Biological characterization of French Potato virus Y (PVY) isolates collected from PVY‐susceptible or ‐resistant tobacco plants possessing the recessive resistance gene va

Improved tobacco cultivars introgressed with alleles of the recessive resistance va gene have been widely deployed in France to limit agronomical consequences associated with Potato virus Y (PVY) infections. Unfortunately, necrotic symptoms associated with PVY have been reported on these cultivars suggesting that PVY is able to overcome the resistance. A field survey was performed in France in 2007 to (i) estimate the prevalence of PVY in tobacco plants showing symptoms and (ii) characterize PVY isolates present in susceptible and va‐derived tobacco cultivars. A serological typing procedure, applied to 556 leaves collected from different French tobacco growing areas, was performed using polyclonal antisera raised against different viral species including PVY. Viral species were detected in 80·8% of leaves and PVY was present in 83·5% of infected samples. However, statistical analysis confirmed that the probability of a tobacco plant being infected with PVY is reduced in va hosts. Eighty‐six PVY isolates were mechanically inoculated on one susceptible and three va‐derived tobacco cultivars used as indicator hosts to define virulence of these isolates against alleles 0, 1 and 2 of the va gene. Both qualitative and quantitative analyses showed that 55 PVY isolates were able to overcome the three va alleles. Moreover, the monitored biological diversity of PVY isolates was higher in the susceptible tobacco hosts than in the va‐derived ones. This study helps to understand consequences of the deployment of the va gene in tobacco on diversity and virulence of PVY isolates.     

Impact of tobacco recessive resistance gene va on biological properties of Brazilian Potato virus Y (PVY) isolates

To investigate the role of environmental conditions on the selection of virulent Potato virus Y (PVY) isolates subject to pressure from the recessive resistance gene va in tobacco, a field survey was performed in Brazil where va‐derived genotypes have been recently introduced and now represent less than one‐third of cultivated tobacco genotypes. A serological analysis of 397 leaves collected from different Brazilian tobacco‐growing areas and mainly from plants with symptoms indicated that 52·4% of samples were infected by at least one of the viral species tested. PVY was present in 72·1% of infected samples. The probability of a plant being infected with PVY was reduced in va hosts. However, the biological characterization of PVY isolates on indicator hosts showed that 20 of the 29 tested isolates were able to overcome the alleles of the va gene. Moreover, the observed biological diversity of isolates was higher in susceptible tobacco genotypes than in va‐resistant ones. Comparison of these data with the PVY diversity in French tobacco fields shows that the use of va‐derived genotypes in two environments with contrasting climatic conditions, local hosts and cultural contexts, leads to a similar outcome: the prevalence of virulent isolates. These results strongly suggest an important role of the va gene in the modification of PVY populations.     

A novel type of Potato virus Y recombinant genome,determined for the genetic strain PVYE

Two Potato virus Y (PVY) isolates collected in Brazil, PVY‐AGA and PVY‐MON, were identified as recombinants between two parent genomes, PVY^NTN and PVY‐NE‐11, with a novel type of genomic pattern. The new recombinants had an ordinary PVY^NTN genome structure for approximately 6·7‐kb from the 5′‐end of the genome whereas the 3′‐terminal 3·0‐kb segment had two fragments of NE‐11‐like sequence separated by another small PVY^NTN‐like fragment. PVY strains are defined based on the hypersensitive resistance (HR) response in potato indicators. Both PVY‐AGA and PVY‐MON isolates did not induce the HR in potato cultivars carrying Ny, Nc, or (putative) Nz genes and thus were able to overcome all known resistance genes to PVY. Only one of the two isolates, PVY‐AGA, induced a vein necrosis reaction in tobacco. The biological responses of the potato indicators and tobacco defined PVY‐MON as an isolate of the PVY^E strain. To distinguish PVY‐AGA and PVY‐MON from other PVY^NTN isolates, an RT‐PCR test was developed utilizing new specific primers from the capsid protein gene area and producing a characteristic 955‐bp band. Serological profiling of these PVY isolates with three monoclonal antibodies revealed an unusual reactivity, where one of the two commercial PVY^N‐specific monoclonal antibodies did not recognize PVY‐AGA. The ability of these new PVY recombinants to overcome resistance genes in potato producing mild or no symptoms, combined with the lack of serological reactivity towards at least one PVY^N‐specific antibody may present a significant threat posed by these isolates to seed potato production areas.     

Field resistance against potato virus Y infection using natural and genetically engineered resistance genes

Commercial tobacco cultivars BB16 (burley) and PBD6 (dark air cured) were transformed with the coat protein gene of lettuce mosaic potyvirus (LMV). Transgenic BB16 plants showed resistance to potato virus Y (PVY) infection, against the necrotic strain PVY-N Versailles, as well as the resistance breaking necrotic strain PVY-N 107. Transgenic PBD6, which carries the recessive va gene conferring resistance to PVY, was also protected against PVY-N 107. In the progenies of most BB16 and PBD6 transformants, 45% to 100% of the inoculated plants were immune to PVY infection. The rest of the plants were tolerant, with atypical attenuated non necrotic symptoms and reduced virus accumulation. No recovery was observed in the tolerant plants, which stably expressed detectable level of LMV coat protein. This suggested a protein-mediated mechanism of heterologous protection.     

Brazilian Potato virus Y isolates identified as members of a new clade facilitate the reconstruction of evolutionary traits within this species

Potato virus Y (PVY) is a plant virus distributed worldwide that causes damage to several species of the Solanaceae family. It was established long ago that groups of PVY isolates defined by phylogenetic analyses correlate strongly with those demarcated by differential biological properties. Consequently, life‐history traits of this viral species can be inferred by phylogenetic analysis. In this study, characteristics of PVY isolates sampled in different tobacco fields in Brazil were analysed and most of the tested Brazilian PVY isolates were assigned to the recently described unconventional serogroup Y^U. The analysis of molecular diversity of the coat protein (CP) cistron from some Y^U isolates made it possible (i) to identify specific amino acid residues in the N‐terminal of the CP protein and (ii) to assign some Y^U isolates to a new PVY clade. The symptoms caused by isolates belonging to this new PVY ‘Brazilian’ clade and their ability to infect selected susceptible hosts led to the conclusion that neither veinal necrosis symptoms expressed on infected tobacco plants nor adaptation to potato or pepper hosts are ancestral characteristics of PVY. These observations suggest that PVY has gained a remarkable new biological property and broadened its host range over time.     

The effects of co‐infection by different Potato virus Y (PVY) isolates on virus concentration in solanaceous hosts and efficiency of transmission

The influence of co‐infection on concentration and accumulation of genetically different isolates of Potato virus Y (PVY) in potato and tobacco plants and the efficiency of transmission by Myzus persicae of PVY isolates from doubly versus singly infected plants were evaluated. The vector ability to simultaneously transmit two virus isolates was examined. Eight PVY isolates represented three strain groups: PVY^O (pathotype and serotype O), PVY^NW (pathotype N and serotype O), and PVY^NTN (pathotype and serotype N). Different diagnostic methods, including DAS‐ELISA, multiplex RT‐PCR, aphid transmission tests and bioassays, were applied to detect the presence of PVY isolates in source and assay plants. Significant reductions in concentrations of certain PVY isolates during co‐infection with other isolates were found both in potato and tobacco plants. The observed effects were both isolate‐ and host‐dependent in form. The highest rates of virus transmission by single aphids were recorded with PVY^NTN isolates, and the lowest ones with PVY^O isolates. Individual aphids of M. persicae were able to simultaneously transmit two PVY isolates. The frequency of transmission was generally low, but it reached as high as 20% for one of the isolate combinations. The findings presented in the work provide proof for antagonistic within‐plant interactions between isolates of PVY, with some implications of these interactions for virus transmission by aphid vectors. Consequently, this research contributes to a better understanding of the epidemiology of the disease caused by PVY.     

A single amino Acid change in viral genome-associated protein of potato virus y correlates with resistance breaking in 'virgin a mutant' tobacco

ABSTRACT Tobacco cultivar Virgin A Mutant (VAM) is reported to have the recessive potyvirus resistance gene va. Varied levels of resistance were observed in VAM plants inoculated with Japanese potato virus Y (PVY) isolates. VAM was highly resistant to most of the PVY isolates tested and tolerant to three necrotic strain isolates of PVY-T. Based on data obtained from tissue printing and press blotting, the resistance appeared to be mainly at the level of cell-to-cell movement. PVY replicated in VAM proto-plasts, but the replication was 30% lower than in susceptible tobacco, suggesting that impairment of replication also contributes to resistance. To identify the viral gene product or products involved in VAM resistance, we isolated spontaneous resistance-breaking mutants by passing vein-banding (O strain) isolates several times through VAM plants. By comparing the amino acid sequences of the mutants with their original isolates, we identified a single amino acid substitution in the viral genome-associated protein (VPg) domain that is correlated with VAM resistance breaking. Together, these results suggest that, in addition to its role in replication, VPg plays an important role in the cell-to-cell movement of PVY.     

Mendelian inheritance of rust resistance to Melampsora larici‐epitea in crosses between Salix sachalinensis and S. viminalis

Two F₁, two F₂ and two backcross (BC) full‐sib families of Salix sachalinensis × S. viminalis were tested for resistance to two pathotypes of Melampsora larici‐epitea in leaf‐disc inoculation experiments. Two single‐pustule isolates, VM and ST, belonging to pathotypes LET1 and LET5, respectively, were used in the tests. Disease was scored based on the number of uredinia, uredinial diameter and inoculum densities. Both F₁ families were completely resistant to both VM and ST. Resistance to VM segregated at a 9:6:1 ratio in the F₂ families and at a 1:2:1 ratio in the BC families, suggesting that two independently segregating genes controlled rust resistance, with resistance dominant over susceptibility. This also indicates incomplete dominance of the resistance alleles over the susceptibility to VM. For ST, the equivalent ratios were 3:1 and 1:1, showing that a single dominant gene was responsible for rust resistance. The broad sense heritabilities were >0·91 for uredinial diameter and 0·1–0·33 for the number of uredinia. There were significant overall correlations between data from inoculations with VM and those from inoculations with ST in the number of uredinia, uredinial diameter and disease scores (Spearman’s rank correlation coefficients = 0·31–0·75).     

A classification of <Emphasis Type="Italic">Pepper yellow mosaic virus</Emphasis> isolates into pathotypes

Pepper yellow mosaic virus (PepYMV) is the most important potyvirus infecting sweet pepper in Brazil. In this study, twenty isolates of PepYMV were obtained from commercial sweet pepper crops. To confirm virus identity, the coat protein gene was completely sequenced for eleven of these isolates, and partially sequenced for the other nine isolates. The amino acid identities obtained were above 93% when compared with the sequence of a characterized PepYMV isolate (AF348610). Extracts of Nicotiana tabacum cv. TNN plants infected with the different isolates were used to inoculate the differential series of Capsicum spp cultivars containing the genes pvr2 ¹ , pvr2 ² , pvr2 ³ , pvr2 ⁴ , and Pvr4. Using the same criteria established for Potato virus Y (PVY), fourteen isolates of PepYMV could be classified as known pathotypes described for PVY, that is: 1.2 (2 isolates), 1.3 (6) and 1.2.3 (6). The remaining six isolates, 1.3 (2) and 1.2.3 (4) could not be classified into the typical pathotypes of PVY because they were also virulent on Serrano Criollo de Morellos—334 (C.M 334) which carries the pvr2 ³ and Pvr4 genes. To classify the PepYMV into pathotypes and counter the biological diversity found in this species we propose the utilization of 2^x for the ability to overcome the correspondent allele of the pvr2 locus and 4 for the capacity to break down the Pvr4 gene. Using this criterion we could classify the PepYMV into five pathotypes: 2¹.2²; 2¹.2³; 2¹.2².2³; 2¹.2³. 4 and 2¹.2².2³. 4.     

Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper: support for the matching allele model in eIF4E resistance–potyvirus interactions

The pathogenic properties of Potato virus Y (PVY; genus Potyvirus, family Potyviridae) isolates collected in naturally infected pepper (Capsicum annuum) fields in Tunisia were evaluated against recessive resistance alleles at the pvr2 locus of pepper. Two pathotypes were observed. Pathotype (0,1,3) isolates were able to infect plants carrying the susceptibility allele pvr2⁺, together with pvr2¹/pvr2¹ and pvr2³/pvr2³ plants, but not pvr2²/pvr2² plants. Pathotype (0) isolates were only able to infect pvr2⁺/pvr2⁺ plants. On the other hand, sequence data and phylogenetic analyses revealed three major groups of isolates, each characterized by particular amino acid residues in the central part of the VPg, the pathogenicity factor towards pvr2. Correspondence between pathogenic properties and phylogeny suggested a single evolutionary step for pathogenicity towards the pvr2¹ and pvr2³ resistances, possibly under the selective pressure of pvr2¹. Indeed, 23% of the pepper plants in this area were shown to carry the pvr2¹ resistance, while pvr2³ was not detected. The data suggested that pathogenicity towards pvr2¹ and pvr2³ were not costly for PVY to infect susceptible pepper genotypes and supported the matching allele model for pepper–PVY interactions.     

大丽轮枝菌效应蛋白VdSRP2的功能分析

为明确效应蛋白VdSRP2在大丽轮枝菌Verticillium dahliae中的生物学功能,从大丽轮枝菌落叶型菌株V592中克隆VdSRP2基因并进行生物信息学分析,利用酵母转化酶分泌系统对其信号肽活性进行测定,采用实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR)技术分析VdSRP2基因在大丽轮枝菌中的表达模式,并以V592菌株为材料获得VdSRP2基因的敲除突变体和过表达体菌株,通过表型分析和致病性测定确定VdSRP2基因的生物学功能。结果显示,VdSRP2基因编码232个氨基酸,含有5个半胱氨酸残基,N-端信号肽具有分泌活性,为真菌的典型效应蛋白;VdSRP2基因主要在大丽轮枝菌菌丝和微菌核中表达,其中经棉花根系诱导培养24 h时表达量最高;与野生型菌株V592相比,VdSRP2基因敲除导致大丽轮枝菌的产孢量和孢子萌发率显著下降,不能形成微菌核,对棉花的致病力明显减弱;但VdSRP2基因敲除不影响大丽轮枝菌的穿透能力;VdSRP2基因在本氏烟和棉花叶片瞬时表达不会诱导细胞死亡。表明VdSRP2是大丽轮枝菌微菌核形成必需...     

Hairpin-based virus resistance depends on the sequence similarity between challenge virus and discrete,highly accumulating siRNA species

Virus resistance can be effectively generated in transgenic plants by using the plant’s silencing machinery. To study the specificity of gene-silencing-based resistance, homozygous tobacco (Nicotiana tabacum L.) plants containing a 597-nt hairpin RNA construct of the Potato Virus Y (PVY) replicase sequence were challenged with a variety of PVY strains. The transgene-carrying tobacco line was immune to five potato PVY strains with high sequence similarity (88.3–99.5%) to the transgene. Infection with more distant tomato and pepper PVY field strains (86–86.8% sequence similarity) caused delayed symptom appearance in the transgenic tobacco. Transgene production of small interfering (si) RNA was detected by northern blot and measured using a custom-designed microarray for the detection of small RNAs. siRNA accumulation peaks were observed throughout the inverted-repeat transgene. In the resistance-breaking tomato and pepper strains there were nucleotide differences in the sequences correlated to siRNA transgene accumulation, indicating the role of siRNA specificity in resistance breaking. The log of transgene siRNA signal intensity increased with probe GC content, indicating that the accumulating siRNA molecules were GC-rich. Sequence similarity of highly accumulating siRNAs with the target virus strain appears to be important for both resistance and resistance-breaking characteristics.     

Virus recognition and pathogenicity: Implications for resistance mechanisms and breeding

The major method of control of virus diseases in crop plants is breeding for resistance. The genetics of resistance, and of matching virulence (the ability of a virus strain to overcome a specific host resistance gene) have been studied less for viruses than for fungal and bacterial pathogens. This paper draws on a survey of the genetics of resistance to a large number of viruses in cultivated crops, and makes some generalisations and predictions about mechanisms. Most resistance to viruses in crops is monogenic. Dominant alleles are associated with virus-localisation mechanisms, which are induced after infection. The nature of the ‘recognition event’ between plant- and virus-coded functions, which triggers resistance plus a cascade of secondary responses, is not yet known. Gene dosage-dependent alleles tend to be associated with non-localising resistance, which allows some virus spread, but inhibits multiplication. Recessive alleles may involve a negative type of resistance mechanism, whereby the resistant plant lacks some function normally required by the virus for pathogenesis. Such resistance tends to be expressed as complete immunity. Many resistance genes have been overcome by virulent isolates of viruses; only 10 % of the sample of resistance genes have proved exceptionally durable. Virulence may involve different viral functions. The production of infectious cDNA clones, and construction of chimaeric recombinants between clones of virulent and avirulent isolates, is now allowing detailed mapping of virulence determinants. Transformation of plants with ‘novel’ genes for virus resistance, based on coat proteins and viral satellites, may allow construction of more robust resistance systems.     

Incidence of potato viruses and characterisation of <Emphasis Type="Italic">Potato virus Y</Emphasis> variability in late season planted potato crops in Northern Tunisia

Surveys were conducted of symptomatic potato plants in late season crops, from the major potato production regions in Northern Tunisia, for infection with six common potato viruses. The presence of Potato leafroll virus (PLRV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus A (PVA), Potato virus S (PVS) and Potato virus M (PVM) was confirmed serologically with virus infection levels up to 5.4, 90.2, 4.3, 3.8, 7.1 and 4.8%, respectively. As PVY was prevalent in all seven surveyed regions, further biological, serological and molecular typing of 32 PVY isolates was undertaken. Only one isolate was shown to induce PVY^O-type symptoms following transmission to tobacco and to react only against anti-PVY^O-C antibodies. Typical vein necrosis symptoms were obtained from 31 samples, six of which reacted against both anti-PVY^N and anti-PVY^O-C antibodies showing they contained mixed isolates, while 25 of them reacted only with anti-PVY^N antibodies. An immunocapture RT-PCR molecular test using a PVY^NTN specific primer pair set in the 5’NTR/P1 genomic region and examination of recombinant points in three genomic regions (HC-Pro/P3, CI/NIa and CP/3’NTR) showed that all 25 serotype-N PVY isolates were PVY^NTN variants with similar recombinations to the standard PVYNTN-H isolate. This is the first report of the occurrence of the PVY^NTN variant and its high incidence in late season potatoes in Tunisia.     

Inheritance of fomesafen and imazethapyr resistance in a multiple herbicide-resistant Euphorbia heterophylla population

Euphorbia heterophylla populations endued with resistance to both imazethapyr (an acetolactate synthase inhibitor) and fomesafen (a protoporphyrinogen oxidase inhibitor) were first detected in Brazil in 2004. The objectives of this study were to determine the number and dominance of genes conferring imazethapyr and fomesafen resistance in E. heterophylla and to establish the types of inheritance of the resistance traits. It was hypothesised that two dominant nuclear genes control this herbicide resistance and each gene confers resistance to a single herbicide. We crossed a susceptible (S) and a multiple-resistant (R) E. heterophylla biotype and generated F₁ families which, in turn, were self-fertilised to produce F₂ families. Backcrosses of the F₁ families with the S- and R parental biotypes resulted in BC_s and BC_r families respectively. The F₁, F₂, BC_s and BC_r families and the S and R parental biotypes were subjected to imazethapyr and fomesafen wherein each herbicide was used as a selecting agent. F₁ plants and the S- and R parental biotypes were used to evaluate the dominance of the resistance trait in multiple-dose bioassays with imazethapyr and fomesafen. The observed frequencies of resistant plants in the F₁, F₂, BC_s and BC_r families did not significantly differ from the expected frequencies for a resistance trait regulated by two dominant genes in which each gene confers resistance to a single herbicide. The lack of difference in herbicide response between the F₁ family and the R parental biotype was taken as evidence for completely dominant resistance to imazethapyr and fomesafen. Herbicide resistance in E. heterophylla presents with dominant monogenic nuclear inheritance for each herbicide mode of action. Our findings underscore the necessity and urgency of adopting integrated strategies to control E. heterophylla and to inhibit the evolution of new herbicide-resistant strains.     

Selection forMeloidogyne incognita virulence against resistance genes from tomato and pepper and specificity of the virulence/resistance determinants

Experiments were designed to analyze the relationships between the root-knot nematodeMeloidogyne incognita and resistant tomato and pepper genotypes. From a natural avirulent isolate, near-isogenic nematode lineages were selected with virulence either against the tomatoMi resistance gene or the pepperMe3 resistance gene. Despite the drastic selection pressure used, nematodes appeared unable to overcome the pepperMe1 gene, therefore suggesting some differences in the resistance conferred byMe1 andMe3 in this species. Nematodes virulent onMi-resistant tomatoes were not able to reproduce onMe1-resistant nor onMe3-resistant peppers, and nematodes virulent onMe3-resistant peppers were not able to reproduce onMi-resistant tomatoes nor onMe1-resistant peppers. These results clearly demonstrate the specificity ofM. incognita virulence against resistance genes from both tomato and pepper, and indirectly suggest that gene-for-gene relationships could occur between these two solanaceous crops and the nematode.     

大麦抗条纹病基因的定位分析

为发掘大麦中抗条纹病的新基因,采用三明治法通过人工接种大麦条纹病菌Pyrenophora graminea强致病力菌株QWC对甘啤2号(免疫)与Alexis(高感)杂交F_1代及F_2代分离群体进行抗性遗传分析,利用群体分离分析法鉴定与抗病基因连锁的SSR标记,并通过QTL IciMapping软件构建遗传连锁图谱完成对抗病基因的定位。结果显示,甘啤2号与Alexis杂交F_1代对大麦条纹病菌强致病力菌株QWC表现为免疫,F_2代表现3∶1抗感分离,表明甘啤2号对菌株QWC的抗性由1个显性抗性基因控制,将该抗病基因暂命名为Rdg3;该基因位于大麦7H染色体上的SSR标记Bmag206和Bmag7之间,与二者的遗传距离分别为1.78 cM和2.86 cM。经与已定位于7H染色体上的抗病基因比较,发现Rdg3是一个新的抗条纹病基因,可作为大麦抗病育种的新种质资源。     

马铃薯Y病毒复制酶基因不同位置cDNA区段介导对PVY的不同抗性

为探究靶序列位置对RNA介导的病毒抗性产生的影响,利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增马铃薯Y病毒(Potato virus Y,PVY)复制酶基因(nuclear inclusion b,NIb)不同位置的cDNA区段,反向插入双元载体pROKII中,构建了发夹RNA(hairpin RNA,hpR-NA)结构的植物表达载体。将构建的植物表达载体采用冻融法转入农杆菌LBA4404,叶盘法转化烟草NC89,获得转基因植株。攻毒试验表明:PVYNIb基因不同位置cDNA区段介导的对PVY的抗性存在显著差异;3′端1/2处和中间位置的序列可介导高水平的病毒抗性,抗性植株的比例在50%以上,而5′端、5′端1/2处和3′端的序列介导的抗性效率较低,抗性植株的比例仅为10%~30%。Northern杂交显示:抗病植株中RNA的积累量明显低于同类型的感病植株,抗性与RNA积累量呈负相关;抗病转基因植株中有siRNA存在,表明病毒抗性是由RNA介导的。