Defense-Related Gene Expression Following an Orthotospovirus Infection Is Influenced by Host Resistance in Arachis hypogaea.

Planting resistant cultivars is the most effective tactic to manage the thrips-transmitted tomato spotted wilt orthotospovirus (TSWV) in peanut plants. However, molecular mechanisms conferring resistance to TSWV in resistant cultivars are unknown. In this study, transcriptomes of TSWV-susceptible (SunOleic 97R) and field-resistant (Tifguard) peanut cultivars with and without TSWV infection were assembled and differentially expressed genes (DEGs) were compared. There were 4605 and 2579 significant DEGs in SunOleic 97R and Tifguard, respectively. Despite the lower number of DEGs in Tifguard, an increased proportion of defense-related genes were upregulated in Tifguard than in the susceptible cultivar. Examples included disease resistance (R) proteins, leucine-rich repeats, stilbene synthase, dicer, and calmodulin. Pathway analysis revealed the increased downregulation of genes associated with defense and photosynthesis in the susceptible cultivar rather than in the resistant cultivar. These results suggest that essential physiological functions were less perturbed in the resistant cultivar than in the susceptible cultivar and that the defense response following TSWV infection was more robust in the resistant cultivar than in the susceptible cultivar.

Arachis virus Y, a new potyvirid from Brazilian forage peanut (Arachis pintoi).

The complete nucleotide sequence of a new member of the family Potyviridae, which we propose to name "Arachis virus Y" (ArVY), is reported from forage peanut plants (Arachis pintoi) exhibiting virus-like symptoms. The ArVY positive-sense RNA genome is 9,213 nucleotides long and encodes a polyprotein with 2,947 amino acids that is predicted to be cleaved into 10 mature proteins. The complete single open reading frame (ORF) of ArVY shares 47% and 34% nucleotide and amino acid sequence identity, respectively, with the closest related virus, soybean yellow shoot virus. Electron microscopic analysis revealed elongated viral particles typical of those found in plant cells infected with potyviruses.

RNA sequence analysis of diseased groundnut (Arachis hypogaea) reveals the full genome of groundnut rosette assistor virus (GRAV).

The complete genome sequences for two variant isolates of groundnut rosette assistor virus (GRAV) have been determined from symptomatic groundnut plants in western Kenya. The sequences of the two GRAV isolates (sc7.1 and sc7.2) are 84.2% identical at the nucleotide level and 98.5% identical at the coat protein level. The variants sc7.1 and sc7.2 comprise 5850 and 5879 nucleotides respectively, and show similar genome organizations with 7 predicted ORFs (P0, P1, P2, P3a, P3 (coat protein, CP), P4 (movement protein, MP) and P5 (coat protein-readthrough protein, CP-RT). Currently, GRAV is an unassigned virus in the Luteoviridae family, due to the fact that only the sequence of the coat protein was previously obtained. The presence of both ORF0 and ORF 4 within the genome sequence determined in the current work suggest that GRAV should be classified as a member of the genus Polerovirus.

A recombination bin-map identified a major QTL for resistance to Tomato Spotted Wilt Virus in peanut (Arachis hypogaea).

Tomato spotted wilt virus (TSWV) is a devastating disease to peanut growers in the South-eastern region of the United States. Newly released peanut cultivars in recent years are crucial as they have some levels of resistance to TSWV. One mapping population of recombinant inbred line (RIL) used in this study was derived from peanut lines of SunOleic 97R and NC94022. A whole genome re-sequencing approach was used to sequence these two parents and 140 RILs. A recombination bin-based genetic map was constructed, with 5,816 bins and 20 linkage groups covering a total length of 2004 cM. Using this map, we identified three QTLs which were colocalized on chromosome A01. One QTL had the largest effect of 36.51% to the phenotypic variation and encompassed 89.5 Kb genomic region. This genome region had a cluster of genes, which code for chitinases, strictosidine synthase-like, and NBS-LRR proteins. SNPs linked to this QTL were used to develop Kompetitive allele specific PCR (KASP) markers, and the validated KASP markers showed expected segregation of alleles coming from resistant and susceptible parents within the population. Therefore, this bin-map and QTL associated with TSWV resistance made it possible for functional gene mapping, map-based cloning, and marker-assisted breeding. This study identified the highest number of SNP makers and demonstrated recombination bin-based map for QTL identification in peanut. The chitinase gene clusters and NBS-LRR disease resistance genes in this region suggest the possible involvement in peanut resistance to TSWV.

Complete genome sequence of peanut virus C, a putative novel ilarvirus.

We determined the complete genome sequence of a putative novel ilarvirus, tentatively named "peanut virus C" (PVC), identified in peanut (Arachis hypogaea). The three segmented genomic RNA molecules of PVC were 3474 (RNA1), 2925 (RNA2), and 2160 (RNA3) nucleotides in length, with five predicted open reading frames containing conserved domains and motifs that are typical features of ilarviruses. The three genomic RNAs shared nucleotide sequence similarity (74% identity and 93% query coverage for RNA1, 75% identity and 85% query coverage for RNA2, and 72% identity and 70% query coverage for RNA3) with the most closely related ilarvirus, parietaria mottle virus. These results suggest that PVC is a novel member of the genus Ilarvirus in the family Bromoviridae.

Comparison of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae) as Vectors for a Peanut Strain of Tomato Spotted Wilt Orthotospovirus.

Tomato spotted wilt orthotospovirus (TSWV) is a major disease in peanut, Arachis hypogaea L., across peanut producing regions of the United States and elsewhere. Two thrips, Frankliniella fusca Hinds and Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), are considered important vectors of TSWV in peanut in the Southeast. We compared the efficiency of acquisition (by larvae) and transmission (adults) of both thrips species for TSWV (Texas peanut-strain) to leaf disks of peanut (Florunner), as well as to Impatiens walleriana Hook. f. (Dwarf White Baby) and Petunia hybrida Juss. 'Fire Chief' using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Both species were competent TSWV vectors in peanut and Impatiens, although F. fusca was the more efficient vector overall, i.e., virus acquisition and transmission rates for F. fusca averaged over several bioassays were 51.7 and 26.6%, respectively, compared with 20.0 and 15.3% for F. occidentalis. Neither species effectively transmitted this TSWV strain to Petunia (i.e., ≤3.6% transmission). We found statistically similar virus acquisition and transmission rates between both sexes for each species. We also detected no differences in TSWV-acquisition and transmission frequency between macropterous and brachypterous (short-wing) forms of F. fusca collected from a field population in south Texas. DAS-ELISA failed to detect low levels of TSWV in a few thrips that subsequently proved to be competent vectors.

Characterization and Genetic Structure of a Tospovirus Causing Chlorotic Ring Spots and Chlorosis Disease on Peanut; Comparison with Iranian and Polish Populations of Tomato yellow fruit ring virus.

A Tospovirus species was isolated from peanut plants showing chlorotic ring spots and chlorosis, and identified as Tomato yellow fruit ring virus (TYFRV) on the basis of its biological, serological, and molecular properties. In host range studies, a broad range of indicator plants was infected by the five isolates studied; all the isolates systemically infected Nicotiana tabacum cultivars and, thus, they were classified into the N-host-infecting type isolates of the virus. These isolates strongly reacted with TYFRV antibodies but not with the specific antibodies of other tospoviruses tested. Recombination analyses showed that the nucleoprotein gene of the peanut isolates and other isolates studied were nonrecombinant. In phylogenetic trees, the virus isolates were clustered in three genogroups: IRN-1, IRN-2, and a new group, POL; the peanut isolates fell into IRN-2 group. Multiple sequence alignments showed some genogroup-specific amino acid substitutions among the virus isolates studied. The results revealed the presence of negative selection in TYFRV populations. Also, the Iranian populations had higher nucleotide diversity compared with the Polish population. Genetic differentiation and gene flow analyses indicated that the populations from Iran and Poland and those belonging to different genogroups were partially differentiated populations. Our findings seem to suggest that there has been frequent gene flow between some populations of the virus in the mid-Eurasian region of Iran.

Influence of Groundnut bud necrosis virus on the Life History Traits and Feeding Preference of Its Vector, Thrips palmi.

The effect of Groundnut bud necrosis virus (GBNV) infection on the life history traits of its vector, Thrips palmi, and its feeding preference on GBNV-infected plants were studied. A significant difference was observed in the developmental period (first instar to adult) between the GBNV-infected and healthy thrips, wherein the developmental period of GBNV-infected thrips was decreased. However, there was no effect on the other parameters such as preadult mortality, adult longevity, and fecundity. Further investigation on a settling and feeding choice assay of T. palmi to GBNV-infected and healthy plants showed that T. palmi preferred GBNV-infected cowpea plants more than the healthy cowpea plants. This preference was also noticed for leaf disks from GBNV-infected cowpea, groundnut, and tomato plants.

Three decades of managing Tomato spotted wilt virus in peanut in southeastern United States.

Southeastern states namely Georgia, Florida, and Alabama produce two-thirds of the peanuts in the United States. Thrips-transmitted Tomato spotted wilt virus (TSWV), which causes spotted wilt disease, has been a major impediment to peanut production for the past three decades. The cultivars grown in the 1980s were extremely susceptible to TSWV. Early yield losses extended to tens of millions of dollars each year (up to 100% loss in many fields). This situation led to the creation of an interdisciplinary team known as "SWAT: Spotted Wilt Action Team". Initial efforts focused on risk mitigation using a combination of chemical and cultural management practices along with a strong investment in breeding programs. Beginning in the mid 1990s, cultivars with field resistance were developed and integrated with cultural and chemical management options. A Risk Mitigation Index (Peanut Rx) was made available to growers to assess risks, and provide options for mitigating risks such as planting field resistant cultivars with in-furrow insecticides, planting after peak thrips incidence, planting in twin rows, and increasing seeding rates. These efforts helped curtail losses due to spotted wilt. The Peanut Rx continues to be refined every year based on new research findings. Breeding efforts, predominantly in Georgia and Florida, continue to develop cultivars with incremental field resistance. The present-day cultivars (third-generation TSWV-resistant cultivars released after 2010) possess substantially greater field resistance than second-generation (cultivars released from 2000 to 2010) and first-generation (cultivars released from 1994 to 2000) TSWV resistant cultivars. Despite increased field resistance, these cultivars are not immune to TSWV and succumb under high thrips and TSWV pressure. Therefore, field resistant cultivars cannot serve as a 'stand-alone' option and have to be integrated with other management options. The mechanism of resistance is also unknown in field resistant cultivars. Recent research in our laboratory evaluated field resistant cultivars against thrips and TSWV. Results revealed that some resistant cultivars suppressed thrips feeding and development, and they accumulated fewer viral copies than susceptible cultivars. Transcriptomes developed with the aid of Next Generation Sequencing revealed differential gene expression patterns following TSWV infection in susceptible than field resistant cultivars. Results revealed that the upregulation of transcripts pertaining to constitutive and induced plant defense proteins in TSWV resistant cultivars was more robust over susceptible cultivars. On the flipside, the long-term effects of using such resistant cultivars on TSWV were assessed by virus population genetics studies. Initial results suggest lack of positive selection pressure on TSWV, and that the sustainable use of resistant cultivars is not threatened. Follow up research is being conducted. Improvements in TSWV management have enhanced sustainability and contributed to increased yields from <2800kg/ha before 1995 to ∼5000kg/ha in 2015.

Identification of major QTLs underlying tomato spotted wilt virus resistance in peanut cultivar Florida-EP(TM) '113'.

BACKGROUND: Spotted wilt caused by tomato spotted wilt virus (TSWV) is one of the major peanut (Arachis hypogaea L.) diseases in the southeastern United States. Occurrence, severity, and symptoms of spotted wilt disease are highly variable from season to season, making it difficult to efficiently evaluate breeding populations for resistance. Molecular markers linked to spotted wilt resistance could overcome this problem and allow selection of resistant lines regardless of environmental conditions. Florida-EP(TM) '113' is a spotted wilt resistant cultivar with a significantly lower infection frequency. However, the genetic basis is still unknown. The objective of this study is to map the major quantitative trait loci (QTLs) linked to spotted wilt resistance in Florida-EP(TM) '113'. RESULTS: Among 2,431 SSR markers located across the whole peanut genome screened between the two parental lines, 329 were polymorphic. Those polymorphic markers were used to further genotype a representative set of individuals in a segregating population. Only polymorphic markers on chromosome A01 showed co-segregation between genotype and phenotype. Genotyping by sequencing (GBS) of the representative set of individuals in the segregating population also depicted a strong association between several SNPs on chromosome A01 and the trait, indicating a major QTL on chromosome A01. Therefore marker density was enriched on the A01 chromosome. A linkage map with 23 makers on chromosome A01 was constructed, showing collinearity with the physical map. Combined with phenotypic data, a major QTL flanked by marker AHGS4584 and GM672 was identified on chromosome A01, with up to 22.7 % PVE and 9.0 LOD value. CONCLUSION: A major QTL controlling the spotted wilt resistance in Florida-EP(TM) '113' was identified. The resistance is most likely contributed by PI 576638, a hirsuta botanical-type line, introduced from Mexico with spotted wilt resistance. The flanking markers of this QTL can be used for further fine mapping and marker assisted selection in peanut breeding programs.

Mapping Quantitative Trait Loci of Resistance to Tomato Spotted Wilt Virus and Leaf Spots in a Recombinant Inbred Line Population of Peanut (Arachis hypogaea L.) from SunOleic 97R and NC94022.

Peanut is vulnerable to a range of diseases, such as Tomato spotted wilt virus (TSWV) and leaf spots which will cause significant yield loss. The most sustainable, economical and eco-friendly solution for managing peanut diseases is development of improved cultivars with high level of resistance. We developed a recombinant inbred line population from the cross between SunOleic 97R and NC94022, named as the S-population. An improved genetic linkage map was developed for the S-population with 248 marker loci and a marker density of 5.7 cM/loci. This genetic map was also compared with the physical map of diploid progenitors of tetraploid peanut, resulting in an overall co-linearity of about 60% with the average co-linearity of 68% for the A sub-genome and 47% for the B sub-genome. The analysis using the improved genetic map and multi-season (2010-2013) phenotypic data resulted in the identification of 48 quantitative trait loci (QTLs) with phenotypic variance explained (PVE) from 3.88 to 29.14%. Of the 48 QTLs, six QTLs were identified for resistance to TSWV, 22 QTLs for early leaf spot (ELS) and 20 QTLs for late leaf spot (LLS), which included four, six, and six major QTLs (PVE larger than 10%) for each disease, respectively. A total of six major genomic regions (MGR) were found to have QTLs controlling more than one disease resistance. The identified QTLs and resistance gene-rich MGRs will facilitate further discovery of resistance genes and development of molecular markers for these important diseases.

Evaluation of Alternatives to Carbamate and Organophosphate Insecticides Against Thrips and Tomato Spotted Wilt Virus in Peanut Production.

Thrips are important pests of peanut. They cause severe feeding injuries on peanut foliage in the early season. They also transmit Tomato spotted wilt virus (TSWV), which causes spotted wilt disease. At-plant insecticides and cultivars that exhibit field resistance to TSWV are often used to manage thrips and spotted wilt disease. Historically, peanut growers used the broad-spectrum insecticides aldicarb (IRAC class 1A; Temik) and phorate (IRAC class 1B; Thimet) for managing thrips and thereby reducing TSWV transmission. Aldicarb has not been produced since 2011 and its usage in peanut will be legally phased out in 2018; therefore, identification of alternative chemistries is critical for thrips and spotted wilt management. Here, eight alternative insecticides, with known thrips activity, were evaluated in field trials conducted from 2011 through 2013. In addition, different application methods of alternatives were also evaluated. Imidacloprid (Admire Pro), thiamethoxam (Actara), spinetoram (Radiant), and cyantraniliprole (Exirel) were as effective as aldicarb and phorate in suppressing thrips, but none of the insecticides significantly suppressed spotted wilt incidence. Nevertheless, greenhouse assays demonstrated that the same alternative insecticides were effective in suppressing thrips feeding and reducing TSWV transmission. Spotted wilt incidence in the greenhouse was more severe (∼80%) than in the field (5­25%). In general, field resistance to TSWV in cultivars only marginally influenced spotted wilt incidence. Results suggest that effective management of thrips using alternative insecticides and subsequent feeding reduction could improve yields under low to moderate virus pressure.

Effects of Thrips Density, Mode of Inoculation, and Plant Age on Tomato Spotted Wilt Virus Transmission in Peanut Plants.

Spotted wilt caused by tomato spotted wilt virus (TSWV; family Bunyaviridae; genus Tospovirus) is a serious disease of peanut (Arachis hypogaea L.) in the southeastern United States. Peanut genotypes with field resistance to TSWV are effective in suppressing spotted wilt. All commercially available genotypes with field resistance to TSWV were developed through conventional breeding. As a part of the breeding process, peanut genotypes are regularly screened under field situations. Despite numerous advantages associated with field screening, it is often limited by inconsistent vector (thrips) and TSWV pressure. A greenhouse transmission protocol would aid in thorough screening of selected genotypes and conserve time. In this study, various parameters associated with TSWV transmission, including tobacco thrips, Frankliniella fusca (Hinds) density, mode of inoculation, and plant age, were evaluated. Greater incidences of TSWV infection were obtained with thrips-mediated inoculation when compared with mechanical inoculation. TSWV inoculation with three, five, and 10 thrips resulted in greater incidences of TSWV infection in plants than inoculation with one thrips. However, incidences of TSWV infection did not vary between plants inoculated with three, five, and 10 viruliferous thrips. With both thrips-mediated and mechanical inoculation methods, incidences of TSWV infection in 1-wk-old plants were greater than in 4-wk-old plants. TSWV copy numbers, as determined by qPCR, also decreased with plant age. Results suggest that using at least three thrips per plant and 1- to 2-wk-old plants would maximize TSWV infection in inoculated plants.

Simultaneous detection of groundnut rosette assistor virus (GRAV), groundnut rosette virus (GRV) and satellite RNA (satRNA) in groundnuts using multiplex RT-PCR.

Groundnut rosette disease (GRD) is the most devastating disease of groundnuts in sub-Saharan Africa. The disease is caused by synergistic interactions between viruses and virus-like pathogens: groundnut rosette assistor virus (GRAV), groundnut rosette virus (GRV) and a satellite RNA (satRNA). The multi-pathogenic nature of GRD requires efficient diagnostic systems for plant breeding and pathology work. Currently, TAS-ELISA and RT-PCR are used to detect all three pathogens. This approach is time-consuming, expensive and not easily amenable to high throughput. A multiplex PCR-based approach was developed to detect all three pathogens at once, reducing diagnostics costs and time by two thirds. The technique is highly robust and amenable to high throughput, with sensitivity and specificity values of 88 % and 100 %, respectively. The positive predictive value for the technique is 100 %, and the negative predictive value is 90.6 %.

Host plant resistance against tomato spotted wilt virus in peanut (Arachis hypogaea) and its impact on susceptibility to the virus, virus population genetics, and vector feeding behavior and survival.

Tomato spotted wilt virus (TSWV) severely affects peanut production in the southeastern United States. Breeding efforts over the last three decades resulted in the release of numerous peanut genotypes with field resistance to TSWV. The degree of field resistance in these genotypes has steadily increased over time, with recently released genotypes exhibiting a higher degree of field resistance than older genotypes. However, most new genotypes have never been evaluated in the greenhouse or laboratory against TSWV or thrips, and the mechanism of resistance is unknown. In this study, TSWV-resistant and -susceptible genotypes were subjected to TSWV mechanical inoculation. The incidence of TSWV infection was 71.7 to 87.2%. Estimation of TSWV nucleocapsid (N) gene copies did not reveal significant differences between resistant and susceptible genotypes. Parsimony and principal component analyses of N gene nucleotide sequences revealed inconsistent differences between virus isolates collected from resistant and susceptible genotypes and between old (collected in 1998) and new (2010) isolates. Amino acid sequence analyses indicated consistent differences between old and new isolates. In addition, we found evidence for overabundance of nonsynonymous substitutions. However, there was no evidence for positive selection. Purifying selection, population expansion, and differentiation seem to have influenced the TSWV populations temporally rather than positive selection induced by host resistance. Choice and no-choice tests indicated that resistant and susceptible genotypes differentially affected thrips feeding and survival. Thrips feeding and survival were suppressed on some resistant genotypes compared with susceptible genotypes. These findings reveal how TSWV resistance in peanut could influence evolution, epidemiology, and management of TSWV.

Second generation peanut genotypes resistant to thrips-transmitted tomato spotted wilt virus exhibit tolerance rather than true resistance and differentially affect thrips fitness.

Spotted wilt disease caused by Tomato spotted wilt virus (TSWV) (family Bunyaviridae; genus Tospovirus) is a major constraint to peanut (Arachis hypogaea L.) production in the southeastern United States. Reducing yield losses to TSWV has heavily relied on planting genotypes that reduce the incidence of spotted wilt disease. However, mechanisms conferring resistance to TSWV have not been identified in these genotypes. Furthermore, no information is available on how these genotypes influence thrips fitness. In this study, we investigated the effects of newly released peanut genotypes (Georganic, GA-06G, Tifguard, and NC94022) with field resistance to TSWV and a susceptible genotype (Georgia Green) on tobacco thrips, Frankliniella fusca (Hinds), fitness, and TSWV incidence. Thrips-mediated transmission resulted in TSWV infection in both TSWV-resistant and susceptible genotypes and they exhibited typical TSWV symptoms. However, some resistant genotypes had reduced viral loads (fewer TSWV N-gene copies) than the susceptible genotype. F. fusca larvae acquired TSWV from resistant and susceptible genotypes indicating that resistant genotypes also can serve as inoculum sources. Unlike resistant genotypes in other crops that produce local lesions (hypersensitive reaction) upon TSWV infection, widespread symptom development was noticed in peanut genotypes. Results indicated that the observed field resistance in peanut genotypes could be because of tolerance. Further, fitness studies revealed some, but not substantial, differences in thrips adult emergence rates and developmental time between resistant and susceptible genotypes. Thrips head capsule length and width were not different when reared on different genotypes.

Identification of expressed resistance gene analogs from peanut (Arachis hypogaea L.) expressed sequence tags.

Low genetic diversity makes peanut (Arachis hypogaea L.) very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat (NBS-LRR) resistance genes (R) have been identified, but a small portion with expressed transcripts has been found. We aimed to identify resistance gene analogs (RGAs) from peanut expressed sequence tags (ESTs) and to develop polymorphic markers. The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases. A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered, and assembled 156 contigs and 229 singletons as peanut-expressed RGAs. There were 69 that encoded for NBS-LRR proteins, 191 that encoded for protein kinases, 82 that encoded for LRR-PK/transmembrane proteins, 28 that encoded for Toxin reductases, 11 that encoded for LRR-domain containing proteins and four that encoded for TM-domain containing proteins. Twenty-eight simple sequence repeats (SSRs) were identified from 25 peanut expressed RGAs. One SSR polymorphic marker (RGA121) was identified. Two polymerase chain reaction-based markers (Ahsw-1 and Ahsw-2) developed from RGA013 were homologous to the Tomato Spotted Wilt Virus (TSWV) resistance gene. All three markers were mapped on the same linkage group AhIV. These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes.

Pathogen-derived resistance using a viral nucleocapsid gene confers only partial non-durable protection in peanut against peanut bud necrosis virus.

Genetic engineering of peanut (Arachis hypogaea L.) using the gene encoding for the nucleocapsid protein (N gene) of peanut bud necrosis virus (PBNV; genus Tospovirus, family Bunyaviridae) was used to impart resistance to bud necrosis disease in peanut (PBND), a disease for which no durable resistance is available in the existing germplasm. Over 200 transgenic lines of peanut var. JL 24 were developed for which integration and expression of the transgenes was confirmed by PCR, Southern hybridization, RT-PCR and western blot analysis. The T(1) and T(2) generation transgenic plants were assayed through virus challenge in the greenhouse by using mechanical sap inoculation at 1:100 and 1:50 dilutions of PBNV, and they showed varying levels of disease incidence and intensity. Greenhouse and field evaluation with T(2) generation plants indicated somewhat superior performance of the three transgenic events that showed considerable reduction in disease incidence. However, only one of these events showed over 75 % reduction in disease incidence when compared to the untransformed control, indicating partial and non-durable resistance to PBND using the viral N-gene.

Analysis of two strains of Peanut stunt virus: satRNA-associated and satRNA free.

Peanut stunt virus (PSV) is a pathogen of legumes, vegetables, trees, and weeds occurring worldwide. The species is characterized by significant genetic variability. PSV strains are classified into four subgroups on the basis of their nucleotide sequence homology. Here, we are presenting two further, fully sequenced PSV strains-PSV-Ag and PSV-G, that could be considered as I subgroup representatives. However, their sequence homology with other typical I subgroups members, similarly as another strain-PSV-P, characterized by our group previously, is lower than 90%. This lead us to propose further subdivision of the I subgroup into IA, IB, and IC units, and to classify PSV-Ag and PSV-G strains to the last one. In this article, we are showing that identity level of PSV-Ag and PSV-G is very high and apart from the presence of satRNA in the first one, they differ only by a few nucleotides in their genomic RNAs. Nevertheless, symptoms they cause on host plants might differ significantly, just as the levels in infected plants. Effect of single amino acid changes between strains on the three-dimensional structure of viral proteins was analyzed. Differences occur mainly on the protein surfaces which can possibly affect protein-protein interaction in infected cells, which is discussed.

Interference in plant defense and development by non-structural protein NSs of Groundnut bud necrosis virus.

Groundnut bud necrosis virus (GBNV) infects a large number of leguminous and solanaceous plants. To elucidate the biological function of the non-structural protein encoded by the S RNA of GBNV (NSs), we studied its role in RNA silencing suppression and in viral pathogenesis. Our results demonstrated that GBNV NSs functions as a suppressor of RNA silencing using the agroinfiltration patch assay. An in silico analysis suggested the presence of pro-apoptotic protein Reaper-like sequences in the GBNV NSs, which were known to be present in animal infecting bunyaviruses. Utilizing NSs mutants, we demonstrated that a Leu-rich domain was required for RNA silencing suppression activity, but not the non-overlapping Trp/GH3 motif of the Reaper-like sequence. To investigate the role of NSs in symptom development we generated transgenic tomato expressing the GBNV NSs and showed that the expression of NSs in tomato mimics symptoms induced by infection with GBNV, such as leaf senescence and necrosis. As leaf senescence is controlled by miR319 regulation of the transcription factor TCP1, we assessed the accumulation of both RNAs in transgenic NSs-expressing and GBNV-infected tomato plants. In both types of plants the levels of miR319 decreased, while the levels of TCP1 transcripts increased. We propose that GBNV-NSs affects miRNA biogenesis through its RNA silencing suppressor activity and interferes with TCP1-regulated leaf developmental pathways.