Rubisco small subunit (RbCS) is co-opted by potyvirids as the scaffold protein in assembling a complex for viral intercellular movement.

Plant viruses must move through plasmodesmata (PD) to complete their life cycles. For viruses in the Potyviridae family (potyvirids), three viral factors (P3N-PIPO, CI, and CP) and few host proteins are known to participate in this event. Nevertheless, not all the proteins engaging in the cell-to-cell movement of potyvirids have been discovered. Here, we found that HCPro2 encoded by areca palm necrotic ring spot virus (ANRSV) assists viral intercellular movement, which could be functionally complemented by its counterpart HCPro from a potyvirus. Affinity purification and mass spectrometry identified several viral factors (including CI and CP) and host proteins that are physically associated with HCPro2. We demonstrated that HCPro2 interacts with both CI and CP in planta in forming PD-localized complexes during viral infection. Further, we screened HCPro2-associating host proteins, and identified a common host protein in Nicotiana benthamiana-Rubisco small subunit (NbRbCS) that mediates the interactions of HCPro2 with CI or CP, and CI with CP. Knockdown of NbRbCS impairs these interactions, and significantly attenuates the intercellular and systemic movement of ANRSV and three other potyvirids (turnip mosaic virus, pepper veinal mottle virus, and telosma mosaic virus). This study indicates that a nucleus-encoded chloroplast-targeted protein is hijacked by potyvirids as the scaffold protein to assemble a complex to facilitate viral movement across cells.

Grapevine vein clearing virus Is Prevalent and Genetically Variable in Grape Aphid (Aphis illinoisensis Shimer) Populations.

Grapevine vein clearing virus (GVCV) causes severe stunting and death of cultivated grapevines and is prevalent in native Vitis spp. and Ampelopsis cordata in the Midwest region of the United States. GVCV can be transmitted from wild A. cordata to Vitis spp. by grape aphid (Aphis illinoisensis) under greenhouse conditions, but its prevalence, genetic composition, and genome number in native grape aphids are unknown. In this study, we collected grape aphids from native Vitaceae across the state of Missouri in 2018 and 2019, and conducted diagnostic, genetic, and quantitative analyses. GVCV was detected in 91 of the 105 randomly sampled communities on 71 Vitaceae plants (87%). It was present in 211 of 525 single grape aphids (40%). Diverse GVCV variants from aphids were present on both GVCV-negative and GVCV-positive plants. Identical GVCV variants were found in grape aphids sampled from wild and cultivated Vitaceae, indicating that viruliferous aphids likely migrate and disperse GVCV variants among wild and cultivated Vitaceae. In addition, we found that the number of GVCV genomes varies largely in the stylet and body of individual aphids. Our study provides a snapshot of GVCV epidemics and genetic structure in its mobile vector and sessile hosts. This presents a good model for studying the epidemiology, ecology, and evolution of a plant virus.

North American Grape 'Norton' is Resistant to Grapevine Vein Clearing Virus.

Grapevines (Vitis spp.) host viruses belonging to 17 families. Virus-associated diseases are a constant challenge to grape production. Genetic resources for breeding virus-resistant grape cultivars are scarce. 'Norton' is a hybrid grape of North American Vitis aestivalis and is resistant to powdery mildew and downy mildew. In this study, we assessed resistance of 'Norton' to grapevine vein clearing virus (GVCV), which is prevalent in native, wild Vitaceae and in vineyards in the Midwest region of the U.S. We did not detect GVCV in 'Norton' as either the scion or the rootstock up to 3 years after it was grafted with a GVCV-infected 'Chardonel' grapevine. Upon sequencing of small RNAs, we were able to assemble the GVCV genome from virus small RNAs in GVCV-infected 'Chardonel' scion or rootstock, but not from grafted 'Norton' scion and rootstock. This study unveils a new trait of 'Norton' that can be used in breeding GVCV-resistant grape cultivars, and to investigate genetic mechanisms of 'Norton' resistance to GVCV.

A Natural Reservoir and Transmission Vector of Grapevine Vein Clearing Virus.

Grapevine vein clearing virus (GVCV) is associated with a vein-clearing and vine-decline disease. In this study, we surveyed wild Ampelopsis cordata from the Vitaceae family and found that 31% (35 of 113) of native A. cordata plants are infected with GVCV. The full-length genome sequence of one GVCV isolate from A. cordata shared 99.8% identical nucleotides with an isolate from a nearby cultivated 'Chardonel' grapevine, suggesting the occurrence of an insect vector. To identify a vector, we collected Aphis illinoisensis (common name: grape aphids) from wild A. cordata plants and detected GVCV in the aphid populations. We found that A. illinoisensis is capable of transmitting GVCV from infected A. cordata to Chardonel grapevines in the greenhouse. Upon transmission, GVCV caused severe symptoms on the infected Chardonel 45 days post transmission. We conclude that wild GVCV isolates from A. cordata are capable of inducing a severe disease on cultivated grapevines once they spread from native A. cordata to vineyards via grape aphids. The discovery of a natural reservoir and an insect vector of GVCV provides timely knowledge for disease management in vineyards and critical clues on viral evolution and epidemiology.

Genetic and Phenotypic Characterization of Grapevine vein clearing virus from Wild Vitis rupestris.

Grapevine vein clearing virus (GVCV), a new member of the genus Badnavirus in the family Caulimoviridae, is associated with a vein clearing and vine decline disease that severely affects grape production and berry quality in commercial vineyards in the Midwest region of the United States. In this paper, the genetic and phenotypic characteristics of GVCV-VRU1 and GVCV-VRU2, two isolates from wild Vitis rupestris grapevines in their native habitat, are described. The GVCV-VRU1 genome is 7,755 bp long while the GVCV-VRU2 genome consists of 7,725 bp, both of which are different from the genome of the GVCV-CHA isolate (7,753 bp), which was originally discovered in the grape cultivar 'Chardonel'. The nucleotide sequence identity among GVCV-VRU1, GVCV-VRU2, and GVCV-CHA ranges from 91.6 to 93.4%, and open reading frame (ORF) II is the most divergent ORF with only 83.3 to 88.5% identity. Sequence analysis of the ORF II indicated that GVCV isolates genetically similar to GVCV-VRU1 and GVCV-VRU2 also are present in commercial vineyards. Symptoms of GVCV-VRU1- or GVCV-VRU2-infected wild V. rupestris grapevine appeared initially as translucent vein clearing on young leaves and progressed to vein necrosis on mature leaves. Inoculation of GVCV-VRU1 or GVCV-VRU2 by grafting onto grape cultivar Chardonel resulted in mild mottle and leaf distortion. The natural range of wild V. rupestris grapevines overlaps with commercial vineyards in the Midwestern United States. Therefore, the discovery of GVCV isolates in wild V. rupestris grapevines has important implications for epidemics and management of the GVCV-associated disease.

Functions of EDS1-like and PAD4 genes in grapevine defenses against powdery mildew.

The molecular interactions between grapevine and the obligate biotrophic fungus Erysiphe necator are not understood in depth. One reason for this is the recalcitrance of grapevine to genetic modifications. Using defense-related Arabidopsis mutants that are susceptible to pathogens, we were able to analyze key components in grapevine defense responses. We have examined the functions of defense genes associated with the salicylic acid (SA) pathway, including ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), EDS1-LIKE 2 (EDL2), EDL5 and PHYTOALEXIN DEFICIENT 4 (PAD4) of two grapevine species, Vitis vinifera cv. Cabernet Sauvignon, which is susceptible to E. necator, and V. aestivalis cv. Norton, which is resistant. Both VaEDS1 and VvEDS1 were previously found to functionally complement the Arabidopsis eds1-1 mutant. Here we show that the promoters of both VaEDS1 and VvEDS1 were induced by SA, indicating that the heightened defense of Norton is related to its high SA level. Other than Va/VvEDS1, only VaEDL2 complemented Arabidopsis eds1-1, whereas Va/VvPAD4 did not complement Arabidopsis pad4-1. Bimolecular fluorescence complementation results indicated that Vitis EDS1 and EDL2 proteins interact with Vitis PAD4 and AtPAD4, suggesting that Vitis EDS1/EDL2 forms a complex with PAD4 to confer resistance, as is known from Arabidopsis. However, Vitis EDL5 and PAD4 did not interact with Arabidopsis EDS1 or PAD4, correlating with their inability to function in Arabidopsis. Together, our study suggests a more complicated EDS1/PAD4 module in grapevine and provides insight into molecular mechanisms that determine disease resistance levels in Vitis species native to the North American continent.

Genetic diversity and tissue and host specificity of Grapevine vein clearing virus.

Grapevine vein clearing virus (GVCV) is a new badnavirus in the family Caulimoviridae that is closely associated with an emerging vein-clearing and vine decline disease in the Midwest region of the United States. It has a circular, double-stranded DNA genome of 7,753 bp that is predicted to encode three open reading frames (ORFs) on the plus-strand DNA. The largest ORF encodes a polyprotein that contains domains for a reverse transcriptase (RT), an RNase H, and a DNA-binding zinc-finger protein (ZF). In this study, two genomic regions, a 570-bp region of the RT domain and a 540-bp region of the ZF domain were used for an analysis of the genetic diversity of GVCV populations. In total, 39 recombinant plasmids were sequenced. These plasmids consisted of three individual clones from each of 13 isolates sampled from five grape varieties in three states. The sequence variants of GVCV could not be phylogenetically grouped into clades according to geographical location and grape variety. Codons of RT or ZF regions are subject to purifying selection pressure. Quantitative polymerase chain reaction assays indicated that GVCV accumulates abundantly in the petioles and least in the root tip tissue. Upon grafting of GVCV-infected buds onto four major grape cultivars, GVCV was not detected in the grafted 'Chambourcin' vine but was present in the grafted 'Vidal Blanc', 'Cayuga White', and 'Traminette' vines, suggesting that Chambourcin is resistant to GVCV. Furthermore, seven nucleotides were changed in the sequenced RT and ZF regions of GVCV from a grafted Traminette vine and one in the sequenced regions of GVCV from grafted Cayuga White but no changes were found in the sequenced regions of GVCV in the grafted Vidal Blanc. The results provide a genetic snapshot of GVCV populations, which will yield knowledge important for monitoring GVCV epidemics and for preventing the loss of grape production that is associated with GVCV.

Fabrication and characterization of smart titanium alloy bolt based on high-frequency piezoelectric thin-film.

We reported here on the fabrication and characterization of a smart titanium alloy bolt based on a high-frequency piezoelectric thin-film sensor. The thin-film sensor was directly deposited on a titanium alloy bolt head with radio frequency magnetron sputtering and characterized by a scanning electron microscope and an atomic force microscope. The ultrasonic characteristics of the smart bolt, which include a pure and broad frequency spectrum peaked at 14.81 MHz, high measurement accuracy below 3%, and high repeatability free from some interference from bolt detection position change, were fully characterized. No obvious frequency shift was observed with the increase in axial preload. Based on the mono-wave method [TOF (time of flight) of longitudinal mode wave], TOF change increased linearly with preload force in the range of 0-20 kN. With the increase in temperature from 22 to 150 °C, the TOF linearly increases while the longitudinal wave velocity linearly decreases. The results indicate the prepared smart titanium alloy bolt is suitable as a smart aviation and automotive fastener.

Small RNA profiling of virus-infected grapevines: evidences for virus infection-associated and variety-specific miRNAs.

Grapevine is one of the economically and culturally important perennial fruit crops. More than 60 viruses infect grapevines and adversely affect their growth and development. Latent infection of most viruses in grapevines leads to chronic modulation of gene expression at transcriptional and post-transcriptional levels. Plant small RNAs (sRNAs) consist of microRNA (miRNA) and small interfering RNA (siRNA). miRNAs are expressed from the plant genome while most siRNAs are derived from double-stranded RNA molecules which are intermediates during virus replication. In a previous study, we constructed four cDNA libraries of sRNAs that were enriched from three virus-infected grapevines and one virus-free grapevine. Majority of siRNAs align most closely with the genomes of DNA viruses in the genus Badnavirus, family Caulimoviridae that led to the discovery of a new Grapevine vein clearing virus in grapevines. In this study, we conducted a comprehensive analysis of miRNAs in the four cDNA libraries and identified novel and stress-related miRNAs. The results indicated that miRNA abundance was influenced by virus infection. A total of 54 new miRNAs were identified and characterized, six of which, VITIS-MIR17, 18, 19, 20, 21, and 22, were detected only in virus-infected samples. One target of VITIS-MIR18 is the gene coding a non-apical meristem protein (GSVIVT00035370001), a transcription factor in the regulation of plant development and stress responses. Among the virus infection-induced known miRNAs, miRNA168 and miRNA3623 likely regulate grapevine's defense response, miRNA319 and miRNA395 modulate the expression of genes that are involved in nutrient metabolisms while miRNA396 plays a role in the regulation of cell division and cell cycle. The abiotic stress-induced miR169 and mi398 were negatively regulated by virus infection in grapevines. In addition, variety-specific miRNAs were discovered and compiled. The newly discovered miRNAs expand the miRNA profiles in the Vitis species. The characteristics of variety-specific and virus infection-associated miRNAs help understand the biology underlying the development and defense response of grapevines.

Berry skin development in Norton grape: distinct patterns of transcriptional regulation and flavonoid biosynthesis.

BACKGROUND: The complex and dynamic changes during grape berry development have been studied in Vitis vinifera, but little is known about these processes in other Vitis species. The grape variety 'Norton', with a major portion of its genome derived from Vitis aestivalis, maintains high levels of malic acid and phenolic acids in the ripening berries in comparison with V. vinifera varieties such as Cabernet Sauvignon. Furthermore, Norton berries develop a remarkably high level of resistance to most fungal pathogens while Cabernet Sauvignon berries remain susceptible to those pathogens. The distinct characteristics of Norton and Cabernet Sauvignon merit a comprehensive analysis of transcriptional regulation and metabolite pathways. RESULTS: A microarray study was conducted on transcriptome changes of Norton berry skin during the period of 37 to 127 days after bloom, which represents berry developmental phases from herbaceous growth to full ripeness. Samples of six berry developmental stages were collected. Analysis of the microarray data revealed that a total of 3,352 probe sets exhibited significant differences at transcript levels, with two-fold changes between at least two developmental stages. Expression profiles of defense-related genes showed a dynamic modulation of nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes and pathogenesis-related (PR) genes during berry development. Transcript levels of PR-1 in Norton berry skin clearly increased during the ripening phase. As in other grapevines, genes of the phenylpropanoid pathway were up-regulated in Norton as the berry developed. The most noticeable was the steady increase of transcript levels of stilbene synthase genes. Transcriptional patterns of six MYB transcription factors and eleven structural genes of the flavonoid pathway and profiles of anthocyanins and proanthocyanidins (PAs) during berry skin development were analyzed comparatively in Norton and Cabernet Sauvignon. Transcriptional patterns of MYB5A and MYB5B were similar during berry development between the two varieties, but those of MYBPA1 and MYBPA2 were strikingly different, demonstrating that the general flavonoid pathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3'-O-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase (F3'5'H), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose:flavonoid 3'-O-glucosyltransferase (UFGT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR) 1 and LAR2 in berry skin of Norton than in those of Cabernet Sauvignon. It was also found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignon berry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulation patterns in Norton berry skin. CONCLUSIONS: This study provides an overview of the transcriptome changes and the flavonoid profiles in the berry skin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of PR-1 and stilbene synthase genes likely contributes to the developmentally regulated resistance during ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase gene in berry development and disease resistance. Transcriptional regulation of MYBA1, MYBA2, MYB5A and MYBPA1 as well as expression levels of their putative targets F3'H, F3'5'H, LDOX, UFGT, ANR, LAR1, and LAR2 are highly correlated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a unique pattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enological characteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties.

Association of a novel DNA virus with the grapevine vein-clearing and vine decline syndrome.

A severe vein-clearing and vine decline syndrome has emerged on grapevines (Vitis vinifera) and hybrid grape cultivars in the Midwest region of the United States. The typical symptoms are translucent vein-clearing on young leaves, short internodes and decline of vine vigor. Known viral pathogens of grapevines were not closely associated with the syndrome. To obtain a comprehensive profile of viruses in a diseased grapevine, small RNAs were enriched and two cDNA libraries were constructed from a symptomatic grapevine and a symptomless grapevine, respectively. Deep sequencing of the two cDNA libraries showed that the most abundant viral small RNAs align with the genomes of viruses in the genus Badnavirus, the family Caulimoviridae. Amplification of the viral DNA by polymerase chain reaction allowed the assembly of the whole genome sequence of a grapevine DNA virus, which shared the highest homology with the Badnavirus sequences. This is the first report of a DNA virus in grapevines. The new DNA virus is closely associated with the vein-clearing symptom, and thus has been given a provisional name Grapevine vein clearing virus (GVCV). GVCV was detected in six grapevine cultivars showing vein-clearing and vine decline syndrome in Missouri, Illinois, and Indiana, suggesting its wide distribution in the Midwest region of the United States. Discovery of DNA viruses in grapevines merits further studies on their epidemics and economic impact on grape production worldwide.

Changes in protein abundance during powdery mildew infection of leaf tissues of Cabernet Sauvignon grapevine (Vitis vinifera L.).

A comparative analysis of differentially expressed proteins in a susceptible grapevine (Vitis vinifera 'Cabernet Sauvignon') during the infection of Erysiphe necator, the causal pathogen of grapevine powdery mildew (PM), was conducted using iTRAQ. The quantitative labeling analysis revealed 63 proteins that significantly changed in abundance at 24, 36, 48, and 72 h post inoculation with powdery mildew conidiospores. The functional classification of the PM-responsive proteins showed that they are involved in photosynthesis, metabolism, disease/defense, protein destination, and protein synthesis. A number of the proteins induced in grapevine in response to E. necator are associated with the plant defense response, suggesting that PM-susceptible Cabernet Sauvignon is able to initiate a basal defense but unable to restrict fungal growth or slow down disease progression.

A functional EDS1 ortholog is differentially regulated in powdery mildew resistant and susceptible grapevines and complements an Arabidopsis eds1 mutant.

Vitis vinifera (grapevine) is the most economically important deciduous fruit crop, but cultivated grapevine varieties lack adequate innate immunity to a range of devastating diseases. To identify genetic resources for grapevine innate immunity and understand pathogen defense pathways in a woody perennial plant, we focus in this study on orthologs of the central Arabidopsis thaliana defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). The family of EDS1-like genes is expanded in grapevine, and members of this family were previously found to be constitutively upregulated in the resistant variety 'Norton' of the North American grapevine species Vitis aestivalis, while they were induced by Erysiphe necator, the causal agent of grapevine powdery mildew (PM), in the susceptible V. vinifera variety 'Cabernet Sauvignon'. Here, we determine the responsiveness of individual EDS1-like genes in grapevine to PM and salicylic acid, and find that EDS1-like paralogs are differentially regulated in 'Cabernet Sauvignon', while two are constitutively upregulated in 'Norton'. Sequencing of VvEDS1 and VaEDS1 cDNA and genomic clones revealed high conservation in the protein-encoding sequence and some divergence of the promoter sequence in the two grapevine varieties. Complementation of the Arabidopsis eds1-1 mutant showed that the EDS1-like gene with highest predicted amino acid sequence similarity to AtEDS1 from either grapevine varieties is a functional ortholog of AtEDS1. Together, our analyses show that differential susceptibility to PM is correlated with differences in EDS1 expression, not differences in EDS1 function, between resistant 'Norton' and susceptible 'Cabernet Sauvignon'.

Viral small RNAs reveal the genomic variations of three grapevine vein clearing virus quasispecies populations.

Viral small RNAs (vsRNAs) include viral small interfering RNAs (vsiRNAs) that are initiators and products of RNA silencing, and small RNAs that are derived from viral RNAs with function still unknown. Sequencing of vsRNAs allows assembling of viral genomes and revelation of viral population variations at genomic levels. Grapevine vein clearing virus (GVCV) is a new member of the family Caulimoviridae whose DNA genome is replicated by reverse transcription of pre-genomic RNA molecules. In this short report, three genomic sequences of GVCV were assembled from vsRNAs that were isolated and sequenced from three individual grapevines in commercial vineyards and compared to the GVCV-CHA reference genome. Profiles of single nucleotide polymorphism among three viral populations indicated a closer relatedness between two populations in different grape cultivars at the same location than those in the same grape cultivar at different locations, suggesting the spread of GVCV populations among vineyards of close proximity. Classic types of vsiRNAs (21-nt, 22-nt, and 24-nt) were found in the three GVCV vsiRNA populations, but these did not produce alignment hotspots on the GVCV-CHA reference genome. The number of 36-nt reads is the highest among vsRNAs, the role of these vsRNAs remains unclear. The analysis of vsRNAs provides a first holistic picture of genomic variations among GVCV viral quasispecies populations that help monitor epidemics and evolution of GVCV populations, an emerging virus that is becoming a threat to grape production in the Midwest region of the USA.

Integrity of nonviral fragments in recombinant Tomato bushy stunt virus and defective interfering RNA is influenced by silencing and the type of inserts.

Recombinant plant viruses have the propensity to remove foreign inserts during replication. This process is virus-specific and occurs in a host-dependent manner. In the present study, we investigated the integrity of foreign inserts in recombinant plant viruses using a model system consisting of Tomato bushy stunt virus (TBSV) and its defective interfering RNA (DI). These were tested in Nicotiana benthamiana plants that were either wild type or transgenic for the green fluorescent protein (GFP) gene. GFP-derived inserts were retained in the recombinant TBSV and DI population that were inoculated onto GFP-transgenic N. benthamiana plants in which silencing of the GFP transgene was initiated, but they were removed from the virus and DIs that were maintained on wild-type plants. A foreign insert derived from an endogenous N. benthamiana gene encoding the H subunit of the magnesium chelatase (NbChlH) was deleted, whereas the fragment of an RNA-dependent RNA polymerase gene (NbRdRP1m) was retained in the recombinant TBSV population. These results demonstrate that the recombination of TBSV to remove nonviral fragments is influenced by silencing and the type of inserts.

The plant gene CCD1 selectively blocks cell death during the hypersensitive response to Cauliflower mosaic virus infection.

The P6 protein of Cauliflower mosaic virus (CaMV) W260 elicits a hypersensitive response (HR) on inoculated leaves of Nicotiana edwardsonii. This defense response, common to many plant pathogens, has two key characteristics, cell death within the initially infected tissues and restriction of the pathogen to this area. We present evidence that a plant gene designated CCD1, originally identified in N. bigelovii, can selectively block the cell death pathway during HR, whereas the resistance pathway against W260 remains intact. Suppression of cell death was evident not only macroscopically but also microscopically. The suppression of HR-mediated cell death was specific to CaMV, as Tobacco mosaic virus was able to elicit HR in the plants that contained CCD1. CCD1 also blocks the development of a systemic cell death symptom induced specifically by the P6 protein of W260 in N. clevelandii. Introgression of CCD1 from N. bigelovii into N. clevelandii blocked the development of systemic cell death in response to W260 infection but could not prevent systemic cell death induced by Tomato bushy stunt virus. Thus, CCD1 blocks both local and systemic cell death induced by P6 of W260 but does not act as a general suppressor of cell death induced by other plant viruses. Furthermore, experiments with CCD1 provide further evidence that cell death could be uncoupled from resistance in the HR of Nicotiana edwardsonii to CaMV W260.

Current understanding of grapevine defense mechanisms against the biotrophic fungus (Erysiphe necator), the causal agent of powdery mildew disease.

The most economically important disease of cultivated grapevines worldwide is powdery mildew (PM) caused by the ascomycete fungus Erysiphe necator. The majority of grapevine cultivars used for wine, table grape, and dried fruit production are derived from the Eurasian grape species Vitis vinifera because of its superior aroma and flavor characteristics. However, this species has little genetic resistance against E. necator meaning that grape production is highly dependent on the frequent use of fungicides. The integration of effective genetic resistance into cultivated grapevines would lead to significant financial and environmental benefits and represents a major challenge for viticultural industries and researchers worldwide. This review will outline the strategies being used to increase our understanding of the molecular basis of V. vinifera susceptibility to this fungal pathogen. It will summarize our current knowledge of different resistance loci/genes that have evolved in wild grapevine species to restrict PM infection and assess the potential application of these defense genes in the generation of PM-resistant grapevine germplasm. Finally, it addresses future research priorities which will be important in the rapid identification, evaluation, and deployment of new PM resistance genes which are capable of conferring effective and durable resistance in the vineyard.

Satellite panicum mosaic virus capsid protein elicits symptoms on a nonhost plant and interferes with a suppressor of virus-induced gene silencing.

The capsid protein (CP) of satellite panicum mosaic virus (SPMV) has been implicated as a pathogenicity factor, inducing severe chlorosis on millet plants co-infected with SPMV and its helper virus, Panicum mosaic virus (PMV). In this study, we tested the effects of SPMV CP on Nicotiana benthamiana, a plant that does not support PMV+SPMV infections. SPMV CP expressed from a Potato virus X (PVX) gene vector elicited necrotic lesions on N. benthamiana. Pathogenicity factors often have the additional feature of acting as suppressors of gene silencing; therefore, several assays were developed to test if SPMV CP could act in such a capacity. The results showed that SPMV CP failed to act as a suppressor of posttranscriptional gene silencing when such tests were performed with transgenic N. benthamiana plants silenced for green fluorescent protein (GFP) expression by agroinfiltration or plant virus vectors. However SPMV CP expressed from the PVX gene vector did interfere with suppressor activity associated with PVX p25. This included a rebounded level of GFP silencing along the vascular tissues, including the veins on upper noninoculated leaves. Therefore, the roles of the SPMV CP now include encapsidation of the SPMV RNA, activity as a pathogenicity factor in both host and nonhost plants, and the enigmatic feature of interfering with suppression of gene silencing.

Tombusvirus P19-mediated suppression of virus-induced gene silencing is controlled by genetic and dosage features that influence pathogenicity.

The p19 protein (P19) of Tomato bushy stunt virus (TBSV) is a pathogenicity determinant with host-dependent effects on virus spread and symptom induction. In addition, results in this study confirm that Potato virus X-mediated delivery of P19 suppresses posttranscriptional gene silencing (PTGS). To study the relevance of this activity for TBSV biology, we evaluated whether TBSV activates virus-induced gene silencing (VIGS) and if this process is suppressed by P19. TBSV vectors with the green fluorescent protein (GFP) gene, either active or inactive for P19 expression, were inoculated onto GFP-transgenic Nicotiana benthamiana plants. In the absence of P19 expression, VIGS was activated, as evidenced by the disappearance of GFP mRNA and green fluorescence. Coexpression of GFP and P19 from the TBSV vector suppressed VIGS, except in the newly emerging leaves. The suppressor activity required a central P19 region that is also known to be essential for host-dependent virus spread and symptom induction. Defective interfering RNAs (DIs) that contained the 3' end of the GFP gene induced silencing very effectively. The concomitant DI-instigated reduction in P19 accumulation failed to suppress this process, analogous to the known P19 dosage effects for other biological activities. In conclusion, (i) TBSV and its DIs are very effective inducers of VIGS, (ii) P19 is a strong suppressor of PTGS, (iii) P19 is a moderate suppressor of VIGS, and (iv) the suppressor activity is influenced by genetic and dosage features that are also important for P19-associated pathogenesis.

A novel co-delivery system consisting of a Tomato bushy stunt virus and a defective interfering RNA for studying gene silencing.

Virus induced gene silencing (VIGS) and suppression are RNA-specific defense and counter-defense circuits in plant-virus interactions. These phenomena have been investigated extensively with an Agrobacterium-mediated transient expression system. In this study, a virus-based transient expression system was developed to study these phenomena. A Tomato bushy stunt virus (TBSV) viral vector with an inactivated P19 suppressor gene, referred to as pHST2-14, was chosen to express the P1 of Tobacco etch virus (TEV). TEV P1 is a component of a well-characterized VIGS suppressor, TEV P1/HC-Pro protein. A TBSV defective interfering RNA (DI) that contains the 3' proximal portion of a green fluorescence protein (GFP) gene, DI-P, was used as a silencing inducer of the homologous GFP gene on GFP transgenic Nicotiana benthamiana (NbGFP) plants. The TEV P1 gene was inserted into pHST2-14 to generate TBSV-P1. Transcripts of TBSV-P1 were then mixed with DI-P transcripts and inoculated onto NbGFP plants. DI-P consistently accumulated in NbGFP plants that were inoculated with TBSV-P1 and DI-P, and efficiently induced silencing of GFP transgene. These results demonstrate that a TBSV-based co-delivery system can provide a new alternative tool to investigate gene silencing and its influence by a TBSV-expressed foreign protein. It also can be used to elucidate functions of endogenous genes in plants.