Transcriptome and small RNAome profiling uncovers how a recombinant begomovirus evades RDRγ-mediated silencing of viral genes and outcompetes its parental virus in mixed infection.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) causes severe disease of cultivated tomatoes. Geminiviruses replicate circular single-stranded genomic DNA via rolling-circle and recombination-dependent mechanisms, frequently generating recombinants in mixed infections. Circular double-stranded intermediates of replication also serve as templates for Pol II bidirectional transcription. IS76, a recombinant derivative of TYLCV with a short sequence in the bidirectional promoter/origin-of-replication region acquired from a related begomovirus, outcompetes TYLCV in mixed infection and breaks disease resistance in tomato Ty-1 cultivars. Ty-1 encodes a γ-clade RNA-dependent RNA polymerase (RDRγ) implicated in Dicer-like (DCL)-mediated biogenesis of small interfering (si)RNAs directing gene silencing. Here, we profiled transcriptome and small RNAome of Ty-1 resistant and control susceptible plants infected with TYLCV, IS76 or their combination at early and late infection stages. We found that RDRγ boosts production rates of 21, 22 and 24 nt siRNAs from entire genomes of both viruses and modulates DCL activities in favour of 22 and 24 nt siRNAs. Compared to parental TYLCV, IS76 undergoes faster transition to the infection stage favouring rightward transcription of silencing suppressor and coat protein genes, thereby evading RDRγ activity and facilitating its DNA accumulation in both single and mixed infections. In coinfected Ty-1 plants, IS76 efficiently competes for host replication and transcription machineries, thereby impairing TYLCV replication and transcription and forcing its elimination associated with further increased siRNA production. RDRγ is constitutively overexpressed in Ty-1 plants, which correlates with begomovirus resistance, while siRNA-generating DCLs (DCL2b/d, DCL3, DCL4) and genes implicated in siRNA amplification (α-clade RDR1) and function (Argonaute2) are upregulated to similar levels in TYLCV- and IS76-infected susceptible plants. Collectively, IS76 recombination facilitates replication and promotes expression of silencing suppressor and coat proteins, which allows the recombinant virus to evade the negative impact of RDRγ-boosted production of viral siRNAs directing transcriptional and posttranscriptional silencing.

Mutation of the conserved late element in geminivirus CP promoters abolishes Arabidopsis TCP24 transcription factor binding and decreases H3K27me3 levels on viral chromatin.

In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements that lie within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but is instead directed to responsive promoters through interactions with host factors, most likely transcriptional activators and/or repressors. In this study, we describe a repressive plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb Repressive Complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, mutation of the TCP24 binding site (the CLE) in tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest that TCP24 binding to the CLE leads to CP promoter repression and H3K27me3 deposition, while TCP24-AL2 interaction may recruit AL2 to derepress and activate the promoter. Thus, a repressive host transcription factor may be repurposed to target a viral factor essential for promoter activity. The presence of the CLE in many begomoviruses suggests a common scheme for late promoter regulation.

Phylogenetic and recombination analysis of Begomoviruses associated with Cotton leaf curl disease and in silico analysis of viral-host protein interactions.

Cotton leaf curl disease (CLCuD), caused by numerous begomoviruses (BGVs), is a highly disastrous disease in cotton crops worldwide. To date, several efforts have shown limited success in controlling this disease. CLCuD-associated BGVs (CABs) are known for their high rate of intra and interspecific recombinations, which raises an urgent need to find an efficient and conserved target region to combat disease. In the present study, phylogenetic analysis of selected 11 CABs, along with associated alphasatellites, and betasatellites revealed a close evolutionary relationship among them. Recombination analysis of 1374 isolates of CABs revealed 54 recombination events for the major players of CLCuD in cotton and the Cotton leaf curl Multan virus (CLCuMuV) as the most recombinant CAB. Recombination breakpoints were frequent in all regions except C2 and C3. C3-encoded protein, known as viral replication enhancer (REn), promotes viral replication by enhancing the activity of replicase (Rep) protein. Both proteins were found to contain significantly conserved domains and motifs. The identified motifs were found crucial for their interaction with host protein PCNA (Proliferating cell nuclear antigen), facilitating viral replication. Interruption at the REn-PCNA and Rep-PCNA interactions by targeting the identified conserved motifs is proposed as a prospect to halt viral replication, after suitable experimental validation.

Single-tube colorimetric loop-mediated isothermal amplification (LAMP) assay for high-sensitivity detection of SLCMV in cassava from southern India.

Sri Lankan cassava mosaic virus (SLCMV) is a major cause for mosaic infections in cassava leaves, resulting in significant economic losses in southern India. SLCMV leads to growth retardation, leaf curl, and chlorosis in the host, with rapid transmission through whitefly insect vectors. Detecting SLCMV promptly is crucial, and the study introduces a novel and efficient colorimetric Loop-mediated isothermal amplification (LAMP) assay for successful detection in 60 min. Three primer sets were designed to target the conserved region of the SLCMV genome, specifically the coat protein gene, making the assay highly specific. The LAMP assay offers rapid and sensitive detection, completing within 60 min in a temperature-controlled water bath or thermal cycler. Compared to PCR techniques, it demonstrates 100 times superior sensitivity. The visual inspection of LAMP tube results using a nucleic acid dye and observing ladder-like pattern bands in a 2 % agarose gel confirms the presence of SLCMV. The assay is specific to SLCMV, showing no false positives or contaminations when tested against other virus. The standardized SLCMV LAMP assay proves technically efficient, providing a rapid, specific, simple, and low-cost solution, streamlining the detection and management of SLCMV.

Transient expression analysis of promoters of okra enation leaf curl virus in Nicotiana benthamiana, cotton and okra plants.

Viral promoters can be used to drive heterologous gene expression in transgenic plants. As part of our quest to look for new promoters, we have explored, for the first time, the promoters of okra enation leaf curl virus (OELCuV), a begomovirus infecting okra (Abelmoschus esculentus). The Rep and CP promoters of OELCuV fused with the gfp reporter gene, were expressed transiently in the natural host okra and the laboratory host cotton and Nicotiana benthamiana. The expression levels of the promoters were quantified through confocal laser scanning microscopy and GFP assay in N. benthamiana and okra. The results indicated that the Rep promoter was more active than the CP promoter, whose activity was similar to that of CaMV 35S promoter. Additionally, the Rep and CP promoters showed increase of expression, probably due to transactivation, when assayed following inoculation of OELCuV and betasatellite DNAs in cotton plants. A moderate increase in promoter activity in N. benthamiana was also seen, when assayed following the inoculation of the heterologous begomovirus Sri Lankan cassava mosaic virus.

First report of Ageratum yellow vein virus infecting papaya in Lampung, Indonesia.

BACKGROUND: Papaya (Carica papaya) is a tropical fruit of great economic and nutritional importance, loved for its sweet and delicious flesh. However, papaya cultivation faces serious challenges in the form of Begomovirus attacks. Begomoviruses are a group of viruses that pose a serious threat to plants worldwide. Including papaya, Begomovirus has become a significant threat to papaya production in various parts of the world and has been identified in several regions in Indonesia. METHODS: DNA was extracted from seven samples representing different papaya growing areas using a Plant Genomic DNA Mini Kit. Genomic DNA from the samples was subjected to PCR using universal primers of AC2, AC1, SPG1 and SPG2. The PCR products then sequenced using the dideoxy (Sanger) approach. The obtained sequence then compared to the gene bank using BLAST software available at NCBI. Multiple sequence alignment and phylogenetic tree construction were analyzed using the MEGA11 program. RESULTS: Detection based on viral nucleic acid in papaya plants in Pesawaran, Lampung Province with seven sampling points using universal primers SPG1/SPG2 showed positive results for Begomovirus infection with visible DNA bands measuring ± 900 bp. Direct nucleotide sequencing using SPG1/SPG2 primers for the AC2 and AC1 genes of the Begomovirus and confirmed by the BLAST program showed that papaya samples were infected with Ageratum yellow vein virus (AYVV). The phylogenetic results show that AYVV from papaya samples has a close relationship with the AYVV group from several other countries, with 98% homology. CONCLUSION: In the papaya cultivation area in Pesawaran, Lampung province, it was identified as Begomovirus, Ageratum yellow vein virus (AYVV) species and is closely related to the AYVV group from several other countries. Overall, our study further suggests that Ageratum acts as an alternative host and reservoir for Begomovirus.

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.

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.

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.

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.

Molecular markers associated with resistance to squash leaf curl China virus and tomato leaf curl New Delhi virus in tropical pumpkin (Cucurbita moschata Duchesne ex Poir.) breeding line AVPU1426.

Virus diseases are a major production constraint for pumpkin. Recessive resistance to squash leaf curl China virus and tomato leaf curl New Delhi virus has been mapped in Cucurbita moschata (Duchesne ex Poir.) breeding line AVPU1426 to chromosomes 7 and 8, respectively. Molecular markers tightly associated with the resistance loci have been developed and were able to correctly predict resistance and susceptibility with an accuracy of 99% for squash leaf curl China virus resistance and 94.34% for tomato leaf curl New Delhi virus in F2 and back cross populations derived from the original resistance source AVPU1426. The markers associated with resistance are recommended for use in marker-assisted breeding.

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

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

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

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

Distinct tomato yellow leaf curl Chuxiong virus isolated from whiteflies and plants in China and its symptom determinant and suppressor of post-transcriptional gene silencing.

A begomovirus isolated from whiteflies (Bemisia tabaci) and tomato, sweet potato in China was found to be representative of a distinct begomovirus species, for which the name tomato yellow leaf curl Chuxiong virus (TYLCCxV) is proposed. The results of genomic identification and sequence comparison showed that TYLCCxV shares the highest complete nucleotide sequence identity (88.3%) with croton yellow vein mosaic virus (CroYVMV), and may have originated from the recombination between synedrella leaf curl virus (SyLCV) and squash leaf curl Yunnan virus (SLCuYV). Agrobacterium-mediated inoculation showed that TYLCCxV is highly infectious for a range of plant species, producing upward leaf curling, leaf crumpling, chlorosis, distortion, and stunt symptoms in Solanum lycopersicum plants. The results of Southern blot indicated that TYLCCxV is capable of efficiently replicating two heterologous betasatellites. The inoculation of PVX::C4 on Nicotiana benthamiana induced upward leaf curling and stem elongation symptoms, suggesting that TYLCCxV C4 functions as a symptom determinant. TYLCCxV V2 is an important virulence factor that induces downward leaf curling symptoms, elicits systemic necrosis, and suppresses local and systemic GFP silencing in co-agroinfiltrated N. benthamiana and transgenic 16c plants. Considering the multifunctional virulence proteins V2 and C4, the possibility of TYLCCxV causing devastating epidemics on tomato in China is discussed.

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

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

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.

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.

From Metagenomics to Ecogenomics: NGS-Based Approaches for Discovery of New Circular DNA Single-Stranded Viral Species.

Viruses comprise the most abundant genetic material in the biosphere; however, global viral genomic population (virome) has been largely underestimated. Recently, high-throughput sequencing (HTS) has provided a powerful tool for the detection of known viruses and the discovery of novel viral species from environmental and individual samples using metagenomics and ecogenomics approaches, respectively. Viruses with circular DNA single-stranded (ssDNA) genomes belonging to the begomovirus genera (family Geminiviridae) constitute the largest group of emerging plant viruses worldwide. The knowledge of begomoviruses viromes is mostly restricted to crop plant systems; nevertheless, it has been described that noncultivated plants specifically at the interface between wild and cultivated plants are important reservoirs leading to viral evolution and the emergence of new diseases. Here we present a protocol that allows the identification and isolation of known and novel begomoviruses species infecting cultivated and noncultivated plant species. The method consists of circular viral molecules enrichment by rolling circle amplification (RCA) from begomovirus-positive total plant DNA, followed by NGS-based metagenomic sequencing. Subsequently, metagenomic reads are processed for taxonomic classification using Viromescan software and a customized Geminiviridae family database, and begomovirus-related reads are used for contigs assembly and annotation using Spades software and Blastn algorithm, respectively. Then, the obtained begomovirus-related signatures are used as templates for specific primers design and implemented for PCR-based ecogenomic identification of individual samples harboring the corresponding viral species. Lastly, full-length begomovirus genomes are obtained by RCA-based amplification from total plant DNA of selected individual samples, cloning, and viral molecular identity corroborated by Sanger sequencing. Conclusively, the identification and isolation of a novel monopartite begomovirus species native to the New World (NW) named Gallium leaf deformation virus (GLDV) is shown.

Harnessing Viral Promoters for Targeted RNAi: Engineering Phloem-Specific Resistance Against Tomato Yellow Leaf Curl Thailand Virus.

Phloem-specific promoter efficiently triggers graft-transmissible RNA interference (gtRNAi). We leveraged a phloem-specific promoter derived from the Rice tungro bacilliform virus, optimizing the RNAi mechanism's efficiency and specificity. Here, we detail the construction of phloem-specific promoter-based gtRNAi system and its application through grafting experiments, demonstrating its effectiveness in inducing tomato yellow leaf curl Thailand virus (TYLCHTV) resistance in non-transgenic scions. This strategy presents a practical application for protecting crops against viruses without genetically modifying the entire plant.

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.