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1 Federal Centre for Breeding Research on Cultivated Plants, Institute of Resistance Research and Pathogen Diagnostics, Theodor-Roemer-Weg 4, Aschersleben D-06449, Germany
2 Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK
3 Life Science Department, Hangzhou Teacher's College, Hangzhou 310006, China
Correspondence
Konstantin Kanyuka
kostya.kanyuka{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Virus sequences have been deposited at EMBL (accession nos AJ517471–AJ517477). Amino acid sequence alignments for the entire polyproteins of RNA1 and RNA2 of all European BaYMV isolates are available as supplementary data at http://vir.sgmjournals.org.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These viruses are widespread throughout Europe (particularly United Kingdom, France and Germany) and east Asia and are major pathogens of winter barley. Yield losses of >50 % may occur when susceptible barley crops are grown on severely infected soils (Plumb et al., 1986). Like other members of the family Potyviridae, bymoviruses have single-stranded positive-sense RNA genomes encapsidated in long, flexuous particles. The other five genera of the family Potyviridae (Ipomovirus, Macluravirus, Potyvirus, Rymovirus and Tritimovirus) have monopartite genomes but the bymoviruses have two separately encapsidated mRNA species. RNA1 and RNA2 are both translated into large polyproteins, which are then processed by virus-encoded proteases into ten structural and non-structural products. Bymoviruses are also distinguished from other members of the Potyviridae by their ability to be transmitted by the root-inhabiting endoparasite, Polymyxa graminis. Chemical control of soil-borne virus diseases is neither efficient nor acceptable for economic and ecological reasons. Moreover, P. graminis produces resting spores that can lie dormant but viable in soil for several decades, and the viruses are protected from the environment within the spores. This makes crop rotation inadequate as a control measure. Often entire fields infected with bymoviruses can only be used to grow non-cereal crops. Therefore, in recent years considerable scientific effort has been directed mainly towards breeding disease-resistant varieties.
One recessive gene, rym4, that confers complete immunity against BaYMV and BaMMV has been extensively used in breeding for resistance against these viruses in Europe, where the majority of resistant commercial barley cultivars carry this gene (Graner & Bauer, 1993). However, in the late 1980s another pathotype of BaYMV, BaYMV-2, which is able to overcome rym4-controlled resistance, was detected in Germany and United Kingdom and later in several other European countries (Adams, 1991; Hariri et al., 1990; Huth, 1989; Steyer et al., 1995). Very little is known about the pathogenicity mechanisms deployed by BaYMV and BaMMV to attack barley or about the mechanism(s) for recessively inherited resistance to these viruses. Therefore, knowledge of the molecular interaction between bymoviruses and cereal crops is urgently required to help develop novel options for control, or even to prevent these soil-borne virus diseases before they become widespread.
Despite serious efforts in the past, pathotype 2 of BaYMV can only be distinguished from the common BaYMV-1 isolates by its ability to infect rym4 genotypes. Several previous attempts to differentiate the pathotypes by serological and molecular techniques have failed (Hariri et al., 1996; Shi et al., 1995, 1996), because there were no consistent differences between the viral coat protein sequences of the two pathotypes and because only selected regions of the genome or limited numbers of virus isolates were examined.
The current study aimed to investigate which of the two genomic RNAs determines virulence towards resistant varieties and which BaYMV-2 cistron(s) are responsible for rym4 resistance breaking.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Isolate BaYMV-1ASL and isolates BaYMV-2ASL and BaYMV-2GB had been maintained in climate chambers by serial mechanical passages for about 10 years on susceptible or resistant (rym4) barley plants, respectively.
Immunocapture (IC)-RT-PCR, cloning and sequencing.
IC-RT-PCR was performed according to Mumford & Seale (1997). To amplify fragments of the BaYMV genome by IC-RT-PCR, sap from infected leaves was incubated overnight at 4 °C in PCR tubes pre-coated with anti-BaYMV IgG. The RNA was reverse-transcribed and then amplified by PCR using sequence-specific primers. The DNA fragments obtained were separated electrophoretically and when required were extracted from the agarose gel using the Wizard PCR Purification kit (Promega) and then cloned into pGEM-T (Promega) according to the manufacturer's instructions. cDNA clones were subsequently sequenced with universal M13 forward and reverse primers in both orientations on the ALF-Express automated sequencer (Pharmacia) using Cy5-labelled primers and the ThermoSequenase Cycle Sequencing Kit (Amersham Pharmacia Biotech). Sequence data were processed using the software package Vector NTI 6 (InforMax Inc.).
Primers FY (5'-AGGAACCAAGACCTGTTACTCA-3') and RY (5'-AGGAGGAAGAAATGGATAATGT-3') were used for differential detection of two mechanically transmitted BaYMV pathotypes. These primers were designed to amplify the P2 cistron fragment corresponding to nt 1480–2903 of the wild-type RNA2 of BaYMV-1MPI (EMBL accession no. D01099; Davidson et al., 1991).
Two pairs of primers (P15, 5'-GCTGTTGAGAGCAAACTATGTG-3', with P16, 5'-GAAACTGTCCTCGGTGTTCT-3', and P17a, 5'-CATCAGCGGAAGCTACTAGAAGAAA-3', with P18, 5'-TGGTTCCTCAATAGCAAAAG-3') were designed for amplification of overlapping DNA fragments in the region of RNA1 polymorphic between BaYMV-2GB and BaYMV-1ASL by IC-RT-PCR in routine co-inoculation experiments. The RT-PCR products were isolated, cloned and sequenced. For each primer pair and each plant RNA sample, five individual clones were sequenced in both orientations.
Cloning and sequencing of full-length viral cDNAs.
Double-stranded cDNAs that corresponded to full-length RNA1 and RNA2 of BaYMV were produced by long-range PCR using the Expand Long Template PCR System (Invitrogen) following reverse transcription with the Expand Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Reverse transcription used either isolated viral RNA or total plant RNA as a template and the specific primers RESK-20 [5'-(T)14GGTCCTCGTAATTGAACTAAGTGAACCACCAT-3'] for RNA1, or RESK-21 [5'-(T)14GTCACATTTCCTGTGTACAAAGGTTGTTGTTC-3'] for RNA2. The long-range PCR was performed with primer RESK-19 [5'-(A)6TAAAACAACCCTAAACCAAAACAAACAAAACGA-3'] in combination with either RESK-20 or RESK-21 for RNA1 and RNA2, respectively. The resulting DNA fragments were separated electrophoretically, extracted from the agarose gel and cloned into the pCR-XL-TOPO vector (Invitrogen) as recommended by the manufacturer. Full-length cDNA clones were sequenced with BaYMV-specific primers using the ABI-377 automated DNA sequencer (Amersham) and the ABI PRISM BigDye Terminators Kit (Applied Biosystems). Sequence data were processed using the UNIX-based Staden software package (Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK) or programs from the Wisconsin (GCG) package. Sequences were aligned using CLUSTALW version 1.8 (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Timpe & Kühne, 1994, 1995). RNA2 molecules of three German mechanically transmitted laboratory isolates of BaYMV (BaYMV-1ASL, BaYMV-2ASL, BaYMV-2GB) were also noticeably shorter than those of field isolates. Cloning and sequencing of the 3' portions of the RNA2 molecules showed, as predicted, that P2 cistrons of these isolates contain large isolate-specific deletions of variable size (Fig. 1 and supplementary data Fig. 1 at JGV Online: http://vir.sgmjournals.org; EMBL accession nos AJ517479–AJ51781). IC-RT-PCR using primers FY and RY, which flank the P2 cistron region prone to deletions, and genomic RNA of BaYMV-1ASL, BaYMV-2ASL and BaYMV-2GB as templates consistently resulted in amplification of DNA fragments of the expected sizes of 497, 560 and 596 bp, respectively (data not shown). To investigate whether RNA2 molecules of these pathotypes could occur together in the same leaf, seedlings of the susceptible barley variety Corona were co-inoculated with BaYMV-1ASL and BaYMV-2GB. IC-RT-PCR with leaves showing typical virus symptoms showed that the two isolates occurred either separately or together in single leaves and that isolate-specific PCR fragments could easily be identified and differentiated by gel electrophoresis (Fig. 2).
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); and (ii) BaYMV-2 is likely to be less virulent than BaYMV-1, as after the mechanical inoculation the proportion of susceptible plants infected with BaYMV-2 (30–50 %) is consistently smaller than that with BaYMV-1 (50–90 %) and as judged by ELISA the BaYMV-2 titre in the infected plants is usually lower that that for BaYMV-1 (data not shown). The latter phenomenon is quite common – the plant pathogen's acquired ability to overcome disease resistance usually comes at a cost of associated reduced fitness (Jones, 2001; Leach et al., 2001).
Eleven plants of variety Express (rym4) out of 25 co-inoculated with both virus isolates also developed typical symptoms of virus infection, which were indistinguishable from those seen on susceptible genotypes: yellow streaking, especially of younger leaves, and also small brown necrotic patches. Individual leaves showing symptoms were tested by IC-RT-PCR for the presence of BaYMV RNA2 (Fig. 3). Based on the size of the amplified DNA fragments, the majority of samples (eight) contained the RNA2 of both BaYMV-2GB and BaYMV-1ASL, one sample contained only the BaYMV-2GB RNA2 and, importantly, in two samples only the BaYMV-1ASL RNA2-specific amplification product was detected. To confirm that the smaller DNA fragments (see Fig. 3) predicted to be BaYMV-1ASL RNA2 were not further deletions of BaYMV-2GB RNA2 or recombinant BaYMV-2GB/BaYMV-1ASL RNA2 molecules, three of these fragments (from sample nos 1, 7 and 18) were recovered from the gel, cloned and sequenced. As expected, all sequences were identical and characteristic of BaYMV-1ASL RNA2 molecules.
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RNA1 of BaYMV is required for pathogenicity
Polymorphism in RNA1 sequences between the two pathotypes was then used to determine whether infection by BaYMV-2 also allows replication and movement of BaYMV-1 RNA1 in the resistant genotypes. Overlapping PCR-derived fragments of RNA1 of both isolates were generated and sequenced, starting at the 5' end of this genome component, to identify polymorphic regions. All primers were designed based on the published sequence of the German isolate BaYMV-1MPI (EMBL accession no. X69757; Peerenboom et al., 1992). This molecular analysis showed that there were a number of consistent nucleotide differences in the VPg and the 5' half of the NIa-proteinase cistrons that could be used to distinguish RNA1 of BaYMV-1ASL and BaYMV-2GB. Several leaf samples of the variety Express (rym4) that had been co-inoculated with two BaYMV pathotypes, including those that had tested positive for the presence of RNA2 from both pathotypes (see Fig. 3
), were subjected to IC-RT-PCR to amplify overlapping DNA fragments in these polymorphic regions of RNA1, as described in the Methods. The sequences of the RT-PCR products obtained from the infected leaves all corresponded to BaYMV-2GB and there were no sequences of BaYMV-1ASL (data not shown).
To rule out the possibility that the elimination of BaYMV-1ASL RNA1 in the co-inoculation experiment was caused by factors other than the presence of rym4, a control co-inoculation of the susceptible variety Corona was performed. Eleven out of 25 co-inoculated plants showed virus symptoms and tested positive by ELISA. IC-RT-PCR on the individual leaf samples was performed with the RNA1-specific P17a/P18 primer pair. The amplified DNA fragments were cloned and five independent clones from each of the 11 RT-PCR reactions were sequenced. The sequencing results demonstrated that BaYMV-1ASL RNA1 was predominant in the samples from the co-inoculated susceptible plants that do not possess the rym4 gene. This was exactly as expected if BaYMV-2 has a reduced fitness.
These experiments therefore show that RNA1 of BaYMV-2 determines its ability to overcome the rym4-controlled resistance.
Pathogenicity towards the rym4 barley genotypes correlates with amino acid changes in the RNA1-encoded VPg protein
To determine whether nucleotide or amino acid substitutions in the BaYMV genome correlate with resistance breaking, the complete, or almost complete, RNA1 molecules were cloned and sequenced from three BaYMV-1 (BaYMV-1EST, accession no. AJ515479; BaYMV-1ROT, AJ515485; BaYMV-1ASL, AJ515484) and five BaYMV-2 isolates (BaYMV-2CRW, AJ515480; BaYMV-2HAT, AJ515481; BaYMV-2ASL, AJ515483; BaYMV-2BRZ, AJ515482; BaYMV-2GB, AJ517478). All sequences had a single predicted ORF of identical size and were very similar to one another and to the published German BaYMV-1MPI isolate (X69757). Nucleotide identity between the different sequences was between 97·9 and 99·4 %, with amino acid identities of 98·3–99·7 %. The most variable regions were the P3 protein and the VPg (Fig. 4).
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at JGV Online: http://vir.sgmjournals.org). These sequencing data are in agreement with the results of virus co-inoculation experiments of rym4 barley varieties described above and strengthen the case against the involvement of RNA2 in resistance-breaking.
However, there was one distinct and consistent amino acid sequence difference between polyproteins encoded by RNA1 of BaYMV-1 and BaYMV-2 isolates. This was at aa 1307, which is in the central region of VPg. To rule out the possibility that this difference might be a spurious coincidence, the VPg region only was cloned and sequenced from four more German BaYMV-1 (BaYMV-1WST, BaYMV-1QLB, BaYMV-1SLA, BaYMV-1HKL) and BaYMV-2 (BaYMV-2QLB, BaYMV-2VIR, BaYMV-2GB, BaYMV-2SLA) field isolates and compared with the corresponding sequence of other isolates (Fig. 5 and supplementary data Fig. 2 at JGV Online: http://vir.sgmjournals.org). All BaYMV-1 isolates examined encoded a lysine at aa 1307, whereas BaYMV-2 isolates encoded an asparagine (or, in BaYMV-2ASL, a histidine) at this position.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
An extensive sequencing analysis of 16 BaYMV isolates was used to identify a region in the VPg protein that is polymorphic between rym4 resistance-breaking and non resistance-breaking isolates (Fig. 5). A substitution of the basic amino acid lysine (K1307) for the uncharged amide asparagine (N1307) is likely to affect the biochemical properties of VPg. We believe that the same hypothesis applies to the VPg protein of BaYMV-2ASL, which contains the weak basic amino acid histidine at position 1307. The isoelectric point (pI) of lysine is 9·7, whereas histidine has nearly a neutral pI of 7·6. Also, lysine and histidine have structurally different side chains with very different pKa values of 10·79 and 6·06, respectively. Moreover, the VPg of BaYMV-2ASL contains two more unique amino acid substitutions (lysine
arginine at position 1216 and valineisoleucine at position 1297), which may additionally contribute to a conformational change and affect the ability of this protein to interact with other molecules. Interestingly, it was recently reported that a single amino acid substitution in the VPg protein of spontaneous mutants of Potato virus Y (PVY) correlates with their ability to overcome tobacco va gene-specified resistance (Masuta et al., 1999).
The VPg protein is a multifunctional protein necessary for potyvirus multiplication and movement in host plants (Rajamäki & Valkonen, 2002; Schaad et al., 1997). Both the recessive resistance gene and the corresponding virus component that determines pathogenicity have been characterized for several other potyviruses. Thus, it was shown that variations in the VPg protein allow Tobacco vein mottling virus to overcome va gene resistance in tobacco (Nicolas et al., 1997) and Tobacco etch virus (TEV) to break resistance controlled by two unlinked recessive genes in the tobacco cultivar V20 (Schaad et al., 1997). The ability of Lettuce mosaic virus (LMV) to overcome mo11 and mo12 resistance genes of Lactuca sativa was also mapped to the 3' half of the LMV genome, which encompasses the VPg cistron (Redondo et al., 2001).
It is becoming clear that in many characterized Potyvirus/plant interaction systems modifications of the VPg protein are responsible for resistance breaking. In the current study, we have shown that this appears also to be the case for the bymovirus BaYMV/rym4 barley interaction. Multifunctionality of potyviral VPg and the recessive nature of resistance suggest it is likely that in susceptible plants there is a functional interaction between this virus protein and plant proteins encoded by the dominant alleles of resistance genes. In resistant plants, this host factor component may be either missing or modified and therefore compromises a particular step of virus infection (Revers et al., 1999). By using the yeast two-hybrid system to test for protein–protein interactions, two laboratories have independently demonstrated that the VPg protein of Turnip mosaic virus (TuMV) and TEV interact with the plant cap binding protein eIF4E (Léonard et al., 2000; Schaad et al., 2000). Importantly, the recessive Arabidopsis mutants that lack eIF(iso)4E were immune to TuMV and TEV (Lellis et al., 2002). Moreover, it was recently demonstrated that a natural recessive resistance gene, pvr2, against PVY corresponds to the eukaryotic translation initiation factor eIF4E (Ruffel et al., 2002). Therefore, eIF4E and eIF(iso)4E play an important role in potyvirus genome expression or replication at the single-cell level. It remains to be seen whether the BaYMV VPg protein interacts with the eIF4E or eIF(iso)4E homologue of barley and whether these genes play an important role in host resistance during bymovirus infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 12 May 2003;
accepted 16 June 2003.
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