A balance between vector survival and virus transmission is achieved through JAK/STAT signaling inhibition by a plant virus.

Viruses pose a great threat to animal and plant health worldwide, with many being dependent on insect vectors for transmission between hosts. While the virus-host arms race has been well established, how viruses and insect vectors adapt to each other remains poorly understood. Begomoviruses comprise the largest genus of plant-infecting DNA viruses and are exclusively transmitted by the whitefly Bemisia tabaci. Here, we show that the vector Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway plays an important role in mediating the adaptation between the begomovirus tomato yellow leaf curl virus (TYLCV) and whiteflies. We found that the JAK/STAT pathway in B. tabaci functions as an antiviral mechanism against TYLCV infection in whiteflies as evidenced by the increase in viral DNA and coat protein (CP) levels after inhibiting JAK/STAT signaling. Two STAT-activated effector genes, BtCD109-2 and BtCD109-3, mediate this anti-TYLCV activity. To counteract this vector immunity, TYLCV has evolved strategies that impair the whitefly JAK/STAT pathway. Infection of TYLCV is associated with a reduction of JAK/STAT pathway activity in whiteflies. Moreover, TYLCV CP binds to STAT and blocks its nuclear translocation, thus, abrogating the STAT-dependent transactivation of target genes. We further show that inhibition of the whitefly JAK/STAT pathway facilitates TYLCV transmission but reduces whitefly survival and fecundity, indicating that this JAK/STAT-dependent TYLCV-whitefly interaction plays an important role in keeping a balance between whitefly fitness and TYLCV transmission. This study reveals a mechanism of plant virus-insect vector coadaptation in relation to vector survival and virus transmission.

Begomoviral ßC1 orchestrates organellar genomic instability to augment viral infection.

Chloroplast is the site for transforming light energy to chemical energy. It also acts as a production unit for a variety of defense-related molecules. These defense moieties are necessary to mount a successful counter defense against pathogens, including viruses. Previous studies indicated disruption of chloroplast homeostasis as a basic strategy of Begomovirus for its successful infection leading to the production of vein-clearing, mosaic, and chlorotic symptoms in infected plants. Although begomoviral pathogenicity determinant protein Beta C1 (ßC1) was implicated for pathogenicity, the underlying mechanism was unclear. Here we show that, begomoviral ßC1 directly interferes with the host plastid homeostasis. ßC1 induced DPD1, an organelle-specific nuclease, implicated in nutrient salvage and senescence, as well as modulated the function of a major plastid genome maintainer protein RecA1, to subvert plastid genome. We show that ßC1 was able to physically interact with bacterial RecA and its plant homolog RecA1, resulting in its altered activity. We observed that knocking-down DPD1 during virus infection significantly reduced virus-induced necrosis. These results indicate the presence of a strategy in which a viral protein alters host defense by targeting modulators of chloroplast DNA. We predict that the mechanism identified here might have similarities in other plant-pathogen interactions.

Genetic analysis of resistance to bean leaf crumple virus identifies a candidate LRR-RLK gene.

Bean leaf crumple virus (BLCrV) is a novel begomovirus (family Geminiviridae, genus Begomovirus) infecting common bean (Phaseolus vulgaris L.), threatening bean production in Latin America. Genetic resistance is required to ensure yield stability and reduce the use of insecticides, yet the available resistance sources are limited. In this study, three common bean populations containing a total of 558 genotypes were evaluated in different yield and BLCrV resistance trials under natural infection in the field. A genome-wide association study identified the locus BLC7.1 on chromosome Pv07 at 3.31 Mbp, explaining 8 to 16% of the phenotypic variation for BLCrV resistance. In comparison, whole-genome regression models explained 51 to 78% of the variation and identified the same region on Pv07 to confer resistance. The most significantly associated markers were located within the gene model Phvul.007G040400, which encodes a leucine-rich repeat receptor-like kinase subfamily III member and is likely to be involved in the innate immune response against the virus. The allelic diversity within this gene revealed five different haplotype groups, one of which was significantly associated with BLCrV resistance. As the same genome region was previously reported to be associated with resistance against other geminiviruses affecting common bean, our study highlights the role of previous breeding efforts for virus resistance in the accumulation of positive alleles against newly emerging viruses. In addition, we provide novel diagnostic single-nucleotide polymorphism markers for marker-assisted selection to exploit BLC7.1 for breeding against geminivirus diseases in one of the most important food crops worldwide.

Identification of a novel monopartite begomovirus associated with leaf curl disease of Citharexylum spinosum in India.

The present study reports the complete genome of a novel monopartite begomovirus, named tentatively as "Citharexylum leaf curl virus" (CitLCuV), associated with leaf curl disease of Citharexylum spinosum in India. CitLCuV genome (2767 nucleotide) contained the typical genome organization of Old World begomoviruses and shared the maximum nucleotide sequence identity of 89.7% with a papaya leaf crumple virus (PaLCrV) isolate. In addition, two small non-canonical open reading frames (C5 and C6) were determined in the complementary strand of CitLCuV genome. Phylogenetic analysis revealed the relatedness of CitLCuV to PaLCrV and rose leaf curl virus. Recombination analysis detected a possible recombination event in CitLCuV genome. Based on begomovirus species demarcation criteria, CitLCuV can be regarded as a novel begomoviral species.

Breakdown of Ty-1-Based Resistance to Tomato Yellow Leaf Curl Virus in Tomato Plants at High Temperatures.

The global dissemination of the Israel (IL) and mild (Mld) strains of tomato yellow leaf curl virus (TYLCV) (family Geminiviridae, genus Begomovirus) is a major threat to tomato production in many regions worldwide. The use of resistant hybrid cultivars bearing the dominant resistance genes Ty-1, Ty-3, and Ty-3a has become a common practice for controlling tomato yellow leaf curl disease (TYLCD) caused by TYLCV. However, TYLCD symptoms have been sporadically observed in resistant cultivars grown in seasons when temperatures are high. In this study, we used TYLCV-resistant cultivars with confirmed presence of Ty-1, which were determined using newly developed allele-specific markers based on polymorphisms within the locus. These Ty-1-bearing resistant tomato plants and susceptible plants were infected with TYLCV and grown at moderate or high temperatures. Under high-temperature conditions, the Ty-1-bearing tomato cultivar Momotaro Hope (MH) infected with TYLCV-IL had severe TYLCD symptoms, which were almost equivalent to those of the susceptible cultivar. However, MH plants infected with TYLCV-Mld were symptomless or had slight symptoms under the same temperature condition. The quantitative analysis of the TYLCV-IL viral DNA content revealed a correlation between symptom development and viral DNA accumulation. Furthermore, under high-temperature conditions, TYLCV-IL caused severe symptoms in multiple commercial tomato cultivars with different genetic backgrounds. Our study provided the scientific evidence for the experientially known phenomenon by tomato growers, and it is anticipated that global warming, associated with climate change, could potentially disrupt the management of TYLCV in tomato plants mediated by the Ty-1 gene.

First report of Tomato interveinal chlorosis virus infecting Rhynchosia minima plants in Brazil.

Tomato interveinal chlorosis virus (ToICV; Begomovirus solanumintervenae, genus Begomovirus, family Geminiviridae) has been described infecting tomato (Solanum lycopersicum) and Macroptilium lathyroides in Northeastern (NE) Brazil for more than a decade (Albuquerque et al., 2012; Silva et al., 2012). During a survey in 2020, plants of the leguminous weed Rhynchosia minima exhibiting virus-like symptoms such as mosaic and interveinal chlorosis were observed in the state of Alagoas, NE Brazil. Symptomatic leaf samples of R. minima were randomly collected (n=15; supplementary figure 1). Total DNA from each sample was used as a template for PCR amplification of partial begomoviral DNA-A sequences using the degenerate primer pair PAL1v1978 and PAR1c496, universal for geminiviruses (Rojas et al., 1993). Amplicons of ~1.2 kbp were observed from 12 samples, although this should not be considered as incidence since only symptomatic plants were collected. To identify the begomovirus associated with R. minima, viral genomes were amplified from PCR-positive samples using rolling circle amplification (RCA) (Inoue-Nagata et al., 2004). The RCA products were digested with HindIII, cloned into the pBluescript II KS+ plasmid vector and bidirectionally Sanger-sequenced (Macrogen Inc., Seoul). BLASTn searches indicated that the clones (n=4) reported here corresponded to a begomovirus DNA-A component, and pairwise comparisons showed that they shared the highest identity with ToICV, at 92.4-94.7% nucleotide sequence identity. Based on the species demarcation criteria of ≥91% nucleotide identity for the genus Begomovirus (Brown et al., 2015), the begomoviruses obtained from R. minima are new isolates of ToICV. The new DNA-A sequences of 2,619-2,623 nt in length were deposited in GenBank under accession numbers PP639092 to PP639095. Multiple nucleotide sequence alignments were prepared using the MUSCLE algorithm implemented in MEGA v.11 (Kumar et al., 2018), and a maximum likelihood (ML) tree was reconstructed in RaxML-NG (Kozlov et al., 2019), assuming a general time reversible (GTR) nucleotide substitution model with a gamma (G) model of rate heterogeneity and 1,000 bootstrap replicates. The DNA-A-based tree showed that the ToICV sequences clustered into a monophyletic group, additionally supporting these isolates as members of the species Begomovirus solanumintervenae. At least two independent interspecies recombination events were predicted among the ToICV isolates, with breakpoints located in the Rep-encoding region and ToICV (GenBank Accession JF803253), tomato mottle leaf curl virus (JF803248) and soybean blistering mosaic virus (MN486865) detected as putative parents. To the best of our knowledge, this is the first report of ToICV infecting R. minima worldwide, expanding the host range of this begomovirus. Non-cultivated plants such as R. minima play a crucial role as reservoirs and sources of inoculum for begomoviruses (Paz-Carrasco et al., 2014), reinforcing their relevance to socioeconomically important crops.

An Interspecies Recombinant Sida Yellow Mosaic China Virus Isolate and Betasatellite Cause a Leaf Curl Disease in Tobacco in Hainan, China.

Tobacco (Nicotiana tabacum) is an herbaceous crop. Cigar tobacco, a group of tobacco cultivars, has recently been planted in a few provinces in China. Since its introduction, symptoms such as leaf curling and vein thickening have appeared. Here we report a begomovirus, Sida yellow mosaic China virus-Hainan isolate (designated SiYMCNV-HN), associated with the betasatellite (designated SiYMCNB-HN) as the causal agent of a leaf curl disease in cigar tobacco (N. tabacum cv. Haiyan101) in Hainan Province, China. Phylogenetic and recombination analyses indicate that SiYMCNV-HN is an interspecies recombinant with a SiYMCNV isolate as the major parent and a Sida yellow vein Vietnam virus isolate as the minor parent. Full-length infectious clones of SiYMCNV-HN and SiYMCNB-HN were generated, which were highly infectious and induced high pathogenicity through agroinfiltration in Nicotiana benthamiana and N. tabacum. This newly reported recombinant begomovirus poses potential threats to tobacco plantations in the region.

Genetic diversity and pathogenicity characterization of tomato-infecting begomoviruses in Taiwan.

The tomato yellow leaf curl disease (TYLCD) caused by whitefly (Bemisia tabaci) transmitted begomoviruses (Geminiviridae) has constrained tomato production in Taiwan since 1981. Lisianthus enation leaf curl virus (LELCV), tomato leaf curl Taiwan virus (ToLCTV), and tomato yellow leaf curl Thailand virus (TYLCTHV) were the major viruses associated with TYLCD. In 2019-2020, we investigated TYLCD throughout Taiwan, with a 10-100% incidence on tomato fields. Begomovirus sequences were detected in 321 out of 506 collected samples by PCR with primers PAL1v1978B and PAR1c71H. In 2015-2016, 59 out of 99 samples collected in Hualien-Taitung areas were also found to have begomovirus sequences. Based on the analysis of 68 viral genomic sequences, six begomoviruses were identified, including LELCV, ToLCTV, TYLCTHV, tomato leaf curl Hsinchu virus (ToLCHsV) and two new begomoviruses, tentatively named tomato leaf curl Chiayi virus (ToLCCYV) and tomato leaf curl Nantou virus (ToLCNTV). Various isolates of LELCV and TYLCTHV were grouped into four and two strains, respectively. Recombinants were detected in LELCV-A, -C, and -D, ToLCCYV, ToLCNTV, and TYLCTHV-F. Based on virus specific detection, the majority of TYLCD-associated viruses were mixed-infected by TYLCTHV-B with either TYLCTHV-F, LELCV-A, -B, or -D, and/or ToLCTV. Meanwhile, viral DNA-B was mostly associated with TYLCTHV and all identified DNA-Bs were highly homologous with previous TYLCTHV DNA-B. The pathogenicity of selected begomoviruses was confirmed through agroinfection and whitefly transmission. All tomato plants carrying Ty-1/3 and Ty-2 resistant genes were infected by all LELCV strains and ToLCCYV, although they appeared symptomless, suggesting these viruses could be managed through the use of the resistance pyramid.

Size Restriction Is Required for Proper Functioning of a Bipartite Begomovirus AC4 Protein.

Many geminiviruses, including members of the genus Begomovirus, produce a protein known as C4 or AC4. Whereas C4/AC4 typically consists of more than 80 amino acid residues, a few are much shorter. The significance of these shorter C4/AC4 proteins in viral infection and why the virus maintains their abbreviated length is not yet understood. The AC4 of the begomovirus Tomato leaf curl Hsinchu virus contains only 65 amino acids, but it extends to 96 amino acids when the natural termination codon is replaced with a normal codon. We discovered that both interrupting and extending AC4 were harmful to tomato leaf curl Hsinchu virus (ToLCHsV). The extended AC4 (EAC4) also showed a reduced ability to promote the infection of the heterologous virus Potato virus X than the wild-type AC4. When the wild-type AC4 was fused with yellow fluorescent protein (AC4-YFP), it was predominantly found in chloroplasts, whereas EAC4-YFP was mainly localized to the cell periphery. These results suggest that ToLCHsV's AC4 protein is important for viral infection, and the virus may benefit from the abbreviated length, because it may lead to chloroplast localization. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Differential expression of genes during recovery of Nicotiana tabacum from tomato leaf curl Gujarat virus infection.

MAIN CONCLUSION: Nicotiana tabacum exhibits recovery response towards tomato leaf curl Gujarat virus. Transcriptome analysis revealed the differential expression of defense-related genes. Genes encoding for cysteine protease inhibitor, hormonal- and stress-related to DNA repair mechanism are found to be involved in the recovery process. Elucidating the role of host factors in response to viral infection is crucial in understanding the plant host-virus interaction. Begomovirus, a genus in the family Geminiviridae, is reported throughout the globe and is known to cause serious crop diseases. Tomato leaf curl Gujarat virus (ToLCGV) infection in Nicotiana tabacum resulted in initial symptom expression followed by a quick recovery in the systemic leaves. Transcriptome analysis using next-generation sequencing (NGS) revealed a large number of differentially expressed genes both in symptomatic as well as recovered leaves when compared to mock-inoculated plants. The virus infected N. tabacum results in alteration of various metabolic pathways, phytohormone signaling pathway, defense related protein, protease inhibitor, and DNA repair pathway. RT-qPCR results indicated that Germin-like protein subfamily T member 2 (NtGLPST), Cysteine protease inhibitor 1-like (NtCPI), Thaumatin-like protein (NtTLP), Kirola-like (NtKL), and Ethylene-responsive transcription factor ERF109-like (NtERTFL) were down-regulated in symptomatic leaves when compared to recovered leaves of ToLCGV-infected plants. In contrast, the Auxin-responsive protein SAUR71-like (NtARPSL) was found to be differentially down-regulated in recovered leaves when compared to symptomatic leaves and the mock-inoculated plants. Lastly, Histone 2X protein like (NtHH2L) gene was found to be down-regulated, whereas Uncharacterized (NtUNCD) was up-regulated in both symptomatic as well as recovered leaves compared to the mock-inoculated plants. Taken together, the present study suggests potential roles of the differentially expressed genes that might govern tobacco's susceptibility and/or recovery response towards ToLCGV infection.

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.

Constant ratio between the genomic components of bipartite begomoviruses during infection and transmission.

The genomic components of multipartite viruses are encapsidated in separate virus particles, and the frequencies of genomic components represent one of the key genetic features. Many begomoviruses of economic significance are bipartite, and the details of the association between their genomic components remain largely unexplored. We first analyzed the temporal dynamics of the quantities of DNA-A and DNA-B and the B/A ratio of the squash leaf curl China virus (SLCCNV) in plants and found that while the quantities of DNA-A and DNA-B varied significantly during infection, the B/A ratio remained constant. We then found that changes in the B/A ratio in agrobacteria inoculum may significantly alter the B/A ratio in plants at 6 days post inoculation, but the differences disappeared shortly thereafter. We next showed that while the quantities of DNA-A and DNA-B among plants infected by agrobacteria, sap transmission and whitefly-mediated transmission differed significantly, the B/A ratios were similar. Further analysis of gene expression revealed that the ratio of the expression of genes encoded by DNA-A and DNA-B varied significantly during infection. Finally, we monitored the temporal dynamics of the quantities of DNA-A and DNA-B and the B/A ratio of another bipartite begomovirus, and a constant B/A ratio was similarly observed. Our findings highlight the maintenance of a constant ratio between the two genomic components of bipartite begomoviruses during infection and transmission, and provide new insights into the biology of begomoviruses.

Association of a novel begomovirus species with fenugreek yellow vein disease in India.

BACKGROUND: Fenugreek (Trigonella foenum-graecum L.) is an annual medicinal and spice crop belonging to the family Fabaceae. The occurrence of a yellow vein disease was recorded in fenugreek in Jodhpur (India) in 2022. The infection of begomoviruses in legume crops results in significant yield loss and major economic loss. The current study reports an association of a novel begomovirus species associated with yellow vein disease in Fenugreek. METHODS AND RESULTS: In symptomatic fenugreek plants, geminivirus-like particles were visible under a transmission electron microscope. Further, nucleotide sequence analysis of the rolling circle amplified product revealed 2743 nucleotide DNA-A genome with close relatedness to French bean leaf curl virus (88.21%) and Senna leaf curl virus (87.63%). It was proposed as a new begomovirus species, Fenugreek yellow vein Rajasthan virus. The genome organization suggested the presence of a typical nonanucleotide sequence along with 7 ORFs in DNA-A. A possible recombination event took place in the coat protein (V1) region with Pedilanthus leaf curl virus and Chilli leaf curl virus as major and minor parents. The recombinant virus poses possible threats to several other legume crops. To the best of our knowledge, this is the first report of the association of FeYVRaV with fenugreek yellow vein disease from northwestern India. CONCLUSIONS: In conclusion, the presence of a novel begomovirus species associated with yellow vein disease in fenugreek is alarming and needs further studies on its infectivity to prevent its spread to legume crops.

Identification of begomoviruses associated with the insect vector Bemisia tabaci and various host plants on Java Island, Indonesia.

Begomoviruses are economically important plant viruses and are transmitted by Bemisia tabaci which is a complex of various cryptic species. However, it is uncertain whether most begomoviruses that infect host plants are transmitted by B. tabaci at a similar rate. We compared the begomovirus profiles that were detected in a total of 37 whitefly populations and 52 host plants on Java Island, Indonesia. Seven begomovirus species were detected in B. tabaci at different rates: pepper yellow leaf curl Indonesia virus (PepYLCIV, 56.8%), tomato yellow leaf curl Kanchanaburi virus (TYLCKaV, 46.0%), tomato leaf curl New Delhi virus (ToLCNDV, 21.6%), squash leaf curl China virus (SLCCNV, 21.6%), ageratum yellow vein China virus (AYVCNV, 2.7%), mungbean yellow mosaic India virus (MYMIV, 2.7%), and okra enation leaf curl virus (OELCuV, 2.7%). The begomoviruses were detected at different rates in three cryptic species of B. tabaci. In addition, six begomovirus species were detected in the various host plants at different rates: PepYLCIV (67.3%), TYLCKaV (53.9%), ToLCNDV (13.5%), MYMIV (11.5%), AYVCNV (3.9%), and Tomato yellow leaf curl Thailand virus (TYLCTHV) (1.9%). By comparing the virus presence between whiteflies and plants, five begomoviruses (AYVCNV, MYMIV, PepYLCIV, ToLCNDV, and TYLCKaV) were detected in both samples, but their sequence similarity was highly variable depending on the begomovirus themselves; TYLCKaV was highest (99.4%-100%) than any other viruses. Our study suggests B. tabaci acquire begomoviruses at different rates from plants. This study provides important information on the potential variation in the begomovirus transmission mechanism.

An Insight into Emerging Begomoviruses and their Satellite Complex causing Papaya Leaf Curl Disease.

Papaya leaf curl disease (PaLCD) was primarily detected in India and causes major economic damage to agriculture crops grown globally, seriously threatening food security. Begomoviruses are communicated by the vector Bemisia tabaci, and their transmission efficiency and persistence in the vector are the highest, exhibiting the widest host range due to adaptation and evolution. Symptoms induced during PaLCD include leaf curl, leaf yellowing, interveinal chlorosis, and reduced fruit quality and yield. Consequently, plants have evolved several multi-layered defense mechanisms to resist Begomovirus infection and distribution. Subsequently, Begomovirus genomes organise circular ssDNA of size ~2.5-2.7 kb of overlapping viral transcripts and carry six-seven ORFs encoding multifunctional proteins, which are precisely evolved by the viruses to maintain the genome-constraint and develop complex but integrated interactions with a variety of host components to expand and facilitate successful infection cycles, i.e., suppression of host defense strategies. Geographical distribution is continuing to increase due to the advent and evolution of new Begomoviruses, and sweep to new regions is a future scenario. This review summarizes the current information on the biological functions of papaya-infecting Begomoviruses and their encoded proteins in transmission through vectors and modulating host-mediated responses, which may improve our understanding of how to challenge these significant plant viruses by revealing new information on the development of antiviral approaches against Begomoviruses associated with PaLCD.

Crinivirus Tomato Chlorosis Virus Compromises the Control of Tomato Yellow Leaf Curl Virus in Tomato Plants by the Ty-1 Gene.

Tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato crops in warm regions of the world, and is associated with infections of several whitefly (Bemisia tabaci)-transmitted single-stranded (ss)DNA begomoviruses (genus Begomovirus, family Geminiviridae). The most widespread begomovirus isolates associated with TYLCD are those of the type strain of the Tomato yellow leaf curl virus species, known as Israel (TYLCV-IL). The Ty-1 gene is widely used in commercial tomato cultivars to control TYLCV-IL damage, providing resistance to the virus by restricting viral accumulation and tolerance to TYLCD by inhibiting disease symptoms. However, several reports suggest that TYLCV-IL-like isolates are adapting to the Ty-1 gene and are causes of concern for possibly overcoming the provided control. This is the case with TYLCV-IL IS76-like recombinants that have a small genome fragment acquired by genetic exchange from an isolate of Tomato yellow leaf curl Sardinia virus, another begomovirus species associated with TYLCD. Here we show that TYLCV-IL IS76-like isolates partially break down the TYLCD-tolerance provided by the Ty-1 gene and that virulence differences might exist between isolates. Interestingly, we demonstrate that mixed infections with an isolate of the crinivirus (genus Crinivirus, family Closteroviridae) species Tomato chlorosis virus (ToCV), an ssRNA virus also transmitted by B. tabaci and emerging worldwide in tomato crops, boosts the breakdown of the TYLCD-tolerance provided by the Ty-1 gene either with TYLCV-IL IS76-like or canonical TYLCV-IL isolates. Moreover, we demonstrate the incorporation of the Ty-2 gene in Ty-1-commercial tomatoes to restrict (no virus or virus traces, no symptoms) systemic infections of recombinant TYLCV-IL IS76-like and canonical TYLCV-IL isolates, even in the presence of ToCV infections, which provides more robust and durable control of TYLCD.

First report of Ludwigia yellow vein Vietnam virus causing leaf curling on tobacco plants in Hainan province, China.

Cigar tobacco (Nicotiana tabacum L.) has been recently introduced into China for various industrial applications. From March 2022, certain symptoms of begomovirus infection, including leaf curling and thickening of veins, were sporadically (disease incidence was approximately 0.2%) observed in several cigar tobacco plantations in numerous counties in Hainan Province, China (Figure 1A). These typical symptoms of begomovirus infection were similar to those caused by the sida leaf curl virus-Hainan (SiLCV-HN) begomovirus and its associated betasatellite, as reported in our previous study on cigar tobacco plants in the same region (Wang et al. 2022). In order to determine whether these symptoms were caused by SiLCV-HN or other begomoviruses, samples of leaves were collected from the diseased tobacco plants for DNA extraction, and the total DNA was extracted for viral metagenomics using an Illumina Sequencing platform at Tiangen Biotech, Beijing. A total of 65711396 filtered reads were obtained, of which 65362322 (99.47%) reads matched to the genome of tobacco. The remaining unmapped 349074 (0.53%) reads were analyzed by BLASTn against the virus Refseq Database of GenBank and subsequently assembled. A total of 8 (5+2+1) enriched contigs of the complete sequence of ludwigia yellow vein Vietnam virus (LuYVVNV) and 9 (8+1) contigs of ludwigia yellow vein virus-associated DNA beta (LuYVB) were finally obtained (Table 1). LuYVVNV belongs to the Begomovirus genus that infects various weeds, including Ludwigia octovalvis and Impatiens balsamina. As far as we know, it was reported earliest on weed in Vietnam (Ha et al. 2008). GenBank contains data pertaining to previously identified isolates of LuYVVNV, and the data revealed that the virus was discovered in Vietnam and the Yunnan province of China currently. However, there are no reports on the infection of crops by LuYVVNV to date. The findings of the present study indicated that LuYVVNV and LuYVB could be responsible for the aforementioned symptoms observed on cigar tobacco. The complete genomes of LuYVVNV and LuYVB were amplified using primer pairs designed based on sequence assembly for viral metagenomics (Table 2). Indeed, two DNA bands with length 2763 bp of LuYVVNV genome and 1348 bp of LuYVB were amplified from leaf samples of diseased tobacco (Figure 1B). The products of polymerase chain reaction (PCR) amplification were analyzed by Sanger sequencing, and the complete nucleotide sequences of LuYVVNV and its associated betasatellite were obtained. Analysis with the BLASTn tool of NCBI revealed that the genome sequence of LuYVVNV isolated from the Hainan province of China had the highest identity of 96.9% to a different isolate of LuYVVNV (GenBank accession number: MN210347.1). These two isolates belong to the same strain, according to the latest revision of Begomovirus taxonomy (Brown et al. 2015). The isolate of LuYVVNV identified in this study was designated as LuYVVNV, Hainan isolate (LuYVVNV-HN, GenBank accession number: OP948731). BLASTn analysis further revealed that the associated betasatellite had the highest sequence identity of 96.9% with an LuYVB (GenBank accession number: AJ965541.1) of a different viral isolate, according to the classification and nomenclature of DNA betasatellites of begomoviruses (Briddon et al. 2008). The sequence of LuYVB obtained herein was therefore designated as LuYVB, Hainan isolate (LuYVB-HN, GenBank accession number: OP948732). The pathogenicity of LuYVVNV-HN and LuYVB-HN was determined using infectious clones that were constructed by ligating two fragments of LuYVVNV-HN or LuYVB-HN to a binary pCAMBIA1300 expression vector, as previously described (Wang et al. 2019). Infectious clones of LuYVVNV-HN, LuYVB-HN, and LuYVVNV-HN plus LuYVB-HN were separately agroinfiltrated into N. benthamiana for determining viral pathogenicity. The typical symptoms of begomovirus infection were observed in N. benthamiana plants inoculated with LuYVVNV-HN alone or LuYVVNV-HN plus LuYVB-HN, and the emerging leaves were mildly or severely down-curled, respectively, at 7 days post inoculation (dpi), with 100% disease incidence (6/6) (Figure 1C). Positive PCR products of the AV1 gene of LuYVVNV-HN were obtained from N. benthamiana plants inoculated with LuYVVNV-HN alone or LuYVVNV-HN plus LuYVB-HN. The ßC1 gene of LuYVB-HN was only obtained from N. benthamiana plants co-infected with LuYVVNV-HN and LuYVB-HN (Figure 1D). No symptoms of viral infection were observed in plants individually inoculated with LuYVB-HN, and the results of PCR were negative (Figure 1C and 1D). These findings indicated that the N. benthamiana plants had been successfully inoculated with LuYVVNV-HN, and that LuYVB-HN was incapable of causing infections on its own, but functioned as a helper and enhanced viral pathogenicity. This report is the first to identify isolates of LuYVVNV and LuYVB from cigar tobacco, which is an economically important crop plant. The findings provide insights into the epidemic threat of begomovirus reservoirs in weeds to crop plants, and emphasize the need for monitoring and controlling whitefly-transmitted viral diseases in tobacco plantations worldwide (Ye et al. 2021).

Characterization of Soybean, Tomato, and Nicandra physalodes as Sources of Inoculum of Tomato Severe Rugose Virus to Tomato Crops.

Tomato severe rugose virus (ToSRV) is one of Brazil's main begomoviruses infecting tomato (Solanum lycopersicum). Recent studies indicate that soybean (Glycine max) crops harboring the whitefly Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) may have epidemiological significance by acting as an asymptomatic amplifier host for the virus. In this study, we gathered experimental greenhouse and field evidence of the role of soybean in the epidemiology of the disease caused by ToSRV. Tomato and Nicandra physalodes, known as good sources of inoculum of this begomovirus, were used as references. The infection rates of soybean, tomato, and N. physalodes with ToSRV in greenhouse no-choice transmission tests with B. tabaci MEAM1 were 50, 71.4, and 64.2%, respectively. The transmission efficiencies of ToSRV to tomato when B. tabaci MEAM1 acquired the virus in ToSRV-infected soybean, tomato, and N. physalodes were 43, 33, and 20%, respectively. Leaves of ToSRV-infected soybean, tomato, and N. physalodes used as sources of inoculum had similar virus titers. In the host preference assay, viruliferous whiteflies preferred to land on tomato rather than soybean and N. physalodes, whereas aviruliferous whiteflies landed indistinctly on these plants. Under experimental field conditions, the transmission efficiency of ToSRV to tomato was higher when tomato was used as a source of inoculum, followed by N. physalodes and soybean. Considering that soybean is extensively cultivated in several Brazilian states that also grow tomato, it can serve as an efficient asymptomatic source of inoculum and support the recent hypothesis that it can also play, under certain conditions, a relevant role as an amplifier host in the epidemiology of the disease caused by ToSRV.

Cajanus Scarabaeoides Yellow Mosaic Virus, a New Bipartite Begomovirus Causing Yellow Mosaic Disease in Cajanus scarabaeoides in India.

Yellow mosaic disease of Cajanus scarabaeoides (L.) Thouars (CsYMD) was observed in up to 46% of C. scarabaeoides plants in the mungbean, urdbean, and pigeon pea fields from 22 districts of Chhattisgarh State, India, during 2017 to 2019. The symptoms were characterized by yellow mosaic on green leaves and yellow discoloration of leaves in advanced stages of the disease. Severely infected plants showed shortened internodal length and reduced leaf size. CsYMD was transmissible to healthy C. scarabaeoides and C. cajan by whitefly (Bemisia tabaci). The infected plants developed typical yellow mosaic symptoms on their leaves within 16 and 22 days of inoculation, respectively, suggesting a begomovirus etiology. Molecular analysis revealed that this begomovirus has a bipartite genome composed of DNA-A (2,729 nucleotides) and DNA-B (2,630 nucleotides). Sequence and phylogenetic analyses revealed that the nucleotide sequence of the DNA-A component had the highest identity of 81.1% with DNA-A of Rhynchosia yellow mosaic virus (RhYMV; NC_038885), followed by mungbean yellow mosaic virus (MN602427; 75.3%). DNA-B had the highest identity of 74.0% with DNA-B of RhYMV (NC_038886). As per ICTV guidelines, this isolate had <91% nucleotide identity with DNA-A of any of the begomoviruses reported; so, it is proposed as a new begomovirus species, tentatively named C. scarabaeoides yellow mosaic virus (CsYMV). After agroinoculation with DNA-A and DNA-B clones of CsYMV, all Nicotiana benthamiana plants developed leaf curl symptoms along with light yellowing symptoms 8 to 10 days after inoculation (DAI), while ∼60% of the C. scarabaeoides plants developed yellow mosaic symptoms similar to those observed in the field 18 DAI, thus fulfilling Koch's postulates. From these agro-infected C. scarabaeoides plants, CsYMV was transmissible to healthy C. scarabaeoides plants by B. tabaci. Apart from these hosts, CsYMV also infected and caused symptoms in mungbean and pigeon pea.

First Report of Tomato Leaf Curl Cebu Virus Associated with Stachytarpheta jamaicensis Golden Yellow Mosaic Disease in Taiwan.

Stachytarpheta jamaicensis (L.) Vahl, also known as snack weed, is an exotic plant in Taiwan. In April 2021, severe golden yellow mosaic leaves (Fig. S1) were observed on S. jamaicensis plants in Taichung City, Taiwan. Samples from eight symptomatic and two asymptomatic plants were collected from the public flowerbed. Total DNA was extracted from each of the collected samples by using a modified CTAB method (Echevarría-Machado et al. 2005). PCR with Begomovirus degenerate primers (PAL1v1978/PAR1c715; Rojas et al. 1993) was conducted. The expected 1.5-kb fragment was amplified only from the 8 symptomatic samples. Two randomly selected amplicons were cloned on pCRII-TOPO TA vector (Invitrogen Co., San Diego, CA, USA) and sequenced with the ABI3730 automatic sequencer (Applied Biosystems, Hammonton, NJ, USA) at National Chung Hsing University (NCHU). After NCBI BLASTn analysis, the sequences were shown to be most closely related to tomato leaf curl Cebu virus (ToLCCeV) isolates (EU487042, EU487025, KU946997), with 92.4-92.5% nucleotide sequence identity by using the CLUSTAL W method of MegAlign program (DNASTAR, Inc., Madison, WI, USA). A ToLCCeV specific primer pair (FJJ2021-165 /166 5'-ACTTACAGGCCCATGTATCG-3' / 5'-GAATGGGTATCCGAGCACG-3') was designed to amplify and sequence the remaining half of viral DNA. The expected 1.6-kb amplicon was amplified only from the symptomatic samples. The full-length of DNA-A consisted of 2.7-kb nucleotides (ToLCCeV isolate stachy, ON525110 and ON525111) and contained six open reading frames (two in viral sense, V1 to V2 and four in the viral complementary sense, C1 to C4) and the conserved nonanucleotide motif (TAATATTAC). The full-length DNA-A of ToLCCeV stachy isolates shared 99.9% nucleotide identity to each other and 91.2-92.4% and 91.3-92.5% nucleotide identities to other ToLCCeV isolates (EU487042, EU487025, KU946997) available in NCBI GenBank. Besides, ToLCCeV is a monopartite begomovirus that harbors no DNA-B. Thus, there were no bands amplified from the degenerate primer pair for DNA-B (DNABLC2 / DNABLV2; Green et al. 2001). Furthermore, the infectious clone was constructed by using phi29 DNA polymerase (New England Biolabs, Ipswich, MA, USA) for rolling circle amplification (RCA). The RCA product was partially digested with ApaI (NEB) and ligated into the binary vector pCambia0380 (AF234290). The resulting recombinant vector was transformed into Agrobacterium tumefaciens C58. A. tumefaciens C58, containing the infectious ToLCCeV-Stachy DNA-A vector, was grown overnight in LB broth containing kanamycine (50 µg/ml) at 28°C. S. jamaicensis and Nicotiana benthamiana (Nb, four to six leaf stage) plants were agroinoculated to confirm the infectivity of the ToLCCeV clone. The leaf curling and blister symptoms were observed on the Nb systemic leaves 17-day post inoculation (dpi) and the golden yellow mosaic symptom noticed on S. jamaicensis systemic leaves 30-dpi. The presence of the viral DNA in the inoculated plants was confirmed by PCR using the specific primer pair of ToLCCeV. To the best of our knowledge, this is the first report of the monopartite begomovirus, ToLCCeV, associated with golden yellow mosaic disease in S. jamaicensis in Taiwan. The existence of ToLCCeV might severely impact the tomato and pepper industry because they are the natural hosts of ToLCCeV (Tsai et al. 2011) and ToLCCeV may be transmitted by the whitefly, Bemisia tabaci, in Taiwan (Ko et al. 2005).