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Transcription factors (TFs) play crucial roles in plant development and pathogen defense. However, plant viruses can exploit TFs to facilitate their infection or transmission. In this study, we confirmed the βC1 proteins, encoded by tobacco curly shoot virus (TbCSV)- and tomato yellow leaf curl China virus (TYLCCNV)-associated betasatellites, interacted with GLABROUS1 enhancer binding protein (GeBP) TFs from solanaceous plants including Nicotiana benthamiana, Solanum lycopersicum, S. tuberosum, and Capsicum annuum. Further analysis verified the nuclear localization, homodimerization, and DNA-binding ability of the GeBP TFs, along with its interaction with βC1 in the nucleus. PVX-mediated overexpression of NbGeBP showed no effect on the accumulation of viral and betasatellite DNAs in N. benthamiana plants after infection with TbCSV and its heterologous betasatellite, malvastrum yellow vein virus associated betasatellite (MaYVB), or its homologous betasatellite, TbCSB. However, both TbCSV and MaYVV caused a decrease in NbGeBP expression during the early stages of infection, regardless of the presence of homologous or heterologous betasatellites, implying that NbGeBP might play a role in virus infection. TbCSV/TbCSB and TYLCCNV/TYLCCNB infect many solanaceous plants, and solanaceous GeBP proteins interact with βC1 proteins from TbCSB and TYLCCNB. The yeast two-hybrid and bimoleccular fluorescence complementation assays showed that AtGeBP from Arabidopsis thaliana could not interact with TbCSB βC1, revealing that the GeBP-βC1 interactions might only exist in GeBP proteins from solanaceous plants. Importantly, the βC1 protein from MaYVB, which was almost not reported on natural infection in solanaceous plants, could not interact with GeBP, suggesting the potential roles of GeBP in monopartite begomovirus infection of solanaceous plants. [ABSTRACT FROM AUTHOR]

Summary: The growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant‐based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large‐scale production due to high operational costs and batch‐to‐batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)‐derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harbouring the gene of interest (GOI) flanked by cis‐acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper‐inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast‐derived CUP2‐Gal4 copper‐responsive transcriptional activator for Rep/RepA expression, and (iv) a copper‐inducible Flp recombinase to minimize basal Rep/RepA expression. CuSO4 application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti‐SARS‐CoV‐2 antibody upon copper treatment. Notably, the system is functional in post‐harvest applications, a strategy with high potential impact for large‐scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost‐effective and scalable production of recombinant proteins in plants. [ABSTRACT FROM AUTHOR]

Abstract Whiteflies (Bemisia tabaci) and the diseases they transmit are a major detriment to crop yields and a significant contributor to world hunger. The highly evolved interactions of host plant, phloem-feeding insect vector with endosymbionts and persistently transmitted virus represent a tremendous challenge for interdisciplinary study. Presented here is the establishment of a colony of axenic whiteflies on tissue-cultured plants. Efficient colony establishment was achieved by a surface sterilization of eggs laid on axenic phototrophically tissue-cultured plants. The transfer of emerging whiteflies through coupled tissue culture vessels to new axenic plants facilitates robust subculturing and produces hundreds of whitefly adults per month. Whitefly proliferation on more than two dozen plant species is shown as well as in vitro testing of whitefly preference for different plants. This novel multi-organism system provides the high-level of biocontainment required by Federal permitting to conduct virus transmission experiments. Axenic whitefly adults were able to acquire and transmit a begomovirus into tissue-cultured plants, indicating that culturable gut microorganisms are not required for virus transmission. The approach described enables a wide range of hypotheses regarding whitefly phytopathology without the expense, facilities, and contamination ambiguity associated with current approaches.

Abstract Background Tomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) is a significant threat to cucumber (Cucumis sativus) production in many regions. Previous studies have reported the genetic mapping of loci related to ToLCNDV resistance, but no resistance genes have been identified. Results We conducted map-based cloning of the ToLCNDV resistance gene in cucumber accession No.44. Agroinfiltration and graft-inoculation analyses confirmed the resistance of No.44 to ToLCNDV isolates from the Mediterranean and Asian countries. Initial mapping involving two rounds of phenotyping with two independent F2 populations generated by crossing the begomovirus-susceptible cultivar SHF and No.44 consistently detected major quantitative trait loci (QTLs) on chromosomes 1 and 2 that confer resistance to ToLCNDV. Fine-mapping of Cy-1, the dominant QTL on chromosome 1, using F3 populations narrowed the candidate region to a 209-kb genomic segment harboring 24 predicted genes. Among these genes, DFDGD-class RNA-dependent RNA polymerase (CsRDR3), an ortholog of Ty-1/Ty-3 of tomato and Pepy-2 of capsicum, was found to be a strong candidate conferring ToLCNDV resistance. The CsRDR3 sequence of No.44 contained multiple amino acid substitutions; the promoter region of CsRDR3 in No.44 had a large deletion; and the CsRDR3 transcript levels were greater in No.44 than in SHF. Virus-induced gene silencing (VIGS) of CsRDR3 using two chromosome segment substitution lines harboring chromosome 1 segments derived from No.44 compromised resistance to ToLCNDV. Conclusions Forward and reverse genetic approaches identified CsRDR3, which encodes a DFDGD-class RNA-dependent RNA polymerase, as the gene responsible for ToLCNDV resistance at the major QTL Cy-1 on chromosome 1 in cucumber. Marker-assisted breeding of ToLCNDV resistance in cucumber will be expedited by using No.44 and the DNA markers developed in this study.

Geminiviridae are ssDNA plant viruses whose control has both economical and agricultural importance. Their capsids assemble into two distinct architectural forms: (i) a T = 1 icosahedral and (ii) a unique twinned quasi-isometric capsid. Described here are the high-resolution structures of both forms of the maize streak virus using cryo-EM. A comparison of these two forms provides details of the coat protein (CP) and CP–CP and CP–genome interactions that govern the assembly of the architecture of the capsids. Comparative analysis of other representative members of Geminiviridae reveals structural conservation of 60–95% compared to a sequence similarity of 21–30%. This study provides a structural atlas of these plant pathogens and suggests possible antiviral-targetable regions of these capsids. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas have been recognized as powerful genome-editing tools in diverse eukaryotic species, including plants, and thus hold great promise for engineering virus resistance in plants. Nevertheless, further attention is required regarding various issues associated with applying new powerful technologies in the field. This mini-review focuses on the recent advances in using CRISPR/Cas9 and CRISPR/Cas13 systems to combat DNA and RNA viruses in plants. We explored the utility of CRISPR/Cas for targeting the viral genome and editing host susceptibility genes in plants. We also provide insights into the limitations and challenges of using CRISPR/Cas for plant virus interference and propose individual combinatorial solutions. In conclusion, CRISPR/Cas technology has the potential to offer innovative and highly efficient approaches for controlling viruses in important crops in the near future. [ABSTRACT FROM AUTHOR]

Whiteflies (Bemisia tabaci) and the diseases they transmit are a major detriment to crop yields and a significant contributor to world hunger. The highly evolved interactions of host plant, phloem-feeding insect vector with endosymbionts and persistently transmitted virus represent a tremendous challenge for interdisciplinary study. Presented here is the establishment of a colony of axenic whiteflies on tissue-cultured plants. Efficient colony establishment was achieved by a surface sterilization of eggs laid on axenic phototrophically tissue-cultured plants. The transfer of emerging whiteflies through coupled tissue culture vessels to new axenic plants facilitates robust subculturing and produces hundreds of whitefly adults per month. Whitefly proliferation on more than two dozen plant species is shown as well as in vitro testing of whitefly preference for different plants. This novel multi-organism system provides the high-level of biocontainment required by Federal permitting to conduct virus transmission experiments. Axenic whitefly adults were able to acquire and transmit a begomovirus into tissue-cultured plants, indicating that culturable gut microorganisms are not required for virus transmission. The approach described enables a wide range of hypotheses regarding whitefly phytopathology without the expense, facilities, and contamination ambiguity associated with current approaches. [ABSTRACT FROM AUTHOR]

Begomovirus infections have threatened the global production of multiple crops, including Solanaceae species. Recombination, a frequent phenomenon in begomoviruses, has profound effects on viral populations, allowing them to adapt to changing environments. We previously isolated pepper yellow leaf curl Aceh virus (PepYLCAV) from pepper (Capsicum annuum), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum) plants in Aceh Province, Indonesia, and determined that it is a recombinant of the widespread pepper yellow leaf curl Indonesia virus (PepYLCIV). To compare PepYLCAV with its major putative parent PepYLCIV in terms of virulence, multiple solanaceous species (Nicotiana benthamiana, pepper, and tomato) were inoculated with the two viruses. Symptom severity, growth inhibition, and viral DNA levels were greater with PepYLCAV than with PepYLCIV infection in N. benthamiana and tomato plants. In pepper plants, PepYLCAV caused severe leaf distortion rather than yellowing and greater growth inhibition than PepYLCIV did but with no increase in viral DNA. Inoculation with PepYLCAV compromised Ty-1-mediated resistance in tomato, resulting in moderate symptoms, which were not present in plants infected with PepYLCIV. A re-analysis of samples collected from fields in Indonesia from 2012 to 2019 using PCR showed that PepYLCAV rather than PepYLCIV has been the predominant species since 2017. The recombinant bipartite begomovirus PepYLCAV is more widespread and more virulent than its major putative parent PepYLCIV and can overcome the Ty-1-mediated resistance of commercial F1 hybrid tomato cultivars. [ABSTRACT FROM AUTHOR]

Mulberry crinkle leaf virus (MCLV), identified in mulberry plants (Morus alba L.), is a member of the genus Mulcrilevirus in the family Geminiviridae. The functions of the V2 protein encoded by MCLV remain unclear. Here, Agrobacterium-mediated infectious clones of a wild-type MCLV vII (MCLVWT) and two V2 mutant MCLV vIIs, including MCLVmV2 (with a mutation of the start codon of the V2 ORF) and MCLVdV2 (5′-end partial deletion of the V2 ORF sequence), were constructed to investigate the roles of V2 both in planta and at the cellular level. Although all three constructs (pCA-1.1MCLVWT, pCA-MCLVmV2, and pCA-MCLVdV2) were able to infect both natural host mulberry plants and experimental tomato plants systematically, the replication of the MCLVmV2 and MCLVdV2 genomes in these hosts was significantly reduced compared to that of MCLVWT. Similarly, the accumulation of MCLVmV2 and MCLVdV2 in protoplasts of Nicotiana benthamiana plants was significantly lower than that of MCLVWT either 24 h or 48 h post-transfection. A complementation experiment further confirmed that the decreased accumulation of MCLV in the protoplasts was due to the absence of V2 expression. These results revealed that MCLV-encoded V2 greatly enhances the level of MCLV DNA accumulation and is designated the replication enhancer protein of MCLV. [ABSTRACT FROM AUTHOR]

We focused on the geminiviral vector systems to develop an efficient vector system for plant biotechnology. Begomoviruses and curtoviruses, which belong to the Geminiviridae family, contain an intergenic region (IR) and four genes involved in replication, including replication-associated protein (Rep, C1), transcriptional activator (TrAP, C2), and replication enhancer (REn, C3). Geminiviruses can amplify thousands of copies of viral DNA using plant DNA polymerase and viral replication-related enzymes and accumulate viral proteins at high concentrations. In this study, we optimized geminiviral DNA replicon vectors based on tomato yellow leaf curl virus (TYLCV), honeysuckle yellow vein virus (HYVV), and mild curly top virus (BMCTV) for the rapid, high-yield plantbased production of recombinant proteins. Confirmation of the optimal combination by co-delivery of each replication-related gene and each IR harboring the Pontellina plumata-derived turbo green fluorescence protein (tGFP) gene via agroinfiltration in Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 3 days. Co-expression with the p19 protein of the tomato bush stunt virus, a genesilencing suppressor, further enhanced tGFP accumulation by stabilizing mRNA. With this system, tGFP protein was produced at 0.7-1.2 mg/g leaf fresh weight, corresponding to 6.9-12.1% in total soluble protein. These results demonstrate the advantages of rapid and high-level production of recombinant proteins using the geminiviral DNA replicon system for transient expression in plants. [ABSTRACT FROM AUTHOR]

Abstract 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.

The Tomato Chlorosis Criniviruses (ToCV) and Tomato yellow leaf curl Geminivirus (TYLCV), which naturally infecting and limited to the phloem, have caused a drastic reduction in tomato yield. The current study aims to determine the incidence of single and mixed viruses using biological, serological and molecular PCR methods in natural tomato plants. The incidence of mixed infection was found more frequently, followed by ToCV and TYLCV (42.3, 28.8, 17.8 in 2020 and 49.1, 29.7 and 19.1% in 2021, respectively). ToCV causes chlorosis, TYLCV causes leaf curl and yellowing, while mixed ToCV & TYLCV cause progress symptoms. By using the heat shock protein 70 (HSP70) and coat protein (CP) genes, ToCV and TYLCV isolates could be identified. These isolates were recorded in GenBank under accession codes "ON951644.1" and "OP265136.1" respectively. Host plants responded differently to severe and common disease density between ToCV and TYLCV. The transmitted whitefly could distinguish between ToCV and TYLCV within 15-20 minutes of the acquisition period. ToCV increases in fields with high whitefly populations, requiring further research to understand effects and reduce harm.

Grapevine red blotch virus (GRBV) is the causative agent of red blotch disease. Limited information is available on the seasonal variation of GRBV titer in relation to disease symptom expression in vineyards across the United States. In this study, no statistically significant difference in GRBV titer was found among asymptomatic infected vines in June (P = 0.451) and among symptomatic infected vines in October (P = 0.068) in a diseased ‘Cabernet franc’ vineyard in California, regardless of the years symptomatic, one to seven, as shown by qPCR. Similarly, there were no statistically significant differences in GRBV titer as it related to isolates of the two phylogenetic clades in asymptomatic infected ‘Cabernet franc’ and ‘Cabernet Sauvignon’ vines in June (P = 0.138 and P = 0.778, respectively) and in symptomatic infected vines in October (P = 0.806 and P = 0.490, respectively). GRBV titer differed among cultivars in diseased California vineyards (P < 0.001) and increased over the course of the growing season in infected ‘Merlot’ and ‘Cabernet franc’ vines, but not in ‘Cabernet Sauvignon’ vines. Patterns observed in California were consistent in New York and Georgia vineyards. In a Geneva double curtain-trellised ‘Cabernet Sauvignon’ vineyard in Georgia, GRBV distribution was uneven between cordons, and virus titer was variable within the vine canopy in June (P = 0.017) but not in October (P = 0.107). This work revealed consistent patterns of GRBV titer during a growing season in different vineyards across the United States. It also highlighted relatively high virus titer in symptomless grapevines in June, when Spissistilus festinus-mediated GRBV transmission is documented in northern California.

Abstract Geminiviruses are an important group of viruses that infect a variety of plants and result in heavy agricultural losses worldwide. The homologs of C4 (or L4) in monopartite geminiviruses and AC4 (or AL4) in bipartite geminiviruses are critical viral proteins. The C4 proteins from several geminiviruses are the substrates of S-acylation, a dynamic post-translational modification, for the maintenance of their membrane localization and function in virus infection. Here we initiated a screening and identified a plant protein ABAPT3 (Alpha/Beta Hydrolase Domain-containing Protein 17-like Acyl Protein Thioesterase 3) as the de-S-acylation enzyme of C4 encoded by BSCTV (Beet severe curly top virus). Overexpression of ABAPT3 reduced the S-acylation of BSCTV C4, disrupted its plasma membrane localization, inhibited its function in pathogenesis, and suppressed BSCTV infection. Because the S-acylation motifs are conserved among C4 from different geminiviruses, we tested the effect of ABAPT3 on the C4 protein of ToLCGdV (Tomato leaf curl Guangdong virus) from another geminivirus genus. Consistently, ABAPT3 overexpression also disrupted the S-acylation, subcellular localization, and function of ToLCGdV C4, and inhibited ToLCGdV infection. In summary, we provided a new approach to globally improve the resistance to different types of geminiviruses in plants via de-S-acylation of the viral C4 proteins and it can be extendedly used for suppression of geminivirus infection in crops.

The article in the Plant Biotechnology Journal discusses the development of a reporter tomato line to track the replication of a geminivirus in real-time and with cellular resolution. Geminiviruses are plant-infecting viruses that cause severe diseases in crops worldwide. The transgenic reporter tomato plants containing a fluorescent protein allow for the monitoring of viral replication in a non-destructive and cost-effective manner, providing valuable insights into the dynamics of viral infection in different plant organs. This tool can be used to study factors impacting viral infection and isolate infected cells for further analysis, making it a valuable resource for plant virus research. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

The Tomato Chlorosis Criniviruses (ToCV) and Tomato yellow leaf curl Geminivirus (TYLCV), which naturally infecting and limited to the phloem, have caused a drastic reduction in tomato yield. The current study aimsto determine the incidence of single and mixed viruses using biological, serological and molecular PCR methods in natural tomato plants. The incidence of mixed infection was found more frequently, followed by ToCV and TYLCV (42.3, 28.8, 17.8 in 2020 and 49.1, 29.7 and 19.1% in 2021, respectively). ToCV causes chlorosis, TYLCV causes leaf curl and yellowing, while mixed ToCV & TYLCV cause progress symptoms. By using the heat shock protein 70 (HSP70) and coat protein (CP) genes, ToCV and TYLCV isolates could be identified. These isolates were recorded in GenBank under accession codes "ON951644.1" and "OP265136.1" respectively. Host plants responded differently to severe and common disease density between ToCV and TYLCV. The transmitted whitefly could distinguish between ToCV and TYLCV within 15-20 minutes of the acquisition period. ToCV increases in fields with high whitefly populations, requiring further research to understand effects and reduce harm. [ABSTRACT FROM AUTHOR]

A tomato‐infecting virus known as Beet curly top Iran virus (BCTIV) cause a significant disease for tomato plants and several other plant species around the world. Chitosan polysaccharide is a natural biopolymer that has been utilised as an exo‐elicitor to enhance plant defence mechanisms against a variety of plant diseases. This study investigates the efficacy of chitosan in combating BCTIV disease on tomato plants and modulating the host–virus interaction under greenhouse conditions. Twenty‐four hours before the virus inoculation, tomato plants were sprayed with a protective chitosan solution at different concentrations (0.5, 1, 1.5, and 2 mg/mL). Tomato plants were inoculated with a BCTIV infectious clone using an Agrobacterium‐inoculation method. The findings clearly demonstrated a reduction in the severity of the disease in chitosan‐treated plants as compared to Mock‐plants, with the percentage decreasing from 61.53% to 75.28% in 1.5 mg/mL treated plants and from 9.01% to 28.43% in 0.5 mg/mL treated plants. In addition, the utilisation of chitosan has the potential to deactivate the accumulation of BCTIV within the host tissues. The virus accumulation was greatly alleviated in 1, 1.5, and 2 mg/mL‐treated plants by 71.29%, 90.11%, and 93.14%, respectively, and over the mock plants. Furthermore, it was found that chitosan applied at all tested concentrations increased the relative expression and mRNA accumulation of genes related to resistance, including the pathogenesis‐related protein gene PR‐1, the HSP90 gene, and the AGO2a antiviral gene. These genes reached their maximum by 22.9‐, 12.93‐, and 4.44‐fold increases, respectively, over the untreated control. According to gas chromatography–mass spectroscop (GC‐MS) fractionation profile, chitosan increased 28 bioactive metabolic components, such as n‐hexadecanoic acid, heptanone, 1,2‐dimethylbenzene, dicarboxylic acid, and cis‐11‐octadecenoic acid methyl‐ester, to improve metabolic pathways. Results reported here revealed that foliar application of chitosan decreases the rate of the disease severity and virus accumulation in BCTIV‐infected tomato plants. This effect is associated with increased gene expression and defence‐related factors, enhancing tomato resistance to BCTIV infection. Consequently, chitosan treatments could be part of an integrated approach for reducing the severity of BCTIV disease in tomato and other host plants. [ABSTRACT FROM AUTHOR]

Geminiviruses are an important group of viruses that infect a variety of plants and result in heavy agricultural losses worldwide. The homologs of C4 (or L4) in monopartite geminiviruses and AC4 (or AL4) in bipartite geminiviruses are critical viral proteins. The C4 proteins from several geminiviruses are the substrates of S-acylation, a dynamic post-translational modification, for the maintenance of their membrane localization and function in virus infection. Here we initiated a screening and identified a plant protein ABAPT3 (Alpha/Beta Hydrolase Domain-containing Protein 17-like Acyl Protein Thioesterase 3) as the de-S-acylation enzyme of C4 encoded by BSCTV (Beet severe curly top virus). Overexpression of ABAPT3 reduced the S-acylation of BSCTV C4, disrupted its plasma membrane localization, inhibited its function in pathogenesis, and suppressed BSCTV infection. Because the S-acylation motifs are conserved among C4 from different geminiviruses, we tested the effect of ABAPT3 on the C4 protein of ToLCGdV (Tomato leaf curl Guangdong virus) from another geminivirus genus. Consistently, ABAPT3 overexpression also disrupted the S-acylation, subcellular localization, and function of ToLCGdV C4, and inhibited ToLCGdV infection. In summary, we provided a new approach to globally improve the resistance to different types of geminiviruses in plants via de-S-acylation of the viral C4 proteins and it can be extendedly used for suppression of geminivirus infection in crops. [ABSTRACT FROM AUTHOR]

Chili pepper cultivation in the Indian subcontinent is severely affected by viral diseases, prompting the need for environmentally friendly disease control methods. To achieve this, it is essential to understand the molecular mechanisms of viral resistance in chili pepper. The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) genes are known to provide broad-spectrum resistance to various phytopathogens by activating systemic acquired resistance (SAR). An in-depth understanding of NPR1 gene expression during begomovirus infection and its correlation with different biochemical and physiological parameters is crucial for enhancing resistance against begomoviruses in chili pepper. Nevertheless, limited information on chili CaNPR genes and their role in biotic stress constrains their potential in breeding for biotic stress resistance. By employing bioinformatics for genome mining, we identify 5 CaNPR genes in chili. The promoter regions of 1,500 bp of CaNPR genes contained cis-elements associated with biotic stress responses, signifying their involvement in biotic stress responses. Furthermore, these gene promoters harbored components linked to light, development, and hormone responsiveness, suggesting their roles in plant hormone responses and development. MicroRNAs played a vital role in regulating these five CaNPR genes, highlighting their significance in the regulation of chili genes. Inoculation with the begomovirus “cotton leaf curl Khokhran virus (CLCuKV)” had a detrimental effect on chili plant growth, resulting in stunted development, fibrous roots, and evident virus symptoms. The qRT-PCR analysis of two local chili varieties inoculated with CLCuKV, one resistant (V1) and the other susceptible (V2) to begomoviruses, indicated that CaNPR1 likely provides extended resistance and plays a role in chili plant defense mechanisms, while the remaining genes are activated during the early stages of infection. These findings shed light on the function of chili’s CaNPR in biotic stress responses and identify potential genes for biotic stress-resistant breeding. However, further research, including gene cloning and functional analysis, is needed to confirm the role of these genes in various physiological and biological processes. This in-silico analysis enhances our genome-wide understanding of how chili CaNPR genes respond during begomovirus infection.

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.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas have been recognized as powerful genome-editing tools in diverse eukaryotic species, including plants, and thus hold great promise for engineering virus resistance in plants. Nevertheless, further attention is required regarding various issues associated with applying new powerful technologies in the field. This mini-review focuses on the recent advances in using CRISPR/Cas9 and CRISPR/Cas13 systems to combat DNA and RNA viruses in plants. We explored the utility of CRISPR/Cas for targeting the viral genome and editing host susceptibility genes in plants. We also provide insights into the limitations and challenges of using CRISPR/Cas for plant virus interference and propose individual combinatorial solutions. In conclusion, CRISPR/Cas technology has the potential to offer innovative and highly efficient approaches for controlling viruses in important crops in the near future.

Abstract 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.

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. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Geminiviruses are a group of circular single-stranded DNA viruses that cause severe diseases in many crop plants. However, there is still no fluorescent protein tag suitable for labeling viral proteins endogenously due to the limited genomic space and structure of geminiviruses for foreign gene fragment insertion. Here, we established a split super-folder green fluorescent protein (sfGFP)-based imaging system that provides a platform to visualize the subcellular localization of geminiviral proteins in Nicotiana benthamiana. A short fragment of the GFP- coding sequence (GFP11) was inserted into a specific locus of the geminiviral genome, while the remainder of the GFP (GFP1–10) was transiently or constitutively expressed in N. benthamiana. Consequently, complementation fluorescence enables the examination of the subcellular localization of viral proteins in particular cells. Using this split sfGFP system, we examined the subcellular localization of the coat protein and BV1 protein of tomato golden mosaic virus (TGMV) and further monitored its intercellular and long-distance movement in N. benthamiana during viral infection. This approach allows us to study endogenously the subcellular localization and viral movement of geminiviruses in N. benthamiana. This new split sfGFP system may also hold the potential to provide orthogonal fluorescent proteins usable for geminiviral genome tagging in plants.

Objective Beet curly top Iran virus (BCTIV) is a destructive virus causing damage to agriculture industry annually. BCTIV has a circular single-stranded DNA genome with five open reading frames (ORFs): V1, V2 and V3 on genomic strand, and C1 and C2 on complementary strand. V2 acts as a suppressor of silencing interfering with this defense pathway. This study was aimed to determine the evolutionary characteristics and genetic structure of V2 among different BCTIV isolates.Materials and methods Total number of 31 BCTIV V2 nucleotide sequences were extracted from the GenBank and analyzed. The nucleotide sequence was aligned and the phylogenetic tree was drawn. Nucleotide polymorphism analysis was determined. Also, the occurrence of insertion-deletion polymorphism (InDel) was investigated. Nonsynonymous (dN) and synonymous (dS) substitution rates and dN/dS ratio were calculated in order to check the selection pressure on nucleotide sequences and V2 codons. Entropy analysis was also performed to investigate possible variations in nucleotide positions.Results Based on the host and geographical location, V2 sequences were placed in different clusters of the phylogenetic tree. 20 haplotypes and 94 polymorphic loci was detected. Haplotype and nucleotide diversity was calculated as 0.963 and 0.06517, respectively. The mean nucleotide differences was 23.463. InDel polymorphism was not observed. The rates of dN and dS were calculated as -0.60699 and 0.03020, respectively, both of which were not significant. The dN/dS ratio was also -20.10201. Total number of 26 positions among the codons showed a positive value of dN/dS ratio, while 34 positions had a negative value. Also, at least 9 recombination events were observed. Entropy analysis showed that nucleotide position 247 has the most changes with an entropy rate of 0.93.ConclusionsThe results suggest that the variation in the nucleotide sequence of BCTIV V2 can be effective in suppressing gene silencing and the pathogenicity of different isolates in different hosts. The variability of this part of the virus genome may in the future lead to an increase in the range of host range of the virus or to overcome the resistance of plant varieties.

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. [ABSTRACT FROM AUTHOR]

Introduction: Citrus chlorotic dwarf-associated virus (CCDaV) is an economically important citrus virus associated with leaf curling, deformation, and chlorosis found in China. Plants have evolved RNA silencing to defend against viral infections; however, the mechanism by which CCDaV suppresses RNA silencing in citrus remains unknown. Methods: Six proteins encoded by CCDaV were ectopically expressed in Nicotiana benthamiana 16c using the pCHF3 vector to identify RNA-silencing suppression activities. Results: V2 protein encoded by CCDaV suppressed local RNA silencing and systemic RNA silencing triggered by GFP RNA, but did not impede short-distance movement of the RNA silencing signal in N. benthamiana 16c. GFP fluorescence observations showed that the ability of V2 protein to suppress RNA silencing was weaker than tomato bushy stunt virus P19. Deletion analysis showed that the putative nuclear localization signal (NLS, 25-54 aa) was involved in the RNA silencing suppression activity of V2 protein. Furthermore, V2 protein cannot block dsRNA-triggered RNA silencing. The subcellular localization assay suggested that V2 protein was localized to nucleus of N. benthamiana. Conclusion: Overall, the results of this study demonstrate that CCDaV-V2 acts as an activity of silencing suppression. This is the first reported RNA-silencing suppressor encoded by Citlodavirus and will be valuable in revealing the molecular mechanism of CCDaV infection. [ABSTRACT FROM AUTHOR]

The global dissemination of the Israel (IL) and mild (Mid) 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 7y-7, 7y-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 7y-7-bearing resistant tomato plants and susceptible plants were infected with TYLCV and grown at moderate or high temperatures. Under high-temperature conditions, the 7y-7-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. [ABSTRACT FROM AUTHOR]

The full‐genome sequences of a bipartite begomovirus were identified in a mungbean (Vigna radiata) crop displaying symptoms such as yellow mosaics and stunting in the Al‐Batinah North region (23.6867° N 57.9058° E), Oman. Initial detection and confirmation of the virus genome were carried out through polymerase chain reaction, followed by obtaining complete genomes using rolling circle amplification. Bioinformatics analysis on both genomic components, DNA‐A and DNA‐B, revealed over 99% nucleotide sequence identity to the mungbean yellow mosaic India virus (MYMIV), previously known to infect tomato plants. Pairwise sequence analysis using the STD tool and subsequent phylogenetic analysis unveiled that the DNA‐A of the MYMIV isolate formed a cluster with closely related DNA‐A sequences of MYMIV from GenBank, while the DNA‐B clustered with the corresponding DNA‐B of the MYMIV isolate. Additionally, no evidence of recombination events was observed in the recombination analysis. Similarly, the nucleotide substitution rates between the DNA‐A and DNA‐B segments of MYMIV did not show significant differences. This finding represents the first documented instance of bipartite MYMIV infecting V. radiata in Oman. [ABSTRACT FROM AUTHOR]

BackgroundGeminiviruses are circular, single-stranded viruses responsible for enormous crop loss worldwide. Rapid expansion of geminivirus diversity outweighs the continuous effort to control its spread. Geminiviruses channelize the host cell machinery in their favour by manipulating the gene expression, cell signalling, protein turnover, and metabolic reprogramming of plants. As a response to viral infection, plants have evolved to deploy various strategies to subvert the virus invasion and reinstate cellular homeostasis.Main bodyNumerous reports exploring various aspects of plant-geminivirus interaction portray the subtlety and flexibility of the host-pathogen dynamics. To leverage this pool of knowledge towards raising antiviral resistance in host plants, a comprehensive account of plant's defence response against geminiviruses is required. This review discusses the current knowledge of plant's antiviral responses exerted to geminivirus in the light of resistance mechanisms and the innate genetic factors contributing to the defence. We have revisited the defence pathways involving transcriptional and post-transcriptional gene silencing, ubiquitin-proteasomal degradation pathway, protein kinase signalling cascades, autophagy, and hypersensitive responses. In addition, geminivirus-induced phytohormonal fluctuations, the subsequent alterations in primary and secondary metabolites, and their impact on pathogenesis along with the recent advancements of CRISPR-Cas9 technique in generating the geminivirus resistance in plants have been discussed.ConclusionsConsidering the rapid development in the field of plant-virus interaction, this review provides a timely and comprehensive account of molecular nuances that define the course of geminivirus infection and can be exploited in generating virus-resistant plants to control global agricultural damage.

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. [ABSTRACT FROM AUTHOR]

Geminiviruses are a group of circular single-stranded DNA viruses that cause severe diseases in many crop plants. However, there is still no fluorescent protein tag suitable for labeling viral proteins endogenously due to the limited genomic space and structure of geminiviruses for foreign gene fragment insertion. Here, we established a split super-folder green fluorescent protein (sfGFP)-based imaging system that provides a platform to visualize the subcellular localization of geminiviral proteins in Nicotiana benthamiana. A short fragment of the GFP- coding sequence (GFP11) was inserted into a specific locus of the geminiviral genome, while the remainder of the GFP (GFP1–10) was transiently or constitutively expressed in N. benthamiana. Consequently, complementation fluorescence enables the examination of the subcellular localization of viral proteins in particular cells. Using this split sfGFP system, we examined the subcellular localization of the coat protein and BV1 protein of tomato golden mosaic virus (TGMV) and further monitored its intercellular and long-distance movement in N. benthamiana during viral infection. This approach allows us to study endogenously the subcellular localization and viral movement of geminiviruses in N. benthamiana. This new split sfGFP system may also hold the potential to provide orthogonal fluorescent proteins usable for geminiviral genome tagging in plants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The color of the chili fruit is an important factor that determines the quality of the chili, as red chilies are more popular among consumers. The accumulation of capsanthin is the main cause of reddening of the chili fruit. Capsanthin is an important metabolite in carotenoid metabolism, and its production level is closely linked to the expression of the genes for capsanthin/capsorubin synthase (CCS) and carotenoid hydroxylase (CrtZ). We reported for the first time that the synthesis of capsanthin in chili was enhanced by using a geminivirus (Bean Yellow Dwarf Virus). By expressing heterologous β-carotenoid hydroxylase (CrtZ) and β-carotenoid ketolase (CrtW) using codon optimization, the transcription level of the CCS gene and endogenous CrtZ was directly increased. This leads to the accumulation of a huge amount of capsanthin in a very short period of time. Our results provide a platform for the rapid enhancement of endogenous CCS activity and capsanthin production using geminivirus in plants. [ABSTRACT FROM AUTHOR]

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

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Sweet potato symptomless virus 1 (SPSMV-1) is a single-stranded circular DNA virus, belonging to the genus Mastrevirus (family Geminiviridae) that was first identified on sweet potato plants in South Korea in 2012. Although SPSMV-1 does not induce distinct symptoms in sweet potato plants, its co-infection with different sweet potato viruses is highly prevalent, and thus threatens sweet potato production in South Korea. In this study, the complete genome sequence of a Korean isolate of SPSMV-1 was obtained by Sanger sequencing of polymerase chain reaction (PCR) amplicons from sweet potato plants collected in the field (Suwon). An infectious clone of SPSMV-1 (1.1-mer) was constructed, cloned into the plant expression vector pCAMBIA1303, and agro-inoculated into Nicotiana benthamiana using three Agrobacterium tumefaciens strains (GV3101, LBA4404, and EHA105). Although no visual differences were observed between the mock and infected groups, SPSMV-1 accumulation was detected in the roots, stems, and newly produced leaves through PCR. The A. tumefaciens strain LBA4404 was the most effective at transferring the SPSMV-1 genome to N. benthamiana. We confirmed the viral replication in N. benthamiana samples through strand-specific amplification using virion-sense- and complementary-sense-specific primer sets.

In mixed infections between begomoviruses, common in tropical and subtropical regions, events such as transcomplementation and transreplication can occur. The aim of this research was to evaluate the possibility of asymmetric transreplication and transcomplementation events between Potato yellow mosaic virus (PYMV) and three begomoviruses previously isolated from tomato crop-associated weeds: Croton golden mosaic virus (CohGMV), Rhynchosia golden mosaic Colombia virus (RhGMCV) and Verbena mottle virus (VeMV). To achieve this goal, tomato leaves were inoculated with artificial mixtures of genomic components from PYMV, CohGMV, RhGMCV, or VeMV by using biolistic. The possible interactions between the viruses at the level of replication, movement and symptom expression were analyzed. Our results showed asymmetric complementation at the level of movement and replication, as well as heterologous transactivation events in mixed infections between PYMV with CohGMV, RhGMCV, or VeMV. To explain the possible asymmetrical complementation at the level of movement and replication between these begomoviruses, a bioinformatic analysis of the viral promoters of PYMV, CohGMV, RhGMCV and VeMV was performed. Similarities in cis-regulatory elements of the analyzed regions in the begomovirus studied were identified in our analysis. Asymmetrical complementation and transactivation events observed under experimental conditions suggest that if mixed infections occur under natural field conditions, new pseudorecombinats could emerge. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Beet curly top virus (BCTV), which is synonymous with curly top virus (CTV), causes significant yield loss in common bean (snap and dry beans) cultivars and several other important crops. Common bean cultivars have been found to be resistant to CTV, but screening for resistance is challenging due to the cyclical nature of epidemics and spotty feeding by the leafhopper that vectors the virus. We used an SNP dataset for the Snap Bean Association Panel (SnAP) agroinoculated with CTV-Logan (CA/Logan) strain to locate the Bct gene region to a 1.7-Mb interval on chromosome Pv07 using genome-wide association study (GWAS) analysis. Recombinant lines from the SnAP were used to further narrow the Bct region to a 58.0-kb interval. A missense SNP (S07_2970381) in candidate gene Phvul.007G036300 Exonuclease V (EXO5) was identified as the most likely causal mutation, and it was the most significant SNP detected by GWAS in a dry bean population (DBP) naturally infected by the CTV-Worland (Wor) strain. Tmshift assay markers developed for SNP S07_2970381 and two linked SNPs, S07_2970276 and S07_2966197, were useful for tracking different origins of the Bct EXO5 candidate gene resistance to CTV in common bean. The three SNPs identified four haplotypes, with haplotype 3-1 (Haplo3-1) of Middle American origin associated with the highest levels of CTV resistance. This SNPhaplotype assay will enable breeders to track resistance sources and to develop cultivars with better CTV resistance. [ABSTRACT FROM AUTHOR]

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 fabac/)-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-1L). 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-I gene either with TYLCV-IL IS76-like or canonical TYLCV-IL isolates. Moreover, we demonstrate the incorporation of the Ty-2 gene in 7v-/-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. [ABSTRACT FROM AUTHOR]

Geminiviruses have mastered plant cell modulation and immune invasion to ensue prolific infection. Encoding a relatively small number of multifunctional proteins, geminiviruses rely on satellites to efficiently re-wire plant immunity, thereby fostering virulence. Among the known satellites, betasatellites have been the most extensively investigated. They contribute significantly to virulence, enhance virus accumulation, and induce disease symptoms. To date, only two betasatellite proteins, βC1, and βV1, have been shown to play a crucial role in virus infection. In this review, we offer an overview of plant responses to betasatellites and counter-defense strategies deployed by betasatellites to overcome those responses. [ABSTRACT FROM AUTHOR]

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM‐DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA‐responsive genes in a repressed state. In the presence of JA‐Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA‐responsive genes. A growing body of evidence has shown that JA signaling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato‐infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock‐down of JAZ8 favors geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8‐silenced plants neither display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus‐encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti‐geminiviral effect. [ABSTRACT FROM AUTHOR]