Identification and functional analyses of host factors interacting with the 17-kDa protein of Barley yellow dwarf virus-GAV.

Barley yellow dwarf viruses (BYDVs) cause significant economic losses on barley, wheat, and oats worldwide. 17-kDa protein (17K) of BYDVs plays a key role in viral infection in plants, whereas the underlying regulation mechanism of 17K in virus infection remains elusive. In this study, we determined that 17K of BYDV-GAV, the most common species found in China in recent years, was involved in viral pathogenicity. To identify the host factors interacting with 17K, the full length coding sequence of 17K was cloned into pGBKT7 to generate the bait plasmid pGBKT7-17K. 114 positive clones were identified as possible host factors to interact with 17K through screening a tobacco cDNA library. Gene ontology enrichment analysis showed that they were classified into 35 functional groups, involving three main categories including biological processes (BP), cellular components (CC), and molecular functions (MF). Kyoto Encyclopedia of Genes and Genome (KEGG) analysis indicated the acquired genes were assigned to 49 KEGG pathways. The majority of these genes were involved in glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms, and glycolysis/gluconeogenesis. The interactions between 17K and the 27 proteins with well-documented annotations were verified by conducting yeast two-hybrid assays and 12 of the 27 proteins were verified to interact with 17K. To explore the putative function of the 12 proteins in BYDV-GAV infection, the subcellular localization and expression alterations in the presence of BYDV-GAV were monitored. The results showed that, under the condition of BYDV-GAV infection, RuBisCo, POR, and PPD5 were significantly up-regulated, whereas AEP and CAT1 were significantly down-regulated. Our findings provide insights into the 17K-mediated BYDV-GAV infection process.

Barley yellow dwarf virus-GAV-derived vsiRNAs are involved in the production of wheat leaf yellowing symptoms by targeting chlorophyll synthase.

BACKGROUND: Wheat yellow dwarf virus disease is infected by barley yellow dwarf virus (BYDV), which causes leaf yellowing and dwarfing symptoms in wheat, thereby posing a serious threat to China's food production. The infection of plant viruses can produce large numbers of vsiRNAs, which can target host transcripts and cause symptom development. However, few studies have been conducted to explore the role played by vsiRNAs in the interaction between BYDV-GAV and host wheat plants. METHODS: In this study, small RNA sequencing was conducted to profile vsiRNAs in BYDV-GAV-infected wheat plants. The putative targets of vsiRNAs were predicted by the bioinformatics software psRNATarget. RT-qPCR and VIGS were employed to identify the function of selected target transcripts. To confirm the interaction between vsiRNA and the target, 5' RACE was performed to analyze the specific cleavage sites. RESULTS: From the sequencing data, we obtained a total of 11,384 detected vsiRNAs. The length distribution of these vsiRNAs was mostly 21 and 22 nt, and an A/U bias was observed at the 5' terminus. We also observed that the production region of vsiRNAs had no strand polarity. The vsiRNAs were predicted to target 23,719 wheat transcripts. GO and KEGG enrichment analysis demonstrated that these targets were mostly involved in cell components, catalytic activity and plant-pathogen interactions. The results of RT-qPCR analysis showed that most chloroplast-related genes were downregulated in BYDV-GAV-infected wheat plants. Silencing of a chlorophyll synthase gene caused leaf yellowing that was similar to the symptoms exhibited by BYDV-GAV-inoculated wheat plants. A vsiRNA from an overlapping region of BYDV-GAV MP and CP was observed to target chlorophyll synthase for gene silencing. Next, 5' RACE validated that vsiRNA8856 could cleave the chlorophyll synthase transcript in a sequence-specific manner. CONCLUSIONS: This report is the first to demonstrate that BYDV-GAV-derived vsiRNAs can target wheat transcripts for symptom development, and the results of this study help to elucidate the molecular mechanisms underlying leaf yellowing after viral infection.

Transcriptome response comparison between vector and non-vector aphids after feeding on virus-infected wheat plants.

BACKGROUND: Plant viruses maintain intricate interactions with their vector and non-vector insects and can impact the fitness of insects. However, the details of their molecular and cellular mechanisms have not been studied well. We compared the transcriptome-level responses in vector and non-vector aphids (Schizaphis graminum and Rhopalosiphum padi, respectively) after feeding on wheat plants with viral infections (Barley Yellow Dwarf Virus (BYDV) and Wheat dwarf virus (WDV), respectively). We conducted differentially expressed gene (DEG) annotation analyses and observed DEGs related to immune pathway, growth, development, and reproduction. And we conducted cloning and bioinformatic analyses of the key DEG involved in immune. RESULTS: For all differentially expressed gene analyses, the numbers of DEGs related to immune, growth, development, reproduction and cuticle were higher in vector aphids than in non-vector aphids. STAT5B (signal transducer and activator of transcription 5B), which is involved in the JAK-STAT pathway, was upregulated in R. padi exposed to WDV. The cloning and bioinformatic results indicated that the RpSTAT5B sequence contains a 2082 bp ORF encoding 693 amino acids. The protein molecular weight is 79.1 kD and pI is 8.13. Analysis indicated that RpSTAT5B is a non-transmembrane protein and a non-secreted protein. Homology and evolutionary analysis indicated that RpSTAT5B was closely related to R. maidis. CONCLUSIONS: Unigene expression analysis showed that the total number of differentially expressed genes (DEGs) in the vector aphids was higher than that in the non-vector aphids. Functional enrichment analysis showed that the DEGs related to immunity, growth and reproduction in vector aphids were higher than those in non-vector aphids, and the differentially expressed genes related to immune were up-regulated. This study provides a basis for the evaluation of the response mechanisms of vector/non-vector insects to plant viruses.

Enhanced heat tolerance of viral-infected aphids leads to niche expansion and reduced interspecific competition.

Vector-borne pathogens are known to alter the phenotypes of their primary hosts and vectors, with implications for disease transmission as well as ecology. Here we show that a plant virus, barley yellow dwarf virus, increases the surface temperature of infected host plants (by an average of 2 °C), while also significantly enhancing the thermal tolerance of its aphid vector Rhopalosiphum padi (by 8 °C). This enhanced thermal tolerance, which was associated with differential upregulation of three heat-shock protein genes, allowed aphids to occupy higher and warmer regions of infected host plants when displaced from cooler regions by competition with a larger aphid species, R. maidis. Infection thereby led to an expansion of the fundamental niche of the vector. These findings show that virus effects on the thermal biology of hosts and vectors can influence their interactions with one another and with other, non-vector organisms.

Modeling Approach Influences Dynamics of a Vector-Borne Pathogen System.

The choice of a modeling approach is a critical decision in the modeling process, as it determines the complexity of the model and the phenomena that the model captures. In this paper, we developed an individual-based model (IBM) and compared it to a previously published ordinary differential equation (ODE) model, both developed to describe the same biological system although with slightly different emphases given the underlying assumptions and processes of each modeling approach. We used both models to examine the effect of insect vector life history and behavior traits on the spread of a vector-borne plant virus, and determine how choice of approach affects the results and their biological interpretation. A non-random distribution of insect vectors across plant hosts emerged in the IBM version of the model and was not captured by the ODE. This distribution led simultaneously to a slower-growing vector population and a faster spread of the pathogen among hosts. The IBM model also enabled us to test the effect of potential control measures to slow down virus transmission. We found that removing virus-infected hosts was a more effective strategy for controlling infection than removing vector-infested hosts. Our findings highlight the need to carefully consider possible modeling approaches before constructing a model.

Molecular and Ultrastructural Mechanisms Underlying Yellow Dwarf Symptom Formation in Wheat after Infection of Barley Yellow Dwarf Virus.

Wheat (Tritium aestivum L.) production is essential for global food security. Infection of barley yellow dwarf virus-GAV (BYDV-GAV) results in wheat showing leaf yellowing and plant dwarfism symptom. To explore the molecular and ultrastructural mechanisms underlying yellow dwarf symptom formation in BYDV-GAV-infected wheat, we investigated the chloroplast ultrastructure via transmission electron microscopy (TEM), examined the contents of the virus, H2O2, and chlorophyll in Zhong8601, and studied the comparative transcriptome through microarray analyses in the susceptible wheat line Zhong8601 after virus infection. TEM images indicated that chloroplasts in BYDV-GAV-infected Zhong8601 leaf cells were fragmentized. Where thylakoids were not well developed, starch granules and plastoglobules were rare. Compared with mock-inoculated Zhong8601, chlorophyll content was markedly reduced, but the virus and H2O2 contents were significantly higher in BYDV-GAV-infected Zhong8601. The transcriptomic analyses revealed that chlorophyll biosynthesis and chloroplast related transcripts, encoding chlorophyll a/b binding protein, glucose-6-phosphate/phosphate translocator 2, and glutamyl-tRNA reductase 1, were down-regulated in BYDV-GAV-infected Zhong8601. Some phytohormone signaling-related transcripts, including abscisic acid (ABA) signaling factors (phospholipase D alpha 1 and calcineurin B-like protein 9) and nine ethylene response factors, were up-regulated. Additionally, reactive oxygen species (ROS)-related genes were transcriptionally regulated in BYDV-GAV infected Zhong8601, including three up-regulated transcripts encoding germin-like proteins (promoting ROS accumulation) and four down-regulated transcripts encoding peroxides (scavenging ROS). These results clearly suggest that the yellow dwarf symptom formation is mainly attributed to reduced chlorophyll content and fragmentized chloroplasts caused by down-regulation of the chlorophyll and chloroplast biosynthesis related genes, ROS excessive accumulation, and precisely transcriptional regulation of the above-mentioned ABA and ethylene signaling- and ROS-related genes in susceptible wheat infected by BYDV-GAV.

The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing.

The plant viral family Luteoviridae is divided into three genera: Luteovirus, Polerovirus and Enamovirus. Without assistance from another virus, members of the family are confined to the cells of the host plant's vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant's viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant's silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant's anti-viral defense.

Luteovirus: insights into pathogenicity.

Luteoviruses are economically important plant viruses, infecting almost all cereals throughout the world. Idiosyncrasies related to this virus group may be a strategic consequence of viral genome compression. However, many fundamental questions have yet to be resolved. This review summarizes selected findings covering molecular aspects of pathogenesis relating to plant-infecting RNA viruses in general, and luteoviruses in specific. These studies enhance our understanding of the replication structures and the virus infection pathways.

The effect of elevated temperature on Barley yellow dwarf virus-PAV in wheat.

Barley yellow dwarf virus-PAV (BYDV-PAV) is associated with yellow dwarf disease, one of the most economically important diseases of cereals worldwide. In this study, the impact of current and future predicted temperatures for the Wimmera wheat growing district in Victoria, Australia on the titre of BYDV-PAV in wheat was investigated. Ten-day old wheat (Triticum aestivum, cv. Yitpi) seedlings were inoculated with BYDV-PAV and grown at ambient (5.0-16.1°C, night-day) or elevated (10.0-21.1°C, night-day) temperature treatments, simulating the current Wimmera average and future daily temperature cycles, respectively, during the wheat-growing season. Whole above-ground plant samples were collected from each temperature treatment at 0 (day of inoculation), 3, 6, 9, 12, 15, 18, 21 and 24 days after inoculation and the titre of BYDV-PAV was measured in each sample using a specific one-step multiplex normalised reverse transcription quantitative PCR (RT-qPCR) assay. Physical measurements, including plant height, dry weight and tiller number, were also taken at each sampling point. The titre of BYDV-PAV was significantly greater in plants grown in the elevated temperature treatment than in plants grown in the ambient treatment on days 6, 9 and 12. Plants grown at elevated temperature were significantly bigger and symptoms associated with BYDV-PAV were visible earlier than in plants grown at ambient temperature. These results may have important implications for the epidemiology of yellow dwarf disease under future climates in Australia.

Agrobacterium-mediated infection of whole plants by yellow dwarf viruses.

Barley yellow dwarf virus-PAV (BYDV-PAV) and cereal yellow dwarf virus-RPV (CYDV-RPV) are only transmitted between host plants by aphid vectors and not by mechanical transmission. This presents a severe limitation for the use of a reverse genetics approach to analyze the effects of mutations in these viruses on plant infection and aphid transmission. Here we describe the use of agroinfection to infect plants with BYDV-PAV and CYDV-RPV. The cDNAs corresponding to the complete RNA genomes of BYDV-PAV and CYDV-RPV were cloned into a binary vector under the control of the cauliflower mosaic virus 35S promoter and the nopaline synthase transcription termination signal. The self-cleaving ribozyme from hepatitis virus D was included to produce a transcript in planta with a 3' terminus identical to the natural viral RNA. ELISA and RT-PCR analysis showed that the replicons of BYDV-PAV and CYDV-RPV introduced by Agrobacterium into Nicotiana benthamiana and N. clevelandii gave rise to a local infection in the infiltrated mesophyll cells. After several weeks systemic infection of phloem tissue was detected, although no systemic symptoms were observed. Three heterologous virus silencing suppressors increased the efficiency of agroinfection and accumulation of BYDV-PAV and CYDV-RPV in the two Nicotiana species. The progeny viruses purified from infiltrated tissues were successfully transmitted to oat plants by aphids, and typical yellow dwarf symptoms were observed. This study reports the first agroinfection of eudicot plants using BYDV-PAV and CYDV-RPV.

The community ecology of barley/cereal yellow dwarf viruses in Western US grasslands.

Research on plant viruses in natural ecosystems has been increasing rapidly over the past decade. This paper reviews recent research on the barley and cereal yellow dwarf viruses (B/CYDVs) in grasslands of the western US, beginning with the evidence that the disease caused by these viruses facilitated the invasion of western US grasslands by European annual grasses. Observational and experimental studies of B/CYDVs were carried out along a latitudinal gradient (33.8-48.8°N) from southern California to southern Canada. The prevalence and community composition of B/CYDVs were assessed over a variety of scales and under a range of biotic and abiotic conditions. The findings indicate that both biotic and abiotic factors are important influences on virus ecology and epidemiology. Introduced annual grasses are high-quality hosts that amplify both virus and vector populations in this system, but our research suggests that endemic perennial grasses are critically important for sustaining virus populations in contemporary grasslands largely composed of introduced species. Experiments indicated that increased phosphorus supply to hosts resulted in greater host biomass and higher virus prevalence. Using experimental exclosures, it was found that the presence of grazing vertebrate herbivores increased the abundance of annual grasses, resulting in increased virus prevalence. The results of these studies suggest that patterns of B/CYDV prevalence and coinfection in western US grasslands are strongly shaped by the interactions of host plants, vectors, vertebrate herbivores, and abiotic drivers including nutrients.

The influence of virus-induced changes in plants on aphid vectors: insights from luteovirus pathosystems.

Plant virus infection can alter the suitability of host plants for their aphid vectors. Most reports indicate that virus-infected plants are superior hosts for vectors compared to virus-free plants with respect to vector growth rates, fecundity and longevity. Some aphid vectors respond preferentially to virus-infected plants compared to virus-free ones, while others avoid infected plants that are inferior hosts. Thus, it appears vectors can exploit changes in host plant quality associated with viral infection. Enhanced vector performance and preference for virus-infected plants might also be advantageous for viruses by promoting their spread and possibly enhancing their fitness. Our research has focused on two of the most important luteoviruses that infect wheat (Barley yellow dwarf virus), or potato (Potato leafroll virus), and their respective aphid vectors, the bird-cherry oat aphid, Rhopalosiphum padi, and the green peach aphid, Myzus persicae. The work has demonstrated that virus infection of host plants enhances the life history of vectors. Additionally, it has shown that virus infection alters the concentration and relative composition of volatile organic compounds in host plants, that apterae of each vector species settle preferentially on virus-infected plants, and that such responses are mediated by volatile organic compounds. The findings also indicate that plants respond heterogeneously to viral infection and as a result different plant parts change in attractiveness to vectors during infection and vector responses to virus-infected plants are dynamic. Such dynamic responses could enhance or reduce the probability of virus acquisition by individual aphids searching among plants. Finally, our work indicates that compared to non-viruliferous aphids, viruliferous ones are less or not responsive to virus-induced host plant volatiles. Changes in vector responsiveness to plants after vectors acquire virus could impact virus epidemiology by influencing virus spread. The potential implications of these findings for virus ecology and epidemiology are discussed.

Spatiotemporal model of barley and cereal yellow dwarf virus transmission dynamics with seasonality and plant competition.

Many generalist pathogens are influenced by the spatial distributions and relative abundances of susceptible host species. The spatial structure of host populations can influence patterns of infection incidence (or disease outbreaks), and the effects of a generalist pathogen on host community dynamics in a spatially heterogeneous community may differ from predictions derived via simple models. In this paper, we model the transmission of a generalist pathogen within a patch framework that incorporates the movement of vectors between discrete host patches to investigate the effects of local host community composition and vector movement rates on disease dynamics.We use barley and cereal yellow dwarf viruses (B/CYDV), a suite of generalist, aphid-vectored pathogens of grasses, and their interactions with a range of host species as our case study. We examine whether B/CYDV can persist locally or in a patch framework across a range of host community configurations. We then determine how pathogen-mediated interactions between perennial and annual competitors are altered at the local and regional scale when the host populations are spatially structured. We find that the spatial configuration of the patch system, host composition within patches, and patch connectivity affect not only the ability of the pathogen to invade a fragmented system, but also determine whether the pathogen facilitates the invasion of a non-native host species. Further, our results suggest that connectivity can interact with arrival time and host infection tolerance to determine the success or failure of establishment for newly arriving species.

Pyramiding of Ryd2 and Ryd3 conferring tolerance to a German isolate of Barley yellow dwarf virus-PAV (BYDV-PAV-ASL-1) leads to quantitative resistance against this isolate.

Barley yellow dwarf virus (BYDV) is an economically important pathogen of barley, which may become even more important due to global warming. In barley, several loci conferring tolerance to BYDV-PAV-ASL-1 are known, e.g. Ryd2, Ryd3 and a quantitative trait locus (QTL) on chromosome 2H. The aim of the present study was to get information whether the level of tolerance against this isolate of BYDV in barley can be improved by combining these loci. Therefore, a winter and a spring barley population of doubled haploid (DH) lines were genotyped by molecular markers for the presence of the susceptibility or the resistance encoding allele at respective loci (Ryd2, Ryd3, QTL on chromosome 2H) and were tested for their level of BYDV-tolerance after inoculation with viruliferous (BYDV-PAV-ASL-1) aphids in field trials. In DH-lines carrying the combination Ryd2 and Ryd3, a significant reduction of the virus titre was detected as compared to lines carrying only one of these genes. Furthermore, spring barley DH-lines with this allele combination also showed a significantly higher relative grain yield as compared to lines carrying only Ryd2 or Ryd3. The QTL on chromosome 2H had only a small effect on the level of tolerance in those lines carrying only Ryd2, or Ryd3 or a combination of both, but the effect in comparison to lines carrying no tolerance allele was significant. Overall, these results show that the combination of Ryd2 and Ryd3 leads to quantitative resistance against BYDV-PAV instead of tolerance.

A single-stranded conformational polymorphism (SSCP)-derived quantitative variable to monitor the virulence of a Barley yellow dwarf virus-PAV (BYDV-PAV) isolate during adaptation to the TC14 resistant wheat line.

A standardized single-stranded conformational polymorphism (SSCP) procedure is proposed as an alternative to the time-consuming biological characterization of Barley yellow dwarf virus-PAV (BYDV-PAV) isolates. Using this procedure, six of 21 overlapping regions used to scan the viral genome gave patterns specific to '4E' (avirulent) or '4T' ('4E'-derived virulent) isolates. The calibration of samples and integration of SSCP patterns corresponding to the nucleotide region 1482-2023 allowed the estimation of P(T) values that reflect the proportions of a '4T'-specific band. Analysis of the biological (area under the pathogen progress curve) and molecular (P(T)) data suggested a positive linear relation between these variables. Moreover, sequence analysis of the nucleotide region 1482-2023 highlighted the presence of a nucleotide polymorphism (C/A(1835)) which can be considered as a candidate for virus-host interactions linked to the monitored virulence. According to these parameters, P(T) values associated with '4E'- and '4T'-derived populations show that: (i) long-term infection of a BYDV-PAV isolate on the 'TC14' resistant host leads to the fixation of virulent individuals in viral populations; and (ii) the introduction of susceptible hosts in successive 'TC14' infections results in the maintenance of low virulence of the populations. Thus, the presented study demonstrates that SSCP is a useful tool for monitoring viral populations during the host adaptation process. The described impact of host alternation provides new opportunities for the use of the 'TC14' resistance source in BYDV-resistant breeding programmes. This study is part of the global effort made by the scientific community to propose sustainable alternatives to the chemical control of this viral disease.

Development of EST-PCR markers for Thinopyrum intermedium chromosome 2Ai#2 and their application in characterization of novel wheat-grass recombinants.

A series of expressed sequence tags-derived polymerase chain reaction (EST-PCR) markers specific to chromosome 2Ai#2 from Thinopyrum intermedium were developed in this study using a new integrative approach. The target alien chromosome confers high resistance to barley yellow dwarf virus (BYDV), which is a severe virus disease in wheat. To generate markers evenly distributed on 2Ai#2, a total of 105 primer pairs were designed based on mapped ESTs from 8 bins of wheat chromosome 2B with intron-prediction by aligning ESTs with genomic sequences of the new model plant Brachypodium distachyon. Eight and seven polymorphic markers on the short arm and the long arm of chromosome 2Ai#2, respectively, were obtained with a polymorphism rate of 14.3%. These chromosome 2Ai#2-specific EST-PCR markers were then used in tracing and exploring the structural variation of the alien chromosome in the population derived from the immature embryo culture of the cross between N452, a 2Ai#2(2D) substitution line, and common wheat CB037. Two centric fusion of translocations involving 2Ai#2 short or long arm with wheat chromosome 2D and some new genetic stocks including telosomes with the alien chromosome short or long arm were identified in the SC(3) generations, which provided basic materials to further study the mechanism of the BYDV resistance. BYDV tests in two field seasons suggest that the BYDV resistance was mainly conferred by the short arm, gene interaction on both arms of the alien chromosome was discussed.

Comparison of Thinopyrum intermedium derivatives carrying barley yellow dwarf virus resistance in wheat.

Resistance to both barley yellow dwarf virus (BYDV) and cereal yellow dwarf virus (CYDV) has been demonstrated in wheat genetic stocks with Thinopyrum intermedium chromatin. A number of resistance-bearing translocations have been reported on chromosome arm 7DL from two independent Th. intermedium sources; one source is the addition line L1 and the other is the spontaneous substitution line P29. Another source of resistance in wheat cytogenetic stocks is available as a 2Ai(2D) substitution line. We used a set of 38 molecular markers and the available deletion stocks to compare the size of the 7DL translocations more comprehensively than has been done previously. We also compared the efficacy of BYDV resistance of the various genetic stocks both before and after transfer to a common genetic background. TC14 was confirmed as carrying the smallest translocation, replacing about 20% of the distal end of 7DL. TC5 and TC10 had 90% of the chromosome arm replaced by Th. intermedium chromatin; the proximal 10% corresponded to wheat chromatin. YW642 appeared to have the whole 7DL replaced by Th. intermedium chromatin, as confirmed by the co-dominant marker cfd68 mapping on the bin nearest the centromere. Translocation line P961341 had bins 3, 7, and 8 replaced by Th. intermedium chromatin, making this the second smallest translocation with BYDV and CYDV resistance. The translocation sizes reported here differ from some of the previous estimates. The translocated Th. intermedium segments appeared to be bigger than the replaced wheat 7DL fragments. All the resistances derived from the L1 and P29 group 7 chromosomes and the 2Ai#2 chromosome were effective in reducing the number of infected plants and the mean virus titre, regardless of the background. Some evidence is discussed suggesting the long arm of the Th. intermedium group 7 chromosome 7Ai#1 carries two resistances, the distal Bdv2 and a proximal second gene.

Molecular characterization of the genomic region harboring the BYDV-resistance gene Bdv2 in wheat.

Barley yellow dwarf virus (BYDV) can cause significant losses of wheat worldwide. The long arm segment of Thinopyrum intermedium chromosome 7Ai#1 carrying the BYDV resistance gene Bdv2 was translocated to the distal region of the long arm of wheat chromosome 7D in translocation line Yw642. In this study, 40 wheat EST sequences located in the distal region of 7DL were explored to identify specific PCR markers for the Bdv2 region on the basis of the homoeologous relationship between wheat chromosome 7D and Th.intermedium chromosome 7Ai#1. Our results revealed 8 novel EST-PCR markers specific to the Bdv2 region, including 5 EST-STS markers of BE404744, BE498985, BE591497, BG606695 and BQ161842, and 3 EST-SSCP markers of BE404953, BG312663 and BE498985. These EST-PCR markers could distinguish Bdv2 from another BYDV-resistance gene located on Th.intermedium chromosome 2Ai-2. These specific bands for the Bdv2 region were further cloned and sequenced. The sequencing analysis indicated that the specific sequences for the Bdv2 region were highly homologous with the original wheat EST sequences that were used to design primers, and encode respectively a protein kinase, P450, centrin, transducin, and a hypothetical protein. This study created a starting point for eventual cloning of the Bdv2 gene and understanding the defense mechanism.

A Chinese isolate of barley yellow dwarf virus-PAV represents a third distinct species within the PAV serotype.

The complete nucleotide sequence of barley yellow dwarf virus (BYDV) PAV-CN genomic RNA was determined. This represents the seventh complete genome sequence of a BYDV-PAV serotype. The genome organization of PAV-CN was comparable to that of other BYDV-PAV serotypes, but the nucleotide sequence of full genome was only 76.9-80.3% similar. Sequence similarity of individual open reading frames and untranslated regions (UTR) between PAV-CN and other PAV isolates ranged from 37.9 to 98.2%. Overall, PAV-CN was most similar to BYDV-PAS, which belongs to one of two distinct species within the PAV serotype of BYDV, although the 5' UTR and ORF1 of PAV-CN was most similar to BYDV-GAV, another member of the genus Luteovirus that is not serologically related to BYDV-PAV. These data suggest that PAV-CN may have undergone a recombination event with GAV and that PAV-CN represents a third distinct species within the PAV serotype of BYDV.

Genetic characterization and mapping of major gene resistance to potato leafroll virus in Solanumtuberosum ssp. andigena.

Major gene inheritance of resistance to Potato leafroll virus (PLRV) was demonstrated in a parthenogenic population derived from the highly resistant tetraploid andigena landrace, LOP-868. This major gene or chromosome region seems to control a single mechanism for resistance to infection and virus accumulation in this source. About 149 dihaploid lines segregated in a ratio of 107 resistant to 32 susceptible, fitting the expected ratio for inheritance of a duplex gene under random chromatid segregation. A tetraploid AFLP map was constructed using as reference the ultra high density (UHD) map. All AFLP markers associated with PLRV resistance mapped to the same linkage group. Map position was confirmed by analysis of previously-mapped SSR markers. Rl (adg) is located on the upper arm of chromosome V, at 1 cM from its most closely linked AFLP marker, E35M48.192. This marker will be used to develop allele-specific primers or a pair of flanking PCR-based markers for their use in marker assisted selection.