Two Novel Geminiviruses Identified in Bees (Apis mellifera and Nomia sp.).

Members of the Geminviridae family are circular single-stranded DNA plant-infecting viruses, some of which impact global food production. Geminiviruses are vectored by sap-feeding insects such as leafhoppers, treehoppers, aphids, and whiteflies. Additionally, geminivirus sequences have also been identified in other insects such as dragonflies, mosquitoes, and stingless bees. As part of a viral metagenomics study on honeybees and solitary bees (Nomia sp.), two geminivirus genomes were identified. These represent a novel citlodavirus (from honeybees collected from Westmoreland, Jamaica) and a mastrevirus-like genome (from a solitary bee collected from Tempe, Arizona, USA). The novel honeybee-derived citlodavirus genome shares ~61 to 69% genome-wide nucleotide pairwise identity with other citlodavirus genome sequences and is most closely related to the passion fruit chlorotic mottle virus identified in Brazil. Whereas the novel solitary bee-derived mastrevirus-like genome shares ~55 to 61% genome-wide nucleotide identity with other mastreviruses and is most closely related to tobacco yellow dwarf virus identified in Australia, based on pairwise identity scores of the full genome, replication-associated protein, and capsid protein sequences. Previously, two geminiviruses in the Begomovirus genus were identified in samples of stingless bee (Trigona spp.) samples. Here, we identify viruses that represent two new species of geminiviruses from a honeybee and a solitary bee, which continues to demonstrate that plant pollinators can be utilized for the identification of plant-infecting DNA viruses in ecosystems.

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.

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.

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.

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.

Unusual outbreaks of curly top disease in processing tomato fields in northern California in 2021 and 2022 were caused by a rare strain of beet curly top virus and facilitated by extreme weather events.

In the western United States, curly top disease (CTD) is caused by beet curly top virus (BCTV). In California, CTD causes economic loss to processing tomato production in central and southern areas but, historically, not in the north. Here, we document unusual CTD outbreaks in processing tomato fields in the northern production area in 2021 and 2022, and show that these were caused by the rare spinach curly top strain (BCTV-SpCT). These outbreaks were associated with proximity of fields to foothills and unusually hot, dry, and windy spring weather conditions, possibly by altering migrations of the beet leafhopper (BLH) vector from locations with BCTV-SpCT reservoirs. Support for this hypothesis came from the failure to observe CTD outbreaks and BLH migrations in 2023, when spring weather conditions were cool and wet. Our results show the climate-induced emergence of a rare plant virus strain to cause an economically important disease in a new crop and location.

Interactions of beet leafhopper (Hemiptera: Cicadellidae), vector of beet curly top virus, and hemp in New Mexico.

The beet leafhopper, Circulifer tenellus (Baker 1896), is the sole vector of beet curly top virus (BCTV). Both the virus and the vector have very wide host ranges, including many crops and weeds. Industrial hemp (Cannabis sativa L.) has been reported as a host for both the virus and leafhopper in the past few years with the legal cultivation of the crop in the United States. This research assessed the interactions of the beet leafhopper and hemp in New Mexico by determining the natural infection of hemp with BCTV in 3 field plots in 2021 and 2022 and monitoring the numbers of leafhoppers using yellow sticky traps. The relative preference of beet leafhopper for hemp types and varieties of hemp was assessed using cafeteria-style choice tests. Higher numbers of beet leafhoppers were trapped in and around hemp fields in 2022 than in 2021 in all 3 locations. BCTV was found to infect all 3 types of hemp (cannabidiol or CBD, fiber, and grain) in 2022 in 1 location and only a single CBD variety of hemp in the other 2 locations. Two BCTV strains were identified in CBD hemp, while an additional BCTV strain was found infecting chile pepper grown at the same location.

Longer cluster hanging time decreases the impact of grapevine red blotch disease in Vitis vinifera L. Merlot across two seasons.

BACKGROUND: Grapevine red blotch virus (GRBV) is a recently discovered virus and a major concern for the wine industry. Prior research indicated that GRBV delays grape ripening by reducing °Brix and anthocyanin concentrations in grapes from infected vines, resulting in higher ethanol concentrations in wines made from healthy fruit compared to diseased vines, which have an impact on sensory properties. In this study, infected fruit (Vitis vinifera L. Merlot) was sequentially harvested (in 2016 and 2017) and chaptalized (in 2017) to ameliorate the impact of GRBV on grape and final wine composition. RESULTS: Chemical parameters including phenolic and volatile profiles of grapes and their subsequent wines were measured. Sensory properties were determined by descriptive analyses. Results demonstrated that GRBV decreased sugar accumulation and anthocyanin synthesis in grapes. Wines from GRBV grapes harvested at later ripening stage produced wines that were more similar chemically and sensorially to wines made from healthy fruit than to wines made from GRBV fruit harvested earlier. CONCLUSION: A longer hang time of GRBV grapes is a potential strategy to mitigate the impacts of GRBV. However, chaptalization of diseased fruit must was inefficient at increasing similarities to wines made from healthy fruit. © 2023 Society of Chemical Industry.

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.

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.

Comparative taxonomical, biological and pharmacological potential of healthy and geminivirus infected leaves of Hibiscus rosa-sinensis L.: First report.

In the present research project, the first report on comparative analysis of the taxonomical, biological and pharmacological potential of healthy and geminivirus infected Hibiscus rosa sinensis (L.) leaves of the family Malvaceae was done by using different micro and macroscopic techniques. First of all, leaves were characterized for Cotton leaf curl Multan virus (CLCuMuV) and its associated betasatellite (Cotton leaf curl Multan Betasatellite; CLCuMB). Different morphological parameters like shape and size of stem, leaves, seeds and roots, presence and absence of ligule, distance between nodes and internodes and type of inflorescence etc. were analyzed. CLCuMuV infected H. rosa-sinensis revealed systematic symptoms of infection like chlorosis of leaves, stunted growth, decrease in size of roots, shoots and distortion etc. Anatomical investigation was performed under light ad scanning electron microscope. Different anatomical features like length and shape of guard cells, subsidiary cells, presence or absence of stomata, secretory ducts and trichomes were examined. In both plant samples anomocytic types of stomata and elongated, non-glandular and pointed tip trichomes were present, but the size (especially length and width) of trichomes and other cells like epidermal, subsidiary, and guard cells were highest in virus infected plants likened to healthy one. In the antibacterial activity, the maximum antibacterial potentail was seen in methanolic extract of K. pneumonea while antifungal activity was shown by methanolic extract of A. solani. Plants interact with different biological entities according to environmental conditions continuously and evolved. These types of interactions induce changes positively and negatively on plant metabolism and metabolites production. Many plant viruses also attacked various host plants consequently alter their secondary metabolism. To overcome such virus infected plants produces many important and different types of secondary plant metabolites as a defense response. Subsequent analysis of this n-hexane plant extract using Gas chromatography mass spectroscopy technique revealed that Hibiscus eluted contained 10 main compounds in Healthy sample and 13 compounds in infected one. Presence of essential secondary metabolites were also analyzed by FTIR analysis. The present study provides a comprehensive and novel review on taxonomy (morphology, anatomy) and antimicrobial potential of both healthy and geminivirus infected H. rosa-sinensis.

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.

Combined Omics Approaches Reveal Distinct Mechanisms of Resistance and/or Susceptibility in Sugar Beet Double Haploid Genotypes at Early Stages of Beet Curly Top Virus Infection.

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid production and genetic selection, BCTV resistant breeding lines have been developed. Using BCTV resistant (R) [KDH13; Line 13 and KDH4-9; Line 4] and susceptible (S) [KDH19-17; Line 19] lines, beet leafhopper mediated natural infection, mRNA/sRNA sequencing, and metabolite analyses, potential mechanisms of resistance against the virus and vector were identified. At early infection stages (2- and 6-days post inoculation), examples of differentially expressed genes highly up-regulated in the 'R' lines (vs. 'S') included EL10Ac5g10437 (inhibitor of trypsin and hageman factor), EL10Ac6g14635 (jasmonate-induced protein), EL10Ac3g06016 (ribosome related), EL10Ac2g02812 (probable prolyl 4-hydroxylase 10), etc. Pathway enrichment analysis showed differentially expressed genes were predominantly involved with peroxisome, amino acids metabolism, fatty acid degradation, amino/nucleotide sugar metabolism, etc. Metabolite analysis revealed significantly higher amounts of specific isoflavonoid O-glycosides, flavonoid 8-C glycosides, triterpenoid, and iridoid-O-glycosides in the leaves of the 'R' lines (vs. 'S'). These data suggest that a combination of transcriptional regulation and production of putative antiviral metabolites might contribute to BCTV resistance. In addition, genome divergence among BCTV strains differentially affects the production of small non-coding RNAs (sncRNAs) and small peptides which may potentially affect pathogenicity and disease symptom development.

Beet Curly Top Iran Virus Rep and V2 Suppress Post-Transcriptional Gene Silencing via Distinct Modes of Action.

Beet curly top Iran virus (BCTIV) is a yield-limiting geminivirus belonging to the becurtovirus genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles Mastrevirus, whereas the virion strand organization is similar to the Curtovirus genus. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) levels. Multiple geminivirus genes have been identified as viral suppressors of RNA silencing (VSR) but VSR activity remains mostly elusive in becurtoviruses. We found that BCTIV-V2 and -Rep could suppress specific Sense-PTGS mechanisms with distinct efficiencies depending on the nature of the silencing inducer and the target gene. Local silencing induced by GFP inverted repeat (IR) could not be suppressed by V2 but was partially reduced by Rep. Accordingly, we documented that Rep but not V2 could suppress systemic silencing induced by GFP-IR. In addition, we showed that the VSR activity of Rep was partly regulated by RNA-dependent RNA Polymerase 6 (RDR6), whereas the VSR activity of V2 was independent of RDR6. Domain mapping for Rep showed that an intact Rep protein was required for the suppression of PTGS. In summary, we showed that BCTIV-Rep and -V2 function as silencing suppressors with distinct modes of action.

A NitroPure Nitrocellulose Membrane-Based Grapevine Virus Sampling Kit: Development and Deployment to Survey Japanese Vineyards and Nurseries.

We developed a NitroPure Nitrocellulose (NPN) membrane-based method for sampling and storing grapevine sap for grapevine virus detection. We devised an efficient nucleic acid extraction method for the NPN membrane, resulting in 100% amplification success for grapevine leafroll-associated virus 2 (GLRaV2) and 3 (GLRaV3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A, grapevine virus B, and grapevine red blotch virus (GRBV). This method also allowed the storage of recoverable nucleic acid for 18 months at room temperature. We created a sampling kit to survey GLRaV2, GLRaV3, and GRBV in Japanese vineyards. We tested the kits in the field in 2018 and then conducted mail-in surveys in 2020-2021. The results showed a substantial prevalence of GLRaV3, with 48.5% of 132 sampled vines being positive. On the other hand, only 3% of samples tested positive for GLRaV2 and none for GRBV.

Inhibition of pepper huasteco yellow veins virus by foliar application of ZnO nanoparticles in Capsicum annuum L.

The Pepper huasteco yellow vein virus (PHYVV) is an endemic geminivirus in Mexico causing partial or total losses in the pepper crop since the damage caused by the virus has not been fully controlled. In this work, we evaluated the effect of ZnO NPs (0, 50, 100, 150, and 200 mM) as a preventive (72 h before) and curative (72 h after) treatment of PHYVV infection in two jalapeño pepper varieties. In this study, we observed a decrease in symptoms, and it could be caused by an induction of the defense system in pepper plants and a direct action on PHYVV by foliar application of ZnO NPs. Our findings suggest that ZnO NP application significantly decreased the viral titer for both varieties at 200 mM by 15.11-fold. However, this effect was different depending on the timing of application and the variety of pepper. The greatest decrease in the viral titer in the preventive treatment in both varieties was at the concentration of 200 mM (1781.17 and 274.5 times, respectively). For curative treatment in cv. Don Pancho at the concentration of 200 mM (333.33 times) and cv. Don Benito at 100 mM (43.10 folds). compared to control. Furthermore, virus mobility was generally restricted for both varieties at 100 mM (15.13-fold) compared to the control. The results possibly delineated that ZnO NPs increased plant resistance possibly by increasing POD (2.08 and 0.25 times) and SOD (0.998 and 1.38) in cv. Don Pancho and cv. Don Benito, respectively. On the other hand, in cv. Don Pancho and cv. Don Benito presented a decrease in CAT (0.61 and 0.058) and PAL (0.78 and 0.77), respectively. Taken together, we provide the first evidence to demonstrate the effect of ZnO NPs on viral symptoms depending on the plan-virus-ZnO NP interaction.

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.