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WO1999058697A1 - Plantes transgeniques resistantes aux maladies - Google Patents

Plantes transgeniques resistantes aux maladies Download PDF

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Publication number
WO1999058697A1
WO1999058697A1 PCT/SG1999/000012 SG9900012W WO9958697A1 WO 1999058697 A1 WO1999058697 A1 WO 1999058697A1 SG 9900012 W SG9900012 W SG 9900012W WO 9958697 A1 WO9958697 A1 WO 9958697A1
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Prior art keywords
gene
plant
cucumovirus
tmv
transgenic plant
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PCT/SG1999/000012
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English (en)
Inventor
Shou-Wei Ding
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Institute Of Molecular Agrobiology
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Priority claimed from PCT/SG1998/000035 external-priority patent/WO1999058696A1/fr
Application filed by Institute Of Molecular Agrobiology filed Critical Institute Of Molecular Agrobiology
Priority to AU32845/99A priority Critical patent/AU3284599A/en
Priority to EP99950356A priority patent/EP1078090A1/fr
Publication of WO1999058697A1 publication Critical patent/WO1999058697A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance

Definitions

  • the present invention relates to methods and materials for conferring disease resistance to plants. More particularly, the invention relates to transgenic plants containing a heterologous nucleic acid which confers disease resistance, particularly against infectious pathogens, such as viruses. The invention further relates to methods and materials for preparation of such transgenic plants.
  • Infectious diseases of cultivated plants cause substantial reductions in food, forage and fiber throughout the world. Control of these diseases has been based primarily on cultural practices that include removal of infected debris, eradication of weed hosts (herbicide applications), prevention of vector transmission (pesticide applications) , indexing for pathogen-free starting material (seed or vegetative propagules) and breeding for disease resistance. Large scale methods for curing plants once they have become infected with viruses do not exist. Thus, the control of diseases is dependent upon methods to prevent or delay the establishment of infection. 2
  • transgenic plants have proven to be a valuable strategy for protecting plants from viral diseases.
  • Stubbs, G. et al.. United States patent 5,723,750 describe transgenic plants expressing genes encoding wild-type and modified coat proteins of different virus groups. These transgenic plants have been shown to have varying levels of resistance to infection by the corresponding virus.
  • the expression of heterologous genes encoding coat proteins does not confer broad resistance to viral infections and has no effect on pathogenesis caused by other infectious agents.
  • Gene-for-gene disease resistance is an important plant defense mechanism against pathogens. It is induced only when the host plant carrying a resistance (R) gene is challenged by a pathogen carrying a matching avirulence (Avr) gene (Keen, 1990) . (A bibliography is provided at the end of the written description.)
  • R resistance
  • Avr avirulence
  • the specific interaction between a matching pair of R-Avr genes usually induces the hypersensitive response ("HR") and results in the local containment of the invading pathogen at sites of attempted ingress.
  • HR hypersensitive response
  • the recognition of a pathogen induces a rapid cell death process that results in the formation of a zone of dead cells (necrosis) around the site of- infection.
  • SAR systemic acquired resistance
  • PR pathogenesis-related proteins
  • SA salicylic acid
  • Lam, E. et al., U.S. patent 5,629,470 have described a process for providing higher plants with enhanced resistance to pathogenic attack by one or more plant pathogens by transforming cells of the plants with the bacterio-opsin (bO) gene.
  • R genes specific for bacteria, viruses, fungi or nematodes have been cloned from a variety of plant species and it is striking to note that these R genes often encode structurally similar proteins, suggesting a high degree of mechanistic conservation among the pathways that plants use to trigger defense responses (Baker et al., 1997; Hammond-Kosack and Jones, 1997) .
  • the Avr genes of pathogens by definition encode or produce signal molecules that can initiate HR in the corresponding resistant plants.
  • Avr and A vr9 from the extracellular growing fungal pathogen Cladosporium sulvum encode pre-proproteins that are processed into small secreted peptides that can elicit R gene-dependent defense responses even in the absence of pathogens (Knogge, 1996) .
  • the bacterial cells encode or produce signal molecules that can initiate HR in the corresponding resistant plants.
  • Avr and A vr9 from the extracellular growing fungal pathogen Cladosporium sulvum encode pre-proproteins that are processed into small secreted peptides that can elicit R gene-dependent defense responses even in the absence of pathogens (Knogge, 1996) .
  • the bacterial pathogens encode or produce signal molecules that can initiate HR in the corresponding resistant plants.
  • AvrD locus of Pseudomonas syrlngae pv. glycinea encodes enzymes involved in the synthesis of exported ⁇ yringolide elicitors which, when injected into the intercellular leaf spaces, can elicit HR in soybean cultivar ⁇ that carry the Rpg4 gene (Leach and White, 1996) .
  • the Avr protein itself is the elicitor (Leach and White, 1996) .
  • These Avr genes generally encode hydrophilic proteins that lack signal sequences and do not induce HR when injected into leaves of plants with the matching R genes.
  • the 126K replicase protein (Padgett et al., 1997) of tobacco mosaic virus (TMV) is the only viral Avr protein for which the cloning of a matching R gene, N, has been reported (Whitha et al., 1994).
  • the N gene product predicted as a cytoplasmic protein, belongs to the NBS-LRR family of resistance genes (Baker et al., 1997) . It has been reported recently that systemic leaves of nepovirus- and caulimovirus-infected plants exhibit a strong virus resistance similar to poet-transcriptional gene silencing ("PTGS”), thus providing evidence for a second type of natural defense mechanism against viruses in plants (Covey et al.,
  • PTGS was first discovered in transgenic plants and involves the degradation of RNA species that are similar to the transcribed part of a silencer transgene. As a result, only a low level of the transgene RNA exists even if it is transcribed at a high level (Depicker and Van Montagu, 1997) .
  • PTGS confers virus resistance in transgenic plants if the silencer transgene is derived from or shares sequence homology with the genome of a plant virus (Lindbo et al., 1993; Guo and Garcia, 1997; Ruiz et al., 1998).
  • transgenic plants that have been stably transformed with the 2b gene of cucu ovirus or an active fragment thereof have been found to possess systemic resistance to pathogenic infectious agents, such as viruses.
  • the protein encoded by this gene activates strong disease resistance responses in host plants.
  • a further aspect of the invention relates to seeds and propagating parts of transgenic plants stably transformed with a cucumovirus 2b gene or an active fragment thereof.
  • the invention further provides methods and vectors for introducing the cucumovirus 2b gene into plants.
  • a further aspect of the invention relates to a plant RNA virus-encoded suppressor of PTGS, which is targeted as an Avr factor by a strong host resistance mechanism resembling the gene-for-gene disease resistance.
  • a molecular dissection of the suppressor indicates a two-domain structure in which one domain is sufficient for resistance activation, while the other was required for triggering hypersensitive cell death.
  • Disarmed Avr genes can be obtained by such means as domain swapping to link an active resistance domain to an inactive cell death domain, or selective deactivation of an Avr cell death domain.
  • a single such disarmed Avr gene can confer to a transgenic plant broad-spectrum pathogen resistance.
  • Figure 1 illustrates the structural features and genome organization of the chimeric viral RNA transcripts from plasmids pTMV-t2b and pPVX-t2b.
  • Figure 2 is a Northern blot hybridization showing the effect of the 2b gene on expression of the pathogenesis-related protein in leaves of tobacco plants infected with tobacco mosaic virus carrying the 2b gene.
  • Figure 3 shows the two-domain structure of Tav2b, as revealed by domain mapping.
  • the figure shows plant response to various 2b chimeras.
  • HR means observation of necrotic lesions and virus multiplication in the inoculated leaves of N. tobaccum cv. Samsun nn only
  • R means observation of virus multiplication in the inoculated leaves only, but with no visible nectrotic lesions
  • S means observation of susceptibility and systemic infection 8
  • FIG. 4 shows the genomic organization of the TMV-30B vector and the inserted fragments from either TAV or CMV. Boxes represent open reading frames (ORF) encoded by TMV, TAV or CMV. Asterisk (*) denotes the amber stop codon which can be read through to give the 1S3K protein.
  • the three sgRNA promoters are represented by arrows. Note that this vector is derived from the infectious cDNA clone of TMV-U except for the 3" -terminal sequence, including the coat protein (CP) coding sequence plus its sgRNA promoter (arrow 3), which is from TMV U5 (W.O. Dawson, pern. comm. ) .
  • T2b TAV
  • C2b CMV
  • FIG. 5 shows the accumulation of viral and plant RNAs in tobacco plants.
  • the Samsun plants were inoculated with TMV-30B (TMV) , TMV-C2b (C2b) , TMV-T ⁇ 2b2 (T ⁇ 2b2), TMV-T ⁇ 2bl, TMV-T2b (T2b or mock-inoculated with buffer alone (M) .
  • TMV TMV-30B
  • C2b TMV-C2b
  • T ⁇ 2b2 TMV-T ⁇ 2b2
  • TMV-T ⁇ 2bl TMV-T2b
  • T2b mock-inoculated with buffer alone
  • M buffer alone
  • dpi total RNAs were extracted from either the inoculated leaves or systemic leaves and subjected to Northern blot analysis using 3i P-labeled DNA probes specific for TMV genomic RNA (TMV) , PR-la mRNA or the 18 ' S RNA (as a loading control) .
  • Figure 6 shows the time course of the accumulation of mRNA encoded by PR-la and PR-5 in the Samsun plants inoculated with TMV-T2b. The time (dpi) when total RNAs were extracted after inoculation is indicated above each lane.
  • FIG 7 shows accumulation of the six TMV recombinants int he inoculated and systemic leaves of Samsun tobacco plants.
  • a probe for the genomic RNA of TMV was used for the hybridization.
  • the plant response (as abbreviated in Figure 3) to each of the recombinants is also indicated.
  • the 2b coding sequence in the progeny virus was obtained by RT-PCR followed by DNA sequencing and found to be identical to the respective inoculum.
  • M mock inoculated
  • Cucumber mosaic virus belongs to a virus genus called Cucumovirus which also includes tomato asper y virus (TAV) .
  • Cucumovirus contains a tripartite single-stranded RNA genome that encodes five genes: la, 2a, 2b, 3a and coat protein. The identification and functional analysis of the 2b gene have been described in previous publications. (Ding et al., 1994; 1995; 1996) . It has been demonstrated that the 2b gene encoded by the cucumoviruses is important for both systemic virus spread and virulence determination.
  • the ribonucleotide sequence of the 2b gene is provided in SEQ ID NO. 1.
  • the present invention relates to a transgenic plant stably transformed with a cucumovirus 2b gene or active fragment thereof operatively linked to a promoter that is capable of effecting expression of said gene in said plant when said plant is infected with a pathogenic organism.
  • the cucumovirus 2b gene used for producing disease-resistant plants 10 advantageously is one to which the nucleic acid of SEQ ID NO. 1 will hybridize under stringent conditions.
  • the present invention provides methods for rendering a plant resistant to disease caused by an infectious pathogenic agent, which comprises stably transforming the plant with a cucumovirus 2b gene or active fragment thereof operatively linked to a plant-active promoter that is capable of effecting expression of the gene in the plant when said plant is infected with a pathogenic organism.
  • the invention provides an expression vector comprising a cucumovirus 2b gene or active fragment thereof operably linked to a plant- active promoter. Mutational analysis has confirmed that the 2b gene is responsible for the resistance response. Point mutations in the gene have been shown to render it nonfunctional and abolish the ability of the gene to activate the resistance response.
  • the C-terminal 26-amino acid and 45- amino acid sequences of the gene are essential for its disease-resistance function, although the codons encoding the four C-terminal amino acids can be removed without losing activity.
  • Transfer of the codon ⁇ encoding the C-terminal 26 amino acids and the C- terminal 45 amino acids of the tomato aspermy virus 2b gene to the corresponding regions of the inactive cucumber mosaic virus 2b gene does not yield an active chimeric gene; therefore, the N-terminal portion of the protein also appears to contain one or more domains that are essential for resistance activation. Accordingly, the invention relates to transgenic plants and vectors that contain an active fragment of the 2b gene . 11
  • the 2b gene also has been found to be dual functional. Depending on the virus genetic background from which it is expressed, 2b can function as either a virulence or an avirulence determinant. These findings suggest that one may search among susceptible cultivars for R-like genes that can potentially recognize virulent proteins by in planta expression from heterologous viral vectors. It is likely that many Avr gene products are virulence factors until a matching R gene evolves in a host cultivar. These results show that the plant gene- or-gene resistance is also effective in targeting as an Avr a virus-encoded suppressor of the plant gene silencing defense mechanism.
  • both TMV-TCl and TMV-TC2 encoding a complete resistance domain, induced strong virus resistance in the challenged tobacco plants.
  • Complete (e.g., TMV-CT1, 2 and 3) or partial (e.g., TMV-TC3) replacements or deletion (e.g., TMV-T ⁇ 2bl) of this domain result in the loss of the activity in resistance 12 activation.
  • the second domain, encompassing amino acids 70-9 of Tav2b, is tentatively referred to as the "cell death" domain because its absence in TMV-TC2 leads to a loss of cell death induction, but without apparent effect on resistance activation.
  • the Cmv2b C-terminal 34 aa fused in TC2 may function as a positive or negative modulator of cell death initiation, leading to a local symptomless phenotype of TMV-TC2.
  • these two functional domains correspond to the overlapping and non-overlapping regions of the cucumoviral 2b genes, as defined previously according to whether or not it overlaps with the 2a gene (Ding et al., 1995). Distinct from the N-terminal resistance domain
  • the cell death domain is dependent on the presence of a functional resistance domain.
  • TMV-CT2 and TMV-CT3 encode a complete cell death domain, but fail to induce necrotic lesions in the inoculated leaves (Fig. 3) .
  • Fig. 3 Other evidence supporting this observation is that the two amino acid substitutions that rendered Tav2b inactive In triggering resistance and cell death are both located within the resistance domain, but outside of the cell death domain. It is interesting that the resistance domain is functionally independent in contrast to a conditional cell death domain.
  • cell death signaling which is usually placed downstream of the Avr-R interaction in the HR network (Hammond-Kosack and Jones, 1996; Lamb and Dixon, 1997), probably involves a subsequent interaction of the pathogen Avr product with another plant protein, possibly via its cell death domain.
  • the 2b gene or its active fragment may be introduced into plants using conventional vectors and procedures. Generally, such techniques involve inserting the gene into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequences and one or more marker sequences to facilitate selection of transformed cells or plants.
  • a number of plant-active promoters are known in the art and may be used to effect expression of the nucleic acid sequences disclosed herein.
  • Constitutive 14 promoters such as the nos promoter or the 35S promoter, of cauliflower mosaic virus, may be used; however, constitutive expression may be harmful to the transgenic plants.
  • inducible promoters especially pathogen-inducible promoters, such as pathogenesis-related protein promoters are preferred.
  • plant cell is intended to encompass any cell derived from a plant including undiff ⁇ rentiated tissues such as callus and suspension cultures, as well as plant seeds, pollen or plant embryos.
  • Plant tissues suitable transformation include leaf tissues, root tissues, meristems, protoplasts, hypocotyl ⁇ , cotyledons, scutellum, shoot apex, root, immature embryo, pollen, and anther.
  • One technique for transforming plants is by contacting tissue of such plants with an inoculum of a bacterium transformed with a vector comprising the gene in accordance with the present invention.
  • this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
  • Bacteria from the genus Agroja teriu/n can be utilized advantageously to transform plant cells. Suitable species of such bacteria include Agrobacterium tumefaclens and Agrobacterium rhlzogens. Agrobacterium tumefa cx&ns (e.g., strains • LBA4404 or EHA105) is particularly useful due to its well-known ability to transform plants.
  • Another approach to transforming plant cells with the nucleic acid of this invention involves propelling inert or biologically active particles into plant 15 cells.
  • This technique is disclosed in U.S. patents 4,945,050, 5,036,006 and 5,100,792 all to Sanford et. al., which are hereby incorporated by reference.
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector comprising the 2b gene.
  • Biologically active particles e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced
  • Biologically active particles can also be propelled into a plant cell tissue.
  • Another method of transforming plant cells is the electroporation method. This method involves mixing the protoplasts and the desired DNA and forming holes in the cell membranes by electric pulse so as to introduce the DNA into the cells, thereby transforming the cells.
  • This method currently has high reproducibility and various genes have been introduced into monocotyledons, especially rice plants by this method (Toriyama et. al., 1988, Shimamoto et al . , 1989 and Rhodes et al., 1988).
  • transgenic plants of the present invention may be used in preparing transgenic plants of the present invention.
  • explants, callus tissues or suspension cultures can be exposed to the appropriate chemical environment (e.g., cytokinin and auxin) so the newly grown cells can differentiate and give rise to embryos which then regenerate into roots and shoots.
  • the appropriate chemical environment e.g., cytokinin and auxin
  • genes of the present invention are useful in enhancing resistance to disease-causing pathogens in both monocotyledonous plants (“monocots”) and dicotyledonous plants (“dicots”), such as corn, wheat, rice, millet, oat, barley, sorghum, sunflower, sweet potato, alfalfa, sugar beet, brassica species, tomato, pepper, soybean, tobacco, melon, squash, potato, peanut, pea, cotton or cacao.
  • TMV tobacco mosaic virus
  • PVX potato virus X
  • TAV ORF 2b coding sequence (nucleotides 2447-2734 O f RNA2) from pQCD2qt (Ding et al., 1996) using the Pfu DNA polymerase (Stratagene) .
  • This sequence was inserted into the genome of TMV and PVX upstream of the respective coat protein (CP) gene.
  • the PCR fragment was blunt-end cloned at the Pmel site of a TMV vector, known as pTMV-30B, to yield TMV-t2b ( Figure 1) .
  • TAV insert in TMV-t2b was excised as an Agei - xhoi fragment (see Figure 1) , and this fragment was end- filled and cloned into Clal-digested and end-filled pPC2S (an expression vector based on potato virus X (Chapman et al., 1992)) to produce PVX-t2b.
  • the 2b gene expression was controlled by independent promoters (arrows labelled as 1 and 3 in Fig. 1) which are recognized only by the respective RNA-dependent RNA polymerase encoded by TMV or PVX.
  • TMV-t2b and PVX-t2b The 2b-expressing derivatives of TMV or PVX (TMV-t2b and PVX-t2b) were used to infect plants and the functional role of the 2b gene was inferred from differences in the induced plant responses between the wild type and its 2b-expresslng derivative.
  • TMV-t2b induced a typical hypersensitive response (HR) in Samsun (nn) tobacco plants.
  • Plasmids pTMV-30B, pPC2S and their derivatives were linearized and transcribed in vi tro as described (Chapman et al., 1992) in the presence of the cap analog (NEB) using the T7 RNA polymerase (Promega) .
  • the capped RNA transcripts were inoculated mechanically onto fully developed leaves of Nicotiana tabacum cv Samsun (nn) .
  • the plants were incubated in the Conviron growth chambers (24°C constant, 75% humidity and 16 hours light/8 hours dark) .
  • Northern blot hybridization was performed using PR-la cDNA as a probe (obtained by PCR amplification from tobacco plants based on the sequence disclosed by Cornelissen, B.J. et al. (1987)). Total RNAs extracted from plants 5 days
  • Lane 1 (lane 1), 7 days (lane 2), 10 days (lane 3), and 13 days (lane 4) showed increasing expression of PR-1.
  • Lanes 5 and 6 were infected with wild-type TMV; however, the tobacco genotype was nn for lane 5 and NN for lane 6. 7
  • N. tabacum Samsun (nn) tobacco contains no resistance gene specific to TMV, and this is confirmed in this study that when infected with the vector TMV-30B alone, the tobacco plants developed systemic mosaic symptoms and no induction of the PR genes was observed. Thus, it is concluded that the resistance responses of the tobacco plants to TMV-t2b challenge is due to the in cis expression of the TAV 2b gene from the TMV genome.
  • TMV-t ⁇ 2bl (SEQ ID NO. 2 ) is predicted not to translate any of the 2b protein in infected plants. In plants infected with TMV-t ⁇ 2b2 (SEQ ID NO. 3), however, a truncated 2b protein missing the C-terminal 52 amino acid residues is expected to be expressed.
  • TMV-t ⁇ 2bl nor TMV-t ⁇ 2b2 induced local necrotic lesions in the inoculated leaves and transcription of mRNA ⁇ for PR proteins was also not induced. Therefore, it is the TAV 2b protein that functions as the activator of resistance responses.
  • C-terminal 52 amino acid sequence of the TAV 2b protein is essential for this activity (see below) .
  • the 2b gene encoded by the Q strain of cucumber mosaic virus (CMV) (SEQ ID NO. 4) was similarly engineered to be expressed from the TMV genome.
  • TMV-q2b syste ically infected Samsun tobacco plants
  • the CMV 2b protein was inactive in resistance activation.
  • the TAV 2b protein as encoded by TMV-t2b was progressively replaced from the C-terminus by the structurally equivalent regions of the CMV 2b protein.
  • Both Nicotiana benthamiana and Physalis floridana plants are similar to the Samsun tobacco in that they are susceptible to TMV, and the infected plants do not develop HR. Infectivity assays showed that challenge inoculation with TMV-t2b induced typical local necrotic lesions in the inoculated leaves of both Nicotiana benthamiana and Physalis floridana plants, while the uninfected parts of the plants remained symptom-free. These results suggest that the TAV 2b gene is also capable of activating resistance in these plant species against TMV. The fact that the TAV 2b gene can activate resistance against TMV in three different plant species of two genera suggests that it will function similarly in a wide range of host species.
  • TAV 2b gene expression in cis from the PVX genome is also capable of triggering the resistance responses in tobacco plants containing no specific resistance gene to PVX.
  • TMV and PVX are distinct plant RNA viruses of different virus genera and the encoded proteins of the two viruses have minimal sequence similarities. Therefore, it is less likely that the resistance activation by the TAV 2b gene requires specific interaction with any of the proteins encoded by the two viral vectors. These results suggests a possibility that the TAV 2b gene will be able to activate resistance mechanisms against a wide range of plant pathogens.
  • TAV 2b gene which is expressed at high levels in infected plants (Shi et al., 1997). It has been shown previously that all three plant species used in this work are fully susceptible to TAV and CMV-qt (Ding et al., 1996), suggesting that these plant species do not contain a resistance gene that recognizes the TAV 2b gene. This result indicates that the resistance activation activity of the TAV 2b gene is not constitutive in these plant species and may require an induction event such as infection with certain vixulent pathogens (e.g., TMV and PVX). This property distinguishes the TAV 2b gene from the known avirulence genes encoded by plant pathogens. 23
  • DNA manipulations and cloning were carried out using standard procedures (Sambrook et al., 1989) unless otherwise described. DNA inserts in all constructs were sequenced in two orientations prior to use.
  • Plasmid pTMV-30B Plasmid pTMV-30B (Pig. 4, a gift from
  • TAV ORF 2b The coding sequence of TAV ORF 2b (nucleotides 2447-2734 of RNA2) was PCR amplified from pQCD2qt (Ding et al., 1996) using the Pfu DNA polymerase (Stratagene) and cloned at the Pmel site of pTMV-30B to yield pTMV-T2b.
  • the plasmid pTMV-T ⁇ 2bl was generated from pTMV-T2b by PCR mutagenesis as previously described (Ding et al., 1995) (incorporated herein by reference) .
  • the plasmid pTMV-T ⁇ 2b2 contained the same two nucleotide mutations as in pQCD2qt2 (Ding et al., 1996) that changed the second codon of ORF 2b to a stop codon.
  • CMV ORF 2b The coding sequence of CMV ORF 2b was obtained from pSK2b (Ding et al., 1994) as a BamHI-Asp718 fragment, which was end-filled and cloned into the Pmel site of pTMV-30B to give pTMV-C2b.
  • Plasmids pTMV-TCl, pTMV-TC2, pTMV-TC3, pT V-CTl, PTMV-CT2 and P TMV-CT3 The previously describe d mutagenesis protocol (Ding et al., 1996) (incorporated herein by reference) that involves three separa t e P CR s 24 was used with templates pQCD2(Ding et al., 1995) or pQCD2qt (Ding et al., 1996) to generate six chimaeric DNA fragments (Fig. 3). The final PCR fragments were cloned at the Pmel 3ite of pTMV-30B to give the six plasmid constructs listed.
  • Plasmid pTMV-T2bC Nucleotide substitutions, AA - -> GT (equivalent to nucleotides 2508-2509 of TAV RNA 2) and CGA --> TCT (nucleotides 2529-2531 of TAV RNA 2) were introduced by PCR into the coding sequence of Tav2b as encoded by pTMV-T2b. The resultant plasmid was called pTMV-T2bC.
  • Plasmids pPVX-T2b and pPVX-T ⁇ 2b2 The inserted TAV sequence in pTMV-T2b and pTMV 2T ⁇ 2b2 (Fig. 4) was obtained as an Agel-Xhol fragment and cloned in the PVX vector pP2C2S.
  • Plasmid pTMV-30B and its derivatives were linearized by Pstl whereas pPVX-T2b and pPVX-T ⁇ 2b2 were linearized by Spel before they were transcribed in vitro in the presence of cap analog using TRNA polymerase (New England Biolabs) .
  • N. tabacum cv. Samsun (nn) plants were grown in C ⁇ nvir ⁇ n growth chambers (22°C constant, 75% humidity and hours photoperiod) .
  • the youngest fully expanded leaves of the plants were dusted with carborundum and inoculated with the capped RNA transcripts (transcribed from l ⁇ g plasmid DNA template/leaf) .
  • leaves were excised and immediately frozen in liquid nitrogen before total plant RNAs were extracted and analyzed by Northern blot analysis as previously described (Ding et al., 1995).
  • RNA transcripts from pPVX-T2b or pPVX-T ⁇ 2b2 were inoculated to the youngest fully expanded leaves of the plants.
  • the following DNA fragments were labeled with ⁇ - 32 p-dCTP by random priming as described (Sambrook et al., 1989).
  • the Sphl-Stul fragment from pTMV-30B corresponds to nucleotides 445 to 1675 of the TMV genome (Goelet et al., 1982).
  • the probes specific for the mRNA of PR-la (Cornelissen et al., 1987), and PR- (Cornelissen et al., 1986), and for the 18S rRNA (Venkateswarlu and Nazar, 1991) were all obtained by PCR according to the published sequences and confirmed by sequencing.
  • For Northern analyses equal amounts of the total RNAs (Stg) were used for all samples and the probe specific for 18S rRNA was employed to monitor RNA loading.
  • the viral progeny RNAs were recovered from plants inoculated with each of the recombinant TMV transcripts and analyzed by RT-PCR and DNA sequencing.
  • the cucumoviral 2b coding sequences cloned at the Pmel site of pTMV-30B were first amplified by RT-PCR using a pair of primers flanking the Pmel site of pTMV-30B.
  • the amplified fragments were then purified from agar ⁇ se gels and either sequenced directly using the same pair of primers or cloned into a plasmid vector before sequencing.
  • Example 8 Induction of hvoersen i i va cell rie ⁇ h 26
  • N. tabacum cv. Samsun does not contain the N gene and is thus susceptible to infection by many TMV strains, including Ul and U5 (Mathews, 1991). Syste ically infected plants show characteristic leaf mosaic symptoms.
  • the vector pTMV-30B (Fig. 4) is similar to the TMV-based in planta expression vectors previously described (Donson et al., 1991; Kumagai et al., 1995), except that expression of the inserted foreign gene was driven by the 22 Ul coat protein (CP) subgenomic RNA (sgRNA) promoter (arrow 2, Fig. 4) whereas the CP gene and its sgRNA promoter (arrow 3, Fig. 1) were obtained from the U5 strain (W.O.
  • CP Ul coat protein
  • sgRNA subgenomic RNA
  • TMV-30B The recombinant viral RNA transcribed from pTMV-30B was named TMV-30B. This nomenclature system is used throughout the text. TMV-30B infected Samsun plants displayed a milder systemic mosaic than did Samsun plants infected with the Ul strain of TMV.
  • pTMV-T2b and pTMV-C2b were cloned downstream of the Ul sgRNA promoter of pTMV-30B to give pTMV-T2b and pTMV-C2b (Fig. 4), respectively.
  • Local necrotic lesions a morphological marker of the HR, appeared on the tobacco leaf inoculated with TMV-T2b about three days post-inoculation (dpi) whereas the rest of the plant remained symptomless for as long as observations were made (five weeks) .
  • TMV-C2b did not induce necrotic lesions on the inoculated leaf, and produced mosaic symptoms on the upper uninoculated systemic leaves. These latter symptoms were similar to those caused by infection with TMV-30B.
  • RNAs were extracted from the inoculated and systemic 27 leaves and analyzed by Northern blot hybridization using a probe specific to the genomic RNA of TMV (indicated on the right of Fig. 5) .
  • high • levels of accumulation of genomic RNAs were detected in both the inoculated leaves (top panel) and the systemic leaves (middle panel) of the plants that were inoculated with either TMV-30B (lane TMV) or TMV-C2b (lane C2b) .
  • TMV-T2b a much lower level of TMV-T2b accumulated in the inoculated leaves (lane T2b, top panel) and no accumulation was detected in the systemic leaves (lane T2b, middle panel) .
  • the presence of the coding sequence of Tav2b or Cmv2b in the progeny viral RNAs extracted from the inoculated leaves was confirmed using sequence-specific probes and by sequencing the cDNA fragments obtained from reverse transcription - polymerase chain reaction (RT-PCR) .
  • RT-PCR reverse transcription - polymerase chain reaction
  • the genomic RNAs of TMV-C2b and TMV-T2b migrated more slowly than the corresponding genomic RNA of TMV-30B during denaturing agarose gel electrophoresis (Fig.
  • RNAs extracted from the tobacco leaves at different time points following TMV-T2b inoculation were subjected to Northern blot analysis using probes specific for PR-la mRNA or PR-5 mRNA. As shown in Fig. 6, the synthesis of both PR-la (top panel) and PR-S 28
  • RNAs were detectable three dpi and reached maximums of steady-state RNA at five (PR-la) and seven (PR-5) dpi, respectively.
  • the timing of mRNA induction at three dpi coincided with the appearance of macroscopic necrotic lesions.
  • the transcription of PR-la mRNA (Fig. 5, lower panel) and PR-5 mRNA was not significantly induced in plants infected by TMV-30B (Fig. 5, lane TMV) or TMV-C2b (Fig. 5, lane C2b) even at ten dpi (or at any other earlier time points) .
  • TMV-30B Fig. 5, lane TMV
  • TMV-C2b Fig. 5, lane C2b
  • TMV-T2b also led to the transcriptional induction of PR-la and PR-5 genes.
  • the hypersensitive cell death, PR gene expression and strong virus resistance induced by Tav2b in the Samsun cultivar are the typical host responses associated with the gene-for-gene disease resistance mechanism.
  • Tav2b when expressed from the TMV genome, functions as an Avr gene.
  • Point mutations were introduced into the coding sequence of Tav2b in the plasmid pTMV-T2b to give pTMV-T ⁇ 2bl and ⁇ TMV-T ⁇ 2b2 (Fig. 4).
  • the recombinant virus TMV-T ⁇ 2b2 was not expected to yield any Tav2b product in infected plants because the second codon (GCA) was converted to a stop codon (TAA) .
  • GCAA codons 44
  • CGA stop codons
  • TMV-T ⁇ 2bl nor TMV-T ⁇ 2b2 induced the formation of necrotic lesions in the inoculated leaves and the inoculated plants developed systemic mosaic symptoms similar to those caused by TMV-C2b.
  • Both TMV-T ⁇ 2bl and TMV-T ⁇ 2b2 accumulated to detectable levels in the 29 inoculated leaves and systemic leaves, as revealed by Northern blot hybridization (Fig. 5, top and middle panels, lanes T ⁇ 2bl and T ⁇ 2b2) .
  • Progeny analysis by RT-PCR and sequencing showed that the introduced mutations in both TMV-T ⁇ 2bl and TMV-T ⁇ 2b2 were stably maintained and no second-site mutations were detected in the inserted TAV sequence.
  • TMV-T2b infection by TMV-T ⁇ 2bl or TMV-T ⁇ 2b2 did not lead to the transcriptional induction of PR-la (Fig. 5, bottom panel, lanes T ⁇ 2bl and T ⁇ 2b2; Fig. 6, left four lanes) or PR-5 (data not shown) genes.
  • PR-la bottom panel
  • PR-5 data not shown
  • Tav2b, T2bC which contained nucleotide substitutions introduced at positions different to those occurred in T ⁇ 2bl and T ⁇ 2b2. Furthermore, as TMV-T ⁇ 2bl infected the Samsun cultivar systemically, expression of the truncated Tav2b of 44 amino acids (aa) must be insufficient to elicit the resistance responses.
  • Cmv2b is not active in this manner when similarly expressed from the TMV genome.
  • Cmv2b and Tav2b are encoded by two different virus species from t h e same Cucumovirus genus (Ding et al., 1994; Shi et al . , 1997).
  • the predicted amino acid sequences of the two proteins are 24% identical (46.2% similarity), the least conserved pair among the known cucumoviral 2b proteins (Ding et al., 1994).
  • results from deletion mutants such as TMV-T ⁇ 2bl may not be informative in mapping functional domains of Tav2b.
  • Tav2b as encoded by pTMV-T2b was progressively replaced from either the N-terminus (pTMV-CTl, 2, and 3) or the C-terminus (pTMV-TCl,
  • CT1, CT2 and CT3 resulted from the replacements of the N-terminal 91, 69 or 50 aa of Tav2b by the equivalent regions of Cmv2b (Fig. 3) .
  • the tobacco plants were susceptible to all three of the TMV recombinants in which the N-terminal 50 aa or more of the encoded 2b protein were derived from Cmv2b (TMV-CT1, 2, and 3) . None of these recombinants induced necrotic lesions (data not shown) and all accumulated in both the inoculated and systemic leaves (Fig. 7, lanes CT1, CT2 and CT3) .
  • the infected plants also displayed systemic mild mosaic symptoms similar to those caused by TMV-C2b.
  • the N-terminal 69 and 91 aa respectively of TCI and TC2 were derived from Tav2b (Fig. 3) . Both induced strong virus resistance in the challenged tobacco plants. This is because both TMV-TCl and TMV-TC2 accumulated to very low levels in the inoculated leaves (Fig. 7, lanes TCI and TC2 of the upper panel) and no accumulation of viral RNAs was detected in the systemic leaves (lanes TCI and TC2 of the lower panel) . The challenged plants remained symptomless for as long as observations were made (five weeks) . Reprobing the filter shown at the upper panel of Fig. 7 with the PR-la probe showed that transcription of PR-la mRNA was induced in the tobacco leaves inoculated with TMV-TCl and TMV-TC2 but not in those inoculated with the remaining four viruses.
  • the function of the N terminal 69 aa of Tav2b in resistance activation is independent of its remaining 26 amino acids at the C-terminus.
  • TC3 contained 50 aa from the N-terminus of the Tav2b protein (Fig. 3) and did not induce virus resistance because TMV-TC3, like TMV-CT3, infected the tobacco plants systemically (Fig. 7, bottom panel) .
  • T ⁇ 2bl encoding a truncated Tav2b of 44 aa
  • Fig. 7 avirulent
  • CT3 contained the same 20 aa from Tav2b, but it failed to induce virus resistance (Fig. 7), indicating that presence in a 2b chimera of this region of Tav2b, unlike the more extended N-terminal region of 69 aa, is not sufficient to activate virus resistance.
  • TMV-TCl induced necrotic lesions in the inoculated leaves identical to those triggered by TMV-T2b. Under the same conditions, TMV-TC2 caused no visible hypersensitive cell death (Fig. 3) . This result, a local and symptomless infection by TMV-TC2, was consistent and reproducible in six independent experiments, involving a total of 37 plants. This indicates that the sequence encompassing amino acids 70-91 of Tav2b is required for triggering hypersensitive cell death. However, TMV-CT2 and TMV-CT3, both of which encode the same 22 amino acids from Tav2b, did not cause necrotic lesions (Fig. 3), suggesting that this sequence alone is insufficient to trigger hypersensitive cell death.
  • TMV-T2bC expressing the resultant Tav2b mutant, did not elicit any of the disease resistance responses associated with wild type Tav2b. Neither necrotic 33 lesions nor transcriptional induction of PR la mRNA were observed in the inoculated Samsun leaves. Furthermore, the TMV-T2bC inoculated plants became systemically infected. Northern blot analysis showed that TMV-T2bC accumulated to similar levels to
  • TMV-T ⁇ 2b2 in both the inoculated and the systemic leaves of the infected plants.
  • RT-PCR and DNA sequencing showed that the introduced mutations were maintained in the progeny viral genome and no additional mutations were found in the Tav2b coding sequence of TMV-T2bC.
  • the two aa substitutions abolished the Avr activity of Tav2b in this tobacco cultivar.
  • Cmv2b expressed from a potato virus X (PVX) vector functions as a virulent determinant and suppresses PTGS of a green fluorescent protein (GFP) transgene in N. benthamiana plants (Brigneti et al., 1998). N. benthamiana plants were also fully susceptible to TMV-T2b. Infected plants displayed a systemic necrosis and began to collapse at 14 dpi and died a few days after. TMV-C2b induced a much milder systemic infection that did not resulted in the death of the infected plants; this difference in virulence between Tav2b and Cmv2b has been observed previously (Ding et al . , 1996).
  • PVX potato virus X
  • GFP green fluorescent protein
  • TMV-T ⁇ 2b2 Plants systemically infected with TMV-T ⁇ 2b2 showed a mild chlorosis similar to that caused by TMV-30B infection. Accumulation of TMV-T2b and TMV-T ⁇ 2b2 in both the inoculated and systemic leaves were confirmed by Northern blot hybridization. 34
  • Tav2b like Cmv2b, was not recognized as an Avr gene in N. benthamiana .
  • the TAV sequences from pTMV-T2b and ⁇ TMV-T ⁇ 2b2 were transferred into a PVX vector (pP2C2S) to give pPVX-T2b and pPVX-TA2b2, from which RNA transcripts were obtained and inoculated to N. benthamiana plants exhibiting complete systemic PTGS of the GFP transgene.
  • PVX-T2b was similarly virulent to TMV-T2b in N. benthamiana and PVX-T2b infection led to a rapid death of the infected plants.
  • PVX-T ⁇ 2b2 was much less virulent than PVX-T2b in N. ben thamiana and no suppression of PTGS of the GFP transgene was observed in PVX-T ⁇ 2b2-infected plants at 18 dpi or at 35 dpi.
  • Tav2b is a virulent determinant and a suppressor of PTGS in N. benthamiana .
  • the coat protein of potato virus X is a strain-specific elicitor of Rxl-mediated virus resistance in potato. Plant J. , 8, 933-941.
  • Potato virus X as a vector for gene expression in Dlants. Plant J. , 2, 549-557.
  • a tobacco mosaic virus induced tobacco protein is homologous to the sweet-tasting protein thaumatin. Na ture, 321, 531-532.
  • RNA virus An interspecies hybrid RNA virus is signi icantly more virulent than either parental virus. Proc. Na t ' l Acad. Sci . USA, 93, 7470-7474.
  • Pirhonen,M.U. Lidell,M.C, Rowley, D.L., Lee,S.W., Jin,S., Liang,Y., Silverstone, S. , Keen,N.T. and Hutcheson,S.W. (1996) Phenotypic expression of
  • Pseudomonas syringae avr genes in E.coli is linked to the activities of the hrp-encoded secretion system. Mol . Plant Microbe Interact . , 9, 252-260.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Tomato asper y virus
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

L'invention concerne des plantes transgéniques transformées à l'aide d'un gène 2b de la mosaïque du concombre ou d'un fragment actif de celui-ci, ou à l'aide d'un gène Avr dont le domaine de mort cellulaire inactif résiste aux maladies provoquées par des agents pathogènes infectieux, par exemple des virus. L'expression de ces gènes déclenche, chez les plantes incapables d'une telle réponse à certains agents pathogènes, l'activation d'une réponse hypersensible et l'expression de protéines liées à la pathogénicité. La transformation d'une grande variété de plantes à l'aide de vecteurs d'expression, dans lesquels le gène est lié de manière opérationnelle à un promoteur actif chez les plantes, permet donc de rendre les plantes résistantes à toute infection pathogène.
PCT/SG1999/000012 1998-05-12 1999-02-12 Plantes transgeniques resistantes aux maladies WO1999058697A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1228230A2 (fr) * 1999-11-12 2002-08-07 University of South Carolina Procede pour accroitre la resistance dans des plantes
JP2011217744A (ja) * 2010-03-24 2011-11-04 Iwate Univ 植物への病原ウイルス感染を防除する組換えａｌｓｖ
WO2012127697A1 (fr) * 2011-03-24 2012-09-27 国立大学法人岩手大学 Alsv recombinant pour la prévention d'une infection virale pathogène d'une plante

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015585A1 (fr) * 1990-04-02 1991-10-17 Rijkslandbouwuniversiteit Wageningen Procede de protection des plantes contre les pathogenes
WO1993025068A1 (fr) * 1992-06-08 1993-12-23 Cornell Research Foundation, Inc. Resistance virale des plantes par transformation a l'aide d'une portion de replicase d'un genome de virus des plantes
WO1996021032A1 (fr) * 1994-12-30 1996-07-11 Asgrow Seed Company Plantes transgeniques presentant une resistance virale heterologue

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015585A1 (fr) * 1990-04-02 1991-10-17 Rijkslandbouwuniversiteit Wageningen Procede de protection des plantes contre les pathogenes
WO1993025068A1 (fr) * 1992-06-08 1993-12-23 Cornell Research Foundation, Inc. Resistance virale des plantes par transformation a l'aide d'une portion de replicase d'un genome de virus des plantes
WO1996021032A1 (fr) * 1994-12-30 1996-07-11 Asgrow Seed Company Plantes transgeniques presentant une resistance virale heterologue

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Title
DING, S-W., ET AL.: "a novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement", THE EMBO JOURNAL, vol. 14, no. 23, 1995, pages 5762 - 5772, XP002089661 *
DING, S-W., ET AL.: "an interspecies hybrid RNA virus is significantly more virulent than either parental virus", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 93, July 1996 (1996-07-01), pages 7470 - 7474, XP002089662 *
DING, S-W., ET AL.: "efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasmid vector", JOURNAL OF GENERAL VIROLOGY, vol. 76, 1995, pages 459 - 464, XP002089663 *
DING,S-W., ET AL.: "new overlapping gene encoded by the cucumber mosaic virus genome", VIROLOGY, vol. 198, 1994, pages 593 - 601, XP002089664 *
LI, H-W., ET AL.: "strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism", THE EMBO JOURNAL, vol. 18, no. 10, May 1999 (1999-05-01), pages 2683 - 2691, XP002110358 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1228230A2 (fr) * 1999-11-12 2002-08-07 University of South Carolina Procede pour accroitre la resistance dans des plantes
JP2011217744A (ja) * 2010-03-24 2011-11-04 Iwate Univ 植物への病原ウイルス感染を防除する組換えａｌｓｖ
WO2012127697A1 (fr) * 2011-03-24 2012-09-27 国立大学法人岩手大学 Alsv recombinant pour la prévention d'une infection virale pathogène d'une plante
CN103562380A (zh) * 2011-03-24 2014-02-05 国立大学法人岩手大学 防除对植物的病原病毒感染的重组alsv

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