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Two phylogenetically unrelated viruses transmitted by different insect vectors, tomato spotted wilt virus (TSWV) and tomato yellow leaf curl virus (TYLCV), are major threats to tomato and other vegetable production. Although co-infections of TSWV and TYLCV on the same host plant have been reported on numerous occasions, there is still lack of research attempting to elucidate the mechanisms underlying the relationship between two viruses when they coexist in the same tomato or other plants. After assessing the effect of four TSWV-coded proteins on suppressing TYLCV in TSWV N transgenic Nicotiana benthamiana seedlings, the TSWV N protein proved to be effective in reducing TYLCV quantity and viral symptoms. Western blot analysis indicated that TSWV N was involved in down-regulating the expression level of the V1, C3, and C4 proteins of TYLCV, among which V1 was the most significantly suppressed one. Moreover, TSWV N was confirmed to reduce TYLCV V1 within both nucleus and cytoplasm, but a greater suppression was observed in cytoplasm. The co-immunoprecipitation and mass spectrometry identified 244 differential proteins from the TYLCV-infected TSWV N transgenic N. benthamiana seedling. These proteins pertaining to energy metabolism pathways were enriched, suggesting that TSWV N could inhibit TYLCV through competing for energy or regulating energy-related metabolism. The evidence presented here offers a novel perspective that will facilitate a comprehensive understanding of virus-virus and virus-host interactions, as well as a potential strategy for plant virus control through using TSWV N in the near future., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Hemipteran insects transmit viruses when infesting plants, during which vectors activate salicylic acid (SA)-regulated antiviral defenses. How vector-borne plant viruses circumvent these antiviral defenses is largely unexplored. During co-infections of begomoviruses and betasatellites in plants, betasatellite-encoded βC1 proteins interfere with SA signaling and reduce the activation of antiviral resistance. βC1 inhibits SA-induced degradation of NbNPR3 (Nicotiana benthamiana nonexpressor of pathogenesis-related genes 3), a negative regulator of SA signaling. βC1 does not bind directly to NbNPR3, but regulates NbNPR3 degradation via heat shock protein 90s (NbHSP90s). NbHSP90s bind to both NbNPR3 and βC1 and suppress SA signaling. This viral success strategy appears to be conserved as it is also documented for viral proteins encoded by two aphid-borne viruses. Our findings reveal an exquisite mechanism that facilitates the persistence of vector-borne plant viruses and provide important insights into the intricacies of the virus life cycle., (© 2024. The Author(s).)

The majority of plant viruses rely on insect vectors for inter-plant transmission. Amid virus transmission, vector-borne viruses such as begomoviruses may significantly modulate host plants in various ways and, in turn, plant palatability to insect vectors. While many case studies on monopartite begomoviruses are available, bipartite begomoviruses are understudied. More importantly, detailed elucidation of the molecular mechanisms involved is limited. Here, we report the mechanisms by which an emerging bipartite begomovirus, the Sri Lankan cassava mosaic virus (SLCMV), modulates plant defenses against whitefly. SLCMV infection of tobacco ( Nicotiana tabacum ) plants significantly downregulated defenses against whitefly, as whitefly survival and fecundity increased significantly on virus-infected plants when compared to the controls. We then profiled SLCMV-induced transcriptomic changes in plants and identified a repertoire of differentially expressed genes (DEGs). GO enrichment analysis of DEGs demonstrated that the term defense response was significantly enriched. Functional analysis of DEGs associated with defense response revealed that four downregulated DEGs, including putative late blight resistance protein homolog R1B-17 ( R1B-17 ), polygalacturonase inhibitor-like ( PGI ), serine/threonine protein kinase CDL1-like ( CDL1 ), and Systemin B , directly contributed to plant defenses against whitefly. Taken together, our findings elucidate the role of novel plant factors involved in the modulation of plant defenses against whitefly by a bipartite begomovirus and shed new light on insect vector-virus-host plant tripartite interactions.

Insect vector-virus-plant interactions have important ecological and evolutionary implications. The constant struggle of plants against viruses and insect vectors has driven the evolution of multiple defense strategies in the host as well as counter-defense strategies in the viruses and insect vectors. Cotton leaf curl Multan virus (CLCuMuV) is a major causal agent of cotton leaf curl disease in Asia and is exclusively transmitted by the whitefly Bemisia tabaci. Here, we report that plants infected with CLCuMuV and its betasatellite CLCuMuB enhance the performance of the B. tabaci vector, and βC1 encoded by CLCuMuB plays an important role in begomovirus-whitefly-tobacco tripartite interactions. We showed that CLCuMuB βC1 suppresses the jasmonic acid signaling pathway by interacting with the subtilisin-like protease 1.7 (NtSBT1.7) protein, thereby enhancing whitefly performance on tobacco plants. Further studies revealed that in wild-type plants, NtSBT1.7 could process tobacco preprohydroxyproline-rich systemin B (NtpreproHypSysB). After CLCuMuB infection, CLCuMuB βC1 could interfere with the processing of NtpreproHypSysB by NtSBT1.7, thereby impairing plant defenses against whitefly. These results contribute to our understanding of tripartite interactions among virus, plant, and whitefly, thus offering ecological insights into the spread of vector insect populations and the prevalence of viral diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

RNA-dependent RNA polymerases (RDRs) are key players in the antiviral defence mediated by RNA silencing in plants. RDR6 is one of the major components of the process, regulating the infection of certain RNA viruses. To better clarify its function against DNA viruses, we analyzed the effect of RDR6 inactivation (RDR6i) in N. benthamiana plants on two phloem-limited begomoviruses, the bipartite Abutilon mosaic virus (AbMV) and the monopartite tomato yellow leaf curl Sardinia virus (TYLCSV). We observed exacerbated symptoms and DNA accumulation for the New World virus AbMV in RDR6i plants, varying with the plant growth temperature (ranging from 16 °C to 33 °C). However, for the TYLCSV of Old World origin, RDR6 depletion only affected symptom expression at elevated temperatures and to a minor extent; it did not affect the viral titre. The accumulation of viral siRNA differed between the two begomoviruses, being increased in RDR6i plants infected by AbMV but decreased in those infected by TYLCSV compared to wild-type plants. In situ hybridization revealed a 6.5-fold increase in the number of AbMV-infected nuclei in RDR6i plants but without egress from the phloem tissues. These results support the concept that begomoviruses adopt different strategies to counteract plant defences and that TYLCSV evades the functions exerted by RDR6 in this host.

In plants, establishment of de novo DNA methylation is regulated by the RNA-directed DNA methylation (RdDM) pathway. RdDM machinery is known to concentrate in the Cajal body, but the biological significance of this localization has remained elusive. Here, we show that the antiviral methylation of the Tomato yellow leaf curl virus (TYLCV) genome requires the Cajal body in Nicotiana benthamiana cells. Methylation of the viral genome is countered by a virus-encoded protein, V2, which interacts with the central RdDM component AGO4, interfering with its binding to the viral DNA; Cajal body localization of the V2-AGO4 interaction is necessary for the viral protein to exert this function. Taken together, our results draw a long sought-after functional connection between RdDM, the Cajal body, and antiviral DNA methylation, paving the way for a deeper understanding of DNA methylation and antiviral defences in plants., Competing Interests: LW, YD, LH, GZ, JZ, RL No competing interests declared, (© 2020, Wang et al.)

In plants, cytosine DNA methylation is an efficient defense mechanism against geminiviruses, since methylation of the viral genome results in transcriptional gene silencing (TGS). As a counter-defense mechanism, geminiviruses encode viral proteins to suppress viral DNA methylation and TGS. However, the molecular mechanisms by which viral proteins contribute to TGS suppression remain incompletely understood. In this study, we found that the C4 protein encoded by tomato leaf curl Yunnan virus (TLCYnV) suppresses methylation of the viral genome through interacting with and impairing the DNA-binding ability of NbDRM2, a pivotal DNA methyltransferase in the methyl cycle. We show that NbDRM2 catalyzes the addition of methyl groups on specific cytosine sites of the viral genome, hence playing an important role in anti-viral defense. Underscoring the relevance of the C4-mediated suppression of NbDRM2 activity, plants infected by TLCYnV producing C4(S43A), a point mutant version of C4 unable to interact with NbDRM2, display milder symptoms and lower virus accumulation, concomitant with enhanced viral DNA methylation, than plants infected by wild-type TLCYnV. Expression of TLCYnV C4, but not of the NbDRM2-interaction compromised C4(S43A) mutant, in 16c-TGS Nicotiana benthamiana plants results in the recovery of GFP, a proxy for suppression of TGS. This study provides new insights into the molecular mechanisms by which geminiviruses suppress TGS, and uncovers a new viral strategy based on the inactivation of the methyltransferase NbDRM2., Competing Interests: The authors have declared that no competing interests exist.

Background: To successfully invade new hosts, plant viruses must break host resistance and be competent to move within and between plant cells. As a means, viral proteins known as pathogenicity determinants have evolved to coordinate a network of protein interactions. The βC1 protein encoded by specific geminiviral satellites acts as a key pathogenicity determinant for this disease-causing family of plant viruses. Post-translational modifications (PTMs) such as ubiquitination and phosphorylation of the βC1 protein have been shown to occur in diverse viruses. However, the relevance of these and other layers of PTMs in host-geminiviral interactions has not been fully understood., Results: Here we identified the significance of a novel layer of PTMs in the βC1 protein of Synedrella yellow vein clearing virus (SyYVCV), a newly identified member of the Begomovirus genus of Geminiviruses. This protein has conserved SUMOylation and SUMO-interacting motifs (SIMs), and we observed SUMOylation of SyYVCV βC1 in host plants as a defensive strategy against ubiquitin-mediated degradation. Counteracting this, SIMs encoded in βC1 mediate the degradation of βC1; however, both these PTMs are essential for the function of βC1 protein since SIM and SUMOylation motif mutants failed to promote pathogenicity and viral replication in vivo. SUMOylation in different motifs of βC1 led to functionally distinct outcomes, regulating the stability and function of the βC1 protein, as well as increased global SUMOylation of host proteins., Conclusion: Our results indicate the presence of a novel mechanism mediating a fine balance between defence and counter-defence in which a SIM site is competitively sought for degradation and, as a counter-defence, βC1 undergoes SUMOylation to escape from its degradation.

In this work, a begomovirus isolated from a bean plant coinfected with the potyviruses bean common mosaic virus and bean common mosaic necrosis virus was characterized. The three viruses were detected by high-throughput sequencing and assembly of total small RNAs, but the begomovirus-related contigs did not allow precise identification. Molecular analysis based on standard DNA amplification techniques revealed the presence of a single bipartite virus, which is a novel begomovirus according to the current taxonomic criteria. Infectious clones were generated and agroinoculated into Phaseolus vulgaris and Nicotiana benthamiana plants. In all cases, viral DNA-A and DNA-B were detected in new growths, but no symptoms were observed, thus indicating that this virus produces asymptomatic infections in both host species.

Geminiviruses are single-stranded DNA viruses that cause devastating diseases in many crops worldwide. The replication enhancer proteins (REn), encoded by the C3 (AC3, and AL3) ORFs of geminiviruses, have critical roles in viral DNA accumulation and symptom development in infected plants. In the current study, we have constructed an infectious clone of the Tobacco curly shoot virus (TbCSV) C3 mutant, TbCSV ΔC3 , that contains two start codon mutations that abrogated C3 ORF expression, but did not alter the amino acid sequence of the C2 ORF. As predicted, the absence of the C3 protein reduced TbCSV DNA accumulation, and over-expression of the C3 protein enhanced TbCSV DNA accumulation in infected leaves of Nicotiana benthamiana. The C3 mutation reduced the expression levels of both virion- and complementary-sense TbCSV genes whereas over-expression of the C3 protein increased TbCSV gene expression. Furthermore, the expression of the wild-type and site-directed mutants of C3 proteins using the potato virus X (PVX) system showed that Y93A mutation reduced the replication enhancement activity of the C3 protein in N. benthamiana. All the available evidence demonstrates that the C3 protein is tightly coupled with TbCSV DNA accumulation. However, the TbCSV ΔC3 mutant was nearly as infectious in N. benthamiana as TbCSV WT and only had slightly delayed and attenuated symptom expression. Our findings demonstrate that TbCSV C3 protein enhances viral replication and gene expression, but has only moderate effects on symptom development in N. benthamiana., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with respect to the data, authorship, or publication of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are non-coding but functional RNA molecules of 21-25 nucleotides in length. MiRNAs play significant regulatory roles in diverse plant biological processes. In order to decipher the relationship between nbe-miR1919c-5p and the accumulations of tobacco curly shoot virus (TbCSV) and its betasatellite (TbCSB) DNAs, as well as viral symptom development, we investigated the function of nbe-miR1919c-5p during TbCSV and TbCSB co-infection in plants using a PVX-and a TRV-based short tandem target mimic (STTM) technology. Suppression of nbe-miR1919c-5p expression using these two technologies enhanced TbCSV and TbCSB co-infection-induced leaf curling symptoms in Nicotiana benthamiana plants. Furthermore, suppression of nbe-miR1919c-5p expression enhanced TbCSV and TbCSB DNA accumulations in the infected plants. Our results can advance our knowledge on the nbe-miR1919c-5p function during TbCSV and TbCSB co-infection.

Begomoviruses can modify their transmission vector, Bemisia tabaci, to benefit their spread, although this may not always be the case. Here, the new begomovirus Ramie mosaic virus (RaMoV) and its vector B. tabaci MED, which is dominant in China and many regions of the world, were used as a model to examine direct and indirect interaction and virus transmission by B. tabaci MED of different sexes. No significant direct or indirect effects of RaMoV were observed in B. tabaci MED females, although RaMoV could shorten the life span of B. tabaci MED females by up to 4 days. A test of RaMoV transmission by different sexes of B. tabaci MED showed that there was higher virus transmission efficiency by females than males. Overall, RaMoV is transmitted by B. tabaci MED in a sex-dependent manner, and further research is needed to uncover the mechanism of the difference in RaMoV transmission by different sexes of B. tabaci.

Background: MicroRNAs (miRNAs) are a class of 21-24 nucleotide endogenous non-coding small RNAs that play important roles in plant development and defense responses to biotic and abiotic stresses. Tobacco curly shoot virus (TbCSV) is a monopartite begomovirus, cause leaf curling and plant stunting symptoms in many Solanaceae plants. The betasatellite of TbCSV (TbCSB) induces more severe symptoms and enhances virus accumulation when co-infect the plants with TbCSV., Methods: In this study, miRNAs regulated by TbCSV and TbCSB co-infection in Nicotiana benthamiana were characterized using high-throughput sequencing technology., Results: Small RNA sequencing analysis revealed that a total of 13 known miRNAs and 42 novel miRNAs were differentially expressed in TbCSV and TbCSB co-infected N. benthamiana plants. Several potential miRNA-targeted genes were identified through data mining and were involved in both catalytic and metabolic processes, in addition to plant defense mechanisms against virus infections according to Gene Ontology (GO) analyses. In addition, the expressions of several differentially expressed miRNAs and their miRNA-targeted gene were validated through quantitative real time polymerase chain reaction (qRT-PCR) approach., Conclusions: A large number of miRNAs are identified, and their target genes, functional annotations also have been explored. Our results provide the information on N. benthamiana miRNAs and would be useful to further understand miRNA regulatory mechanisms after TbCSV and TbCSB co-infection.

Alphasatellites, formerly known as DNA 1, are a satellite-like components associated with begomoviruses (the family Geminiviridae) that require betasatellite for symptom induction but depend on DNA-A for systemic movement. We have converted alphasatellite into gene-silencing vector (modified alphasatellite (∆DNA 1)) by deleting its A-rich region that does not affect the replication nor the movement of the helper virus. Insertion of a transgene green florescence protein (GFP) into ∆DNA 1 resulted in the silencing g of the cognate gene in Nicotiana benthamiana. The silencing persisted for more than one and half month and was associated with the decreased level of mRNA of the target gene. This satellite-like DNA vector induced gene silencing (VIGS) promises to be applicable to other begomovirus/alphasatellite systems, thereby providing the powerful approach to gene discovery and the analysis of gene functions in malvaceous crops. Keywords: cotton; begomovirus; alphasatellite; RNAi.

Whitefly-transmitted viruses of the genus Begomovirus (the family Geminiviridae) have become a limiting factor for agricultural productivity in many warmer parts of the world. The economies of Pakistan and India have, since the early 1990s, suffered losses due to cotton leaf curl disease (CLCuD). The disease is caused by begomoviruses, the most important of which at this time is cotton leaf curl Kokhran virus strain Burewala (CLCuKoV-Bu), and a disease-specific betasatellite, cotton leaf curl Multan betasatellite (CLCuMuB). Efforts to minimize losses due to CLCuD rely mainly on the use of insecticides to kill the whitefly vector; no resistant cotton varieties are currently commercially available. The study described here has investigated RNA interference technology for its potential to yield resistance against CLCuKoV-Bu and three other begomoviruses; CLCuKoV, tomato leaf curl New Delhi virus (ToLCNDV) and Pedilanthus leaf curl virus (PeLCV). Three fragments of the virion-sense V2 gene of CLCuKoV-Bu were transformed into Nicotiana benthamiana in antisense orientation and transgenic lines expressing virus-specific short RNAs were assessed for their ability to yield resistance. Only CLCuKoV-Bu with the V2 sequence closest to the promoter was resistant. Inoculation of CLCuKoV-Bu with CLCuMuB into transgenic plants did not significantly affect the outcome, although viral DNA was detected in number of plants, suggesting that the betasatellite may impair RNAi resistance. Overall the results indicate that targeting the 5' end of V2 gene using antisense-RNA has the potential to deliver resistance against begomoviruses and that RNAi-based resistance imparts some degree of resistance to heterologous viruses. Keywords: geminivirus; begomovirus; RNAi; resistance; CLCuKoV-Burewala; CLCuMuB.

Begomoviruses are important crop viral disease agents, and they are transmitted by whiteflies of the Bemisia tabaci complex. Although the transmission of begomoviruses by whiteflies has been studied for many years, the mechanisms governing differential transmission of begomoviruses by different species of the Bemisia tabaci complex remain largely unknown. Here we firstly compared the transmission efficiency of tobacco curly shoot virus (TbCSV) by four species of the B. tabaci complex and found that Asia II 1 transmitted this virus with the highest efficiency, whereas MEAM1 transmitted it with the lowest. Next, by performing quantitative analysis of virus and immune-fluorescence detection, we found that the efficiency of TbCSV to cross the midgut wall was higher in Asia II 1 than in MEAM1. Finally, we set the quantities of virions in the haemolymph to the same level in Asia II 1 and MEAM1 via injection and then compared their capacity in TbCSV transmission, and found that the difference in TbCSV transmission between them became smaller. Taken together, our findings suggest that the efficiency of a begomovirus to cross the midgut wall of a whitefly to reach the vector's haemolymph plays a significant role in determining transmission of the virus.

RNA silencing is an integral part of the cellular defense mechanisms in plants that act against virus infection. However, the specific role of RNA silencing and the interplay between host and virus components during recovery from geminivirus infection remains unknown. Hence, in this study we aimed to examine the mechanism behind the host-specific recovery of Nicotiana tabacum infected with Tomato leaf curl Gujarat virus (ToLCGV). Unlike Tomato leaf curl New Delhi virus (ToLCNDV), ToLCGV infection resulted in symptom remission in N. tabacum, and we found that this was mainly due to cross-talk between the pre-coat protein (encoded by the AV2 ORF) of the virus and the host RNA-silencing component RNA-dependent RNA polymerase 1 (encoded by NtRDR1) of N. tabacum. Moreover, apart from the AV2 mutant, other mutants of ToLCNDV developed severe symptoms on a transgenic NtRDR1-overexpression line of N. benthamiana. In contrast, inoculation with ToLCGV resulted in symptom remission, which was due to enhanced methylation of the ToLCGV promoter. Our study reveals a novel 'arms race' in which the pre-coat protein of ToLCNDV selectively blocks the recovery process through inhibiting host-specific RDR1-mediated antiviral silencing in tobacco.

Tomato yellow leaf curl virus (TYLCV; genus Begomovirus; family Geminiviridae) infects mainly plants of the family Solanaceae, and the infection induces curling and chlorosis of leaves, dwarfing of the whole plant, and reduced fruit production. Alternatives for direct control of TYLCV and other geminiviruses have been reported, for example, the use of esterified whey proteins, peptide aptamer libraries or artificial zinc finger proteins. The two latter alternatives affect directly the replication of TYLCV as well as of other geminiviruses because the replication structures and sequences are highly conserved within this virus family. Because peptides and proteins offer a potential solution for virus replication control, in this study we show the isolation, biochemical characterization and antiviral activity of a peptide derived from globulins of amaranth seeds (Amaranthus hypochondriacus) that binds to the replication origin sequence (OriRep) of TYLCV and affects viral replication with a consequent reduction of disease symptoms in Nicotiana benthamiana. Aromatic peptides obtained from papain digests of extracted globulins and albumins of amaranth were tested by intrinsic fluorescent titration and localized surface resonance plasmon to analyze their binding affinity to OriRep of TYLCV. The peptide AmPep1 (molecular weight 2.076 KDa) showed the highest affinity value (Kd = 1.8 nM) for OriRep. This peptide shares a high amino acid similarity with a part of an amaranth 11S globulin, and the strong affinity of AmPep1 could be explained by the presence of tryptophan and lysine facilitating interaction with the secondary structure of OriRep. In order to evaluate the effect of the peptide on in vitro DNA synthesis, rolling circle amplification (RCA) was performed using as template DNA from plants infected with TYLCV or another begomovirus, pepper huasteco yellow vein virus (PHYVV), and adding AmPep1 peptide at different concentrations. The results showed a decrease in DNA synthesis of both viruses at increasing concentrations of AmPep1. To further confirm the antiviral activity of the peptide in vivo, AmPep1 was infiltrated into leaves of N. benthamiana plants previously infected with TYLCV. Plants treated with AmPep1 showed a significant decrease in virus titer compared with untreated N. benthamiana plants as well as reduced symptom progression due to the effect of AmPep1 curtailing TYLCV replication in the plant. The peptide also showed antiviral activity in plants infected with PHYVV. This is the first report, in which a peptide is directly used for DNA virus control in plants, supplied as exogenous application and without generation of transgenic lines., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Tomato yellow leaf curl virus (TYLCV) causes great losses in tomato production. In addition to tomato, TYLCV infects many crops or weeds as alternative hosts. These alternative hosts may serve as reservoirs for TYLCV survival and spread. Here, we tested the capability of cultivated, flue-cured tobacco to act as a reservoir host plant for TYLCV. TYLCV DNA was detected in nine flue-cured tobacco cultivars inoculated with an infectious TYLCV clone, although no visible symptoms developed on TYLCV-infected tobacco plants. The percentage of whiteflies with viral DNA increased with an increasing acquisition access period (AAP) and reached 100% after a 12 h AAP on infected tobacco plants. Using infected tobacco plants as virus resources, TYLCV was capable of being transmitted to tobacco and tomato plants by whiteflies, and typical symptoms of TYLCV infection were observed on infected tomato plants but not on infected tobacco plants. Our results suggest that flue-cured tobacco can serve as a reservoir host plant for TYLCV and may play an important role in the spread of TYLCV epidemics in China.

Viruses reshape the intracellular environment of their hosts, largely through protein-protein interactions, to co-opt processes necessary for viral infection and interference with antiviral defences. Due to genome size constraints and the concomitant limited coding capacity of viruses, viral proteins are generally multifunctional and have evolved to target diverse host proteins. Inference of the virus-host interaction network can be instrumental for understanding how viruses manipulate the host machinery and how re-wiring of specific pathways can contribute to disease. Here, we use affinity purification and mass spectrometry analysis (AP-MS) to define the global landscape of interactions between the geminivirus Tomato yellow leaf curl virus (TYLCV) and its host Nicotiana benthamiana . For this purpose, we expressed tagged versions of each of TYLCV-encoded proteins (C1/Rep, C2/TrAP, C3/REn, C4, V2, and CP) in planta in the presence of the virus. Using a quantitative scoring system, 728 high-confidence plant interactors were identified, and the interaction network of each viral protein was inferred; TYLCV-targeted proteins are more connected than average, and connect with other proteins through shorter paths, which would allow the virus to exert large effects with few interactions. Comparative analyses of divergence patterns between N. benthamiana and potato, a non-host Solanaceae , showed evolutionary constraints on TYLCV-targeted proteins. Our results provide a comprehensive overview of plant proteins targeted by TYLCV during the viral infection, which may contribute to uncovering the underlying molecular mechanisms of plant viral diseases and provide novel potential targets for anti-viral strategies and crop engineering. Interestingly, some of the TYLCV-interacting proteins appear to be convergently targeted by other pathogen effectors, which suggests a central role for these proteins in plant-pathogen interactions, and pinpoints them as potential targets to engineer broad-spectrum resistance to biotic stresses., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Recent work reported that Tomato yellow leaf curl virus (TYLCV) is seed-transmissible in tomato, contrary to previous belief. In this work, we explore whether TYLCV is also a seed-borne virus in another member of the Solanaceae family, the experimental host Nicotiana benthamiana.

Geminivirus infection often causes severe vein clearing symptoms in hosts. Recently a betasatellite has emerged as a key regulator of symptom induction. To understand the host-betasatellite interactions in the process of symptom development, a systematic study was carried out involving symptoms induced by a betasatellite associated with radish leaf curl disease (RaLCB) in Nicotiana benthamiana. It has been found that βC1 protein localized to chloroplasts of host cells, and RaLCB lacking βC1, which failed to produce symptoms, had no effect on chloroplast ultrastructure. Vein flecking induced by transiently expressed βC1 was associated with chloroplast ultrastructure. In addition, the betasatellite down-regulates expression of genes involved in chlorophyll biosynthesis as well as genes involved in chloroplast development and plastid translocation. Interestingly, the expression of key host genes involved in chlorophyll degradation remains unaffected. Betasatellite infection drastically reduced the numbers of active reaction centres and the plastoquinol pool size in leaves exhibiting vein clearing symptoms. Betasatellite-mediated impediments at different stages of chloroplast functionality affect the photosynthetic efficiency of N. benthamiana. To the best of the authors' knowledge, this is the first evidence of a chloroplast-targeting protein encoded by a DNA virus which induces vein clearing and structurally and functionally damages chloroplasts in plants., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The begomovirus Tomato yellow leaf curl China virus (TYLCCNV) can benefit its vector, the whitefly Bemisia tabaci, through suppressing the defences of their shared host plants. However, the mechanisms of this vector-virus mutualism remain largely unknown on the insect side of the interaction. Here, we compared the transcriptional profiles of female adult whiteflies of B. tabaci Middle East-Asia Minor 1 feeding on TYLCCNV-free and TYLCCNV-infected tobacco plants using the next-generation sequencing technique and quantitative real-time PCR. Interestingly, the genes involved in the oxidative phosphorylation (OXPHOS) pathway and detoxification enzyme were down-regulated in whiteflies feeding on virus-infected plants. Decreased detoxification activity costs less energy, which may reduce OXPHOS activity. Moreover, the genes involved in redox activity were also down-regulated, which may indicate that the reduced OXPHOS activity decreased reactive oxygen species production. Reduced detoxification activity is likely to attenuate energy costs, thereby enhancing the performance of whiteflies on virus-infected plants. These results provide further insight into the mechanisms of the plant-mediated whitefly-virus mutualism. Moreover, our study suggests that investigating the transcriptional profiles on the insect side of the interaction can advance our understanding of the tripartite interactions., (© 2013 Royal Entomological Society.)

Vectors often perform better on plants infected with pathogens, and this promotes the spread of pathogens. However, few studies have examined how plant defensive compounds mediate such mutualistic relationships. Although tobacco plants are relatively poor host plants for the whitefly Bemisia tabaci, tobacco's suitability to the whitefly was substantially increased when infected by the begomovirus Tomato yellow leaf curl China virus. The change in suitability was associated with induced terpenoid synthesis in whitefly-infested plants and repressed terpenoid synthesis in virus-infected plants. Elevation of terpenoid levels via exogenous stem applications reduced the performance of whiteflies. In contrast, suppression of terpenoid synthesis via gene silencing improved whitefly fitness. By integrating genomics, transcriptomics and metabolomics, this study demonstrated that virus infection depleted the terpenoid-mediated plant defence against whiteflies, thereby favouring vector-virus mutualism. These data suggest that plant terpenoids play a key role in shaping vector-pathogen relationships., (© 2012 Blackwell Publishing Ltd/CNRS.)

• Geminiviruses are plant viruses with circular, single-stranded (ss) DNA genomes that infect a wide range of species and cause important losses in agriculture. Geminiviruses do not encode their own DNA polymerase, and rely on the host cell machinery for their replication. • Here, we identify a positive effect of the curtovirus Beet curly top virus (BCTV) on the begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) infection in Nicotiana benthamiana plants. • Our results show that this positive effect is caused by the promotion of TYLCSV replication by BCTV C2. Transcriptomic analyses of plants expressing C2 unveil an up-regulation of cell cycle-related genes induced on cell cycle re-entry; experiments with two mutated versions of C2 indicate that this function resides in the N-terminal part of C2, which is also sufficient to enhance geminiviral replication. Moreover, C2 expression promotes the replication of other geminiviral species, but not of RNA viruses. • We conclude that BCTV C2 has a novel function in the promotion of viral replication, probably by restoring the DNA replication competency of the infected cells and thus creating a favourable cell environment for viral spread. Because C2 seems to have a broad impact on the replication of geminiviruses, this mechanism might have important epidemiological implications., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Background: Geminiviruses mainly infect terminally differentiated tissues and cells in plants. They need to reprogramme host cellular machinery for DNA replication. This process is thought to be mediated by inactivation of cell-cycle repressor proteins and by induction of host DNA synthesis protein expression through actions of the geminviral replication initiator protein (Rep)., Findings: Exploiting a Nicotiana benthamiana pOri2 line, which is transformed with a transgene consisting of a direct repeat of the African cassava mosaic virus (ACMV)-replication origin (Ori) flanking a non-viral DNA region, and virus-induced RNA silencing (VIGS), the impact of host gene expression on replication of the ACMV-derived replicon was investigated. The ACMV Rep trans-replicated the viral episomal replicon in leaves of young but not older pOri2 plants. Upon VIGS-mediated down-regulation of N. benthamiana NbRBR1, the retinoblastoma-related protein gene coding for a negative cell-cycle suppressor, recovered the ability of ACMV Rep for trans DNA replication, whereas the silencing of NbPCNA coding for the sliding clamp of DNA polymerase had no effect., Conclusions: These results suggest that the cellular machinery for DNA replication in differentiated tissues of older leaves cannot be reprogrammed by Rep alone but may need other uncharacterised viral and plant factors., (© 2011 Bruce et al; licensee BioMed Central Ltd.)

Mungbean yellow mosaic geminivirus (MYMV) causes severe yellow mosaic disease in blackgram, mungbean, Frenchbean, pigeonpea, soybean and mothbean. We attempted to induce resistance against this virus using the transcriptional activator protein gene deleted in the C-terminal activation domain (TrAP-∆AD) and Agrobacterium tumefaciens virE2. MYMV is known to replicate in agroinoculated tobacco leaf discs. Three transgenic tobacco plants which harboured a truncated MYMV transcriptional activator protein gene and two tobacco plants transformed with the octopine type A. tumefaciens virE2 gene were agroinoculated with an A. tumefaciens strain which harboured the partial dimers of both DNA A and DNA B of MYMV. The level of viral DNA accumulation in leaf discs of transgenic plants correlated inversely to the level of the MYMV TrAP-∆AD transcript. Two VirE2-transgenic plants, which complemented tumorigenesis of a virE2 mutant A. tumefaciens strain, effectively reduced MYMV DNA accumulation in the leaf disc agroinoculation assay.

Background: Begomoviruses are single-stranded DNA viruses that cause economically important diseases of many crops throughout the world and induce symptoms in plants, including enations, leaf curling and stunting, that resemble developmental abnormalities. MicroRNAs (miRNAs) are small endogenous RNAs that are involved in a variety of activities, including plant development, signal transduction and protein degradation, as well as response to environmental stress, and pathogen invasion., Results: The present study was aimed at understanding the deregulation of miRNAs upon begomovirus infection. Four distinct begomoviruses African cassava mosaic virus (ACMV), Cabbage leaf curl virus (CbLCuV), Tomato yellow leaf curl virus (TYLCV) and Cotton leaf curl Multan virus/Cotton leaf curl betasatellite (CLCuV/CLCuMB), were used in this study. Ten developmental miRNA were studied. N. benthamiana plants were inoculated with begomoviruses and their miRNA profiles were analysed by northern blotting using specific miRNA probes. The levels of most developmental miRNA were increased in N. benthamiana by TYLCV, CLCuMV/CLCuMB and CbLCuV infection with a common pattern despite their diverse genomic components. However, the increased levels of individual miRNAs differed for distinct begomoviruses, reflecting differences in severity of symptom phenotypes. Some of these miRNA were also common to ACMV infection., Conclusions: Our results have shown a common pattern of miRNAs accumulation upon begomovirus infection. It was found that begomoviruses generally increase the accumulation of miRNA and thus result in the decreased translation of genes involved in the development of plants. Identification of common miRNAs that are deregulated upon begomovirus infection may provide novel targets for control strategies aimed at developing broad-spectrum resistance.

The geminivirus Abutilon mosaic virus (AbMV) encodes two proteins which are essential for viral spread within plants. The nuclear shuttle protein (NSP) transfers viral DNA between the nucleus and cytoplasm, whereas the movement protein (MP) facilitates transport between cells through plasmodesmata and long-distance via phloem. An inducible overexpression system for epitope-tagged NSP and MP in plants yielded unprecedented amounts of both proteins. Western blots revealed extensive posttranslational modification and truncation for MP, but not for NSP. Ultrastructural examination of Nicotiana benthamiana tissues showed characteristic nucleopathic alterations, including fibrillar rings, when epitope-tagged NSP and MP were simultaneously expressed in leaves locally infected with an AbMV DNA A in which the coat protein gene was replaced by a green fluorescent protein encoding gene. Immunogold labelling localized NSP in the nucleoplasm and in the fibrillar rings. MP appeared at the cell periphery, probably the plasma membrane, and plasmodesmata.

In Brazil, at least eight begomoviruses including Tomato rugose mosaic virus (ToRMV) and Tomato yellow spot virus (ToYSV) infect tomatoes. ToYSV symptoms in tomato and Nicotiana benthamiana appear earlier and are more severe compared to those of ToRMV. We investigated the role of several factors in this differential adaptation. To analyze infection kinetics, a single leaf was inoculated and subsequently detached after different periods of time. Viral DNA accumulation was quantified in plants, viral replication was analyzed in protoplasts, and tissue tropism was determined by in situ hybridization. Results indicate that ToYSV establishes a systemic infection and reaches a higher concentration earlier than ToRMV in both hosts. ToRMV negatively interferes with ToYSV during the initial stages of infection, but once systemic infection is established this interference ceases. In N. benthamiana, ToYSV invades the mesophyll, while ToRMV is phloem-restricted. During dual infection in this host, ToYSV releases ToRMV from the phloem.

Different Nicotiana benthamiana lines stably transformed with Abutilon mosaic virus (AbMV) dimeric DNA B were capable of systemically spreading complete bipartite AbMV genomes, following agroinoculation of DNA A alone. Constitutively expressed viral movement protein (BC1) did not induce any persistent disease phenotype, but plants developed transient morphological abnormalities such as radially symmetric leaves after kanamycin withdrawal. Systemic AbMV infection produced symptoms and virus titers indistinguishable from those in non-transgenic plants. In systemically invaded leaves, the begomovirus remained phloem-limited, whereas the plants' susceptibility to mechanical transmission of AbMV was enhanced by a factor of three to five, as compared to non-transgenic controls. Hence, DNA B-encoded movement functions can complement local movement to the phloem after mechanical transmission, but fail to support viral invasion of non-phloem cells in systemically infected organs, indicating that the phloem restriction of AbMV does not result predominantly from a lack of transport competence in mesophyll tissues.

Replication of the begomovirus Abutilon mosaic virus (AbMV) is restricted to phloem nuclei, generating moderate levels of virus DNA. Co-infection with Cucumber mosaic virus (CMV) evidently increased AbMV titers in Nicotiana benthamiana, tobacco, and tomato, resulting in synergistic symptom enhancement. In situ hybridization revealed that in double-infected leaves an increased number of nuclei contained elevated amounts of AbMV. Additionally, the begomoviral phloem-limitation was broken. Whereas CMV 3a movement protein-expressing tobacco plants did not exert any similar influence, the presence of CMV 2b silencing suppressor protein lead to enhanced AbMV titers and numbers of infected vascular cells. The findings prove that AbMV can replicate in nonvascular cells and represent the first report on a true synergism of an RNA/ssDNA virus combination in plants, in which CMV 2b protein plays a role. They indicate considerable consequences of mixed infections between begomo- and cucumoviruses on virus epidemiology and agriculture.

Bhendi yellow vein mosaic disease (BYVMD) is caused by a complex consisting of a monopartite begomovirus BYVMV and a satellite DNA beta component. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction. Here we report the results of the transient expression of green fluorescent protein (GFP) fused with the betaC1 and coat protein (CP) coding regions, in the epidermal cells of Nicotiana benthamiana. GFPCP was found to be targeted into the nucleus whereas GFPbetaC1 was localized towards the periphery of the cell. The sub-cellular localization of the betaC1 protein has been compared with that of the CP in yeast cells using a genetic system for detection of protein nuclear import and export. Expression of betaC1 ORF in transgenic N. benthamiana under the control of the Cauliflower mosaic virus 35S promoter produced severe developmental abnormalities in the plant, like distorted stem, leaves and stunting of the plant. We also present the results on the interaction of CP and betaC1 proteins using yeast two hybrid analysis, suggesting a collaborative role in the inter- and intracellular dynamics of BYVMD.