Patent Grant
7732665
This is a 371 National Stage application of International application no. PCT/IT04/00287, filed May 19, 2004, which claims priority to Italian application no. RM2003A000242, filed May 19, 2003. The entire contents of the above-referenced applications are hereby incorporated by reference in their entirety.
The present invention relates to a method for the preparation of transgenic plants lasting resistant to geminiviruses.
More particularly the invention concerns a method for the preparation of transgenic plants lasting resistant to geminiviruses, wherein the transgene consists of a polynucleotide sequence, derived from the pathogen, suitably modified in order to result in an ineffective target of the post-trascriptional gene silencing induced by geminiviruses.
It is known that geminiviruses are a wide and diversified class of plant viruses that infect several plants of agronomic interest causing serious harvest losses. Such viruses are characterised by virions consisting of two geminate icosahedric particles. Their genome, consisting of one or two circular single-stranded DNA molecules (ssDNA), replicates in the nucleus of infected cells through double stranded intermediates (Hanley-Bowdoin et al., 1999).
The Geminiviridae family is divided in four genera named Mastrevirus, Begomovirus, Curtovirus and Topocuvirus based on the insect vector, the host spectrum and the genome structure (Briddon et al., 1985; Fauquet et al., 2003).
A serious disease of the tomato plant, transmitted by the whitefly Bemisia tabaci, is from a long time known as “tomato yellow leaf curl” in the areas of the Middle East, Asian South East and Africa, (Czosnek et al., 1997). This disease, that can cause harvest losses of 100% (Picò et al., 1996; Czosnek et al., 1997), successively spread both throughout the Western Mediterranean, reaching Sardinia, Sicily and Spain (Czosnek et al., 1997), and America (Polston et al., 1997).
Recently the agents of the disease have been identified and isolated, being viruses belonging to the Geminiviridae family, genera Begomovirus. Phylogenetic studies have highlighted the presence of different viral species related to different geographical origins of the Begomovirus: Asia, Africa and America (Czosnek et al., 1997).
The genoma of the Tomato yellow leaf curl Sardinia virus (TYLCSV) species, is monopartite (Kheyr-Pour et al., 1991). The DNA is transcribed bidirectionally and contains six open reading frame (ORF), two on the viral strand (V): V1 and V2, and four on the complementary strand (C): C1, C2, C3 and C4, as shown in
Strategies used until now in order to control the infection of the geminiviruses transmitted by the Bemisia tabaci are based on the use of expensive fine mesh nets (for the cultivation of fresh-market tomato) and particularly on repeated insecticide treatments (cultivations of both fresh-market and processing tomato). Such strategies result in an increase of the production expenses and represent a serious danger for the health of the agricultural operators and consumer. Furthermore the onset of Bemisia tabaci populations resistant to the insecticide imidacloprid has been already reported (Cahill et al., 1996; Williams et al., 1996).
The development of resistant cultivated species represents the most practical and economic way to control viral infections. Classical breeding programs for introducing resistance to geminiviruses that cause the tomato yellow leaf curl were based on the transfer of resistance genes from wild species of Lycopersicon to species of cultivated tomato. Thereby lines with variable levels of resistance to TYLCSV have been obtained and commercialized, the best lines showing reduced symptoms and low viral replication. However plants with low and mean levels of resistance represent a potential receptacle for further infections.
Another important aspect to be considered is that the agronomic characteristics of the lines obtained are not always optimal and however reflects those of the genotype of cultivated tomato used in breeding programs.
A tomato line immune to the viruses causing the tomato yellow leaf curl disease, namely, with neither symptoms nor viral DNA replication has not been released yet.
With the advent of genetic engineering new perspectives were opened up for the introduction of resistance characters against plant viruses. Most strategies are based on the introduction and expression of pathogen-derived sequences in the plant of interest, Pathogen Derived Resistance (PDR) (Sanford & Johnson, 1985; Abel et al., 1986; Tavazza and Lucioli, 1993).
Although such strategies have been successfully applied for the introduction of resistance characters to plant viruses with RNA genome (Beachy, 1997), in the case of geminiviruses, with a DNA genome, the expression of pathogen-derived sequences has produced plants with no lasting resistance and/or tolerance.
The mechanisms that induce virus resistance achieved through the expression of pathogen-derived sequences can be grouped in two wide classes:
The post-trascriptional gene silencing is a ubiquitary process in eukaryotes, involving the degradation of specific RNAs following the formation of double strand RNA (dsRNA) molecules having sequences homologous to the target RNA.
Although there may be different contexts able to induce the production of dsRNA homologous to the transgene (transcription of aberrant transgenic RNAs, presence in the transgenic RNA of sufficiently long inverted and repeated sequences, integration of the transgene in the plant genome in inverted and repeated multiple copies), once the dsRNA is produced, the latter is recognised and degraded in short molecules of dsRNA of about 21-26 nucleotides, referred to as siRNA.
The siRNAs are then integrated in a multiprotein complex named RISC, that is able to degrade all RNAs having sequence homology with the siRNAs. The latter ones represent therefore the determining factors of RNA silencing specificity and their presence related to a determined sequence establishes univocally that this RNA sequence is post-transcriptionally silenced.
Therefore, transgenic plants post-transcriptionally silenced for sequences derived from viral RNA genome, are resistant to the homologous virus and to viruses with nucleotide sequences closely related to the transgene.
The transgene silencing can be also induced following virus infection.
In fact, viral replication is able to induce silencing of a transgene, initially not silenced, if the nucleotide sequence of the transgene is homologous to a portion of the infecting virus genome. The activation of the silencing mechanism involves the specific degradation of the RNA molecules having sequence homology with the inducer RNA.
As direct consequence, the silencing activation by the virus is associated with a degradation of both transgenic mRNA sequences homologous to the virus and viral genome. This results in the host recovery after an initial infectious step, so that the new vegetative part is proved to be virus free. A peculiar characteristic of the plant tissues that develop subsequently to the recovery phenomenon is that they are highly resistant to a following infection by the same virus.
The resistance mediated by post-transcriptional gene silencing, since based on recognition at the nucleotidic level, confers resistance only against viral isolates closely homologous to the virus genome from which the transgene was derived. Instead, strategies based on the expression of a pathogen protein normally produce plants resistant also to viral strains or isolates not-closely related from a nucleotide point of view.
It is also been shown that the transgene silencing is influenced by the temperature, being inactive at temperatures below 15° C. (Szittya et al., 2003). Therefore plants exposed in field conditions at temperature range below 15° C. can lose the silencing-mediated resistance.
It must be borne in mind that, although from several years transgenic plants resistant to RNA genome viruses have been achieved through mechanisms based on transgene silencing, so far it is not reported that such strategy can be successfully applied to the geminiviruses (DNA genome-viruses).
It's clear that the best strategy in order to obtain plants resistant to a wide spectrum of geminiviruses is the one in which the interfering product is the protein. It is clear that the width of the resistance spectrum increases the agronomic and commercial value of the produced plant.
Thereby the expression in transgenic plants of dysfunctional variants of geminivirus replicative Rep protein has been used in order to obtain plants with greater levels of resistance or immunity against the geminiviruses.
It's known in literature that the expression of a truncated replicative Rep protein (Rep-210) of TYLCSV is able to confer resistance against viral infection, although such resistance is not lasting because the virus is able to overcome it over time.
In tables 1 and 2 are shown the results of the analysis of the resistance of TYLCSV-agroinoculated Rep-210 expressing transgenic plants of Tomato 47×wt (Brunetti et al. 1997) and of N. benthamiana line 102.22 (Noris et al. 1996) respectively.
Lycopersicon
esculentum
Nicotiana
benthamiana
From the results reported in tables 1 and 2, it can be clearly inferred that the resistance against TYLCSV mediated by the transgenic expression of a pathogen-derived sequence, is overcome with time.
Similarly, also the resistance induced by the transgenic expression of a dominant negative mutant of Rep of the bipartite geminivirus “African Cassava Mosaic Virus” is overcome with time (Sangaré et al., 1999).
Another example is represented by the transgenic expression of the TYLCV capsid protein in a tomato interspecific hybrid (Lycopersicon esculentum X L. pennellii) which confers a partial resistance against viral infection (Kunik et al., 1994). Even in this case the resistance mediated by the expression of the capsid protein is not long lasting and it results to be poorly useful from an agronomic point of view.
In the light of the above, is clear the need to have new methods that would allow to use successfully the polynucleotide sequences derived from the geminiviruses in order to obtain long lasting resistant plants against geminiviruses.
The authors of the present invention have now prepared polynucleotide sequences encoding pathogen-derived viral proteins and able to confer virus resistance to the host, suitably modified in order to be ineffective targets of the virus-induced post-transcriptional gene silencing to obtain transgenic plants with lasting levels of resistance against geminiviruses.
In fact during the experiments the authors show that the overcoming of the resistance, and therefore the difficulty to achieve lasting resistance against geminiviruses, is due to the unexpected abilities of the geminiviruses to silence post-transcriptionally the transgene and to spread in a plant in which the transgene, with sequences homologous to the infecting virus, is post-transcriptionally silenced.
As shown in
The TYLCSV ability to spread in a plant in which the transgene Rep-210 is post-transcriptionally silenced, is further circumstantiated as set forth in
The results show that the transgenic tomato plants 47×10D (Brunetti et al., 1997), post-transcriptionally silenced before agroinoculation, as shown by the absence of the Rep-210 protein and by the concurrent presence of the transgene-homologous siRNAs, are susceptible to the TYLCSV infection as well as the controls.
From the above it results that, contrary to RNA viruses, the geminivirus is not blocked by an active silencing of viral gene sequences. The above said is not limited to the kind of transgenic plant to be used or the way the virus should be inoculated, through agroinfection or Bemisia tabaci. In fact, as shown in table 3, using a reduced number of viruliferous bemisia per plant, so as to infect between 90% and 100% of the control plants, about 40% of transgenic plants (line 201) whose transgene is post-trascriptionally silenced, are not or late infected, while at a higher inoculum concentration, all the plants challenged with viruliferous insects are infected similarly to the experiments carried out using agroinoculation.
aSeven viruliferous insects per plant for 2 days
bThirty-five viruliferous insects per plant for 5 days
cWeeks after inoculum
Therefore it's important to consider that the viral agroinoculation conditions used for testing the resistance and assessing persistence over time (as shown in
Accordingly, the introduction of resistance characters against geminiviruses through the expression of pathogen-derived sequences is limited due to the unexpected ability of the geminiviruses to silence post-trascriptionally the transgene and to spread in the silenced plant.
Furthermore the authors show that the transcripts both of positive (V1 and V2) and negative strand (C1, C2, C3 and C4) of TYLCSV are subjected, during a normal infection on wild-type plants, to the viral post-trascriptional silencing, as shown in
Therefore it is an object of the present invention a polynucleotide sequence encoding an amino acid sequence derived from geminiviruses, said polynucleotide sequence being characterised in that it is not a target or it is an ineffective target of the viral post-trascriptional silencing and having:
a) a nucleotide homology lower or equal to 90% with respect to the corresponding gene sequence of the geminiviruses against which a resistance is required, preferably lower or equal to 80%, more preferably lower or equal to 70%;
b) a continuous homology in the RNA transcript, with respect to the corresponding gene sequence of the geminiviruses against which a resistance is required, lower or equal to 17 nucleotides, preferably lower or equal to 8 nucleotides, more preferably lower or equal to 5 nucleotides;
c) a maximum length of the sequence containing a single substitution with respect to the corresponding gene sequence of the geminiviruses no longer than 30 nucleotides, preferably no longer than 20 nucleotides, more preferably equal or lower than 9 nucleotides;
said polynucleotide sequence being able to confer to the whole plants, tissues or plant cells therewith transformed, a lasting resistance against the geminiviruses.
The polynucleotide sequences according to the invention can be wild-type or synthetic or produced by mutagenesis and the geminivirus-derived amino acid sequences encoded by them are wild-type or mutant sequences that interfere with the viral infection.
Therefore the invention includes polynucleotide sequences of geminivirus either suitably changed or wild-type, such as to differ, at the nucleotidic level, with respect to the corresponding genomic sequence of the geminivirus against which it is required to introduce resistance according to the principles above defined and specified in a), b) and c).
Further object of the present invention is a polynucleotide sequence encoding a geminivirus-derived amino acid sequence, said polynucleotide sequence being characterised in that it is not a target or it is an ineffective target of the post-trascriptional silencing and having homology even equal to 100% with respect to the sequence of the geminivirus against which it is required a resistance and being shortened so as to be underrepresented in the siRNAs population with respect to the original sequence, even if maintaining similar interfering abilities.
The gene sequences from which constructing the polynucleotide sequence according to the invention can derive from the geminiviruses such as, Mastrevirus, Curtovirus, Begomovirus, Topocuvirus and particularly can be derived from the species shown in table 4 and their isolates, more particularly from the species of Tomato yellow leaf curl and their isolates shown in table 5.
Preferably the species of Begomovirus are TYLCCNV, TYLCGV, TYLCMaIV, TYLCSV, TYLCTHV, TYLCV, ACMV, BGMV, CaLCuV, ToCMoV, TGMV, ToGMoV, ToMHV, ToMoTV, ToMoV, ToRMV, ToSLCV, ToSRV, Cotton leaf curl (CLCrV, CLCuAV, CICuGV, CLCuKV, CLCuMV, CLCuRV), East African cassava mosaic (EACMCV, EACMMV, EACMV, EACMZV), Potato yellow mosaic (PYMPV, PYMTV, PYMV), Squash leaf curl (SLCCNV, SLCV, SLCYV), Sweet potato leaf curl (SPLCGV, SPLCV), Tobacco leaf curl (TbLCJV, TbLCKoV, TbLCYNV, TbLCZV), Tomato leaf curl (ToLCBV, ToLCBDV, ToLCGV, ToLCKV, ToLCLV, ToLCMV, ToLCNDV, ToLCSLV, ToLCTWV, ToLCVV, ToLCV) and isolates thereof.
Other species of preferred geminivirus, belonging to the other genera Mastrevirus, Curtovirus, Topocuviruses, are WDV, MSV, SSV, BYDV, TYDV, BCTV and their isolates.
The gene sequence belonging to the genome of the geminiviruses can be the sequence C1/AL1/AC1, C2/AL2/AC2, C3/AL3/AC3, C4/AL4/AC4, V1/AR1/AV1, V2/AR2/AV2, BC1/BL1 and BV1/BR1, particularly, the sequence C1/AL1/AC1 of the previously described geminiviruses and their isolates.
The amino acid sequence encoded by the polynucleotide sequence object of the present invention is a pathogen-derived protein able to confer resistance against the geminiviruses to the plants expressing it. Said interfering protein since, according to the invention, is stably expressed, confers a lasting resistance independently from the molecular mechanism by which the protein product is able to induce resistance.
The pathogen-derived protein can be a capsid protein, replication-associated viral protein (Rep), proteins encoded by the genes C2/AL2/AC2, C3/AL3/AC3, C4/AL4/AC4, V2/AR2/AV2, BC1/BL1 and BV1/BR1.
An example of a possible polynucleotide sequence satisfying the above reported requirement is set forth in
The plants, tissues or plant cells that can be transformed with this polynucleotide sequences can be tomato, pepper, tobacco, sweet potato, cotton, melon, squash, manioc, potato, bean, soybean, mung bean, beet, sugar cane, corn, wheat.
It is a further object of the present invention a construct comprising an heterologous polynucleotide sequence containing in 5′-3′ direction:
a) a polynucleotide sequence acting as promoter in said plant or tissue or transformed cells;
b) a non-translated polynucleotide sequence positioned at 5′ of the encoding region, belonging or not to the intergenic region of geminivirus;
c) a polynucleotide sequence according to the invention or a fragment or a variant thereof;
d) a sequence acting as terminator of transcription, positioned at the 3′ with respect to said polynucleotide sequence.
A further object of the present invention is an expression vector comprising the previously described construct.
Further it is an object of the present invention a plant, tissue or transgenic plant cells, progeny thereof as well as seeds comprising in their genome a polynucleotide sequence according to the present invention.
Finally, it is an object of the present invention a method for the preparation of transgenic plants, tissues or plant cells thereof long-lasting resistant to the geminiviruses that comprises the following steps:
a) “identification” or “selection” of a viral gene sequence encoding an amino acid sequence able to confer resistance against geminiviruses;
b) mutagenesis or “choice” of the viral gene sequence so as to make it an ineffective target of the post-trascriptional silencing induced by infecting geminivirus;
c) insertion of the geminivirus mutated or chosen gene sequence obtained in step b) through a construct as described previously, in the plant, tissue or plant cell thereof.
With reference to step a) of the method according to the present invention, the term “identification” means the experimental recognition of said viral gene sequence able to confer resistance against geminiviruses, while the term “selection” means the recognition of an already available viral gene sequence able to confer a not lasting resistance against geminiviruses. Accordingly, the method according to the present invention provides furthermore the solution to the problem of the loss of resistance against geminiviruses that occurs through the employment of known sequences.
Particularly, the mutagenesis predicted in step b) is carried out maintaining a nucleotide homology, with respect to the corresponding gene sequence of the geminiviruses against which it is required to obtain a resistance, lower or equal to 90%, preferably lower or equal to 80%, more preferably lower or equal to 70%, distributed so as the continuous homology in the transcribed RNA with respect to the corresponding sequence of geminiviruses is lower or equal to 17 nucleotides, preferably lower or equal to 8 nucleotides, more preferably lower or equal to 5 nucleotides and the maximum length of the sequence containing a single substitution with respect to the native gene sequence is not more than 30 nucleotides, preferably not more than 20 nucleotides, more preferably lower or equal to 9 nucleotides.
As the amino acid sequence encoded by the polynucleotide sequence identified or selected in step a), according to the present invention, it can be a protein having homology of 100% with respect to the viral wild-type protein.
This mutagenesis includes all those mutations on the nucleotide sequence that don't decrease the ability of the protein to confer resistance against geminivirus. Possible mutations are both silent point mutations and those leading to the substitution with amino acids having similar biochemical characteristics, or deletions and/or insertions and/or substitutions.
Alternatively, the mutagenesis in step b) of the method according to the present invention consists of deletions of the polynucleotide sequence at the extremities so as said sequence, while maintaining similar interfering abilities, is under-represented with respect to the original sequence, in the natural population of the siRNAs produced by the infecting virus.
Alternatively the “choice” in step b) of the method according to the present invention consists in the recognition of geminivirus wild-type sequences that differ at the nucleotidic level from the geminivirus against which it is required resistance so as not to be a target or to be an ineffective target of the post-trascriptional silencing.
Particularly, the mutagenesis action in step b) of the method according to the present invention can consist of deletions of the 3′ or 5′ region of the viral gene sequence of step a), until it is identified the minimum region of said gene sequence that is under-represented with respect to the sequence encoding a wild-type protein, in the population of the siRNAs and that said truncated protein maintains the ability to confer resistance against geminiviruses.
Moreover, the viral gene sequence of step a) of the method according to the present invention can be that of TYLCSV C1/AL1/AC1 gene and the amino acid sequence can be a protein truncated relatively to the viral wild-type protein such as, for instance, Rep-130.
Among various agronomic applications of the synthetic polynucleotide sequences according to the present invention, of particular interest is their use for obtaining tomato plants resistant to TYLCSV. In this particular embodiment, the transgenic polynucleotide sequence encoding the truncated viral Rep protein (Rep-210) has been modified through a 3′deletion resulting in an ineffective target of TYLCSV-induced post-trascriptional gene silencing, while maintaining the ability to confer resistance.
In particular, using stringent hybridizations with radioactive RNA probes, it was identified a transcribed region of the TYLCSV genome that is under-represented in the population of viral-origin siRNAs produced during the infection of the TYLCSV in wild-type plants, as shown in
Therefore, in a particular embodiment of the invention, the amino acid sequence of geminiviruses, such as the TYLCSV, encoded by the polynucleotide sequence according to the invention can be the truncated Rep-130 protein (SEQ ID No 9). In this case the viral gene sequence made an ineffective or non target of the post-trascriptional silencing, is the SEQ ID No 8.
It's a further object of the present invention a method as described above wherein the mutagenesis in step b) consists of silent point mutations of the viral gene sequence of step a) that maintain the ability of the encoded amino acid sequence, to confer resistance against geminiviruses and to be an ineffective or non target of the post-trascriptional silencing.
Particularly, the viral gene sequence of step a) can be the V1/AR1/AV1 (CP) gene for instance of TYLCSV (SEQ ID No 12), and in a particular embodiment the viral gene sequence made ineffective or non target of the post-trascriptional silencing is the SEQ ID No 6.
In addition, the viral gene sequence of step a) can be the TYLCSV C1/AL1/AC1 gene, and in this case the viral gene sequence which was made ineffective or non target of the post-trascriptional silencing can be the SEQ ID No 2 or the SEQ ID No 4.
The present invention now will be described by way of illustrating but not limiting way, according to preferred embodiments thereof, with particular reference to the figures of the enclosed drawings, wherein:
In a natural infection by TYLCSV of wild-type plants, the viral sequences transcribed by both strands of TYLCSV genome are target of post-trascriptional gene silencing as pointed out by the presence of siRNAs homologous to different portions of the genome (
In order to evaluate if some regions of the TYLCSV genome constitute a target of post-trascriptional gene silencing less effective than others, it was performed a systematic study of the siRNA distribution with respect to their position on the viral genome. Therefore the TYLCSV genome has been divided in nine contiguous fragments, each of about three hundred base pairs (as drawn in
In order to identify a region of the TYLCSV C1 gene under-represented in the siRNAs population, total RNAs (Brunetti et al., 1997) both from healthy and TYLCSV-infected tomato plants have been extracted.
Thirty micrograms of such RNAs have been submitted to 8% denaturing polyacrylamide gel electrophoresis and transferred by capillarity on nylon filter through Northern blot. Two identical replicas have been produced and for each it has been carried out an hybridization with probes corresponding to different portions of the 5′ region of the C1 gene, as shown in
In order to quantitatively compare the results obtained by the two different probes (deriving from two independent labelling), scalar amounts of a same 40mer oligonucleotide complementary to both probes have been loaded on both replicas. Columns 100 and 50 correspond to 100 and 50 picograms of such oligonucleotide, respectively. The panels showing the oligonucleotide migration have been set close to the respective panels containing the siRNAs but their position in the figure doesn't correspond to the position on gel, because the oligonucleotide and the siRNAs have different molecular weights.
Both probes after in vitro transcription have been submitted to alkaline hydrolysis (Cox et al., 1984) in order to obtain from them fragments with an average length of 75 nucleotides.
The hybridizations have been performed for 16 hours at 39° C. in the buffer described by Dalmay et al., 2000. After hybridization the filters have been washed in 2×SSC, 0.2% SDS twice for 10 minutes at 40° C., twice for 10 minutes at 45° C. and once for 10 minutes at 50° C.
It is remarkable how the proximal 5′ region of the C1 gene in the siRNAs population is under-represented. Particularly, the quantitative analysis of the results performed through the TYPHOON apparatus (Amersham-Pharmacia) revealed that the siRNAs corresponding to this 5′ region are about 25% (probe B) with respect to those corresponding to the region extended up to nucleotides encoding the 210 amino acid (probe A). Said 5′ region constitutes therefore an ineffective target for the virus-induced post-trascriptional gene silencing.
These results have been confirmed using the method described in example 1, i.e., where PCR fragments corresponding to the two different regions of the C1 gene were hybridised with the population of siRNA extracted from tomato plants infected by TYLCSV.
As previously pointed out (Brunetti et al., 1997), the Rep-210 transgenic plants show a not long lasting resistance and an altered phenotype.
As can be noticed in
It is shown that the transgenic expression of geminivirus C4 gene induces phenotype alterations (Krake et al., 1998).
Therefore, it has been designed several truncated C1 constructs unable to express C4 ORF.
In order to obtain C4 (−) mutants, a stop codon has been introduced in the C4 sequence through the introduction of two point mutations. Particularly, referring to the pTOM130 sequence set forth in
Thereby the translation of the C4 protein is interrupted after only 10 amino acids, while the amino acid sequence of the C1 protein remains unchanged. The two introduced mutations have been chosen among many possible mutations based on the criterion to generate a “strong” stop codon in the C4 reading frame, maintaining in the C1 reading frame a leucine codon compatible with codon usage in plants.
Mutagenesis has been performed by PCR with the following mutated oligonucleotides:
Each of the two mutated primers has been used along with an external primer in two separate PCR reactions using pGEM102 as template (Brunetti et al., 2001).
Particularly, the external oligonucleotides are Rev and Univ (M13/pUC sequencing primer n.1233 and 1224). From the reaction performed with Univ/C4plus it has been obtained a 537 by fragment, while from the reaction with Rev/C4minus a 351 by fragment.
The obtained products have been used as templates for a following amplification reaction carried out using two external primers.
The obtained PCR product has been digested with EcoRI and BamHI restriction enzymes and cloned into the corresponding sites of pJIT60, thus obtaining pJITR210. In both cases it has been carried out the sequencing to verify clones.
In order to define the minimal 5′ terminal region of C1 gene able to confer resistance against TYLCSV, a series of 3′-terminal deletion mutants of C1 gene was cloned in pJIT60 expression vector, resulting in a pJTR series.
The viral sequences have been amplified by PCR with Pfu DNA polymerase (Stratagene), using specific primers containing restriction sites at the ends.
The previously described pJITR210 plasmid, which encodes Rep-210, and contains a stop codon for the internal C4 protein, has been used as template. The fragments obtained by amplification reactions have been digested with BamHI and EcoRI enzymes and cloned in the corresponding sites of pJIT60 resulting in the pJTR series.
All final clones have been sequenced in order to confirm the amplification fidelity and vector-insert junctions. The length and the precise positions of every amplified sequence are set forth in table 6.
The ability of each Rep deletion mutant to confer resistance against TYLCSV has been evaluated through cotrasfection assays of N. benthamiana wild-type protoplasts with a TYLCSV infectious clone (pTOM6) along with each mutant, and following analysed for the replication level of the viral genome through Southern blot. The obtained results are set forth in
The protoplast cotransfection, total nucleic acid extraction and Southern analysis have been performed according to already described methods (Brunetti et al. 2001).
Total nucleic acids extracts from each protoplast sample have been analysed through Southern blot with a digoxigenin-labelled RNA probe corresponding to the sequence encoding Rep-210, and the pGEM-P plasmid used as control. In particular
For an accurate quantitative analysis of the effect of the expression of several truncated forms of Rep on the replication of TYLCSV genome, a Southern analysis has been performed with a 32P-labelled DNA probe corresponding to the region encoding the first 54 N-terminal amino acids of Rep and the radioactivity level corresponding to each band detected on filter has been evaluated, through analysis with the Istant Imager apparatus (Canberra, Packard).
Each mutated construct has been assayed in duplicate, in three independent experiments and the value set forth in
The level of TYLCSV replication in the cotrasfection experiments performed with pTOM6 along with pGEM-P control plasmid was considered equal to 100%.
Particularly,
As pointed out by observing
The analysis of the ability to inhibit TYLCSV replication by the Rep mutants assessed through transient expression in protoplasts, has revealed that the shortest mutant still effective encodes Rep-130 (SEQ ID No 9) as described in the preceding example.
Also it has been previously revealed in other examples that the proximal 5′ portion of C1 gene encoding Rep-130 is a less effective target of post-trascriptional gene silencing compared to sequence encoding Rep-210.
Therefore it has been obtained N. benthamiana transgenic plants expressing Rep-130. For this purpose, the pTOM130 plasmid represented in
N. benthamiana has been transformed with the A. tumefaciens pGV2260 C58 strain containing pTOM130 plasmid and plants resistant to kanamycin have been regenerated as described (Noris et al. 1996).
The primary transformants have been analysed for the presence of transgene by PCR analysis and for the expression of Rep-130 protein through Western blot, as shown in
The protein extracts obtained from transgenic (300-309) or wild-type control (wt) plants have been analysed by Western blot using an anti-TYLCSV Rep rabbit polyclonal primary antibody as described (Noris et al., 1996).
In order to early evaluate the resistance conferred by Rep-130, protoplasts isolated from several primary transgenic Rep-130 expressing N. benthamiana plants were transfected with a TYLCSV infectious clone (pTOM6).
Transgenic lines have been chosen for their high Rep-130 expression, as revealed by Western blot analysis (
The level of TYLCSV replication in such transgenic protoplasts has been compared with that observed in N. benthamiana wild-type and in transgenic protoplasts expressing Rep-210 (line 102.22).
Particularly,
In order to compare the level of TYLCSV replication in the Rep-130 transgenic protoplasts with that observed in wild-type protoplasts, the total nucleic acids extracted from wild-type protoplasts have been also loaded following 1:10 and 1:50 dilutions, as shown in
The analysis of the ability to inhibit TYLCSV replication by Rep mutants, assessed by transient expression in protoplasts, has revealed that the shortest mutant still effective encodes Rep-130, as described in the previous example.
As previously shown, the proximal 5′ portion of C1 gene encoding Rep-130 is an ineffective target of post-trascriptional gene silencing compared with the sequence encoding Rep-210.
Therefore it has been carried out the production of transgenic tomato plants (Lycopersicon esculentum cv. Moneymaker) expressing Rep-130.
The tomato has been transformed using A. tumefaciens pGV2260 C58 strain containing pTOM130 plasmid (
The primary trasformants have been analysed for the presence of the transgene by PCR analysis and for the expression of Rep-130 protein by Western blot (
The protein extracts obtained from transgenic (lines 400) or wild-type control (wt) plants have been analysed by Western blot using an anti-TYLCSV Rep polyclonal rabbit primary antibody as described (Noris et al. 1996).
All transgenic tomato plants expressing Rep-130 protein are phenotypically impossible to distinguish from wild-type plants (
In order to assess the lasting of the resistance against TYLCSV conferred by Rep-130 expression, the N. benthamiana R1 transgenic plants expressing Rep-130 have been agroinoculated with the A. tumefaciens LBA4404 strain containing the TYLCSV infectious clone.
As previously reported, the viral delivery through agroinoculation, used to assay the resistance and evaluate stability over time, corresponds to high or very high viral pressure conditions.
Infection of plants has been assessed at weekly intervals by a “tissue printing” assay, using a digoxigenin-labelled probe specific for the coat protein gene.
The results in table 7 show that, unlike the results described in table 2 concerning transgenic plants expressing Rep-210, transgenic N. benthamiana plants expressing Rep-130 protein show a long-lasting resistance when agroinoculated with TYLCSV. This can be deduced by comparison of the resistant plants at 2 and 6 weeks following inoculation.
In addition, it was assessed the stability of the resistance against TYLCSV conferred by Rep-130 expression in transgenic R2 tomato plants.
The plants have been agroinoculated with the A. tumefaciens C58C1+pCH32 strain containing TYLCSV infectious clone. The agroinoculation conditions used for assaying the resistance and evaluating stability thereof over time, correspond to high or very high viral pressure conditions.
The infection has been assessed at intervals of one or two weeks through dot-blot assay, using a radioactively labelled probe specific for the coat protein gene.
The results in table 8 point out that tomato transgenic plants expressing Rep-130 show a long lasting resistance when agroinoculated with an TYLCSV infectious clone. This can be deduced from the comparison of the resistant plants at 3 and 12 weeks after inoculation.
Therefore the resistance is associated to the presence of Rep-130 protein (SEQ ID No 9) and to the ability of TYLCSV-inoculated transgenic plant to stably express Rep-130, because the sequence encoding it is an ineffective target of virus-induced post-trascriptional gene silencing and Rep, even if further mutated, maintains its ability to confer virus resistance.
In order to achieve the long lasting expression of TYLCSV Rep-210 protein in transgenic plants, it has been produced, employing the method according to the present invention, a synthetic polynucleotide sequence, able to encode for Rep-210 protein, that is not or is an ineffective target of the post-trascriptional gene silencing induced by the infecting virus.
In addition, the following criterions have been followed:
Following the above described criterions, two synthetic sequences encoding Rep-210 have been designed (
A non-translated leader sequence at the 5′ and a stop codon at the 3′ have been added to the sequence of the synthetic Rep-210 silencing minus B gene (SEQ ID No 4).
Particularly, the polynucleotide sequence containing in the 5′-3′ order the non-translated leader sequence, the synthetic sequence encoding Rep-210 (
The synthetic gene has been subsequently cloned in pJIT60 plasmid under the transcriptional control of 35S promoter of the Cauliflower mosaic virus (CaMV) and the transcription termination sequences of the CaMV 35S, producing the pJT60Syn. Then the cassette containing in the 5′-3′ order: 35S promoter, Rep-210 synthetic gene, 35S terminator, has been removed from pJT60Syn plasmid by restriction with KpnI-BglII and cloned in the KpnI-BamHI sites of the binary plasmid pBIN19 generating pTOM102Syn.
The correct expression of Rep-210 protein, encoded by the synthetic gene, has been checked through agroinfiltration of N. benthamiana leaves, with A. tumefaciens C58C1/pCH32 transformed with pTOM102Syn. The strain C58C1/pCH32 transformed with pTOM102 (C4−) has been used as a positive control, while as negative control the strain C58C1/pCH32 transformed with the binary plasmid pBIN19 was used. Western blot analysis (
In order to assess the ability of the Rep-210 protein, encoded by pTOM102Syn, to inhibit TYLCSV replication, a transient co-agroinfiltration assay has been carried out. N. benthamiana leaves have been co-agroinfiltrated with A. tumefaciens C58C1/pCH32 strain containing the TYLCSV infectious clone (pTOM6) along with the A. tumefaciens C58C1/pCH32 strain containing: a) pTOM102Syn plasmid; b) pTOM102 (C4−) plasmid; c) pBIN19 binary plasmid. The TYLCSV replication has been assessed through Southern analysis of the total nucleic acids extracted from the co-agroinfiltrated tissues 72 hours after the infiltration. This analysis has pointed out that Rep-210 protein expressed by the synthetic gene (pTOM102Syn) and by pTOM102(C4−) wild-type gene, inhibits TYLCSV replication in a similar manner (
In order to obtain transgenic N. benthamiana plants expressing the synthetic gene for the Rep-210, N. benthamiana leaf-discs have been transformed using the A. tumefaciens LBA 4404 strain containing pTOM 102Syn plasmid and the kanamycin-resistant plants have been regenerated as described (Noris et al. 1996).
The primary trasformants have been analyzed for the expression of Rep-210 protein by Western blot analysis. Four primary trasformants, 506, 508, 517 and 537 lines accumulating intermediate levels of Rep-210 have been selected for further studies.
The authors have previously shown (Noris et al. 1996; Brunetti et al. 1997) that there is a direct correlation between the amounts of Rep-210 protein produced by the transgenic plants and resistance against TYLCSV. Transgenic plants transformed with the pTOM102 construct accumulating intermediate levels of Rep-210 protein are susceptible to viral infection, like non-transformed plants (Noris et al. 1996 and unpublished data). The low level of Rep-210 protein in these plants is not enough to completely inhibit viral replication, thus allowing the establishment of an early virus-induced post-trascriptional silencing leading to a drastic reduction in Rep-210 protein accumulation which causes lack of resistance.
In order to assess if Rep-210 protein encoded by the synthetic gene is not or is an ineffective target of virus-induced post-trascriptional gene silencing and therefore to control over time the viral infection, line 102.22 transgenic plants (R3) and line 506 transgenic plants (R0) expressing similar amount of Rep-210 (
The results described in the examples point out that it is possible to obtain a long lasting resistance against geminiviruses by expressing in plant a transgene consisting of a pathogen-derived polynucleotide sequence, if the latter is suitably selected or modified in order not to be a target or to be an ineffective target of the post-trascriptional gene silencing by the infecting virus.
As above reported, the transgenic expression of the TYLCV capsid protein in a interspecific tomato hybrid (Lycopersicon esculentum X L. pennellii) confers a partial resistance against viral infection (Kunik et al., 1994). Also in this case, the resistance mediated by the expression of the capsid protein is not long lasting.
In order to obtain a stable expression of the TYLCSV capsid protein (CP) by transgenic plants, it has been produced a synthetic polynucleotide sequence, able to encode the CP, which results in an ineffective target of virus-induced post-trascriptional gene silencing.
The synthetic polynucleotide sequence has been designed so as to satisfy the requisite not to be or to be an extremely ineffective target of virus-induced post-trascriptional gene silencing employing the method according to the present invention.
In addition, the following criterions have been followed:
Following the above described criterions a synthetic sequence encoding CP has been designed (SEQ ID No 12) (
| Number | Date | Country | Kind |
|---|---|---|---|
| RM2003A0242 | May 2003 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IT2004/000287 | 5/19/2004 | WO | 00 | 2/27/2006 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2004/101798 | 11/25/2004 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20050125862 | Polston et al. | Jun 2005 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 0043520 | Jul 2000 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20060206959 A1 | Sep 2006 | US |
