~5.1X) ~R~BI~PS ASV^ ~02 51~10r1ty: 106 ~ 34.9Y.) . ~.

~bte VI shclws ~n nl~gnm~n~ of t~ lt~ c1d~;cq~ nc~ c~rr~s~ondir~
to the ~ruct-lra1 gene ~o~ th~ ;~cldLc chlclnasc ~SE~ d ~IC .~tLn~
ncid sequel,c~. Or ~ cùcwmber lycozy~u~/chitlnas~ (EP O 392 225 and M~trnux, ~t ~I, 1989~ nnd ~n Arnbl~op~M~ lyyo7~y~e/chi~lAnl~Je (~a~
~1., I~90). ~t appe~ fro~ thi~ th~tt there t~ a hou:ology ~f about 452 when ~tll tre~ nC u~r~ eo~pared. Wh~n ~SE~ i~ comp~re~ with the cucus~er ~y~v~y~/chirin~se ~ ho~olog~ o~ abollt 602 ~a~ ob~r~d.

IJ ~ ~ ~c 1~ F~ F l [li ll J~ Er~ I - r l~

115 2(~18~77 ExA!~-pLE 8 C~.R,ACTERIZATION AN~ DETERMINATION O~ ~E PAR~ L ~ll~70 ACID ~.7.~U.~i`7CF
FO2 ~HE SUGf~R BEET ~^1,}-CI.U(A~ S~S 3 ~tD 4 The S~tga;: beet B-1,3-g1UCana~.e~ 3 an~ 4 Wer~ ~O1~led f~O~ Cer~ospor~
S i~feCt~d ~Ugar beet 1e~eS a~S ~ ed in ~h~ Ve ~M~rials an~
M~thDd~" . They ar~ b~SiC PrOte~ h~ing a S~'L'On~ 3frini~y fOr ~-1, 3-g1UCAn. Th~ aminO aCid c~mposiri~n ~ the SU~ar beet ~B--,3-g1~Canas~
3 and 4 iS~eT~ nleS {~re ~ r t~ One g~ n ~C,r ~ , 3~ Uc~nase~;
from tobacc~ and b~lr.~ey ~5 shown ~n Table VII.

8~4soc~OOllLSlJKMlAl2~ S^SS

15 ;s~ F~-,lil Flt~ er~F ~ t.l~1~ .'ll;,'.' F.

~r~sLE VII 2~8477 A~no acsd compo~ition of tob~c~o ~n~ sug~r beor. ~ 1,3 glucariases A~ino ~rid Toba~co~) Sugar ~eet 3 Sugdr b~ 4 B~rl~y Gsb) Aspar~ic A 3~ 46.4 53.4 39 S rhreonine10 12.~ 12.1 14 Serlne 23 25.0 27.4 23 Glut~mic A. 20 23.4 26.7 20 Proline 19 18.4 21.7 lS
Clycine 26 27.8 32.2 31 Alanin~ 20 31.5 35.1 43 Cystein~ 1 0 0 7 V~llne 18 21.3 2S.6 18 Mothio~line 7 5.1 6.6 4.8 Isole~l.ln~ 17 15.9 19.2 14.9 L~ucine 23 22.7 2l.0 22.1 Tyrosine16 13.5 IS.~ 15.4 PhenylAlanino 13 12.8 14.6 12.9 H~seldine5 :3.1 1.9 1.2 Lys~ne 13 12.9 16.2 9.-i 2V Ar8inîn~ 12 12.7 15.0 12.9 T~yptoph.~ne 4 ND N~ ND

MW (KD) 32 32.8 31.6 32 pl 9.9 9.S 9.5 9.N

~) D~ca taken from 8hinshi H. e~ ~l., 1983 2~ b) From Kr~gh ee ~1., 1991 EU945~e~.001/1,S/JlCM~Al~lg91 07 ~ 5S

F ~ r,~ r ~ , FFE ~ J_'7~il F' .

lli' S~S-PA~E c~f ~-1 3-~luc~n8~e ~ 7 7 ~e ~pperene molecular w~ight vf B-1.3-gl~c~n~e 3 and 4 dete~ttinet ~n ~ 10-15X ~radient ~DS-~,el ~Ph~st-Systes~, ~h~rm~c~) w~xe ~3 and 38 kl)a, r~spec~ively~ ~e isoel~c~ric poin~ ~as gre~er th~tn or equ~l to 5 9. S . Wh~n 3nalyz~d by ~:h~n lay~r ~:hr~tatogx~ph~. the ;QPLjor re~ction produ.ts liber~ted frosn l~inarin a~te~ 24 hour~ vf incub~tion wich ~he t~o ~1,3~ tca~ase Isoen~t~es 3 ~nd 4 w~re the dlsner, Iamin~ri-bLose. ~his strongly stlr,~,ests that the ~-1,3-gluc~nase 3 etnd ~ iso-zymes are endo~lucan~.ses.

~mino RCid 3eq~en~ing of ~ 3-gluc~t~5~ 3 ~l~ 4 rhe purified ~-I,3-glnca~ases 3 and 4 were subjec~ed ~o tryptic di~,estion using ehe metslod described in the above "M&tcrt~ls and Methods" and selected peptides were furcher p~t~iri~d and sequenced ns descri~Jed in "M~ter~ls ~nd Methodsr ~tn~ in r:xa~ c 3 above. The lS p~ptides were ~elected on ~'s~e ~asis of th~ S~SDe Crir.~l`ia ;~S the ones used in cc~nnect~on w~th the select;on cf thc tr~pL~c pept.i~1es o~
chi t.~nase 4 (see Ex~mple 3) . ~te .25tt~t-0 ac.id se4uencr < f the pep~icles ar~? shown in Tahl~ VIIl /A~ 9 15~S

r. ~ r ~ F, . ~ FF T~ i F . ~3 TABLE V~II 2~8~77 Amino acià sequ~nce~; for G- l, 3-~luc~n~se 3 and 4 iso ~ At~d from suga~ beet leaves Peptide 3.15. ~-V-Q-N-N-V~YI-P Y

Pep~ide 3.17. (A)^G-A-P-N-V-P-I-V-V-S-E-S~ -P-S-A-C~G

Peptide 3.16. L-Q-G-K-V-S

Peptide 4.25.1. L-G-N^N-L-P-S ~-E-D-V-V-S-L-Y

Pep~ide 4.26.3 L-D Y-A-~-F

P~?ptide /1~27~1~ Y I-A V-C-N-E-I-~I-P N-D-A-R-A-(;-S-I-V-P-~-M-~-N-V
(~)-(Q)-(A)-(P)-(R) Peytid~ 8.2. W-V-Q~N-N-V-V-P-Y

~XAMP~.~ 9 IS~LA~l~N ~ND C~R~C~RI7~rl0~ 0~ rl~E c~NA FGR ~ 1,3-CI.~ NASE ~ ~N`D

t5 It- c~2~ ? ~all~l~?r .~S d~?.~Cr~b~ OV~ ~1 conrl~c~ion wi~h ~", olii;v-n~cl~?ocid~ proht~.~; corr~:~pond;n~ to ~-ept:i-les Iro~ ~le ,6 l . 3-gl-~c~na.~;e 3 ~nd 4 polypeptides w~r~ :;ynt.hesiz~d. ~r~ S'~im~?~ ed the fol lowin~ t.wo 5~qUCn~?~ in the fi~st round of PCR For isol~tion of B-1,3-~lu~anas~ 4:

8~9450cx.001tl.$/JX.~/A12/1991 0~ 55 :' !, FF~ r l r~ " F~- T~ F . ~

120 2~8477 SEP~OL~GICA~ Ac~rERl2ATIo~ 0~` ~U~AR ~EET ~'HIII~`1ASES t. ~

The se~olo~ir~l re1aeion~hit bet~en chitin3se 2 and 4 t~s analyz~d by immuno~lo~tir,g. Uhe~l ~ protei~ ~mple ronra~ning both chitin~se 2 5 (~IW 32 kDa) ~nd 4 (~ 27 kDa) t~ sep. r~te~ by SI~S-PAGE before im-munob1orelng ttle follow.irl6 results t~ere ohser~.e.~t (~ee ~ig. 10~.
Chi~in~e 4 antibod~es ~etect c~nly a 27 kD~ protein ~chitin~s~ 4~, b~ not the ~2 kDa proteln (&h~e,n~s~ 2 iso2yme) ~lthough it is al~o pre.~en~ on the same ni~rocellulo~;e n!er~br~ne In oonel~s~ chi~inase 2 1(~ antihody r~cogrll2es only ~ 32 kD~ proeeln (ohicin~se 2~, b~-t not the 27 kD prote;ll of chitinasc 4 Ihi.r~ ~;trongly l;mo.~ rar.~.c rhe pres~nce of ~wo sc~oloy,ic~l diff~!r¢n~. gr~Jps o cilitinas~ hi.~ ohserv~tion î~ furehor .suh.citanti~te~ h the i~munoblott~ ng i~n.~lv.~.s ~,~ the p~
chitirli~se 2 an~l 4 slrttigen~i. Anti1~odies to Chiti~lil'i~ 4 ~3et:oct oltty i.S ch1ti7~ ;e 4, wherorts ~ntl~odics ~lire~ec7 ~g.i~ls; chitirtas~! 2 orlly rcco2ni;~o chit.it~ 2 ~tn~l no cro~ reactivi~y i~C :~1l W~5 ohs~r~t~d~
'I'he bbvve resul~.r, ~ugge~t that s~ r ~1!1`~ COni'ilin t.WO ~I.ffct~nt .cla~ s vf ba~iic o.ki~.ina~ie~. This ol~ser~.a~io n ;.. nl~ .s~pp;,7~.ed l~y eh~ inform~tiorl obtn~n-~d from th~ :mino :.lcid s~quer:cinl~, nnd tho anll.no ~0 ~cid cc,mpc,.~i~ti¢7n (.~t~e Ti~lc I irl Exnmplo ~ n~!o~l~) ¢f rh~ iic c~liti-n.~ 7-l~l 4. Ttl~ d~ff~ irl~ t~ t~ hc f~ nl~.r. i od{n2, f<~;
chi~.ina~c 2 ~nd 4 conr;LiLuL-~ ~70 d1.stinc~ gene .unilic~;.

A.~i f;~r r~ the pl e.s~nt in~rcllt.o~ s nl ~ W.-t-~, ri * f~l~t th.~ W~
f.~ronr cl.~ o~. of hAcic s~ .. r bcc~ chitinn~ci ~xis; h~ erl o not 25 bocn r~p~r ::ed in ~he ~ i tor~4rllre, Dcfin1t~n of sug~r bt~t. ci~tL~n~i~c 2 cln.c.~

When the N~ J~ .ICi~ uen~ o r iJee hl~ a~ 2 w;~s allgnecl with ~he f~7~owirl~ chl~in"se.~ fro~ be..1rl. ~o~Qeco~ pea A~, pea A2, pe.~ B ~Va~ c~ ~tl ., 19~t), ~a~-ley ~ (Jacob~en e~ al ., l990~.
and barley K ~Kragil ~ al.. 1990), a strong ho,~olog~ becween ~lle~e ba~c ~hltinases w~re ob~e1ved (see Table iX). I~ti~ s~gest~ t~tt rhese ch1tin~.~es belo~t~ tv the s~e chiti~#se clAss~ This was ~ur~h~r substantlate~ b~ se~lo~icQl ~ros~ re~c~ivity c~rrie~ o~t w~th ~n-001/L5/JICM/AI2/29~10729 155 il L ~ FF~ F I [ ~ r ~ I FEE ~ 7-l F .

Pep. ~.15: W, V, Q N, N (V)... 2048~77 Oligos~q. TC-1; 5'TGGGT~C~AACT~A~GT 3' ln ~he seco~d r~n~ of ~CR the follo~in~ seq~c~e w~s used ~s 5'pri-~er:
Pep, 4.27,1: ...... N, E, I, M, P, N, Oli~oseq. TG-2; 5'Ah~GAAATMTGCC

By ~o~paring r.he .~inv ;~cid sequ~nc~s fYom ~-I,3-gl~lcanases in harle~
(~incher, 1986) ~nd tobacco (Shln~hi ~t ~l.,1988), ~ c~n~ensus ~e-quence w~s selectQd and ~sed for consrr~c.i~n o~ a 3'pr;mer ~ith the lS following rons~rls~s sequence:

Pep. seq: ~.a.M.F,D/N,~.
Oligo.seq. TG-3: ~TcgTcr~AAcATTcG~AA

2~ This .c~ nc~ wnc ~Ise~l in the ~con~ PCR ro~n~ wherea~ thc 270 ~ e~
u~ed fo~ clonin~ of ~SF.~ wa~; ~scd ~n the ~ir!;t ro~lnd. To i:;olat~
1,3-~lucnn~sc ~ clone, th~ TC. 1 pr;m~r ~ u:;od .cince pc~ e ~.~8.2 - pep~ 3.1~ T~bl~ ~ll ir- E~mp1c 8) ~li~. prim~r ~a~ u~cd :~s t.he ~' prim~r or bor.l- the PCR r~actions. ~ ~ht` 3' prim~r, .~e TC 3 ~nd ?70 o1i~nncleotidc.~ wer~ u~.;cd f->r the ~ir.~r ~nd ~ccon(l ro~ o PCK, rcsp~.~t.i~e1y.

The ~esult~n~ PCR products WCIC e~ployrd t~ ~c~ccrl tbe .Ibo~c dcscri-bed s~lgar bcct c~A ~-?.,~P libr.lr; to i~o1tltc clo~les harbc~in~ cDN~
encoding, ~ l,3-~,1uc.~n~e~ 3 ~nd 4, rcsp~cti~cl~f. ()ne o~ the c.r)~A se-quences and Ch~ deduce~ ~ino acid sequcnce are shown ~n Sequence 9.

~so~l/Ls/n~M/Al2~ 5 121 ~ 77 tibod1e~ raised ~ainst su~t~ b~et chitin~se 2 . rn~ y ~ ~o~-nized not only su~Rr be~t ~h~tina~e 2, but in ~dditior, ~lso chitina~e P (2~ . 5 kl~?, Q (28 . 5 k~), Gh. 32 ~nd Ch 34 fro~ ~bac~ ol and Linthorst, l~90). chitin~sr~ T, K ~n~ C f~o~ ba~ey ~d chi~ina~e ~i, A2 and ~ from Pea. W~)en antibodi~s raise~ ~gainst b~rl~y ch t;n~s~ K
cr ~he~t genm chitlnase were employed, s~ r ser~ ic~l ~rvss reacti~iti~ ~ere ob~et~ed. Therefore the chi~ina~es desc~-ibed a~ov~
were defined as b~longing t~ a rhitina~e c~ass serologi~ally rel~ted ~ o sug~r b~t chitin~ses ?, e . ~ g3r ~e~t ~hi~in;~se 2 clas~
chitinase.

TABLE IX

~ter~Inal amino ~Cid seq~1~nCe of eh~tinar~e ~so~v~ b~lon~,ing to the su~ar he~t chitinase 2 c1~Ss:

Chitina~e 2 ELcGNQAGGALc1NGLccsQ~GwcGNT~pycc~
15 Bean EQCGRQAGCALCPGGNCCSQFGWCGS~T~YCGP
Tobacco EQCGSQ~CCARCASGI.CCSKFGW
Pea B EQCGRQAGGA~CPNNI,CCSQYG~
P~a ~1 EQCGNQACGXVPY~G
Pea A2 EQCC'l`QAGGALCPCGL
20 B~rley K EQCGSQ~GGATCPNXLCCSRF'~
Bnl ley T xQQcsQAGGAl(pNxLc(~sxF(~w Defin~eion of n s~lg~r bc~t chItln~se ~ clAss When nnt.i~odie.~ r.~is~d .q~in.t .~u8ar beet c~ nA~p 4 wns el~ploycd, non~ of ehe chit~rl;~scs from ~he ~hitin~.se 2 clnss describe~ ~bovc ~5 ~o~lld be r~co~ni~ed. Ghiti-)asc /, fr~ s~g.~r bccts ~h~s b~longs ~o a new chitina~r class ~o far no~ dctected in other pl.~nt s~cics c~l~n sugsr beets. However, ~ecent ~di~ ha~e indicated t:hnr chitinase~
belon~i.ng to the ~e new class exist in rape ~eed. Thus, protein extrscts o~ r~pe seed obtained by a ~etho~ si~ilar t~ the one out-l~ned ~bo~e for ~ugar beet chitinases ~ere ShOWTI to r~acc ~lt'n theabcve mentioned polyclonal antib~dies dlrected ngai~st chitinas~ 4 from s~nr beets.

1~450c~0~1tLStJl~i/Al~/1~91 ~ ~9 15:5S

~XAMRL 11 ~48~ 77 ~Y~MINATIO~ 9F ~'HE HOHOLOGY ~E~WF.EN I'~IE ~ p,~ ~ cl)NA ~r~ 0~1 CHITIN~S ~SI~ T~E HYBRI~IZATION TECHNIQUE

Ber.~de~s exa~lning the homology b~eween the ~u~e en~y~ s, the ~omol-S ogy betwP~n the ~DNA encoding ~he ch;tin~se 4 en~y~e ahd ~NA encoding othe~ chitin~ie enzymes w~5 ex~mi~.*~ ng the hyb~ t~on ~echni-~ue~ de.~cr~bed j.n t~e ~bo~e Ma~e~ials and Metho~s~ ~nder the he~ding ~Identific~ion of ~NA belon~in~ t.o the c'~i~inase 4 ~ene f~mily~.

It ~ppe~r~ fr~m Fig, 11 th~t there i~ a ve~ lo~ de~ee c.f he~olegy 10 ex~tnined ~e 5S~(~ between the c~NA encodinf, ~he sugar ~ee~ cnitinslse 4 en;~yme~i s~nd Dt~A enco~l~n~, e.hirilla~i~ i f~orn other ~ nt~i sucll dS pea, t-.bi~cco ~nd b~ns ~; wt!ll Q!i r)N~ Fo~ ;t~ y bt!e- enc(>tlin~ ~.hl? ctllt.l-nnst 1 .~n~ n2y~e~i. The~;e rt!su1L~; ~he~cforc f~, tht r indics~te ths~ hc chLt~na.~e '~ ~rl~:yme belor-~,s tv ~ n~w class o chi.t. i~s ses .

15 The hir,ll dc~ret~ of hornolo~y benw~ the cD~A ~:nco~ y, che chitinase eni~.yme ~nd the DN~ encod~n~ .h~' t.hi~:in~ frvtr r.~ 5ee~ ~hitilli3se shown by ~he hi~h de~,rie c)f l)N~ hyliri~ii2a~.ion furth~. in~lic~tes thi~t th~ ~,en~s encodin~, chitinf~ce A It- c~ t~r~ ntl rh~ l!;er~ ncodin~g t~t~ ~h;.tilli~st~ ) rdl~c ~ ;ip,n;fir,~n~ o.~tor,t~ d th~
20 beLon~ tt~ t;he ~istme ~ene c1.~.s. Tl~ uL~yor~ l t~y itle resu1~i d~.c~ o.~d in ~xa-n~>1e 10 ~iho~ hi~,h 1c~,r- - of s~ rolo~ l hi>mo1G~y etw~en the mnt~re t~n~.ym-~ fr-,m t~\e t~o p1;An~

EXA~Pt.E 1 'rRt~N~;FORt1AT~O~l OF BACT~RIA (,1~

25 ~v~ct~rium ~Itm~?f~ ?ns JDM ???? wrls tr~in~form.~d wi rh r.l~ Jl~ t t:rnn.~fol~ation ~ tor, pBKI ht~ pl~?pdxation o~ whlch is descr~hed ln Ex.lm~,le 1~. ??~?? tt~ g a r~e2e~tl-c-w m~tlloct essenti~ly as ~e-~crihed by An c~ ~I, (1988). For ~he .~ezc~th~w mechGd che ba~erii-i to be t:ransfo~ed were etJlt;~ate~ In I~-medi~, pH 7.4, ~ve~nl~ht ~t ~0 28C. 280 rpm. The ne~t day th~ b~c~ia w~re s~b~ul~ ate~ in 50 ml 8~e~1 0~1~15/JK.M/A12/1991 07 P~ ' 15 ~S

of LB-medium, pH 7.4, and grown for about 4 hours un?
(OD600) was 0.5-1Ø Tje ci;tire was cooled on ice and centrifuged for 5 minutes at 10.000 x g at 4°C. The supernatant was removed and the bacteria were carefully suspended in 1 ml of icecold 20 mM CaCl2.
0.1 ml of the bacteria suspension was pipetted off in icecold cryo tubes and the bacteria were frozen in liquid nitrogen and maintained at -80°C.
For transformation of the bacterial 1 µg of plasmid DNA was first added to a cryo tube with the frozen bacteria. The bacterial were incubated in a 37°C water bath for 5 minutes, 1 ml of LB-medium, pH
7.4, was added to the cryo tube, and the mixture was incubated for 4 hours at room temperature using mild agitation (agitiation table, 100 x rpm). The cryotube was centrifuged for 30 sec. at 10,000 x g, 4°C.
The supernatant was removed and the bacteria were resuspended in 0.1 ml of LB-medium, pH 7.4. The bacteria were plated on to a YMB-dish with 50 mg/l kanamycin and incubated for 2 to 4 days at 28°C until colonies appeared. The presence of a proper plasmids in the bacteria are verified be restriction analysis of the extracted plasmid prior to the use of the bacteria in the transformation of the plants.
In a similar manner, bacterial transformation with other genetic constructs of the invention may be performed, e.g. as shown in Figs.
18, 19, and 22 and explained in Example 18.

PREPARATION OF GENETICALLY TRANSFORMED TOBACCO (Nicotiana benthamiana and N. cahacum) PLANTS
Plant material Leaves form plants to be genetically transformed were obtained from plants grown in vitro or in vivo. In the latter case, the leaves were sterilized prior to transformation. Sterilization was performed by placing the leaves for 20 min. in a solution of 5% Ca-hypochlorite containing 0.1 ml Tween 80 per 1 followed by washing 5 times in 829450ex.001/LS/JKM/A12/1991 07 29 15:55 124 ~ 7 7 sterile wa~:er rn ~ rc~ pl~n~ we:~e gro"n in cont~ , .`.lOCi~, induc~n~ dfum (1~ ~S) ~urashige ~ Skoog, ;9~2).

Th~ lea~es were pla~d or~e ae a time in ~ 14 e~ ~et~i ~iis~ y w~re then c.~t Into ~quares of ~b~ut 1 CDd2, all 4 side~ co~sr.in~ of S tiss~e which h~d becn cut.. Any cut ~ slle ~hiCh ~d b~en ~leached ~y h5~pos~.hlor~te sterili2~tion w~s remov*d.

Cultivation of b~erf~

24 hour~ b~for~ rran~Or.DatiOn a ~uspens;on o~ A~ro~c ~:~r ~a crAn~for-me~ les-ribed abvve w~s sta~d by ~noc~lAe:ing 2 ~ mt m~ wich 10 appropriat~ nn~:lbiotic~ wlth th~ tr.-nsforn)ed A~robacter ia . The bac teria are ~,rown at 28-C with ~eitaTinn ~30~ ~ rprn).

T~ sforma t f on Tr~r~storll~ation of th~ plant. wa~ <lon~ ~ss~nti~ 5 des~::ri',ed by R.B. Iiorsc'T, e~ ~1. (1986). rh~ bflc:t-~rin c~ltr.ur~? w~c .lil~lr~ Ox wit:h 15 1/l.() MS in~m~diat:~ly b~for~ r.~nsform.~r i~n APPI~Y~ e1Y 11) m1 o ~h~
diturrd hact~r~.~ st~spension was poured into n ~ em P~eri di.Ch, ~nd th~ le.~l piecc~ w~r~ rred in this s~sp~r-siol) for ;~,b~u~ 15 nin. Th~
leaf ~liec~i wer~ thcn rcmov~?d nnd ~xc~c hAeter-t~ pen~ i~,rl w.~;
r~?rnov~ ith ~it~ril~ ~iltc- p,~p~.

20 Co - cu t ~ I v.~ n Thc di~y b- fore tratl~ ~xrn~ t lo~ c<> - cul t l~nt ic.r l~c tl ~ d~ ~.he ~ nta l n i ll~;
l~t() M.~ ~nedi~lnl were corlt:cd ~ h ~c~- r~syringo~ 00 ~lj Ql) the d.ly .~ ttat~f~rrn.3ti~t~ ~ pi~c~ ~>f s~e~ik filtl~r ra~e~- wa~; pl.c~cl 0~ t~
cv-cultlv~ltic.n tll~hes, ~nd the lcrl picce.s whlch had l~e e n llpped lr~
25 tll~ b;3cte~ir .suspcnsion were plac~d upsid~ down on tht fllt~r p~p-r Th~ f piccc~ wcre i2~cubated in a ~,roweh ch~mb~r in werlk lig,ht, e . F~ . 12 h~urs o~ ;ht ancl 12 hollrs of d~rkt~e~is for 2- 3 days .

8~9450~x.(~0t/lS/JlCMfA12tl9~ 29 IS SS

:`

~8~77 l~S
Sct~ I eo t i on/regener~ c l ot?

The leef piece., were transferred to P~ri dishe; c3nt~irlin2 shoot-indtl-:ing MS-medi~ w'th ~00 mg~ of kan~ycSn and ~0 ~ 1 of carbe-niciltin and sub-culti~7~ted ~v~ry e~ weeXs ~c~ th~ $am~ d~

Sh~ots ~tlich appear on shoot-inducino MS-r.ie~iunl 300 k~c dlsh~s w~
~r~nsferred to contair ers with l/2 ~SO 30Q k/c~ shoot~ -~ere ~u~-c~ltivate~ when n~eded. AftcL approxi~tely ~ we~cs, the ~xpress;~n of th~ ,8 gluccoron~A~e actlvity ~ rlg ~he GUS-assay (see "Material~;
~nd ~Jet~odsn) was perfo~ed on tl~e tesf t.ips of green shoots.

PI~nrin~ out Genetically transfoxmed shoot~; formeli roots t~nd tl-le r~.sultirl~; yl~rl~:s which w~re (;I~S-po.c~it~ve werc plnnted out in a ~,rowth e~larnber in wae.*r s< i~kecl con~poc~t. They we~e then covered with pl~scic t.:~g~ .~nct ~r~wn or abotlt 1 wcek~ after which thc two corners of th~ pla~;tic h~&`:
IS were cut oi. Aft~r anot~eL week t~le yl;1<;tl~ h.~ . we~-~ re.~noved.

EXAMPI.~ 1~

PREPARATION OF G~NETlCALLY T~A~Sl~ORMI~ K l~ rT~ t ,~l S ~ MEANS C~F
TRA~SFORMATIO~ WITH ~ACTERIA

Tr~n.~orm.~t.i~l w~i r.~,~rr~ll our. ~Ic~n~ c~tyl~doT~ y :x~ t', ;~.
?O seribed beIo~. ~eeds ~ere r,er~innted ~o~ 4 clily!: in ~ kn~ss orl ,~
.~ub.ct.rnt;e conti~ininv, O . ~ t~,/l o ~ ro~ nd 2 ~;/1 o~ crose . l~
sP~ ngs were r.hen tr~nQ.ferretl r.o .q Nunc~ eontr~ r, C:OTIt ~inill~; 1/2 .
M~ h.~:tr~te .~nd c~ll.ured fc>r ~ d.Iys 1n the li~h~ r~ tyledons were remGved from th~ ~eedI in~,, rmd the cotyll!~ion e:~T)l.tn~s ~r~ r.hen brllsheci on the pc!c;olc~ ~rith ~ small ~>rush ~onr~inil)g R .sllsperls;c)n (OD~60-1,0) of A~ohac~eril~ trtnsfor~ed ~,s de~c~ibed a~,o.e iT` EY~a~plr~
1~. The evtyle~ons were then co^culeivated fo~ 4 d.~ys on a ~ubstrate ~vntainlng 1/10 MS sub~tr~te. The t~nsfor~ed ex~>la;~ we~ trans-fer~ed to ~ ~S sllbstr~te ~lpplemenred with 0,25 m~ l of ~APt ~JO
~8,/1. of kfm~mycin, 8~0 mg~l of car~enieillin ar~d $90 ~g~l of ~f3-6~$~001/~5/~/A12j~91 07 29 lSSS

IJ~ r.. l~ F~ [~ F-~r T,~ t-.~ F

126 ~ 57~
tAxîme ar~d ~he e~pl~ts ~re lncub~te~1 fo~ 14 da~s ~ i.te.
The .r~gener~te~ shoot~ were the~ a~s~t~r~ed ~o c~ntilin~J~~.i wlth MS
conta:in~ng 0.25 m~l of BAP, ~00 ~g/l o~ kanamycin/ a~d ~00 ~1 of c~rbenicillin ~ th~ .s~b$trate. ~le iso3~ted ~hoots ~ere tran~~rred to fresh subs~rate~ ~L~h '~ w~eks i~terv~ls f~~ s~leerlon ~n~ multi-plic~ti~n. Selected shoots we~ r~bte~ cln 1~2 MS ~iubstr~t~ C~ot~ini~g 1 ~g/l IBA.

~ Ti~x~e ~rom su~ar bee~ flnd tobacc~ ha~e ~een transfor~ed ~ith a i genetics cQnstructs containing either ch.~tlnase 1, chi.inase 4 or chltinase 76 a~d the se1ective markers, NPT^II and GUS. Selection of -: the callus and shoots or. kananycin has provecl rha~ th~ ~bra;ned t1sslle expresses the GUS ~arker an~ th~s thar thf~ ~ransforrnat:i~n ha~
occurred .
., EXAMP~ 15 lS AN~l.YXIS 0~ CHITI~RXE ~D ~ 1,3-Gl.UCANAX~. IN T~l;SGE~I~ P~ANTS

c~;pre~ .ion lev~l~ for chitinase an~l a~ luc~.na~e ;~oenzyme~.
c~.n h~ e~atu..,tted ei~h~r ~)y u1-~us~in~, the eot..~lt~ enr;~te uc~lu,ity hy rhe two r.~.<llnchem;~ .ay~., b~ nc.~t~;tlrin~, ~h~ .~n, l f~ln~l acti~ ity tlsing chc biolo~,îc.!tl m~th~d~ <n- by mcn~t2rirle ~ of rhe dif-2~ f~renr. i.so~n2yrtc~. by iintnunc-bl~>~ti~ Isin~ ~,pf~1T~Ic ,tn~ibo..li~, ...11 of ~h~ ir.o~ ls '.~ e, le~c~ V~ tM.~t~!r~iMI!. ~t~ .h~A~.". T1-.
fin~tl t~.~;t o thP r~sulr.in~ t:runr~,en,;c pl;3n~ . the an;lt~ S.~ of tl~ deer ce of rc.ci ct-2nc~ eo yhy~oy.l~hvge.nic fl2r~,i t2 ;inp, ~he infec -t.~ y~ecm d-~ccr ibt~d in '~Mu~ri;21~: ,stc.d M~thod.~" .

25 Usiny, tll~! biologi~.al m~r.hod.c I -TIT, the antifun~ ct l~i t.;~ of the ~r.7.yn~f ~; in thc gcrl~!t ic.~ transform; ~I pl,~2n~.c can '~e d~te~it-ir.e~l. f\
r~rd*d &r,owth of the fun,~i hyph~ ~how~; chat. the tr~tn-~for3ativn h.a.~. r e.sul~d ;n a pl~nt huvin~ ~n iraprvved ~olerancs~ i . e . an increa^
~ed anr.iflmgal flcti~rity to the phyt<~patog,enlc f~m~,i co~yared to 30 non~ tr~n6forme~ plant .

:
:, .~
B29450~x.001JLS/JKMJA12/199~ 07 29 ISSS

~`
~ .

127 ~ 8 ~7 In the ~adioche~iccl ~s~ys, 3~-ehitin ~r 3H-lami~
gub~tr3tes for either chltinase or ~-1,3-~luc~nc~se, respec~ively.
~5in~ stand~r~ curvex ~f product f~r~at~on v~ ~ erL~y~t A~Uft, ~he ~etivity fo~ both chitin~se and ~-1,3-gl~canase in c n~de plan~ eY~-tr~cts can be determ~ned. ~hi5 is i~luseratYd f~rth r in ~ t~m~
co~lrs~ experi~e~t whe-~e ehe level of either chitin~e tFi~. 12a) or ~-1,3~glucan~se (~ig. 12b) Is quane~fie~ gAr bee~ le~ve~ Ae specified ti~e intervAls ~fter infection with ~. betic~I2. A1~hough the en~y~e l~vel of both ~he ch1tinase ~n~ the ~ t,3-gl~canase is ~ery low in ehe control plant it ig rPadily deter~1ned hy the very gensiCiv~ radiochemical techniques. I~ the in~e.ted plAn~s, ~n ~n-h~nced product~on of both enzy~es wa~ firse observed 3-9 d~ys ~fter the infcccion with the ftlngaI paeh~gen, ~ith the~e ~echn~que.c, ehe cGn~t~t-le~ve level of ch;c.ina.~e a~ well a~
~ 1,3-gl~cana~e in tr~nseenic plant~ crtn easi;y be re~orded.

Those tec.~lniques, hhwev~r, do not dierenti~te between the ~ariouci chi~lrl;Ase arld ~-1 3-,zluc.lnrise ~.~07.ymes. Only the t~ l en7.y~ne ac tivitie~ for all ttle chiti~ .se or all Lhe B-l 3-~lrtcan;ise isoen.:ytlles are decermine~l. How~ r t.he pr~*ence of th~ v~riO~I~ ehit:;n..~sP ~ricl ,~-20 1 3~gluc~n~st.~ iso~n~ c~in ~nsily l;t! d~tcctcd -.~pnr~t~ly by anrlly7.-in~ the crude prote~n -xt.r.qct~ by ln~ ,ol~l<)tr.~ne ~fter ie~ r~loll hy SDS - PAGE .

e ;~n~ od; ~o ~-1,3-y,lucuni~c 3 re~o~nizcd only on. sin~le protein ~i~ the (~tco.~por:~ ln~ct*t~ 1~ ~; teri. l (Fi~,. 13! . In cont:r.~cit, n~
25 3n~ n WA.'; tietet.ttt~ ~n rh~ t~tnt.rol l~avei. l?li.: is i-l .3c$r~ enr wlt.h the lcw constitutivt lev-~l. of ex~reci~ik)~l ohs~rv~ cor,~.rol ~ln-lrC.
for ~-1. 3-gl~loarl~is~ t~sine the r~,dlocht~miclil tlS!: ~y. t~ien ~ntîbodltn r~ised ng iin~t either chitin~i~ie ? or / w~re e~llployed, t~o ~ajl,r proCein b 1ntls were ln~luced in thc infe~cLecl l~?af tis!.ue.s. Chi tintl.s(- 230 antlbodi.e.s detect a 26 ~n~! a 32 kD~ band wh~ cns two p~ oteins hRvîng mo1ec.~lAr we~hes of 29 and ?7 kD~ were obse~ed ~lth th~ chitinase 4 Anti~ody. t~en purifi~d chlt1nase~ w~re anal~zed by SDS-PhG~ ~nd no~lotting, the protei~ b~ds recognized by chitin~e 2 ~Inti~d-ie~ were chitina~e t (26 kDa) ~n~ ch~ein~5e 2 ~32 kD~ speç~ ly.
Simi}Arly, the antibo~y to chitinase 4 detected the auth~ntir chi~in-82945&~tOOlJ~i/3KM/A12/lY91 07 24 15~

~ ~~lr ~ t ~ f-FI-J~ I Fi tl-J~ HlE~EE 1~ ,.i F~ L-I~

12~ 2 ~ 7 7 ase ~ ig~n (27 kDa), but ir- ~sldit~c!n slso the "SE ~r This ~AS u~l~xpecte~ .~{nce no ~in~ ~cid se~er~e ho~olo~ b~tw~erl ~: chi~inase 4 ar.d ~SE" has b&en o~s~rved ~ee S~quence 1 and S~quence ~: 8). The Y3-D~ structure of chitinase 4 ~nd "SE" on the n1tro~ell~lo me~brane ~ay create s~fficiene ep~tepe recegni~i~n ~ cw the ~ntî~en-~rl~ibody ir,te~-Ac~ion becween ~he "SE" antigeri ~n~ t~e chit~-~` n~se 4 antibody. The ~eaction bet~een the YSE" anti,~en and th~ chti-nase ~ antibody Wfl5 only pronOunce~ ~hel ~.he ~ntibody sol~e~on t~
diluted 1:100 or 1:2QO. A ~u~h ~eaker re~ccion ~as ob~e~ed when the anti~cdy is d1luted 1:5000 or 1:10,000.

: Tran~gen1c tohacco pl3nts c~ntaining either chitina~e 4 or chitinase 7t; hflve been p~od~lced. In ~d~ition to the GUS positi~e renction, the plat1t~ also expressed ~he chi~inase and or eh~tinase 7~ when ana-Iyz~d by immunoblo~tin~ ~Ising the ehitinAs~ 6 .~ntibod~n TYO prote;n bands (27 kD ~n-l 21 kD) were p~esent on the nitrocellulo~e membrAlle.
~o po~itive reaction was observed ~ith the cotlcrol plants. The rc~t-~sons for the double b~nd is not: knvwn ~c pre~ent, huc ~ay indic~r.e th~t ehitina~e 4 ~ ty be sroc~:;.c~d nt t~.~o ~,ite~ in t~-b.~eco during tLte : tr~l~sl~c.~tion proce.~s when ehe le;lder seq~eslc~ ic r~moved~ In ad~i-~i~n t~ thc nor~n~l processiog ~it~ .~t. t.he l~u V~1 V~L1 A1;- G1ll A~
Cys in cllitiT.-~se 4 (.~ino A-~id position 23-24 i-- Sv~luence t), fl s*ccnd putAr.i~ tob~cco y~o~s!:isl~ sit~ is loc~ ~d ~t t.?l-.~ d~il~O
ac~d .cequenc~ Ser A1M Ser - CY~; A1; (Ph~1t;~S- R~ ~G ~ 9U~ .e t). ~ ~ICaVMSr,~ his site ~ e ri~ to the 21 kD ~,olypipt.ide 2'~ se~v~d in rhe 'mmlmohlottinp, ~n~lysi!;. So~lttl~r~ ;i.. e~1rrie~
ont. n.~ des~ribed in M.~elia1s ~sl~l M~tb~k .~h~-v. conf'rm~: t?,;1t the tr.~n.~ene ~oba~o ~l;tot~i coor~in cht-in,tce ~ cl ~?)i~ 7~, resp~ct.iv~ly.

: EXAMPLE 16 M0~ .ICATION OF THE ~sJGAR REsvl' Clll.'rlNA~F. 4 ~Y SITE DIREGTE~ MUTA~NE
SIS

Site ~recte~ ~uta~enesi~ o~, 3 DNA svquenc~ encoding the su~ar beet chItin3se ~, e.~. ~he chitln~se 4 ~e~e, ~ay ~e calried ou~ by ~se of ':

~50e~ LSJ~/A12/1991 07 29 15~5 .

r~ t 1 ~ lr~ FF~ F l r~ lT I l~l l`F~E r~ r,._, ~ '7 :1 F.i.?

1~9 2~8~77 ; ~Cg rea~tions (describe~ ln ~M~terlals and Me~hocls~ 1 ~PCR tlsed in the constru~tion of genetic ~onscruets of thP iI;verltio ~nd in site-directPd ~utagerlesis on elle basis of clone~ D~7A te~pl~-eeS~ sing specific 3' and 5~ prim~rs for ~ch ~ite directed ~uta~e-S nesls. The cho~ce of the 5p~eific 3~ ~nd ~' p~i~er~ ~.o be used depend : on the p~sitlon ln the D~A seq~enc2 In which the ~odification is to ' be carried out.
:`
Typic~lly, stlitahle ~ino ~cid~ r.o be mod~fl~d, elther by substit~-. tion, deletion or i~sertion are ~elect.ecl ~r~ t~e ~asi~ of an ~naly~is of the amino ~cid sequence o the ~ alre chitina~e 4 enzyme ~ op-t.ionally in ~.ombinAtion with ~n anAlysi~ of the enzym~'5 3-D seru~-ture. Especially amino aclds for~ing part v th~ ~ceive ~ite of the enzyme or of epit~pes thereof as well as amino acid~ of l~portance for .5u~trate specifi~ity and s~bstr~te bindlng are of interest in . lS this co~mection.
.,:
The acCiv~ slte of sugar beet ~hitir~ase 6 ., ; The position c~f the es.senr.ial n~ino acid resi~ues il~el~lded in the - ~c~ive slte of chitin~se 4 have been ~entaeivel~ ident;fie~ by ~h~
followin~ obselvations. Firstly, recerlt invest.igat;.ons w;~.h barley chitinAse C ~emonstr~e~ ~h~r chrmlc~ odific~Li~n wi.rh c~rbodii~de and N-bror40succinimi~e (NBS) .o~pl~clv inhihi~ rn~matic ~ett vity (ra.~ul~s r.o~ sho~1). Si~ r ~xperim~nts e.~rri/~d o~t wSth gluco nmyluse ~rom A.~pcrg~ s ni~cr (Sicrk~ r .,lqqO~ h.~lv- eIuci~ red th~ mo~l~ of nc~iol1 by wh~ch crlrbo~ilmt~e ~nd ~BS ~r~Ac~ at~s ~.hl~
enzyrllf. Carbo~iimide ~.c cov~lenr.~y 11nke~l to che thre-! essen~
~cidic ~,ro~ lucamic l~n~ p~rtic aci~i re.c1ducs) ~onst1tuting ehe .at~lytic. site of &luco.lmyl~e. ~BS oxidI.7cs Ttp r~.;d~as ill~port.~n~
: in eitheI st.sbilizin~ the rr~n~i~iorl sr.. ~r.~ in~ermedi.~r.e of ~he c~ta : iy.~ or Trp te5i-~1es involve~ in sub~trute ~In~l~nr, nt a ~li.srnnce from tne c;~t~ly~ic si~e. ~e experi~enr.~ wi~h chitinase C indic~e tha~ thre~ ~cIdic und two Trp-residue.s Are very ~mpor~nt conctit~
ent~ of the ~ctve site. Secondly, by comp~rison eo ~he nctive sites o oth~r enzy~es which hydro}y7e oIigo~cchari~e ~hains lnclu~ln~ t~
gl~coa~ylase described abo~e, ~he ~eIve siee of chitinase 6 is c~nte~plate~ to be constituted by a~ino sc~d residue 1~4 ~Asp) and `' ~Ool,~/JKM/A12/1991 ~ lSSS

:

~ r~ F~ Fl~ lT ~ ,F,EE r~ .?~ F l~l 130 2~8~77 190 (G1U?~ In oontr~st, chitlns~e ~ fro~ b~rl~y and at~~ ?lant chit~nases of ehe ~ame serolo~ic~ s~ {the sug~r beet chi~l~$e 2 class) h~ve three a~part~c aeid residues S~orre~pondin~ to a~ino ac~d residues 184, 190 ~rld 1~5 of chi~inase 4) in the ~ctive slte.
The positlon 3f the two import~nt ~rp ~esidues lnv~lved in th~ ac~ive slee of chitinase C ~ave no~ been elucid~ed. Since chi~i~ase only cc>n~ain ~hree ~rp resid~e~ in contr~st to the 6 pres~n~ in chltina~.e ~ lmportant ~rp res~dues may ~e ~ore easil~ identifiecl ~n chiti~
nase 4.

The two acidic ~e~idues 1~4 Asp ~nd 190 G1u for~in~ the active site of chitinase 4 is conr.~lned in ~he peptide .22: S1GFDC,LNAP~TV~NA~.
Import~nt Trp-residues of the active 5ites ~ay be contained in pep-tide 4.19.3: GPLQI'rW arld peptide 4,26: rA~F~s.

The active sl~e of the chltinase 4 dlffers from the act;ve sir.es of other pl~nt ch~tina~es, e.g. tob~cco, which has th~ following corre-spon(ling .~no ~cid sequences AIGVDLL~PDLVATDPV, C,PIQI~H ~nd SALWFW-~TPQSP, ~nd it woulct ~e interestinp~ to Iook At the syecific AminO
~cids ~ ;idue~ of chlt.in~se t~ which differ fro~l the correspondin~
~m;no Acids ~esidues o~ ~obacco in o~der to obt~in ~urther infosn?~i 2~ tion a~out thc active site ~n~l pocsibly id~ntif~ suic~b1e mo~if?ea-cic~rt~ res~ltin~ ;n Smproved propcrties o~ t~e modif;ed cn~e. ~?C
Acidic ~l~islo a~id residues ;incl ~h~ ~rp r~siducs llre ~onr.~mpl.~d to be p.lItic~tl;~rty ;n~cr~.~t;np~ ln this rcr.pct~.

~ccordin~ly, ~n in~eres~ing mo~ rit,n ~s c~nr in ~hich ~he, ~ t.~M~C
aci~ in positi~n 1~0 ~b.~r;tllr.--d w{th ~pflrEin.~ flild~ol th~ .?s?~?rti.c acicl i.n poS~t~ol? ~. nr~ sub.c~ uted wlth ~,1utamil~e. Ch~neinz ~hc c.arboxyl eroups ~sp 184 to ~so tm~ for Gl~ 1~0 to (.~,n ~n ch~tin~sc 4 ~re ~n itsc1~ cxpectcd o ha~e ~ neg.?t.iv- ?nfluencc ~n the ~n~ymatic ~?ct~Vity, but is contc~pl~ted to re~ult ln u~the.~ know1cdge of the 6?0de of Action of the chitin~se 4 c~zy~e.

~e .subst~tution of Trp ~n posit~ons 17~, 2~5 .~nd 207 to Tyr ~fl--change th~ binding of the substrate ~ch~.t~n) to the cflta1ytie s~e ~nd ~eshaps the ~ubstrate speciicity. The s~hedu1ed subs~it~tion g~ven above Is onl~ sh~wn as exr~mples, and ~umerous ch~n~ ls ln^

gXY~ U~/AI~Jî~î ~ ~ Is:ss 11 FFl.lil FIL~JLlT; lltllE'EF TCI lt~ lt.-' 1~--7~1 r'.

~8~77 f~rr~d tv achieve st IDor~ pot~nt ~nrifung,al chitinas~
accompli.~hed by sit~?-directed ~Uttgenesis e.~;. u~n~, ~he ID.'~.h5C~
outllned below.

Site dir~cCed mlle~genes~s 5 For all the PCR ~eactions ~ugge~ted here ~ri~ner~ a~ ehosen either r.he3~selves contc~ining r~triction s~es or bein~ looctted ne~r re^
strictiosl sites in ~ m~tnner cre~ g thf~ po~;bility of ~xchanF;in~
the PST' prod~lct ~qith a corre~ponding ~quence in the ~;ene bv restr~c-tion ~nzyme di~ tion followed by ligation of the ~elev;~nt fragmen~s.

1~ The 5' primer to be u~ed in the followi~g examples is termed SD 0 (see Fig. 14).

Whesl Trpl70 of the chitlnase 4 amino acid ~quence is ro b~ ~r~bsti tuted by the ahtino ncid Tyr, ~he ollowing proceclure m:~y be c~trried out:

1~ For the PCR reaction ~he 3' prime~r Sr)l i.~t ur~d (see Fig. t~

Th~ res~llr.ing PCR produ~t ~fron~ bp 301 t:o 538) i~. di~e;~r.ed w~th B~snHI
~nd P~rII nnd is~te~ch~n~ed wieh ~h~ cor~i~spon~l~n~ fr~ment ;~f the chitirl~se 6 g~.ne by conven~;onn1 met~lod~; (S.~mhroQ~. c~ <-t, 1990~.

When (`1nl~0 i~ ~o b~ ~u~stit.~-rr~ with ~he ~mislo ~ Cln, the 3' 20 prim~tr S~ u~ ig. 1~

Wherr l~splS4 i!ti to be substitut~d ~ith t.he .~mino ,~cid A~n, th(? 3' prim~ SD3 i.s ~secl (~

~ e ~CR produc~s ase di~,est~ w~th B~lmHI ur~d ~s~MII ;~r~d interc~lan~ecl with the BnmHI-BspMlI fr;~ment of chc chitin;ls~ ~ &-~ne in a similar 25 Dlann~r as deseribed ~bov~ for exchan&e of Trpl~0.

Whç~n l'rp207 is t~ b~ sub.stitut~d Wit:h the ~mlno acld 'ryr, the 3' primer SD4 is used ~Fig. 14~.
:, 8~50e~,~1JLS/~M/A12~1991 ~!? 29 IS:SS

FF~ F ~ ; l E~EE T~ Ic~.~ F~

132 21~ 77 Whe~ rrp2Q~ is to be substltu.ed wi~h ~he a~ino r.~id ~ n .
primer Sl)5 ~ used (Fig. Ib).

PCR product~ ~e diges~ed with ~a~ll and B~l T ~nd Inte~chan~ed w~rh she BamHI-~alI fr~ent in the chitin~se ~ gene ~t5 described abo~e.

In a sitnila~ r~anner, ot}ier dPsir~blf~ modi~io~tion~ m~; be carricd o~t.
' ' : EXAMPLE l?

CGNST~IJ(,TTO~S OF C.~N~'IC CO~T~UCTS WITH S~ITAB~E C-TF.~.MINAL ~TEN~
: SION

C-terlllinal amin~ acld ~ ucnccs foun~ in eonn~ction ~ir.h ~'AT'ioUs plarlt chi~lnuscs ~Ind ~,lucftn~scs are ex~mplificd in t.hc .~pecificae;on and arf? belie~etl to rrovf~ u5eful ln modification of one or mor~. of ; the .ll~tifun~,~l cnzy~es ~ncode(J by thc e~nf~t:ic constnlcts according to the p~e.sent invcntion whicll do not ~ompri~ t:-scrmin<ll ~.en.~ion i~ .`.0 n.5 tO nllow hese en~.yTn~.~ to bc tr;ln.~locAt~d to tlle v~cuolc.

~rh~ C,-t~rmin~ c~.n.~Son May be in~roduccd in the DN~ .~,oqucnces CO~iTI~ on~ or ~ore of the ~nr.lfun~al ~tor~in~ o~ ~hL~ ve~nt~on by any suitnblc techni~ue cuch a~: PC~.

Fig. I$.~2 i1lustr~ ; th~ .çu~ar bccL a-1,3-~lueann~ (DNA ~ th r ~O tobn~co C-terTnintl extension ~hiCh 1.~ ~nderlille-.l in r.he 'i~,ure.

Fiy~. 15~ .str.t-~s PCR pri~n~r.~ wh;ch e;~ tlsed ~o ehang~ the stop codon an~ ~o iIItro~Rl~e a ~nr~ o~ th~ ~ t.erm~n~l1 eY.t~ns~on, a ~ral sIte i3 cre~r.ed ~t the 3' end.

Fi~. 15c illttstr~tes 4 annealed syn~hetic o~gc~n~cleot1de~ c~n~inin~
the last pRrt of ~he C-t~rnin~l e~tensi~n, ~ stop codon, ~ 5~aI ~ite and an ~coRI site.
~' 8~945~ 001~15/fKM/A12/19'?1 07 2~ lS-SS

~1:` r~ FFi-lll F'iLiL.IlT :: !IHIEEE r ~ L.~~ F.l--.

~he C-terminal ex~ensiori c~n be in~roduced by ~ch~r~
EooRI fr~g~el~ in th~ ~-1,3~ c~na~e gene ~1 th the PCP. pro~llct di~s~ed with X~aI ~n~ Dr~I ~n~ the ~nne~l~d synth~tic ~ligonuclço-tides di~esced with S~ nd EcoRI usin~ con~-~nt~onal ~ethods (Sam-: 5 brook et al, 1990).

Fig 16a 111ust~ees the chiti~ase 4 ge~e with a r.obacco C-tenmin~l extensio~ ~the underllned s~quence in ~he fl~1re).

; Fig 16b illu:~trates P~ primers which can be used to ~ntrod~ce ~ S~a~
~ite near the ~top co~or- in the chitinase 4 gene.

Fig 16c illustrates fout anne~led s~nthetic oli~on~lc~eotides cont~in-in~ the sequence for the C-ter~in~l extension a c~anged stop codon~
a Sm~l ~lte and a Eco~l ~it~.

~e C~terminal exten~:ion ean ~e ~ntrod~cefl b~ exchanging the B~nHI -~:coi~1 fr~gment with the PCR ~rGduct d~.~ested with B.~m~l and S~aI and 15 the ~nne~led synthctic oligonucleo~1dcs ~1 ~ested w~ r.h S1nal anJ EeoRI
like.~ise ~-ng conv~ntion~l m~thod~.

I,ikewis~ other C-termin.~ equences l~k~ rhe ones ~emp1ifie~ in the desc.rlption ¢an bc Q~cl~l to ~!le chlt~islas~ 76, chir.innse ~, 'tSE~ ~n~
~ lue~nn~;c sequen~e.~ e N-.~rminal r.cquenc~ may in ~ xiln~lar mAns~e.l bP e~eh~n~e~ Wi~l orher l~ rm5na1 sæquenee!;. Of p;lrricntar i--tf.~rf.:s~ m~y h~ thc ~ termillal ~e quenc~ nf chit ina~ hf.~wn i~l t.h~
Se~u~ncc 7. Othf.?r ~ntcre~tln~ ~-rerm~na1 ~ftuenc~.s mny b~ rh~ onc~;
shown in 'rable IX.

EXAMPt~F. 18 2S Gene~ic construct.s The excixe~ reco~binant ~Bluexcript c~ntainln~ e ch$tina.se 4 cDNA
8ene (Bl5 chitin~se 4~ was subclone~ in order to supply the ~ene with ~n enhanced 35S promoeer ~nd ~ 35S terminator. ~hi~ c~nstruct ~as .

8294$0~X.O~ M/Al2/1991 07 :

J:~ F~ , I r" jl !7 ~ F r~ lt,~ F, ] .~

~8~77 trAnsf~rr~d to the plant tr,~n~;form~t~o~! vec~or pBKl~
NPTI I and the G~lS genes .

More sp~cifically, a PCR ~mplific~iun reaction -~as p~rfor~ed in order ~o in~roduce th~ ATG 5ite, a r~bosome blnding ste ~d two re~tricti~n sit~s ~ind1II and ~glIl) 5' ~o th~ cD~A ~eq~en~e.

The ~ligonucleotide KB3:
~S CCGAhGCTTAGATÇT~AAC M CAAC~ TCTTCT~>T(~)GGACC,3 ) lS

: lO cont.~Ining th~ two restr~ction site~, a ribosome binding site, the ATC (underllncd) ~n~1 the first- tS nucl~or.ideg of rhe ~15 c.h;e 4 clo~e ucleo~ide 8 and 10 were mix~d (~ nuclcotid~s due to t.he f.~ct that.
ch~ K~ primer w~s us~d ~or the chit J6 clone ~, well ~ ~A5 u~ed ns the ~' PCR pri~er ~nd ~:he oligonucleotid~ KB4 15 ~h~ o~igonucleotlde KB4-: (5 ~CACACGTAGC~ TGG3 ) : I **
261 Nhel 241 was; .Isied as th~ 3' P(;R prim~r (~ clootide ~5S nr~d 2')6 wn~ interchnn-20 g~cl ln ordcr t.o decitroy ~he ~ccon~l ~heT .~i~e).

The 1~C~ ~rodu~:~ wn~ xtr.~.ted twi~e ~ith yh~nol nnd t~ n with chlo roform L~n~ tOH pr~cipir..ltcd. A~rer ro:iuri~n.~ion in ~ r.hc DNA wns dige~:ed with llind~ nd llhel . The ~l~ndIII-Nh~ t f~ nenr. from pP~l.S
chi~ 4 W~ xoh~n~,e~l wlth ~le Ill.ndIIl-~hei PCR Lra~lrel~ (Fig. I7).
:' .~ 25 Th~? con~;~r~lcr. W~!; sequenccd l ith the Tl sequenci~lg primer (corr~spc~
ding to ~he pB1ue~cript Tt promo~:r) .~nd prim~r 3~ 0 CATCGGACCATCCACl'ACC ) 31) and ie was confirme~ th~t the ent~re exch~riged se~u~n~e wa~ ~orre~t.
Furth~r~ore, irl the 5 ' se~uence the or~na1 nucleotide ~ s a T an~

8;~9~.X.~ A/Al2~ ~ lS55 ~ .
"' ' r ~ I L~ . r j~, ." ~ t.L' :~t,'-_' . lJ ~ r' . 1 ~

135 2~8~77 nuo1eot:k~e 10 ~as ~ ~ a~ in th-? pB15 chit ~- elone ~rè~
~i ~e.~ ~t posi~ion ~ 5 and 2Sl were st~ll presen~.

I~e constr.~ot was digested with EcoP~I And a fill in re~ct~on ~as per~or~d with ~leno~ en~yme ~n the presenc~ of dA~P and TTP, ~he S con~cl-~ct was fur.~her digested with ~glII aft~r remov~l of the Klenow en7y~e. The ~A fr~ent ~lII^EcbRl cont~inine the entir~ ohi~inase 4 se~ence w~s clGne~ into ehe rec~or pPS4R ont~.inin& ~n erh~noed 3SS promoter ~nd a 3SS termin~tor. Th~ e,hitin~se 4 ~ene ~a~ inserted in the correct orientati~,n hy digest~ the pPS48 vector with B~
Sm~I (Fiy7. 17). Ttl~ ch~t~nase 4 gene ~ith t~e enhanced 35S promoter ~nd 35S ter~i.nator w,~s ~ran3ferred to ~he plant t~an~f~rm~tion vector pBKlh (~ig. 17) a~ a ~lindltI frag~ent (Fig. ~7! ~e resu1tLng vec-tor pbK~K~, h~rboured in an E . col l DHS has be~n de.~pos i ted wi th the Deursche S~n~n~ung von Mikroorgani~l~,et) und ?elllcul turen ~ H, Masch~rocler Weg, ll D-3300 ~raunschweig (DS~) on 3(t July 1991 under the provisiolls of tl-e Paldapes~ Trea~y un~r rhe p. o~ isional lccession ~c . ....

rh~ "SE~ genc~ was t.hen introd~ccd into the ~.onstr~lct pBKlhK4 tFi~.
17). A l~ll len~th ~SE~ ~onc ~'dS con.ctruccc~ ~y con~ in~ the 5' end of the ~ene LIom ~he p~l~rl clone (EcoRI-KpnI) w~th tho re~t of the ~,enf~ L~m pSE?? (Kpnt-~indIII) in rh~ ~tor~inE ~oc~or pllClq (Fig.
18~ SE~ r,eno wi~S s~ loned ~n the Sm;lI site of pPS4~ a~ a EcoR~ HlndIII ra~m~nt f~lled ;n wi~}~ Kl~ow pot~mc~;Jie in th~ pKe~-sence of nll ~o~lr nl~cl~t~de~ The ori~rl~at.lon of ~hi~ e w~th ?.S rf~ip~ o ~ nh.~n~ p~ o~-~r .~ t~:rmir~.,t~ xn~in-~d by rcstrict~on en,.ym~ .~nalyii~j antl fllrrher oonfirm~l hy sequorloe analysls.

~o ~SE~ grne wlth thc c~ crd 35S pro~otcr ~rl(l 3S~ t.rmiT1.t(>r wa.
c]onod ir~ t~e ~pnl .~lte o~ pBKLhK4 ~s n HndIII ragment in the 3~ presenee ot n ~.indIIJ-KpnI ~d~pt~r (Fi~. 18). The HindIII frag~ent wa~ f~r~hermore clo~led in the ~indIII siC~ bf pBKL4.

Similarly to ~he chitirlase 4, the chi~inase 7~ g~ne wa~ cloned ir~
pBXL4 ~Fi~. 19).

8~Xkx~S~s/nuulAl21l~lo7 ~ ~S~S

136 ~ 7 7 In ~ ~.imilar ~anner, ehe gluc~nase gene can be int~o~
c~nst~-uct pB~L~, pBY~L4K6, pBK~4KSE, or pBK~KK4KSE (~i~. 2~).

~ full leng..h cDNA clone (Sequence 4~ ~s dig~ste-d ~ith EcoRI and : BglII, ~he sticky ends were ~lled ~n with ~len~w poly~er~se tn the 5 pr~sence of all four ~TP' s. The ~lueana~e ~ene Is th~n subcloned ~n the SraaI .~ite Q~ pPS4SMQ~. The orient~ion of ~he g~nQ with r~pect t~ .he ~nhanced 35S ~ro~ot~r and the 35~ cerminator, respectl~ly, ~ay be ex~m~ned by resericticn en2~ nalys~s and further confirmed by sequence analysis.
, The ~lucan~se gene ~ith the enhanced 35S promote~ and the 35S ecn~-naeor i5 cloned in ~he EcoRI si~ of pBKL4, pBKI.~4, pBKL4~SE.
pBKL4~4KSE.

~;~k~ ~/rKM/AI21l~l ~ 29 ~55

Claims (5)

1. A DNA sequence comprising the sugar beet chitinase 4 DNA sequence shown in Sequence 1 or an analogue thereof, the analogue bieng a DNA
sequence encoding a polypeptide having the antifungal activity of the sugar at chitinase 4 as defined herein and i) being a characteristic subsequence of the DNA sequence shown in Sequence 1, ii) hybridizing with the DNA sequence shown in Sequence 1 at 55°C
under the conditions specified in the "Materials and Methods" section under the heading "Identification of DNA belonging to the chitinase 4 gene family", iii) encoding a polypeptide having the amino acid sequence of the sugar beet chitinase 4 shown in Sequence 1, or iv) encoding a polypeptide being reactive with an antibody raised against sugar beet chitinase 4.

2. A DNA sequence according to claim 1, comprising nucleorides 71-793 of the chitinase 4 DNA sequence shown in Sequence 1 and encoding the hevein dom? the functional domain of the sugar beet chitinase 4 enzyme, or an analogue of said DNA sequence.

3. A DNA sequence according to claim 1, comprising nucleotides 176-793 of the chitinase 4 DNA sequence shown Sequence L encoding the functional domain of the sugar beet ? 4 ?me, or an analogue of said DNA sequence.

4. A DNA sequence comprising sug beet chitinase ? gene.

5. A DNA sequence encoding a chitinase is?me which is at least 60% homologous with the sugar beet chitina? enz? encoded by the DNA sequence Sequence 1 and at the most 40% ?molog?us with the sugar beet chitinase 1 encoded by the DNA sequence shown in Sequence