CA2134784A1 - Novel plant virus sequences - Google Patents

Abstract

The amino acid sequence of the major coat protein of barley mild mosaic virus (BaMMV) and part of a non-structural protein is disclosed, together with the nucleotide sequence of the portion of RNA by which they are encoded. Also disclosed is a method of increasing the resistance of plants to disease caused by the BaMMV and transgenic plants having increased resistance to disease.

Description

WO 93t22345 2 13 4 7 ~ ll Pcr~GBg3/oo9lo Title Novel Plant Virus Se~uences Field of the Invention _ _ This invention relates to novel plant virus sequences and concerns novel amino acid sequences, novel nucleotide sequences, vectors comprising the novel nucleotide sequences, plants into which the novel sequences have been introduced, a method of increasing the resistance of plants to disease and transgenic plants having increased resistance to disease.

Backqround of the Invention Barley yellow mosaic disease is one of the most important diseases of winter barley in central E~ropean and Asiatic countries (Inouye & Saito, 1975 Descriptions of Plant Viruses, No. 143). It has recently been recognised that the disease is caused by two viruses: barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV;
Kashiwazaki et al., 1989 Annals Phytopathology Society Japan 55, 16-25; Huth and Adams, 1990 Intervirology 31, 38-42; Prols et al., 1990 Journal of Phytopathology 130, 249-259), although it was previously thought that BaMMV
was a mechanically transmissible isolate of BaYMV. The two viruses have identical particle morphology being composed of flexuous rods with two modal lengths of approximately 275nm and 550nm and produce similar symptoms (Huth et al., 1984 Phytopathology Z 111, 37-S4). The particles encapsidate bipartite, polyadenylated single :, .r~ . ~ S~-.'EET

W O 93t2234~ 2 13 ~ 7 8 4 PC~r/G B93/0091~

~ 2 -stranded RNA genomes of similar size (approximately 8kb and 4kb; Huth et al., lg84; Prols et al., 1990). However, cross-hybridisation studies and restriction enzyme analysis have suggested that the two viruses are substantially different at the nucleotide sequence level (Batista et al., 1989, Plant pathology 38, 226-229 Prols et al., 1990~. Furthermore, there is no cross-reaction in serological tests (Jianping and Adams, 1991, Plant Pathology 40, 226-231, Kashiwazaki et al., 1989a) Since both barley mosaic viruses are transmitted by the soil-borne fungus Polymyxa qraminis, chemical control measures are ineffective and yield losses can only be prevented by cultivating resistant varieties. The resistance of many European varieties to barley yellow mosaic disease is likely to be due to one recessive gene tym4) derived from the spring barley "Ragusa" ~Kaiser &
Friedt, 1989, Theor. Appl. Genetics 77, 241-245).
However, a resistance-breaking strain of BaYMV has recently been reported in both Germany and the UK which overcomes the effect of the ym4 gene and consequently, it has become necessary to broaden the base of resistance to I the barley yellow mosaic disease complex. At least three other resistance genes have been described in Asiatic barley germplasm (Gotz et al., 1991, Proceedings of the Sixth International Barley Genetics Symposium, Kashiwazaki et al., 1989a) and are being actively incorporated into European conventional breeding programs.

A complementary approach is to create resistance via a j genetic engineering route once gene transfer methods have been developed for cereal crops. A variety of molecular techniques have been used to generate plants resistant to a particular virus, including coat-protein mediated cross-wog - 21347~4 3/2234~ PCT/GB93/00910 protection.

Coat-protein mediated cross-protection was first described by Powell-Abel et al., (1986, Science 232, 738-743) who found that transge,nic tobacco plants expressing the TMV
coat protein displayed a significant delay in symptom development. ThiS form of resistance has now been demonstrated for many different viruses including AlMV, CMV, TRV, TSV, PVX, PVY and PVS (reviewed by Nelson et al., 1990, in "Genetic Engineering of Crop Plants" edited by Lycett & Grierson, published by Reed International plc;
Beachy et al., 1990, Ann. Rev. Phytopathology 28, 451-474) and may prove to be a generic approach for creating resistance to RNA viruses.

In addition, Golemboski et al. (1990, PNAS 87, 6311-6315) have reported that tobacco plants transformed with part of the tobacco mosaic virus (TMV) genome encoding a puta ive component of the replicase complex were resistant to infection with closely-related strains of the same virus.
Thus it may be that transgenic plants expressing other virus non-structural protein sequences might also exhibit increased resistance to that particular virus or closely-related strains.

Furthermore, it has been found that it is not even necessary in all cases for the viral genes transformed into the host plant to be translated in order for the transgenic host plant to become resistant. In fact, simple transcription of the viral sequences can suffice to confer resistance. This has been demonstrated for transgenic tobacco plants which have been made resistant to tomato spotted wilt virus (TSWV: de Haan et al., 1992 Bio/Technology 10, 1133-1137) and to the potyvirus, W093J22345 21 3 4 7 8 4 PCT/GB93/0091~;

tobacco etch virus ~TEV; Lindbo and Dougherty, 1992 Virology 189, 725-733). This phenomenon of RNA-mediated resistance induced by the transcription of viral genes ~without translation) has been described for another potyvirus: potato, virus Y ~PVY; van der Vlugt et al; 1992 ~ !
Plant Molecular ~iology 20, 631-639).

Summary of the Invention In one aspect the invention provides an amino acid sequence comprising the amino acid sequence of residues 1-123 (Seq. ID No. 1) of Figure 4 or functional equivalents thereof.

This sequence is the C-terminus of a BaMMV polypeptide which is analogous to the putative replicase component of TMV. Thus the sequence may protect plants in a similar manner to that described by Golemboski et al. (1990).

In one aspect the invention provides an amino acid sequence comprising the amino acid sequence of residues -~ 124-374 (Seq. ID No. 2) of Figure 4 or functional equivalents thereof.

This sequence encompasses the coat polypeptide of BaMMV.
As is known from the prior art (e.g. Powell-Abel et al., 1986), expression of viral coat proteins by plants can increase the resistance of those plants to disease caused by, that virus.

In one embodiment the invention provides an amino acid sequence comprising the amino acid sequence of residues 1- "
374 of Figure 4 (Seq. ID No. 3) or functional equivalents thereof.

W093~22345 213 ~ 7 8 ~ PCT/GB93tO0910 In another aspect the invention provides a nucleotide sequence which comprises a sequence encoding the amino acid sequence of amino acids 124-374 of Figure 4 or functional equivalents thereof.

It is a preferred feature that the nucleotide sequences encoding the amino acid se~uenc~ of amino acids 124-374 of Figure 4 comprise an in-frame ATG translation start codon and an appropriate initiation consensus sequence at the 5' end of the sequence.

It will be apparent~ however, to those skilled in the art that translation signals (such as an ATG start codon) are not essential for~ nucleotide sequences of t.~ invention to possess utility. As explained previously, transcription of viral sequences can be sufficient to confer resistance on a plant containing those sequences: translation need not be essential~

In one embodiment, the nucleotide sequence of the invention comprises the nucleotide sequence of nucleotides ;r' 370-1122 of the sequence of Figure 4~(Seq. ID No. 4).

In a preferred embodiment, the nucleotide sequence of the invention further comprises the sequence of nucleotides 1123-1462 (Seq. ID No. 5) of the nucleotide sequence of Figure 4 or functional equivalents thereof.

In a preferred embodiment therefore, the invention provides the sequence of nucleotides 370-1462 (Seq. ID No.
6) of the nucleotide sequence of Figure 4 or functional equivalents thereof.

~ `

W~93/22345 PCT/GB93/009ln 213 17g;4 In still another aspect, the invention provides a nucleotide sequence comprising a sequence encoding the amino acid sequence of amino acids 1-123 of Figure 4, or functional equivalents thereof.

Preferably the nucleotide sequence in accordance with this aspect of the invention comprises the sequence of nucleotides 1-369 (Seq. ID No. 7) of Figure 4.

It is a further preferred feature that the nucleotide sequences of the invention comprise a 3' poly A tail.
-In another aspect the invention provides a vectorcomprising one or more of the nucleotide sequences of the invention.

Preferably the vector defined above includes a promoter suitable for the intended host and therefore should be capable of expressing a polypeptide encoded by the nucleotide sequence(s) of the invention.

Use of the term "functional equivalents" is intended to mean those nucleotide (RNA and DNA) and amino acid sequences which are essentially similar to the sequence shown in Figure 4 but which contain trivial sequence variations which do not significantly àlter the properties of the protein which they encode or represent. Examples of such functional equivalents are demonstrated in example 1 (and in Figure 7), where several clones obtained by the inventors contained nucleotide sequence differences, which may result in expected amino acid sequence differences.
In particular, the term functional equivalents is intended to encompass sequences which hybridise, under standard conditions, to the complement of the nucleotide sequence - W 0 93~22345 2 1 3 ~ 7 8 'i P ~ /G~93/00910 shown in Figure 4. Typically, such functional equivalents would be expected to show at least 80% nucleotide sequence homology with the sequence shown in Figure 4, and preferably at least 90%. Other functional eauivalents are deletion mutants and truncated forms of the nucleotide and amino acid sequences of the invention, which may be readily prepared by the skilled worker on the basis of the teaching disclosed herein. Such truncated forms have been demonstrated to possess protective activity (e.g. Lindbo &
Dougherty 1992, Molecular Plant-Microbe Interactions 5, 144-153).

-In another aspect the invention provides a plant or partthereof into which a nucleotide sequence of the invention has been artificially introduced.

It has already been shown by other researchers that transgenic plants containing viral sequences (from a particular virus) which are transcribed only, or transcribed and then translated, are rendered more resistant to disease caused by that particular virus. One of reasonable skill in the art would therefore expect that plants transcribing or translating the sequence of the invention would be more resistant to disease caused by barley mild mosaic virus. The increase in resistance may be exhibited as total immunity to the virus or as the delayed onset of symptoms, or any stage between those two extremes te.g. reduced severity of symptoms~.

Thus in another aspect the invention provides a method of rendering plants more resistant to disease caused by barley mild mosaic virus comprising transforming a host W093/22345 ~13 4 7 8 4 PCT/GB93/00910;

plant or part thereof with a vector comprising a nucleotide sequence of the invention, such that the sequence of the invention is transcribed within the host plant.

In one embodiment, the nucleotide sequence of the invention may be operably linked to a promoter sequence recognised by that plant, such that the plant translates the sequence.

In a further aspect, the in~ention provides a transgenic plant genetically engineered so as to have increased resistance to disease caused by barley mild mosaic virus.

In a particular embodiment therefore, the invention provides a method of rendering plants more resistant to disease caused by barley mild mosaic virus comprising transforming a host plant or part thereof with a vector comprising a nucleotide sequence encoding the amino acid sequence of amino acids 124-374 of Figure 4.

Preferably the vector comprises the nucleotide sequence of nucleotides 370-1122 of Figure 4.

Other research has shown that host plant expression of non-structural viral protein sequences may also reduce the severity of symptoms exhibited by virus-infected plants (Golemboski et al., 1990).

Thus in another embodiment the invention provides a method of rendering plants more resistant to disease caused by barley mild mosaic virus comprising transforming a host plant or part thereof with a vector comprising a nucleotide sequence encoding the amino acid sequence of .W093/22345 213 ~ 7 8 '1 PCT/GB93/00910 g _ ~
amino acids 1-123 of Figure 4.

Preferably the vector comprises the sequence of nucleotides 1-369 of Figure 4. .

In a particular embodiment the invention provides a transgenic plant, into which has been artificially introduced a nucleotide sequence encoding the amino acid sequence of Figure 4 or functional equivalents thereof, with increased resistance to disease caused by barley mild mosaic virus.

The lnvention may be better understood by reference to the following illustrative examples and drawings, in which:
.
Figure 1 shows a photograph of SDS-PAGE analysis of BaMMV
coat protein polypeptides from purified virus particles;

Figure 2 shows a photograph of Northern Blot analysis of RNA extracted from purified BaMMV virus particles probed with radiolabelled oligonucleotide probes; ,~

Figure 3 is a schematic representation of the sequencing strategy used to determine the DNA sequence of the BaMMV
coat protein;

Figure 4 shows the nucleotide sequence of the 3' terminal 1,462 nucleotides of BaMMV RNAl, and poly (A) tail;

Figure 5 shows the differences between the predicted amino acid sequence of the 3' terminal of BaMMV and BaYMV RNAl;

~igure 6 shows a comparison of the predicted amino acid sequence of the coat proteins of BaMMV, BaYMV, PVY and W093/22345 PCT/GB93/00gl0 PPV; and Figure 7 shows the sequence variations detected in the partially sequenced CDNA clones. t ExamPle 1 The Streatley strain of BaMMV was originally obtained from Dr M J Adams ~Rothamsted, UK) and was propagated in Hordeum vul~are cultivar Maris Otter by mechanical inoculation using an artist's airbrush (Adams et al., 1986 Annals of Applied Biology 109, 561~572). Virus was purified as described by ~uth et al., (lg84) but with the addition of lmM phenyl methyl sulphonyl fluoride to prevent degradation. BaMMV RNA was isolated from the purified virus by incubation in 0.1mg/ml proteinase K, 0.1% SDS for 20 minutes at room temperature followed by phenol-chloroform extraction and ethanol precipitation.

A virus preparation, purified by the method described above, containing 120 ug protein, was dried on a Univap concentrator and resuspended in 100ul of 70% formic acid containing 2.5mg/ml cyanogen bromide. This was then incubated at room temperature for 16 hours in the dark with continuous stirring. The cyanogen bromide-cleaved peptides were separated either by blotting onto a PVDF
membrane (Matsudaira, 1987 Journal of Biological Chemistry 262, 10,035-10,038) or by HPLC using a Brownlee Aquapore C8 RP300 cartridge column (2.1 x 30mm). Samples were diluted 10X with distilled water prior to loading onto the HPLC column previously equilibrated in 0.1%
trifluoroacetic acid (TFA). Samples were eluted with a 0-70% linear gradient of 90% acetonitrile/0.085% TFA in water over 70 minutes. Selected peaks were subjected to W093t2234~ 213 4 7 ~ 4 PCT/GB93/00910 further chromatography over the same column eluted with a linear gradient of 90% acetonitrile/0.1% rFA as before but the slope of the gradient was adjusted to be 3 times more shallow over the part of the gradient from which the peak had been eluted i~ the first run. N-terminal protein sequencing was performed on an Applied Biosystems model 470 protein sequencer. SDS PAGE was performed according to Laemmli (1970 Nature 227, 680-685).

SDS polyacrylamide gel electrophoresis of purified BaMMV
revealed three polypeptide bands of 32.5kDa, 26kDa and 25kDa. The resulting Coomassie Blue - stained gel is shown in Figure 1 (molecular weight markers, Mr, are shown in kiloDaltons). The proportion of these bands differed between virus preparations. Tn some experiments all three bands were present in roughly equal proportions but in others the upper band was predominant. All three bands cross-reacted with antibodies affinity-purified to the 32.5 kDa band. Together the data suggest that the 32.5kDa band is the major coat protein polypeptide and that the two minor bands are likely to be degradation products.

Attempts to sequence the N-terminus of the three coat protein bands were unsuccessful and, therefore, we chose to use cyanogen bromide cleavage. The peptide digestion products were separated by gel analysis and PVDF blotting or by HPLC. A total of three peptide sequences were obtained (see Figure 4) and two areas of limited redundancy determined from these sequences were selected for the synthesis of oligonucleotide probes for the coat protein gene.

Since BaMMV consists of two RNA components of approximately 8kb and 4kb (Usugi et al., 1989, Annals W O 93~22345 213 ~ 7 8 4 PC~r/GB93/00910' Phytopathology Society Japan 55, 26-31; Batista et al., 1989), the location of the coat protein gene was investigated by Northern blotting and probing with ~radioactively-labelled coat protein oligonucleotides CP-l, and CP-2 (Figure 2).

Total BaMMV RNA prepared as described previously, was separated on formaldehyde agarose gels and transferred to Hybond N nylon filters (Amersham) according to the manufacturer's instructions. The low redundancy 14mer oligonucleotides CP-l and CP-2 (5'-GGRTCXGGYTCYTC-3' and 5'-GTRAAYTGRTCXGG-3', Seq. ID Nos. 8 and 9 respectively, where R=A or G, X= A,C,G or T and Y=C or T) were synthesised to be complementary to BaMMV RNA in regions of the coat protein where the amino acid sequence had been determined. CP-l and CP-2 were radioactively labelled with 32p using T4 polynucleotide kinase and blots were probed at 5C below the predicted Tm as described by Sambrook et al. (1990, Molecular Cloning, a laboratory manual, 2nd edition, Cold Spring Harbor Press, USA).

Both oligonucleotides hybridised predominantly with RNA 1, although there was a slight cross-reaction of CP-2 with RNA 2 (Figure 2: The position of RNA standards (Mr) is shown, with their size in kilobases). This suggests that the coat protein is encoded on the longer of the two RNA
components. It is likely that the cross-reaction of CP-2 with RNA2 is due to conditions of low stringency since hybridisation to the RNA markers was also observed.

To clone the gene for the BaMMV coat protein CDNA clones were syntnesised and probed with CP-l and CP-2.

Briefly, oligo dT primed CDNA was synthesised from total W093/22345 2 1 3 4 7 ~ 4 PCT/CB93/OO9lo BaMMV RNA using the c~NA Synthesis System Plus (Amersham International) following the instructions provided, except that first strand synthesis was performed using Moloney murine leukemia virus RNAse H , reverse transcriptase, and buffer from GIBCO 'BRL . CDNA synthesis was monitored by incorporation of 32p alpha CTP into both first and second strands. EcoRI linkers were ligated to the cDNA following EcoRI restriction site methylation protection (Sambrook et al, 1990). CDNA was fractionated on a sucrose gradient preformed by freezing-thawing tubes containing 20% w/v sucrose in O.lM NaCl, 20mM Tris. HCl pH 8; lOmM EDTA and centrifugation was at 35,000rpm in a Sorvall AH650 rotor for 16 hours at 20C. Aliquots of fractions were separated on a 1% agarose gel, dried and exposed to autoradiographic film. Fractions containing fragments of greater than 500bp were pooled, ligated into EcoRI-digested pUC13 and transformed into E. coli DH5 alpha cells (GIBCO BPL). Plasmid DNA from ampicillin-resistant colonies was prepared by the method of Holmes and Quigley (1981 Anal. Biochem. 114, 193-197) and screened by restriction mapping. Gels were also Sou~hern blotted and probed with CP-l and CP-2.

Four overlapping cDNA clones (p~M 217, -55, -249 and -322) containing sequences homologous to the coat protein gene were identified using CP-l and CP-2 as probes, as shown in Figure 3: the region containing the open reading frame is indicated by the shaded box and the putative cleavage site is shown by an arrow. All four clones were fully sequenced in both strands. Regions of partial single -~
stranded sequence data were also obtained from a further eight cDNA clones tFigure 7) such that all regions of the 3' 1462 nts of RNAl were covered by at least two independent clones. Recombinant clones containing the - 14 ~
coat protein gene were sequenced using the dideoxy method of Sanger et al., ~1~77 PNAS 74, 5463-5467) with the Sequenase Kit from United States Biochemicals. A
combination of exonuclease III deletions (Sambrook et al., 1990), subcloning'and custom made oligonucleotide primers were used to obtain full length sequence from both strands of clones pBM-217, -55, -249 and -322. ~NA sequence was compiled and analysed using the DNASTAR Inc. computer package. Protein sequences were compared using version 1.68 of the Amino Acid Align (AANW) program with a gap penalty of 2 and a deletion penalty of 12.
-The sequence of the 3' terminal 1,462 nucleotides of BaMMVRNA 1 is shown in Figure 4 together with the amino acid sequence for which it encodes. The arrow indicates the presumed site of cleavage resulting in the production of the mature coat protein. The peptide sequences obta;ned by direct sequencing of purified virions are underlined.
A third sequence (LGF TVPID, Seq. ID No. 10) could not be located within any possible ORFs present in this sequence and might be derived from host components which co-purified with the virus preparation. TwO amino acid sequence motifs which are conserved among potyviruses are boxed. A number of sequence variations were detected, especially in those clones which were partially sequenced (e.g~ pBM 616). The sequencing strategy employed is shown in Figures 3 and 7. Four clones (pBM 217, -322, -55 and -249) were sequenced completely. In addition, eight clones (pBM 627, -621, -609 , -616, -604, -608, -614 and -624) were partially sequenced. The direction and length of .
sequence data obtained from these latter clones is shown by arrows. The nucleotides that differed from the consensus sequence (shown in Figure 4) are indicàted.

Computer analysis revealed one large putative ORF
consisting of 1122nts in the (+) strand virion polarity terminated by a stop codon located 340nts upstream of a polyA tail. T~e two peptide sequences from which the oligonucleotide c~at protein probes had been derived were identified within the large ORF as indicated (Figure 4).
The peptide beginning with the amino acid sequence AGHEEPDP, (Seq. ID No. 11) is likely to be at the N-terminus of the coat protein since it does nat follow a methionine residue which usually precedes a cyanogen bromide cleava~e site (~llen, 1981). In addition, there is a putative LQ/A cleavage site which is characteristic of several potyvirus cleavage sites as well as BaYMV
polyprotein cleavage (Kashiwazaki et al, 1989b Journal of General Virology 70, 3015-3023, Shukla et al., 1991 Canadian Journal of Plant Pathology 13, 178-191).

The predicted molecular mass of the BaMMV coat protein, calculated from the DNA sequence, is 28.5Kdal which is less than that estimated from SDS PAGE (Figure 1). This may reflect either aberrant mobility or post-translational modification of the coat protein.

To assess the relationship of the BaMMV coat protein sequence to other viral coat proteins, computer-aided searches were made using the predicted amino acid sequence. The highest level of homology (36~ identity overall) was to the coat protein of BaYMV and the homology was lowest at the N-terminus (Figures 5 and 6a).

Figure 5 shows an alignment of the predicted amino acid sequences at the 3' end of RNA 1 of BaMMV and BaYMV. The top line shows the sequence of BaMMV RNAl, the bottom line shows the sequence of BaYMV RNAl and the middle line shows W093/2234~ 2 1 3 ~17 ~ 4 PCT/GB93/00910;

the areas of homology. Gaps (-) have been introduced to maximise the homology.

Figure 6 shows a dot plot comparison of the predicted ~-amino acid sequence of the coat protein of; a) BaMMV
(BMCP.PRO) and BaYMV (BYCP. PRO?, b) BaMMV and potato virus Y (PVY), (PVYCP.PRO) and c) BaMMV and plum pox virus (PPV), (PPVCP.PRO).

Lower levels of homology (20-22% identity) were found to potyviruses such as PVY and PPV and, again, the homology was weakest at the N-terminus (Figure 6b and 6c). The lack of conservation of the N-terminus of the coat protein of potyviruses has been noted previously (Shukla and Ward, 1988 Archives of Virology 106, 171-200). The sequence motifs NGTS and FDF, known to be conserved within the potyvirus group and between potyviruses and BaYMV
(Kashiwazaki et al, 1989b; Timmerman et al., 1990, Journal of General Virology 71, 1869-1872 Niblett et al., 1991 Journal of General Virology 72, 499-S04), were also , . ~
tl~ conserved between BaMMV and potyviruses (Figure 4).

Computer calculations of the hydropathy profile of the ~- BaMMV coat protein revealed that the N-terminal 60 amino acids and the C-terminal 25 amino acids were hydrophilic in nature. This suggests a similar surface location of ~-~ corresponding regions in potyviruses, BaYMV and BaMMV
(Kashiwazaki et al., 1989b; Shukla et al, 1988 Journal of General Virology 69, 1497-1508).
,; ' i We observed approximately 55% homology between the ,~ predicted amino acid sequence of the BaMMV ORF upstream of the coat protein ~from BaMMV amino acids 1 to 123) and the putative NIb product of BaYMV which is also located ;
:: .

213~784 W093/2234~ PCT/GB93/00910 immediately upstream of the coat protein gene and has homology to RNA dependent RNA polymerases of positive strand RNA viruses (Kashiwazaki et al., 1990 Journal of General Virology 71, 2781-2790). In view of the results of Golemboski et al. (1990 , expression of this sequence (independently from expression of the coat protein) by transgenic plants may increase the resistance of the plant against BaMMV. However, we could detect no significant homology between ~aMMV and BaYMV in the 3' untranslated region. The 3' untranslated region of BaMMV (340nts) is longer t~an that of BaYMV (231nts) and contains a direct repeat of approximately 120nts with short palindromic sequences. Repeats within the 3' untranslated region have been observed in other potyviruses (Dougherty et al , 19~5, Virology 146, 282-291; Hay et al, 1989 Archives of Virology 107, 11-122). In addition, there is a potential polyadenylation motif (UAUGU) 85nts upstream from the poly ~A) tail (Figure 4) which has also been noted in the sequence of potyviruses (Maiss et al, 1989 Journal of General Virology 70, 513-524).

Example 2 Modification of BaMMV Coat Protein Gene for Expression in Plants.
.
Since the coat protein of BaMMV is formed by proteolytic cleavage of a larger precursor, it was necessary to provide a translational start codon in front of the codon coding for the N terminal amino acid. This was achieved using a PCR based technique as outlined below:

A forward primer (Bam 3, Seq. ID No. 12), was designed to contain a BamHI site, a plant initiation consensus W093~Z2345 PCT/GB93/00910`

sequence (Lutcke et al., 1987 EMBO Journal 6, 43-48) t an ATG start codon and sequences homologous to the 5' end of the coat protein gene:

BamHI
Bam 3: 5'-CGC GGATCC AACA ATG GCA GGG CAT GAG GAA CCA-3' A reverse primer (BMCP-l, Seq. ID No. 13) was designed with homology to the BaMMV coat protein between nts 842-~58.

BMCP-l: 5' -GGAATAACAGCGGAAGA-3' PCR was performed using Bam 3 and BMCP-l on cDNA and the products cloned as a BamHI-BqlII (using the BqlII site in coat protein at nt 821) fragment into the BamHI site of Bluescript (Stratagene).

Clones were then sequenced in full to verify addition of the extra sequences and to confirm that PCR had not introduced any base changes. The co~t protein region of the resulting clone (pMP-5) was found to be identical in sequence to ~he same region of pBM-217 and the extra cases at the 5' end had been correctly added.

Clone pBM-217 (containing the full-length coat protein) was digested with MluI (nt 637) and SalI (using the SalI
site present in the polylinker of pUC 13 at the 3' end of the coat protein gene) and the fragment containing the 3' region (nt637-1462) of the coat protein gene t~us released was gel-purified.

Clone pMP-5 was also digested with MluI and SalI
(polylinker) and the large fragment (containing plasmid -~- W093/22345 ~13 4 7 8 l1 PCT/GB93/00910 sequences and the 5' end of the coat protein) was gel-purified. This was ligated with the MluI-SalI fragment from the step described above to re-form the full-length coat protein with a modified 5' end.
. , .
Many other strategies, in addition to those descrlbed above, have been developed to provide resistance to viral pathogens (reviewed by Gadani et al., 1990 Archives of Virology 115, 1-21). These strategies are also based on using the genetic information of the virus pathogen or associated elements to give protection against the particular virus or related viruses.

We suggest that an alternative strategy, which also uses vectors containing viral sequences, might be developed such that protection is provided against a broad range of pathogens, including fungi and nematodes. The strategy is based on cultivar resistance. Cultivar resistance occurs within a host species when some cultivars, but not others, are resistant to infection with certain strains of a virus. The resistance is generally expressed as a hypersensitive response (HR) which results in the formation of a local lesion and pathogen localisation.
This response is known to be a general response and can be induced by fungi, nematodes and bacteria and viruses (reviewed by Bowles, 1990 Ann. Rev. Biochem 59, &73-907).
If a host species that contains a resistance gene to a given viral pathogen is transformed with a DNA clone of that virus (or parts of the virus) under the control of an inducible promoter, it is possible that a localised ~R
could be produced on induction of that pxomoter. The promoter can be chosen such that the virus is only expressed at the site of infection of an invading pathogen. The expression of the virus should trigger an PCr/GBg3/0091~/00 WO 93t2234~ 2 1 3 4 7 8 ~

HR and limit the spread of both vi rus and the invading pathogen .

Claims (15)

Claims

1. An amino acid sequence comprising the amino acid sequence of residues 124-374 of Figure 4 or functional equivalents thereof.

2. An amino acid sequence comprising the amino acid sequence of residues 1-123 of Figure 4 or functional equivalents thereof.

3. An amino acid sequence comprising the amino acid sequence of residues 1-374 of figure 4 or functional equivalents thereof.

4. A nucleotide sequence encoding the amino acid sequence of any one of claims 1-3 or functional equivalents thereof.

5. A nucleotide sequence according to claim 4, further comprising an in-frame ATG translation start codon and an appropriate initiation consensus sequence at the 5' end of the sequence.

6. A nucleotide sequence according to claim 4 or 5, further comprising a 3' polyA tail.

7. A nucleotide sequence according to any one or claims 4, 5 or 6, comprising the sequence of Figure 4.

8. A nucleotide sequence according to any one of claims 4, 5 or 6, comprising the sequence of nucleotides 1-369 of Figure 4.

9. A nucleotide sequence according to any one of claims 4, 5 or 6, comprising the sequence of nucleotides 370-1462 of Figure 4.

10. A vector comprising the nucleotide sequence of any one of claims 4-9.

11. A vector according to claim 10, capable of expressing a polypeptide having the amino acid sequence of Figure 4 or functional equivalents thereof.

12. A method of rendering plants more resistant to disease caused by barley mild mosaic virus, comprising transforming a host plant or part thereof with a vector comprising the sequence of any one of claims 4-9 operably linked to a promoter sequence recognized by said host plant, such that the transformed host produces an RNA
transcript of the sequence of any one of claims 4-9.

13. A method according to claim 12, wherein at least part of said RNA transcript is translated into an amino acid sequence in the host plant.

14. A transgenic plant genetically engineered so as to have increased resistance to disease caused by barley mild mosaic virus.

15. A transgenic plant according to claim 14, produced by the method of claim 12 or 13.