Tomato yellow leaf curl virus manipulates Bemisia tabaci, MEAM1 both directly and indirectly through changes in visual and volatile cues.

The sweetpotato whitefly, Bemisia tabaci MEAM1, is one of the most devastating pests of row-crop vegetables worldwide, damaging crops directly through feeding and indirectly through the transmission of many different viruses, including the geminivirus Tomato yellow leaf curl virus (TYLCV). Y-tube olfactometer tests were conducted at different stages of TYLCV infection in tomatoes to understand how TYLCV affects B. tabaci behavior. We also recorded changes in tomato hosts' color and volatile profiles using color spectrophotometry and gas chromatography-mass spectrometry (GC-MS). We found that the infection status of B. tabaci and the infection stage of TYLCV influenced host selection, with uninfected whiteflies showing a preference for TYLCV-infected hosts, especially during the late stages of infection. Viruliferous B. tabaci attraction to visual targets significantly differed from non-viruliferous B. tabaci. Late-stage infected hosts had larger surface areas reflecting yellow-green wavelengths and higher emissions of methyl salicylate in their volatile profiles. These findings shed new light on several critical mechanisms involved in the viral manipulation of an insect vector and its economically important host.

Multi-GWAS reveals significant genomic regions for Mungbean yellow mosaic India virus resistance in urdbean (Vigna mungo (L.) across multiple environments.

KEY MESSAGE: Unraveling genetic markers for MYMIV resistance in urdbean, with 8 high-confidence marker-trait associations identified across diverse environments, provides crucial insights for combating MYMIV disease, informing future breeding strategies. Globally, yellow mosaic disease (YMD) causes significant yield losses, reaching up to 100% in favorable environments within major urdbean cultivating regions. The introgression of genomic regions conferring resistance into urdbean cultivars is crucial for combating YMD, including resistance against mungbean yellow mosaic India virus (MYMIV). To uncover the genetic basis of MYMIV resistance, we conducted a genome-wide association study (GWAS) using three multi-locus models in 100 diverse urdbean genotypes cultivated across six individual and two combined environments. Leveraging 4538 high-quality single nucleotide polymorphism (SNP) markers, we identified 28 unique significant marker-trait associations (MTAs) for MYMIV resistance, with 8 MTAs considered of high confidence due to detection across multiple GWAS models and/or environments. Notably, 4 out of 28 MTAs were found in proximity to previously reported genomic regions associated with MYMIV resistance in urdbean and mungbean, strengthening our findings and indicating consistent genomic regions for MYMIV resistance. Among the eight highly significant MTAs, one localized on chromosome 6 adjacent to previously identified quantitative trait loci for MYMIV resistance, while the remaining seven were novel. These MTAs contain several genes implicated in disease resistance, including four common ones consistently found across all eight MTAs: receptor-like serine-threonine kinases, E3 ubiquitin-protein ligase, pentatricopeptide repeat, and ankyrin repeats. Previous studies have linked these genes to defense against viral infections across different crops, suggesting their potential for further basic research involving cloning and utilization in breeding programs. This study represents the first GWAS investigation aimed at identifying resistance against MYMIV in urdbean germplasm.

Tomato yellow leaf curl virus: Characteristics, influence, and regulation mechanism.

Tomato yellow leaf curl virus (TYLCV), a DNA virus belonging to the genus Begomovirus, significantly impedes the growth and development of numerous host plants, including tomatoes and peppers. Due to its rapid mutation rate and frequent recombination events, achieving complete control of TYLCV proves exceptionally challenging. Consequently, identifying resistance mechanisms become crucial for safeguarding host plants from TYLCV-induced damage. This review article delves into the global distribution, dispersal patterns, and defining characteristics of TYLCV. Moreover, the intricate interplay between TYLCV and various influencing factors, such as insect vectors, susceptible host plants, and abiotic stresses, plays a pivotal role in plant-TYLCV interactions. The review offers an updated perspective on recent investigations focused on plant response mechanisms to TYLCV infection, including the intricate relationship between TYLCV, whiteflies, and regulatory factors. This comprehensive analysis aims to establish a foundation for future research endeavors exploring the molecular mechanisms underlying TYLCV infection and the development of plant resistance through breeding programs.

Identification of mungbean yellow mosaic India virus and susceptibility-related metabolites in the apoplast of mung bean leaves.

KEY MESSAGE: The investigation of MYMIV-infected mung bean leaf apoplast revealed viral genome presence, increased EVs secretion, and altered stress-related metabolite composition, providing comprehensive insights into plant-virus interactions. The apoplast, an extracellular space around plant cells, plays a vital role in plant-microbe interactions, influencing signaling, defense, and nutrient transport. While the involvement of apoplast and extracellular vesicles (EVs) in RNA virus infection is documented, the role of the apoplast in plant DNA viruses remains unclear. This study explores the apoplast's role in mungbean yellow mosaic India virus (MYMIV) infection. Our findings demonstrate the presence of MYMIV genomic components in apoplastic fluid, suggesting potential begomovirus cell-to-cell movement via the apoplast. Moreover, MYMIV infection induces increased EVs secretion into the apoplast. NMR-based metabolomics reveals altered metabolic profiles in both apoplast and symplast in response to MYMIV infection, highlighting key metabolites associated with stress and defense mechanisms. The data show an elevation of α- and ß-glucose in both apoplast and symplast, suggesting a shift in glucose utilization. Interestingly, this increase in glucose does not contribute to the synthesis of phenolic compounds, potentially influencing the susceptibility of mung bean to MYMIV. Fructose levels increase in the symplast, while apoplastic sucrose levels rise significantly. Symplastic aspartate levels increase, while proline exhibits elevated concentration in the apoplast and reduced concentration in the cytosol, suggesting a role in triggering a hypersensitive response. These findings underscore the critical role of the apoplast in begomovirus infection, providing insights for targeted viral disease management strategies.

A Coiled-Coil Nucleotide-Binding Domain Leucine-Rich Repeat Receptor Gene MeRPPL1 Plays a Role in the Replication of a Geminivirus in Cassava.

Disease resistance gene (R gene)-encoded nucleotide-binding leucine-rich repeat proteins (NLRs) are critical players in plant host defence mechanisms because of their role as receptors that recognise pathogen effectors and trigger plant effector-triggered immunity (ETI). This study aimed to determine the putative role of a cassava coiled-coil (CC)-NLR (CNL) gene MeRPPL1 (Manes.12G091600) (single allele) located on chromosome 12 in the tolerance or susceptibility to South African cassava mosaic virus (SACMV), one of the causal agents of cassava mosaic disease (CMD). A transient protoplast system was used to knock down the expression of MeRPPL1 by clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9). The MeRPPL1-targeting CRISPR vectors and/or SACMV DNA A and DNA B infectious clones were used to transfect protoplasts isolated from leaf mesophyll cells from the SACMV-tolerant cassava (Manihot esculenta) cultivar TME3. The CRISPR/Cas9 silencing vector significantly reduced MeRPPL1 expression in protoplasts whether with or without SACMV co-infection. Notably, SACMV DNA A replication was higher in protoplasts with lower MeRPPL1 expression levels than in non-silenced protoplasts. Mutagenesis studies revealed that protoplast co-transfection with CRISPR-MeRPPL1 silencing vector + SACMV and transfection with only SACMV induced nucleotide substitution mutations that led to altered amino acids in the highly conserved MHD motif of the MeRPPL1-translated polypeptide. This may abolish or alter the regulatory role of the MHD motif in controlling R protein activity and could contribute to the increase in SACMV-DNA A accumulation observed in MeRPPL1-silenced protoplasts. The results herein demonstrate for the first time a role for a CNL gene in tolerance to a geminivirus in TME3.

The Role of Satellites in the Evolution of Begomoviruses.

Begomoviruses have emerged as destructive pathogens of crops, particularly in the tropics and subtropics, causing enormous economic losses and threatening food security. Epidemics caused by begomoviruses have even spread in regions and crops that were previously free from these viruses. The most seriously affected crops include cassava; cotton; grain legumes; and cucurbitaceous, malvaceous, and solanaceous vegetables. Alphasatellites, betasatellites, and deltasatellites are associated with the diseases caused by begomoviruses, but begomovirus-betasatellite complexes have played significant roles in the evolution of begomoviruses, causing widespread epidemics in many economically important crops throughout the world. This article provides an overview of the evolution, distribution, and approaches used by betasatellites in the suppression of host plant defense responses and increasing disease severity.

Dominance of Cotton leaf curl Multan virus-Rajasthan strain associated with third epidemic of cotton leaf curl disease in Pakistan.

Cotton (Gossypium hirsutum) is an economically potent crop in many countries including Pakistan, India, and China. For the last three decades, cotton production is under the constant stress of cotton leaf curl disease (CLCuD) caused by begomoviruses/satellites complex that is transmitted through the insect pest, whitefly (Bemisia tabaci). In 2018, we identified a highly recombinant strain; Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Raj), associated with the Cotton leaf curl Multan betasatellite-Vehari (CLCuMuBVeh). This strain is dominant in cotton-growing hub areas of central Punjab, Pakistan, causing the third epidemic of CLCuD. In the present study, we have explored the CLCuD diversity from central to southern districts of Punjab (Faisalabad, Lodhran, Bahawalpur, Rahimyar Khan) and the major cotton-growing region of Sindh (Tandojam), Pakistan for 2 years (2020-2021). Interestingly, we found same virus (CLCuMuV-Raj) and associated betasatellite (CLCuMuBVeh) strain that was previously reported with the third epidemic in the central Punjab region. Furthermore, we found minor mutations in two genes of CLCuMuV-Raj C4 and C1 in 2020 and 2021 respectively as compared to its isolates in 2018, which exhibited virus evolution. Surprisingly, we did not find these mutations in CLCuMuV-Raj isolates identified from Sindh province. The findings of the current study represent the stability of CLCuMuV-Raj and its spread toward the Sindh province where previously Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Shahdadpur virus (CLCuShV) have been reported. The findings of the current study demand future research on CLCuD complex to explore the possible reasons for prevalence in the field and how the virus-host-vector compatible interaction can be broken to develop resistant cultivars.

Begomovirus Transmission to Tomato Plants Is Not Hampered by Plant Defenses Induced by Dicyphus hesperus Knight.

Plants can respond to insect infestation and virus infection by inducing plant defenses, generally mediated by phytohormones. Moreover, plant defenses alter host quality for insect vectors with consequences for the spread of viruses. In agricultural settings, other organisms commonly interact with plants, thereby inducing plant defenses that could affect plant-virus-vector interactions. For example, plant defenses induced by omnivorous insects can modulate insect behavior. This study focused on tomato yellow leaf curl virus (TYLCV), a plant virus of the family Geminiviridae and genus Begomovirus. It is transmitted in a persistent circulative manner by the whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), posing a global threat to tomato production. Mirids (Hemiptera: Miridae) are effective biological control agents of B. tabaci, but there is a possibility that their omnivorous nature could also interfere with the process of virus transmission. To test this hypothesis, this study first addressed to what extent the mirid bug Dicyphus hesperus Knight induces plant defenses in tomato. Subsequently, the impact of this plant-omnivore interaction on the transmission of TYLCV was evaluated. Controlled cage experiments were performed in a greenhouse setting to evaluate the impact of mirids on virus transmission and vector acquisition by B. tabaci. While we observed a reduced number of whiteflies settling on plants exposed to D. hesperus, the plant defenses induced by the mirid bug did not affect TYLCV transmission and accumulation. Additionally, whiteflies were able to acquire comparable amounts of TYLCV on mirid-exposed plants and control plants. Overall, the induction of plant defenses by D. hesperus did not influence TYLCV transmission by whiteflies on tomato.

Role of aminopeptidase N-like in the acquisition of begomoviruses by Bemisia tabaci, the whitefly vector.

Sri Lankan cassava mosaic virus (SLCMV) is a prominent causative agent of cassava mosaic disease in Asia and relies on the whitefly Bemisia tabaci cryptic complex for its transmission. However, the molecular mechanisms involved in SLCMV transmission by B. tabaci have yet to be understood. In this study, we identified an aminopeptidase N-like protein (BtAPN) in B. tabaci Asia II 1, an efficient vector of SLCMV, which is involved in the SLCMV transmission process. Through the use of glutathione S-transferase pull-down assay and LC-MS/MS analysis, we demonstrated the interaction between BtAPN and the coat protein (CP) of SLCMV. This interaction was further confirmed in vitro, and we observed an induction of BtAPN gene expression following SLCMV infection. By interfering with the function of BtAPN, the quantities of SLCMV were significantly reduced in various parts of B. tabaci Asia II 1, including the whole body, midgut, hemolymph, and primary salivary gland. Furthermore, we discovered that BtAPN is conserved in B. tabaci Middle East-Asia Minor 1 (MEAM1) and interacts with the CP of tomato yellow leaf curl virus (TYLCV), a begomovirus known to cause severe damage to tomato production. Blocking BtAPN with antibody led to a significant reduction in the quantities of TYLCV in whitefly whole body and organs/tissues. These results demonstrate that BtAPN plays a generic role in interacting with the CP of begomoviruses and positively regulates their acquisition by the whitefly.

Systemic resistance induced in tomato plants by Beauveria bassiana-derived proteins against tomato yellow leaf curl virus and aphid Myzus persicae.

BACKGROUND: Tomato (Solanum lycopersicum L.) is an economically important vegetable crop around the globe. Tomato yellow leaf curling (TYLC) is the most devastating viral disease posing a serious threat to tomato production throughout the tropical and subtropical world. Induction of microbe-mediated systemic resistance in plants has been of great interest in recent years as a novel microbiological tool in disease and insect pest management. This in-vitro study aimed to determine the effectiveness of different strains (BB252, BB72 and ARSEF-2860) of a hypocreal fungus Beauveria bassiana against TYLCV disease and aphid Myzus persicae. Potted tomato plants exogenously treated with conidial and filtrate suspensions of B. bassiana strains and of their partially purified or purified proteins were exposed to TYLCV inoculum and aphid M. persicae. RESULTS: Results showed a significant suppression of TYLCV disease severity index by the exogenous application of conidial, filtrate and protein treatments of all B. bassiana strains and this response was directly proportional to the treatment concentration. Similarly, mean fecundity rate of M. persicae was also significantly reduced by the highest concentration of ARSEF-2860-derived elicitor protein PeBb1, followed by the highest concentrations of BB252- and BB72-derived partially purified proteins. Moreover, these B. bassiana-derived proteins also caused a significant upregulation of most of the plant immune marker genes associated with plant defense. CONCLUSION: Overall, the study findings suggest that these B. bassiana strains and their partially purified or purified elicitor proteins could be effective biological tools for the management of TYLCV and aphid infestation on tomato plants. © 2023 Society of Chemical Industry.

Phenotype and signaling pathway analysis to explore the interaction between tomato plants and TYLCV in different organs.

Tomato yellow leaf curl disease (TYLCD), caused by Tomato yellow leaf curl virus (TYLCV), is one of the most destructive diseases in tomato cultivation. By comparing the phenotypic characteristics and virus quantities in the susceptible variety 'Cooperation 909 Red Tomatoes' and the resistant variety 'Huamei 204' after inoculation with TYLCV infectious clones, our study discovered that the root, stem and leaf growth of the susceptible variety 'Cooperation 909 Red Tomatoes' were severely hindered and the resistant variety 'Huamei 204' showed growth inhibition only in roots. TYLCV accumulation in roots were significantly higher than in leaves. Further, we examined the expression of key genes in the SA and JA signalling pathways in leaves, stems and roots and found the up-regulation of SA-signalling genes in all organs of the susceptible variety after inoculation with TYLCV clones. Interestingly, SlJAZ2 in roots of the resistant variety was significantly down-regulated upon TYLCV infection. Further, we silenced the SlNPR1 and SlCOI1 genes individually using virus induced gene silencing system in tomato plants. We found that viruses accumulated to a higher level in SlNPR1 silenced plants than wild type plants, and the virus quantity in roots was significantly increased in SlCOI1 silenced plants. These results provide new insights for advancing research in understanding tomato-TYLCV interaction.

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.

The essential role of clathrin-mediated endocytosis and early endosomes in the trafficking of begomoviruses through the primary salivary glands of their whitefly vectors.

IMPORTANCE: Many plant viruses are transmitted by insect vectors in a circulative manner. For efficient transmission, the entry of the virus from vector hemolymph into the primary salivary gland (PSG) is a step of paramount importance. Yet, vector components mediating virus entry into PSG remain barely characterized. Here, we demonstrate the role of clathrin-mediated endocytosis and early endosomes in begomovirus entry into whitefly PSG. Our findings unravel the key components involved in begomovirus transport within the whitefly body and transmission by their whitefly vectors and provide novel clues for blocking begomovirus transmission.

Differential Effects of RNA-Dependent RNA Polymerase 6 (RDR6) Silencing on New and Old World Begomoviruses in Nicotiana benthamiana.

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.

Genome-wide identification and expression analysis of wall-associated kinase (WAK) and WAK-like kinase gene family in response to tomato yellow leaf curl virus infection in Nicotiana benthamiana.

BACKGROUND: Tomato yellow leaf curl virus (TYLCV) is a major monopartite virus in the family Geminiviridae and has caused severe yield losses in tomato and tobacco planting areas worldwide. Wall-associated kinases (WAKs) and WAK-like kinases (WAKLs) are a subfamily of the receptor-like kinase family implicated in cell wall signaling and transmitting extracellular signals to the cytoplasm, thereby regulating plant growth and development and resistance to abiotic and biotic stresses. Recently, many studies on WAK/WAKL family genes have been performed in various plants under different stresses; however, identification and functional survey of the WAK/WAKL gene family of Nicotiana benthamiana have not yet been performed, even though its genome has been sequenced for several years. Therefore, in this study, we aimed to identify the WAK/WAKL gene family in N. benthamiana and explore their possible functions in response to TYLCV infection. RESULTS: Thirty-eight putative WAK/WAKL genes were identified and named according to their locations in N. benthamiana. Phylogenetic analysis showed that NbWAK/WAKLs are clustered into five groups. The protein motifs and gene structure compositions of NbWAK/WAKLs appear to be highly conserved among the phylogenetic groups. Numerous cis-acting elements involved in phytohormone and/or stress responses were detected in the promoter regions of NbWAK/WAKLs. Moreover, gene expression analysis revealed that most of the NbWAK/WAKLs are expressed in at least one of the examined tissues, suggesting their possible roles in regulating the growth and development of plants. Virus-induced gene silencing and quantitative PCR analyses demonstrated that NbWAK/WAKLs are implicated in regulating the response of N. benthamiana to TYLCV, ten of which were dramatically upregulated in locally or systemically infected leaves of N. benthamiana following TYLCV infection. CONCLUSIONS: Our study lays an essential base for the further exploration of the potential functions of NbWAK/WAKLs in plant growth and development and response to viral infections in N. benthamiana.

Zinc finger protein 330 regulates Ramie mosaic virus infection in the whitefly Bemisia tabaci MED.

BACKGROUND: The whitefly, Bemisia tabaci (Gennadius) is one of the most economically important pests that cause serious damage to agricultural production by transmitting plant pathogenic viruses. Approximately 90% of the virus species transmitted by the whitefly are members of the genus begomovirus. Ramie mosaic virus (RaMoV) is a new bipartite begomovirus that causes severe damage to ramie and several other economic crops in China. In previous studies, we have demonstrated that RaMoV had no obvious direct or indirect effects on B. tabaci. However, whether B. tabaci affects RaMoV infection and the molecular mechanisms of their interaction remain unclear. RESULTS: Here, we identified a zinc finger protein 330 (ZNF330) in B. tabaci MED interacted with the coat protein (CP) of RaMoV by the yeast two-hybrid assay. Then the interaction between ZNF330 and RaMoV CP was further verified by glutathione S-transferase (GST) pull-down assay. The expression of ZNF330 gene was continuously induced after RaMoV infection. ZNF330 negatively regulated RaMoV replication in the B. tabaci MED. Furthermore, the longevity and fecundity of RaMoV-infected female adults were significantly decreased after silencing of ZNF330. CONCLUSIONS: Our results indicated that the ZNF330 protein was involved in the negative regulation of RaMoV replication in the B. tabaci MED. High viral accumulation caused by ZNF330 silencing is detrimental to fecundity and longevity of the B. tabaci MED. These findings provided a new insight into identifying the binding partners in whitefly with viral CP and fully understanding the complex interactions between begomoviruses and their whitefly vector. © 2023 Society of Chemical Industry.

How To Be a Successful Monopartite Begomovirus in a Bipartite-Dominated World: Emergence and Spread of Tomato Mottle Leaf Curl Virus in Brazil.

Begomoviruses are members of the family Geminiviridae, a large and diverse group of plant viruses characterized by a small circular single-stranded DNA genome encapsidated in twinned quasi-icosahedral virions. Cultivated tomato (Solanum lycopersicum L.) is particularly susceptible and is infected by >100 bipartite and monopartite begomoviruses worldwide. In Brazil, 25 tomato-infecting begomoviruses have been described, most of which are bipartite. Tomato mottle leaf curl virus (ToMoLCV) is one of the most important of these and was first described in the late 1990s but has not been fully characterized. Here, we show that ToMoLCV is a monopartite begomovirus with a genomic DNA similar in size and genome organization to those of DNA-A components of New World (NW) begomoviruses. Tomato plants agroinoculated with the cloned ToMoLCV genomic DNA developed typical tomato mottle leaf curl disease symptoms, thereby fulfilling Koch's postulates and confirming the monopartite nature of the ToMoLCV genome. We further show that ToMoLCV is transmitted by whiteflies, but not mechanically. Phylogenetic analyses placed ToMoLCV in a distinct and strongly supported clade with other begomoviruses from northeastern Brazil, designated the ToMoLCV lineage. Genetic analyses of the complete sequences of 87 ToMoLCV isolates revealed substantial genetic diversity, including five strain groups and seven subpopulations, consistent with a long evolutionary history. Phylogeographic models generated with partial or complete sequences predicted that the ToMoLCV emerged in northeastern Brazil >700 years ago, diversifying locally and then spreading widely in the country. Thus, ToMoLCV emerged well before the introduction of MEAM1 whiteflies, suggesting that the evolution of NW monopartite begomoviruses was facilitated by local whitefly populations and the highly susceptible tomato host. IMPORTANCE Worldwide, diseases of tomato caused by whitefly-transmitted geminiviruses (begomoviruses) cause substantial economic losses and a reliance on insecticides for management. Here, we describe the molecular and biological properties of tomato mottle leaf curl virus (ToMoLCV) from Brazil and establish that it is a NW monopartite begomovirus indigenous to northeastern Brazil. This answered a long-standing question regarding the genome of this virus, and it is part of an emerging group of these viruses in Latin America. This appears to be driven by widespread planting of the highly susceptible tomato and by local and exotic whiteflies. Our extensive phylogenetic studies placed ToMoLCV in a distinct strongly supported clade with other begomoviruses from northeastern Brazil and revealed new insights into the origin of Brazilian begomoviruses. The novel phylogeographic analysis indicated that ToMoLCV has had a long evolutionary history, emerging in northeastern Brazil >700 years ago. Finally, the tools used here (agroinoculation system and ToMoLCV-specific PCR test) and information on the biology of the virus (host range and whitefly transmission) will be useful in developing and implementing integrated pest management (IPM) programs targeting ToMoLCV.

The tomato yellow leaf curl virus C4 protein alters the expression of plant developmental genes correlating to leaf upward cupping phenotype in tomato.

Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus in the family Geminiviridae, is efficiently transmitted by the whitefly, Bemisia tabaci, and causes serious economic losses to tomato crops around the world. TYLCV-infected tomato plants develop distinctive symptoms of yellowing and leaf upward cupping. In recent years, excellent progress has been made in the characterization of TYLCV C4 protein function as a pathogenicity determinant in experimental plants, including Nicotiana benthamiana and Arabidopsis thaliana. However, the molecular mechanism leading to disease symptom development in the natural host plant, tomato, has yet to be characterized. The aim of the current study was to generate transgenic tomato plants expressing the TYLCV C4 gene and evaluate differential gene expression through comparative transcriptome analysis between the transgenic C4 plants and the transgenic green fluorescent protein (Gfp) gene control plants. Transgenic tomato plants expressing TYLCV C4 developed phenotypes, including leaf upward cupping and yellowing, that are similar to the disease symptoms expressed on tomato plants infected with TYLCV. In a total of 241 differentially expressed genes identified in the transcriptome analysis, a series of plant development-related genes, including transcription factors, glutaredoxins, protein kinases, R-genes and microRNA target genes, were significantly altered. These results provide further evidence to support the important function of the C4 protein in begomovirus pathogenicity. These transgenic tomato plants could serve as basic genetic materials for further characterization of plant receptors that are interacting with the TYLCV C4.

Topical Spray of dsRNA Induces Mortality and Inhibits Chilli Leaf Curl Virus Transmission by Bemisia tabaci Asia II 1.

Chilli leaf curl virus (ChiLCV; genus: Begomovirus), transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in a persistent-circulative manner, is a major constraint in chilli production. The present study demonstrates for the first time that a topical spray of naked double-stranded RNA (dsRNA) on chilli plants causes mortality and inability to acquire and transmit ChiLCV in B. tabaci. dsRNA targeting heat shock protein 70 (hsp70) and fasciclin 2 (fas2) of B. tabaci Asia II 1 was first assessed under controlled conditions through oral delivery. Hsp70 and fas2 dsRNA resulted in up to 82.22% and 72% mortality of B. tabaci and around 12.4- and 8.5-fold decreases in mRNA levels, respectively, 24 h post-ingestion. ChiLCV copies in hsp70 dsRNA-fed B. tabaci steadily decreased with an increase in dsRNA concentration and were undetectable at a higher concentration of dsRNA. However, ChiLCV copies significantly increased in fas2 dsRNA-fed B. tabaci. Transmission of ChiLCV by B. tabaci was completely inhibited post-24 h feeding on hsp70 dsRNA at 3 µg/mL. Naked hsp70 dsRNA was topically sprayed on ChiLCV-infected chilli plants like an insecticide. 67.77% mortality of B. tabaci, 4.6-fold downregulation of hsp70 mRNA, and 1.34 × 1015-fold decreased ChiLCV copies in B. tabaci were recorded when adults were exposed to the dsRNA-treated plants under semi-field conditions. Foliar application of naked dsRNA reduced the ChiLCV transmission by 75% without any visible symptoms in the inoculated plants. A total of 2 consecutive sprays of dsRNA provided significant protection to B. tabaci for up to 20 days under semi-field conditions.

Genotyping-by-sequencing-based QTL mapping reveals novel loci for Pepper yellow leaf curl virus (PepYLCV) resistance in Capsicum annuum.

Disease caused by Pepper yellow leaf curl virus (PepYLCV) is one of the greatest threats to pepper (Capsicum spp.) cultivation in the tropics and subtropics. Resistance to PepYLCV was previously identified in a few Capsicum accessions, but no resistance QTLs have been mapped. This study aimed to elucidate the genetics of PepYLCV resistance in C. annuum L. Augmented inoculation by the viruliferous whitefly Bemisia tabaci was used to evaluate parental lines and an F2 segregating population derived from a cross between resistant C. annuum line LP97 and susceptible C. annuum line ECW30R. Final evaluation was performed six weeks after inoculation using a standardized 5-point scale (0 = no symptoms to 4 = very severe symptoms). A high-density linkage map was constructed using genotyping-by-sequencing (GBS) to identify single-nucleotide polymorphism (SNP) markers associated with PepYLCV resistance in the F2 population. QTL analysis revealed three QTLs, peplcv-1, peplcv-7, and peplcv-12, on chromosomes P1, P7, and P12, respectively. Candidate genes associated with PepYLCV resistance in the QTL regions were inferred. In addition, single markers Chr7-LCV-7 and Chr12-LCV-12 derived from the QTLs were developed and validated in another F2 population and in commercial varieties. This work thus provides not only information for mapping PepYLCV resistance loci in pepper but also forms the basis for future molecular analysis of genes involved in PepYLCV resistance.