CA2017783A1 - Recombinant rhizobium bacteria inoculants - Google Patents

Abstract

Abstract The sequences of a Rhizobium bacteria responsible for competitiveness with respect to plant nodulation have been isolated and permanently transferred to superior nodulat-ing Rhizobium genome. This has resulted in a stable construct that can form a plant inoculant that yields effective nodulation, while reducing the risk of suppres-sion by other bacteria in the environment.

Description

7~3 RECONBINANT RHIZOBIUM ~ACTERIA INOCULANTS

Back~round Of The Invention A. Field Of The Invention The pressnt invention relates to recombinant DNA
technology. It appears especially useful for improving the nodulation (and thus nitrogen fixation) capability of plants.

. DescriEætion Of The Art Root nodule Rhizobium bacteria are reYpon~ible for symbiotic nitrogen fixation in the nodules of certain plants (e.g. legume~). Where natural bacterial activity is ineffective, the plants must rely on the existing nitrogan in the soil or on fertilizers. Where the former occurs, the auality of the soil is reduced. Where the latter occurs, the cost to the farmer (and ultimately the public) can be ~ub~tantial. Purther, the use of fartilizers often raises environmental concern~.
It is now known that the presence of certain ~inferior~ strains of Rhizobium in 80il can depress the productivity of not only other natural bacteria, but also of ~'superior" bacteria added by inoculation of seeds.
This can frustrate attempts to inoculate seeds prior to planting or to inoculate roots during plant gxowth. When inoculation has been successful, it Ls usually because the indigenous bacterial populations have been small.
Many investigators have studied the factors involved in determining nodule occupancy by trains of Rhizobium.
See e.a. D. Dowling st al., 40 Annu. Rev. Microbiol. 131-7~7~;~
157 (1986) (the disclosure of thiC article and of allother ar,ticles referred to h rein are incorporated by reference as if fully set forth). Despite thi~ work, no solutions to the ab'ove described Rhizobium competition problem have been developed.
In E. Triplett et al., 85 Plant Physiology 335-342 (1987) and 11th North American Rhizobium Conference Abstract GP4 (1987), my laboratory reported on the fact that the Rhizobium legu~_L~e~E~ bv. trifolii bacterial strain T24 appeared to have genes in its coding respon-sible for a Yuppres~or of other Rhizobium (I named the substanc2 trifolitoxin) and other genes coding for T24~s own resistance to trifolitoxin~s effects. Unfortunately, I also have found thst trifolitoxin production by transcon~ugant bacterial cella that I had constructed was readily lost in the absence of tetracycline. Thus, the earlier Rhizobium transcon~ugant~ were not likely to be able to effectivaly limit nodulation by trifolitoxin-sensitive indigenou~ strains of Rhizob~um under agrLcul-' tural condition~ (where tetracycline application is impractical).
It wa8 therefore desired to more specifically i~olate and characterize the genes responsible for the T24 suppressor and resistance characteristics and use informa-tion developed therefrom to find a mean~ for stablyinserting such gene~ in the genome of "~uperior" Rhizobium ~o a~ to ultimately lead to a Rhizobium that can form effective nodules notwithstanding the presence of indigenous strains.

Summary~5~f The Invention The invention provides a recombinant Rhizobium bacteria capable of a ~i~ting in the formation of nitrogen fixation nodule~ on at least ~ome plants. The bacteria has a foreign ~equence expre~sibly coding for trifoli-toxin. The bacteria preferably al~o has a ~equence coding for re~i~tance to trifolitoxin Yuppre~sion, with both foreign sequence~ being in the bacterial genome. The term ~genome~ is u~ed herein to refer to either the bacterial chromosome or other bacterial genetic ~equences in the bacteria.
Inoculant~ can be provided that u~e these bacteria.
Thu~, plant ~eeds (or the roots of young plants) can be inoculated with the bacteria.
Further, plant cell~ can be formed that incorporate these ~equences ( 80 that the plant strain produce~ its own trifolitoxin). In the alternative, a production host can produce trifolitoxin on a commercial scale. In either case, the trifolltoxin can be used a~ a tra~ inoculant by having the superlor ~train have the re~i~tance gene only.
It will be appreciated that the invention provide the ability to effectively create nitrogen fixation nodule~ in the presence of inferior ~train~.
The ob~ects of the Lnvention therefore include:
A. provLding a recombinant bacteria of the above kind;
B. providing a recombinant ho~t of the above kind;
C. providing a plant seed inoculated with a bacteria of the above kind; and D. providing a plant inoculant using a bacteria of the above kind.
These and ~till other ob~ectq and the advantages of the present invention will be apparent from the de~cription that follows.

Descri~tion Of The Preferred Embodiments General Overview Rh~ obium leouminosarum bv. trifolii T24 induces ineffective nodules but produces a potent anti-rhizobial compound, trifolitoxin. As a re~ult of trifolitoxin production, T24 prevents root nodulation by trifolitoxin-sen~itive bacterial strains. The main ob~ective of this work was to identify and isolate the trifolitoxin produc-tion and resistance genes and permsnently transfer those gene to other strains of Rhizobium that produced ~superior~ noduies.
To achieve this, a gsnomic library of T24 was prepared in tho prior art cosmid vector pLAFR3. One cosmid clone wns 1dentified that restored trifolitoxin production and nodulation competitivene~s. We formed a recombinant plasmid from thi~ cosmid clone, p~FXl, that conferred trifolitoxin production and re~istance on other bacteria (slbeit in an unstable fa~hion).
Transpo~on mutagenesis and restriction analysis was then used to map and subclone the insert of pTFXl. A
4.4 kb region of DNA, referred to as tfx was found to be necessary for the expression of trifolitoxin production and resistance in Rhizobium. Another portion was found to have Rufficient homology to Rhizobium genome to permit the u~e of a technique for in~ertion into the genome. Several mutants of pTFXl (with Tn5 insertion3 out~ide the trifoli-toxin region) were therefore u~ed to permanently insert the trifolitoxin gene~ into ~everal strain~ of Rhizobium.
This re ulted in a stable construot having the desired characteristic~.

Meth d~ ~nd Material~

The identification of the precur~or cosmid clone, and the formation of pla~mid pTFXl is de~cribed in detail in my article, E. Triplett, 85 P.N.A.S. USA 3810-3814 (June 1988) (not prior art). I then made a restriction map of pTFXl. I did this by restriction analy~is of Tn5 inser-tions in pTFXl. This map was used to determine the size and location of the trifolitoxin genes a~ well a~ to develop a ~trategy to subclone the trifolitoxin genes into the broad ho~t range vector, pRR415, N. Reen et al., 70 Gene 191-197 (1988). I found that the ability of pTFXl to confer trifolitoxin production as well as resistance in trifolitoxin-sens1tive strains of Rhi~obium were located within a 4.4 kb region of pTPXl, this knowledge, plus my analysi~ of the other portions of the insert in turn ed to selectiQn of the marker exch~nge technique for insert-ing these gene~ in a bacteri~l genome.
In my work, Rhizobium str~ins were cultured at 28C
on Berger~en's ~ynthetic medium (BSM) as described by F.
Bergersen, 14 Aust. J. Biol. Sci. 349-360 (1961). Strains of E. coli were cultured at 37C on Luria-Bertani (LB) medium. Antibiotics were added as needed at the following final concentr~tionst kanamycin (~m), 50 ug/ml; tetra-cycline (Tc), 12.5 ug/ml; ~pectinomycin (sp)/ 50 ug/ml;
~treptomycin (Sm), 50 ug/ml; gentamycin (Gm), 25 ug/ml;
nalidixic acid (Nal), 10 ug/ml; and neomycin (Nm), 75 ug/ml.
Con~ugation of the plasmid mutants (e.g. pTFXl::Tn5) into Rhizobium was performed using procedures analogous to those described in E. Triplett et al., 85 Plant Phy~iol.
335-342 (1987) with Yome modifications. In this regard, the donor, recipient, and helper strains were mixed in a 1:1:1 ratio in water each at a cell density of approxi-mately 5 x 107 per ml. After vortexing, a 5 ul su pen~ion of thi~ mixture is placed on a Y~/RB (see E. Triplett (1987), ~uDra) plate with 3% agar. After incubation for two days at 28 C, each mating was resuspended in 0.1 ml water and spread plate on a BSN plate prepared with noble agar and supplemented with tetracycline and streptomycin.
The use of noble agar in the interruption media eliminated the background of growth on the plates. After five days, transcon~ugants were observed.
Con~ugations involving the transfer of plasmid DNA
between strains of E. coli were done as described above except that 5 ul of the mixture of donor, recipient, and helper strains were placed on an LB plate and incubated at 37C overnight. Interruptions wore done ~8 described above with the appropriate selective media on solid LB
medium.
In the transfer of pla~mid DNA from E. coli to Rhizobium, E. coli DHSa (Bethesda Research Labs) (pRX2013), D. Figurski et al., 76 P.N.A.S. USA 1648-1652 (1979), was used a~ the helper strain. In the tran~fer of pla~mid DNA between two strain~ of E. coli, E. coli HB101, H. Boyer et al., 41 J. Mol. Biol. 459-472 (196g), (pRR2073) (S. Leong et al., 257 J. Biol. Chem. 8724-8730 (1982)) served as the helper strain.
Large scale pla~mid preparations were purified by the boiling method described by D. Holmes e~ al., 114 Anal.
Biochem. 193-197 (1981). For restriction analysis of small amounts of plasmid DNA, plasmids were purified from cells grown on sold medium by the alkaline lysis miniprep method described by F. Ausubel et al., Current Protecols In Molecular Biology (1987).
The recombinant plasmid, pTFX1, was mutagenized with Tn5 by ~he method of G. Ditta, 118 Meth. Enzmol. 519-528 (1986) with slight modification~. The pla~mid pTFXl was transformed into E. coli cell line HBlOl::TnS as described by D. Hanahan, 166 J. Mol. Biol. 557-580 (1983) using LB
medium supplemented with kanamycin and tetracycline for ~election of transformants. The transform~nts were pooled and con~ugated with HB101 ~pRR2073) and C2110nal (Ditta, supra). The triparental matinqs were incubated overnight at 37C. Cells were rQsuspended in water and a dilution series plated on LB medium suppl6mented with kanamycin, tetracycline, and nalidixic acid. Transcon~ugants were pooled and pla~mid DNA isolated by an alkaline lysis miniprep procedure as described by F. Ausubel et al., Current ProtocoLs In ~olecular Biolo~Y, John Wiley & Sons, New York (1987). Fourteen separate matings were performed in order to enhance the prospects of obtaining independent mutations. Plasmid DNA was transformed into E~ coli DH5a r'~
and the subsequent tr~nsformants sQlected on LB medium with kanamyc$n and tetracycline.

Re~triction Analy iR

Restriction analysis of three hundred and thirty-six pTFXl::TnS mutants was done to provide the information nece~ary to construct a restriction map of pTFXl. Each mutant was also con~ugated into R. lequminosarum bv.
trifolii strain TAl as described above to determine the trifolitoxin phenotype. (The tfx genes are not expressed in E. coli.) Plasmid DNA of each pTFXl::TnS mutant wa cleaved with the following restriction enzymes: Eco RI, Kpn I, Dra I, and Mlu I. To accurately map Tn5 insertions within each restriction fragment, selected pTFXl::TnS plasmids were cleaved with Hpa I, an enzyme with two symmetrical restriction sites within the inverted repeat sequence elements of TnS. Hpa I ha~ two restriction sites in pTFXl. This enzy~e waY used for this purpose rather than Bgl II since there are no Bgl II restriction 3ites in pTFXl. Plasmid DNA wa~ electrophoresed in 0.6% agarose at 100 v. For the separation of fragment ~izes greater than 15 kb, field inversion electophore~is was used. At 0.3 8 intervals, the electric field wa~ inverted between 100 v toward the anode and 60 v toward the cathode. Field inversion gels were run for 16 hours at 4C. All gels were 10 cm in length.
The ability of a ~train to produce trifolitoxin was determined by bioassay; Southern analysis was determined with biotinylated probes of either pLAFR3 or pTFXl; and trifolitoxin wa~ partially purified from the cell culture supernatant~ of T24 and various Rhizobium transcon~ugant~
containing pTFX2 by reverse phase chromatography; thece steps all being done as described in the general technique portion of E. Triplett, 85 P~N~AoS~ USA 3810-3814 (June 1988) (not prior art).
From thiY analyYis, I determined that my pxe~iou~
e timate of the size of the in~ert in pTFX1, 24.2 kb was inaccurate. The insert Yize in pTFXl is now known to be 29.5 kb.
The restriction analysi~ showed that two enzyme~, Dra I and Mlu I, did not have restriction sites in either Tn5 or tfx. The tfx region resides on a 10 kb Dra I frag-ment and a- 7.5 kb Mlu I fragment. Since tfx is present on a ~maller fragment in the Mlu I dige~t than in the Dra I
dige~t, Mlu I fragments were chosen for subcloning tfx.

Subclonina One mutant of pTFX1 wa~ chosen whose TnS insertion was located within the 7.5 kb Mlu I fragment of pTFX1, yet did not affect the expression of trifolitoxin production in h~zobium. Ligation of the Mlu I fragments from which contains both th~ intact trifolitoxin productlon genes and a Tn5 in~ertion on the ~ me fragment, to the broad host-range vector, pRK415, allow~ for ~election against the other possible ligation products.
An Mlu I dige~t of plasmid DNA from a pTFXl::Tn5 mutant was blunted with T4 DNA polymerase using technique~
described by F. Ausubel et al., Current Protocols In Molecular Bioloqy, John Wiley & Sons, New York (1987).

~r ~

These fragment~ were ligated to an alkaline phosphata~e-treated Xmn I digest of pRK415. The resulting ligated DNA
was transformed into DH5a competent cellq and tranq-formants selected on LB solid medium supplemented with kanamycin and tetracycline. Re~triction analysis of the plasmid DNA of a selected transformant showed an insert size of 13.2 kb (as was predicted based on the size of an Mlu I fragment of pTFX1 with a Tn5 insertion). This plasmid i9 referred to a~ pTFX2.
A restriction map of pTFX2 wa~ prepared based on the restriction site~ known to be pre~ent in pRK415, the Eco RI restriction sites present in the Mlu I fragment in pTFXl, and on double restriction dige~ts of pTFX2 with Sst I, Eco RI, and Hpa I. The Xmn I site in pRR415 and the Mlu I sites in the insert were eliminated by the blunt end ligation of the insert into the vector.
To determine whether pTFX2 pos~essed functional tfx, this plasmid wa~ con~ugated into Rhizobium. Trifolitoxin production wa8 observed by the resulting transcon~ugants and confirmed using technique~ described previously for pTFSl transcon~ugants in E. Triplett 1988, su~ra (not prior art).

Insertion Into Bacterlal Genome As an example of inserting tfx into a selected bacterial genome, the method of G. Ditta, 118 Meth.
Enzmol. 519-528 (1986) was adapted for ~. leaumino~arum bv. trifolii TAl. (A. Gibson, CSIR0) The technique starts from the idea that certain plasmids may be incom-patible with certain other plasmids in certain hosts, and ~ V ~
that under antibiotic stres~ the host will tend to either drive one out (or hopefully where homology exicts take in the unwanted genetic material as part of the bacterial genome). The incompatible plasmid pPHlJI (J. Beringer, 276 Nature 633-634 (1978)) wa~ con~ugated into several TA1 transcon~ugants with my pTFXl::Tn5. It will be appreciated that the host Rhizobium can be other ~superior~ ho~ts of intere~t. The con~ugation wa~ inter-rupted on BSM prepared in noble agar and ~upplemented gentamycin, kanamycin, and spac~inomycin. The resulting exconlu~ant~ (with the gene in the cell genome) were replica-plated on 3SM with tetracycline. The tetxa-cycline-resistant strains were di~carded.
Bac~erial strains T24, TAl (pTFXl), and trifolitoxin-producing TAl (pTFXl::Tn5) tran~con~ugants and TAl::TFX::Tn5 excon~ugant~ were streaked to single colonies on BSM medium in the absence of selective anti-biotics. Ater two day~ of incubation at 28C, a portion of the confluent growth on the plate was suspended in water and 5 ul of that su~pension spotted in the center of a BSM plste for the assay of trifolitoxin production. A
single colony from the initial plate was used to inoculate a second plate. After two days, confluent growth on the second plate wa~ used to assay trifolitoxin. The assay~
continued for 10 "generations" or until trifolitoxin pxoduction was no longer observed. TAl::T~XsTn5 showed 3tability through ten generation~.
It will be appreciated that the pre~ent invention involves, inter alia, the location of the trifolitoxin production and resistant geneff, ths cloning of them, and ~ r ~
.

the development of a way to in~ert them permanently in the bacterial genome.
Cultures of pTFXl::TnS (a/k/a pTFXl:10-15) in E. coli and Rhizobium TAl::TFX:TnS (a/k/a TAl::10-lS) are on S deposit at the American Type Culture Collection, Rockville, Naryland, U.S.A., with ATCC numbers 67990 and 53912 respectively. They will be made available upon issuance of this patent and as provided under U.S. and other applicable patent laws. However, this availability is not to be construed as a licen~e to u~e the invention.
The preferred way to u~e the preferred bacteria is to streak the deposited TAl::10-15 on BSN solid AGAR and wait for 2-3 days. One then 3treaks the growth product into BS~ uid broth. After several more days one can pour lS the liquid broth on peat and u~es the peat as a carrier to surround the ~eeds or roots. Note al~o that other known commercial inoculant techniques can readily be adapted for use with these bscteria. See e.q. R. Roughley et al. in Nitrogen Pixation In Legumes, pl93-209 (1982); resulting in incculants and inoculated seeds. Thi~ invention appears most likely to be useful on clover, peas, beans, vetch, and soybeans, but may well have utility wherever Rhizobium created nodule~.
Another possible use of the invention iY to insert only the resistance gene in a bacteria and then add trifolitoxin to the soil ~or transform a plant cell so it produces the trifolitoxin)~ In this regard, several vectors are already known that can expressibly transform a plant genome, and many commercial production hosts are known.

It will be appreciated that various other changes to the preferred embodiment may be made. For example, various other strains besides T24 may produce trifoli-toxin, and thus their sequences could be used (e.g. after location with a hybridization probe ba~ed on pTFXl)~
Also, means of inoculating the roots of live plants (a~
opposed to ~ust ~eeds) during transplantation can easily be developed using known technique~. Further, other means for inserting the foreign genes in the bacterial chromosome may prove useful. See e.g. G. Barry, 4 Bio/
Technology 446-449 (1986) and 71 Gene 75-84 (1988). The claims should therefore be looked to to ~udge the full scope of the invention and the preferred embodiment ic not to be considered as representing the full scope of the invention.

Claims (8)

Claims I claim:

1. A recombinant Rhizobium bacteria that is capable of assisting in the formation of nitrogen fixation nodules on at least some plants, the bacteria having a foreign sequence in the bacterial genome expressibly coding for trifolitoxin production.

2. The bacteria of claim 1, wherein the foreign sequence also comprises a sequence coding for resistance to trifolitoxin.

3. A plant seed inoculated with the recombinant Rhizobium bacteria of claim 1.

4. The plant seed of claim 3, wherein the foreign sequence of the recombinant bacteria also comprises a sequence coding for resistance to trifolitoxin.

5. An inoculant for a plant comprising:
a carrier; and the recombinant Rhizobium bacteria of claim 1.

6. The inoculant of claim 5, wherein the foreign sequence of the bacteria further comprises a sequence coding for resistance to trifolitoxin.

7. A recombinant host having a foreign sequence in the host's genome expressibly coding for trifolitoxin.

8. A recombinant Rhizobium bacteria that is capable of assisting in the formation of nitrogen fixation nodules in at least some plants, the bacteria having a foreign sequence in the bacterial genome coding for resistance to trifolitoxin.