Vector transmission of parsley yellow leaf curl virus by the leafhopper Austroagallia sinuata.

Parsley yellow leaf curl virus (PYLCV) is a new member of the family Geminiviridae that has not yet been assigned to an established genus due to limited information about its biological properties. In this study, the ability of Austroagallia leafhoppers, which are commonly found on vegetable farms in Kerman province (Iran), to transmit this virus was studied. After a two-day acquisition access period, Austroagallia sp. successfully transmitted the virus from PYLCV-infected parsley to healthy seedlings. On the basis of male genitalia morphology, the species of leafhopper was identified as A. sinuata. This is the first report of a transmission of plant virus by a member of the genus Austroagallia.

Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

Molecular characterization and pathogenicity of a novel monopartite geminivirus infecting tobacco in China.

The occurrence of geminiviruses causes significant economic losses in many economically important crops. In this study, a novel geminivirus isolated from tobacco in Sichuan province of China, named tomato leaf curl Chuxiong virus (TLCCxV), was characterized by small RNA-based deep sequencing. The full-length of TLCCxV genome was determined to be 2744 nucleotides (nt) encoding six open reading frames. Phylogenetic and genome-wide pairwise identity analysis revealed that TLCCxV shared less than 91% identities with reported geminiviruses. A TLCCxV infectious clone was constructed and successfully infected Nicotiana benthamiana, N. tabacum, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants. Furthermore, expression of the V2, C1 and C4 proteins through a potato virus X vector caused severe chlorosis or necrosis symptom in N. benthamiana. Taken together, we identified a new geminivirus in tobacco plants, and found that V2, C1 and C4 contribute to symptom development.

The C4 Protein of TbLCYnV Promotes SnRK1 ß2 Degradation Via the Autophagy Pathway to Enhance Viral Infection in N. benthamiana.

Geminiviruses are a group of single-stranded DNA viruses that have developed multiple strategies to overcome host defenses and establish viral infections. Sucrose nonfermenting-1-related kinase 1 (SnRK1) is a key regulator of energy balance in plants and plays an important role in plant development and immune defenses. As a heterotrimeric complex, SnRK1 is composed of a catalytic subunit α (SnRK1 α) and two regulatory subunits, ß and γ. Previous studies on SnRK1 in plant defenses against microbial pathogens have mainly focused on SnRK1 α. In this study, we validated the interaction between the C4 protein encoded by tobacco leaf curl Yunnan virus (TbLCYnV) and the regulatory subunit ß of Nicotiana benthamiana SnRK1, i.e., NbSnRK1 ß2, and identified that the Asp22 of C4 is critical for TbLCYnV C4-NbSnRK1 ß2 interactions. NbSnRK1 ß2 silencing in N. benthamiana enhances susceptibility to TbLCYnV infection. Plants infected with viral mutant TbLCYnV (C4D22A), which contains the mutant version C4 (D22A) that is incapable of interacting with NbSnRK1 ß2, display milder symptoms and lower viral accumulation. Furthermore, we discovered that C4 promotes NbSnRK1 ß2 degradation via the autophagy pathway. We herein propose a model by which the geminivirus C4 protein causes NbSnRK1 ß2 degradation via the TbLCYnV C4-NbSnRK1 ß2 interaction to antagonize host antiviral defenses and facilitates viral infection and symptom development in N. benthamiana.

Genetic Diversity, Evolutionary Dynamics, and Ongoing Spread of Pedilanthus Leaf Curl Virus.

Pedilanthus leaf curl virus (PeLCV) is a monopartite begomovirus (family Geminiviridae) discovered just a few decades ago. Since then, it has become a widely encountered virus, with reports from ca. 25 plant species across Pakistan and India, indicative of its notable evolutionary success. Viruses mutate at such a swift rate that their ecological and evolutionary behaviors are inextricably linked, and all of these behaviors are imprinted on their genomes as genetic diversity. So, all these imprints can be mapped by computational methods. This study was designed to map the sequence variation dynamics, genetic heterogeneity, regional diversity, phylogeny, and recombination events imprinted on the PeLCV genome. Phylogenetic and network analysis grouped the full-length genome sequences of 52 PeLCV isolates into 7 major clades, displaying some regional delineation but lacking host-specific demarcation. The progenitor of PeLCV was found to have originated in Multan, Pakistan, in 1977, from where it spread concurrently to India and various regions of Pakistan. A high proportion of recombination events, distributed unevenly throughout the genome and involving both inter- and intraspecies recombinants, were inferred. The findings of this study highlight that the PeLCV population is expanding under a high degree of genetic diversity (π = 0.073%), a high rate of mean nucleotide substitution (1.54 × 10-3), demographic selection, and a high rate of recombination. This sets PeLCV apart as a distinctive begomovirus among other begomoviruses. These factors could further exacerbate the PeLCV divergence and adaptation to new hosts. The insights of this study that pinpoint the emergence of PeLCV are outlined.

Transcriptional and epigenetic changes during tomato yellow leaf curl virus infection in tomato.

BACKGROUND: Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). RESULTS: We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. CONCLUSION: We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.

H3.1K27me1 loss confers Arabidopsis resistance to Geminivirus by sequestering DNA repair proteins onto host genome.

The H3 methyltransferases ATXR5 and ATXR6 deposit H3.1K27me1 to heterochromatin to prevent genomic instability and transposon re-activation. Here, we report that atxr5 atxr6 mutants display robust resistance to Geminivirus. The viral resistance is correlated with activation of DNA repair pathways, but not with transposon re-activation or heterochromatin amplification. We identify RAD51 and RPA1A as partners of virus-encoded Rep protein. The two DNA repair proteins show increased binding to heterochromatic regions and defense-related genes in atxr5 atxr6 vs wild-type plants. Consequently, the proteins have reduced binding to viral DNA in the mutant, thus hampering viral amplification. Additionally, RAD51 recruitment to the host genome arise via BRCA1, HOP2, and CYCB1;1, and this recruitment is essential for viral resistance in atxr5 atxr6. Thus, Geminiviruses adapt to healthy plants by hijacking DNA repair pathways, whereas the unstable genome, triggered by reduced H3.1K27me1, could retain DNA repairing proteins to suppress viral amplification in atxr5 atxr6.

Fast and High-Efficiency Synthesis of Capsanthin in Pepper by Transient Expression of Geminivirus.

The color of the chili fruit is an important factor that determines the quality of the chili, as red chilies are more popular among consumers. The accumulation of capsanthin is the main cause of reddening of the chili fruit. Capsanthin is an important metabolite in carotenoid metabolism, and its production level is closely linked to the expression of the genes for capsanthin/capsorubin synthase (CCS) and carotenoid hydroxylase (CrtZ). We reported for the first time that the synthesis of capsanthin in chili was enhanced by using a geminivirus (Bean Yellow Dwarf Virus). By expressing heterologous ß-carotenoid hydroxylase (CrtZ) and ß-carotenoid ketolase (CrtW) using codon optimization, the transcription level of the CCS gene and endogenous CrtZ was directly increased. This leads to the accumulation of a huge amount of capsanthin in a very short period of time. Our results provide a platform for the rapid enhancement of endogenous CCS activity and capsanthin production using geminivirus in plants.

Replication mechanisms of circular ssDNA plant viruses and their potential implication in viral gene expression regulation.

The replication of members of the two circular single-stranded DNA (ssDNA) virus families Geminiviridae and Nanoviridae, the only ssDNA viruses infecting plants, is believed to be processed by rolling-circle replication (RCR) and recombination-dependent replication (RDR) mechanisms. RCR is a ubiquitous replication mode for circular ssDNA viruses and involves a virus-encoded Replication-associated protein (Rep) which fulfills multiple functions in the replication mechanism. Two key genomic elements have been identified for RCR in Geminiviridae and Nanoviridae: (i) short iterative sequences called iterons which determine the specific recognition of the viral DNA by the Rep and (ii) a sequence enabling the formation of a stem-loop structure which contains a conserved motif and constitutes the origin of replication. In addition, studies in Geminiviridae provided evidence for a second replication mode, RDR, which has also been documented in some double-stranded DNA viruses. Here, we provide a synthesis of the current understanding of the two presumed replication modes of Geminiviridae and Nanoviridae, and we identify knowledge gaps and discuss the possibility that these replication mechanisms could regulate viral gene expression through modulation of gene copy number.

Post translational modifications at the verge of plant-geminivirus interaction.

Plant-virus interaction is a complex phenomenon and involves the communication between plant and viral factors. Viruses have very limited coding ability yet, they are able to cause infection which results in huge agro-economic losses throughout the globe each year. Post-translational modifications (PTMs) are covalent modifications of proteins that have a drastic effect on their conformation, stability and function. Like the host proteins, geminiviral proteins are also subject to PTMs and these modifications greatly expand the diversity of their functions. Additionally, these viral proteins can also interact with the components of PTM pathways and modulate them. Several studies have highlighted the importance of PTMs such as phosphorylation, ubiquitination, SUMOylation, myristoylation, S-acylation, acetylation and methylation in plant-geminivirus interaction. PTMs also regulate epigenetic modifications during geminivirus infection which determines viral gene expression. In this review, we have summarized the role of PTMs in regulating geminiviral protein function, influence of PTMs on viral gene expression and how geminiviral proteins interact with the components of PTM pathways to modulate their function.

Amesuviridae: a new family of plant-infecting viruses in the phylum Cressdnaviricota, realm Monodnaviria.

The phylum Cressdnaviricota comprises viruses with single-stranded, circular DNA genomes that encode an HUH-type endonuclease (known as Rep). The phylum includes two classes, eight orders, and 11 families. Here, we report the creation of a twelfth family in the order Mulpavirales, class Arfiviricetes of the phylum Cressdnaviricota. The family Amesuviridae comprises viruses that infect plants and is divided into two genera: Temfrudevirus, including the species Temfrudevirus temperatum (with temperate fruit decay-associated virus as a member), and Yermavirus, including the species Yermavirus ilicis (with yerba mate-associated circular DNA virus as a member). Both viruses encode Rep proteins with HUH endonuclease and SH3 superfamily helicase domains. Phylogenetic analysis indicates that the replicative module of amesuviruses constitutes a well-supported monophyletic clade related to Rep proteins from viruses in the order Mulpavirales. Furthermore, both viruses encode a single capsid protein (CP) related to geminivirus CPs. Phylogenetic incongruence between the replicative and structural modules of amesuviruses suggests a chimeric origin resulting from remote recombination events between ancestral mulpavirales and geminivirids. The creation of the family Amesuviridae has been ratified by the International Committee on Taxonomy of Viruses (ICTV).

The selection and validation of reference genes for quantitative real-time PCR studies in near-isogenic susceptible and resistant tomato lines, infected with the geminivirus tomato curly stunt virus.

Quantitative real-time PCR (qPCR) is a sensitive and commonly used technique for gene expression profiling and provides insight into biological systems. Successful qPCR requires the use of appropriate reference genes for the normalization of data. In the present study, we aimed to identify and assess the best-suited reference genes in near-isogenic resistant (R) and susceptible (S) tomato lines infected with begomovirus Tomato curly stunt virus (ToCSV). Ten candidate reference genes namely, Actin7 (ACT), ß-6 Tubulin (TUB), Ubiquitin 3 (UBI), Clathrin adaptor complexes medium subunit (CAC), Phytoene desaturase (PDS), Expressed protein (EXP), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Adenine phosphoribosyl transferase-like protein (APT1), TAP42-interacting protein (TIP41) and Elongation factor 1-alpha (EF1α) were selected and evaluated for their expression stability in resistant and susceptible tomato leaves using the analytical tools geNorm, NormFinder, BestKeeper, and RefFinder. After ranking the reference genes from most to least stable, the results suggested that a combination of ACT, EXP, and EF1α in the S lines and a combination of TIP41, APT1, and ACT in the R line is appropriate for qPCR normalization. Furthermore, to validate the identified reference genes, iron superoxide dismutase (SOD), heat shock protein 70 (HSP70) and Glutathione-S-transferase (GST) were selected as targets for normalization. The relative expression of the target genes varied when normalized against the most stable reference genes in comparison to the least stable genes. These results highlight the importance of careful selection of reference genes for accurate normalization in qPCR studies.

Geminiviral betasatellites: critical viral ammunition to conquer plant immunity.

Geminiviruses have mastered plant cell modulation and immune invasion to ensue prolific infection. Encoding a relatively small number of multifunctional proteins, geminiviruses rely on satellites to efficiently re-wire plant immunity, thereby fostering virulence. Among the known satellites, betasatellites have been the most extensively investigated. They contribute significantly to virulence, enhance virus accumulation, and induce disease symptoms. To date, only two betasatellite proteins, ßC1, and ßV1, have been shown to play a crucial role in virus infection. In this review, we offer an overview of plant responses to betasatellites and counter-defense strategies deployed by betasatellites to overcome those responses.

Geminivirus C5 proteins mediate formation of virus complexes at plasmodesmata for viral intercellular movement.

Movement proteins (MPs) encoded by plant viruses deliver viral genomes to plasmodesmata (PD) to ensure intracellular and intercellular transport. However, how the MPs encoded by monopartite geminiviruses are targeted to PD is obscure. Here, we demonstrate that the C5 protein of tomato yellow leaf curl virus (TYLCV) anchors to PD during the viral infection following trafficking from the nucleus along microfilaments in Nicotiana benthamiana. C5 could move between cells and partially complement the traffic of a movement-deficient turnip mosaic virus (TuMV) mutant (TuMV-GFP-P3N-PIPO-m1) into adjacent cells. The TYLCV-C5 null mutant (TYLCV-mC5) attenuates viral pathogenicity and decreases viral DNA and protein accumulation, and ectopic overexpression of C5 enhances viral DNA accumulation. Interaction assays between TYLCV-C5 and the other eight viral proteins described in TYLCV reveal that C5 associates with C2 in the nucleus and with V2 in the cytoplasm and at PD. The V2 protein is mainly localized in the nucleus and cytoplasmic granules when expressed alone; in contrast, V2 forms small punctate granules at PD when co-expressed with C5 or in TYLCV-infected cells. The interaction of V2 and C5 also facilitates their nuclear export. Furthermore, C5-mediated PD localization of V2 is conserved in two other geminiviruses. Therefore, this study solves a long-sought-after functional connection between PD and the geminivirus movement and improves our understanding of geminivirus-encoded MPs and their potential cellular and molecular mechanisms.

Functional identification of a novel C7 protein of tomato yellow leaf curl virus.

Tomato yellow leaf curl virus (TYLCV) is a monopartite geminivirus, and one of the most devastating plant viruses in the world. TYLCV is traditionally known to encode six viral proteins in bidirectional and partially overlapping open reading frames (ORFs). However, recent studies have shown that TYLCV encodes additional small proteins with specific subcellular localizations and potential virulence functions. Here, a novel protein named C7, encoded by a newly-described ORF in the complementary strand, was identified as part of the TYLCV proteome using mass spectrometry. The C7 protein localized to the nucleus and cytoplasm, both in the absence and presence of the virus. C7 was found to interact with two other TYLCV-encoded proteins: with C2 in the nucleus, and with V2 in the cytoplasm, forming conspicuous granules. Mutation of C7 start codon ATG to ACG to block the translation of C7 delayed the onset of viral infection, and the mutant virus caused milder virus symptoms and less accumulations of viral DNAs and proteins. Using the potato virus X (PVX)-based recombinant vector, we found that ectopic overexpression of C7 resulted in more severe mosaic symptoms and promoted a higher accumulation of PVX-encoded coat protein in the late virus infection stage. In addition, C7 was also found to inhibit GFP-induced RNA silencing moderately. This study demonstrates that the novel C7 protein encoded by TYLCV is a pathogenicity factor and a weak RNA silencing suppressor, and that it plays a critical role during TYLCV infection.

Betasatellite-encoded ßC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of geminivirus-encoded replication initiator protein.

Geminivirus-betasatellite disease complexes are an epidemic threat to the majority of economically important crops across the world. Plant virus satellites including betasatellites are maintained by their associated helper virus. Geminivirus-betasatellites influence viral pathogenesis by substantially increasing or decreasing their helper virus accumulation. In the present study, we attempted to understand the mechanistic details of the geminivirus-betasatellite interaction. Here, we used tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl Patna betasatellite (ToLCPaB) as a model system. This study reveals that ToLCGV can efficiently trans-replicate ToLCPaB in Nicotiana benthamiana plants, but ToLCPaB greatly reduced the accumulation of its helper virus DNA. For the first time, we have identified that the ToLCPaB-encoded ßC1 protein is able to interact with ToLCGV-encoded replication initiator protein (Rep). In addition, we demonstrate that the C-terminal region of ßC1 interacts with the C-terminus of Rep (RepC) protein. Our previous study had established that ßC1 proteins encoded by diverse betasatellites possess a novel ATP hydrolysis activity and the conserved lysine/arginine residues at positions 49 and 91 are necessary for this function. Here, we show that mutating lysine at positions 49 to alanine of ßC1 (ßC1K49A) protein did not affect its ability to interact with RepC protein. Biochemical studies performed with ATP hydrolysis activity-deficient K49A mutated ßC1 (ßC1K49A) and RepC proteins revealed that Rep-ßC1 interaction interferes with the ATP hydrolysis activity of Rep protein. Further, we demonstrate that ßC1 protein is able to interact with D227A and D289A mutated RepC proteins but not with D262A, K272A or D286A mutated RepC proteins, suggesting that the ßC1-interacting region of Rep protein encompasses its Walker-B and B' motifs. The results of docking studies supported that the ßC1-interacting region of Rep protein encompasses its motifs associated with ATP binding and ATP hydrolysis activities. Docking studies also provided evidence that the Rep-ßC1 interaction interferes with the ATP binding activity of Rep protein. Together, our findings suggest that ßC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of helper virus Rep protein.

Transcription start site mapping of geminiviruses using the in vitro cap-snatching of a tenuivirus.

Geminiviruses are a family of single-stranded DNA viruses that cause significant yield losses in crop production worldwide. Transcription start site (TSS) mapping is crucial in understanding the gene expression mechanisms of geminiviruses. However, this often requires costly and laborious experiments. Rice stripe virus (RSV) has a mechanism called cap-snatching, whereby it cleaves cellular mRNAs and uses the 5' cleavage product, a capped-RNA leader (CRL), as primers for transcription. Our previous work demonstrated that RSV snatches CRLs from geminiviral mRNAs in co-infected plants, providing a convenient and powerful approach to map the TSSs of geminiviruses. However, co-infections are not always feasible for all geminiviruses. In this study, we evaluated the use of in vitro cap-snatching of RSV for the same purpose, using tomato yellow leaf curl virus (TYLCV) as an example. We incubated RNA extracted from TYLCV-infected plants with purified RSV ribonucleoproteins in a reaction mixture that supports in vitro cap-snatching of RSV. The RSV mRNAs produced in the reaction were deep sequenced. The CRLs snatched by RSV allowed us to locate 28 TSSs in TYLCV. These results provide support for using RSV's in vitro cap-snatching to map geminiviral TSSs.

WRKY1 represses the WHIRLY1 transcription factor to positively regulate plant defense against geminivirus infection.

Geminiviruses constitute the largest group of known plant viruses and cause devastating diseases and economic losses in many crops worldwide. Due to limited naturally occurring resistance genes, understanding plant antiviral defense against geminiviruses is critical for finding host factors of geminiviruses and development of strategies for geminivirus control. Here we identified NbWRKY1 as a positive regulator of plant defense against geminivirus infection. Using tomato yellow leaf curl China virus/tomato yellow leaf curl China betasatellite (TYLCCNV/TYLCCNB) as a representative geminivirus, we found that NbWRKY1 was upregulated in response to TYLCCNV/TYLCCNB infection. Overexpression of NbWRKY1 attenuated TYLCCNV/TYLCCNB infection, whereas knockdown of NbWRKY1 enhanced plant susceptibility to TYLCCNV/TYLCCNB. We further revealed that NbWRKY1 bound to the promoter of the NbWHIRLY1 (NbWhy1) transcription factor and inhibited the transcription of NbWhy1. Consistently, NbWhy1 negatively regulates plant response against TYLCCNV/TYLCCNB. Overexpression of NbWhy1 significantly accelerated TYLCCNV/TYLCCNB infection. Conversely, knockdown of NbWhy1 led to impaired geminivirus infection. Furthermore, we demonstrated that NbWhy1 interfered with the antiviral RNAi defense and disrupted the interaction between calmodulin 3 and calmodulin-binding transcription activator-3. Moreover, the NbWRKY1-NbWhy1 also confers plant antiviral response toward tomato yellow leaf curl virus infection. Taken together, our findings suggest that NbWRKY1 positively regulates plant defense to geminivirus infection by repressing NbWhy1. We propose that the NbWRKY1-NbWhy1 cascade could be further employed to control geminiviruses.

The Three-Cornered Alfalfa Hopper, Spissistilus festinus, Is a Vector of Grapevine Red Blotch Virus in Vineyards.

Spissistilus festinus (Hemiptera: Membracidae) transmit grapevine red blotch virus (GRBV, Grablovirus, Geminiviridae) in greenhouse settings; however, their role as a vector of GRBV in vineyards is unknown. Following controlled exposures of aviruliferous S. festinus for two weeks on infected, asymptomatic vines in a California vineyard in June and a 48 h gut clearing on alfalfa, a nonhost of GRBV, approximately half of the released insects tested positive for GRBV (45%, 46 of 102), including in the salivary glands of dissected individuals (11%, 3 of 27), indicating acquisition. Following controlled exposures of viruliferous S. festinus for two to six weeks on GRBV-negative vines in vineyards in California and New York in June, transmission of GRBV was detected when two S. festinus were restricted to a single leaf (3%, 2 of 62 in California; 10%, 5 of 50 in New York) but not with cohorts of 10-20 specimens on entire or half shoots. This work was consistent with greenhouse assays in which transmission was most successful with S. festinus exposed to a single leaf (42%, 5 of 12), but rarely occurred on half shoots (8%, 1 of 13), and never on entire shoots (0%, 0 of 18), documenting that the transmission of GRBV is facilitated through the feeding of fewer S. festinus on a restricted area of grapevine tissue. This work demonstrates S. festinus is a GRBV vector of epidemiological importance in vineyards.