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The begomoviral V2 protein is known to be multifunctional, including its interaction with and inhibition of CYP1, a papain‐like cysteine protease (PLCP). However, the effect of this interaction on viral pathogenicity remains unclear. Cotton leaf curl Multan virus (CLCuMuV), a typical monopartite begomovirus associated with a betasatellite, is one of the main pathogens responsible for cotton leaf curl disease. This study verifies the interaction between CLCuMuV V2 and NbCP15, a PLCP homologue in Nicotiana benthamiana. The results show that V2 can be cleaved by NbCP15 in vitro, with the N‐terminal cleavage site located between the second and third amino acids. Using an Agrobacterium‐mediated inoculation method, we investigated the influence of cleavage sites on viral pathogenicity. The findings indicate that mutation of the third amino acid in V2 (V2D3A) reduced the pathogenicity of both heterologous PVX and CLCuMuV. Additionally, the NbCP15 gene mutation in N. benthamiana (nbcp15) also resulted in reduced CLCuMuV pathogenicity. These results suggest that CLCuMuV V2 may promote viral infection through its interaction with plant PLCPs. Cotton leaf curl Multan virus uses its V2 protein to promote viral infection through its interaction with a plant papain‐like cysteine protease (NbCP15), which can cleave V2 after the first two amino acids at the N‐terminus.

DNA methylation is a fundamental epigenetic modification that regulates gene expression and represses endogenous transposons and invading DNA viruses. As a counter-defense, the geminiviruses encode proteins that inhibit methylation and transcriptional gene silencing (TGS). Some geminiviruses have acquired a betasatellite called DNA β. This study presents evidence that suppression of methylation-mediated TGS by the sole betasatellite-encoded protein, βC1, is crucial to the association of Tomato yellow leaf curl China virus (TYLCCNV) with its betasatellite (TYLCCNB). We show that TYLCCNB complements Beet curly top virus (BCTV) L2⁻ mutants deficient for methylation inhibition and TGS suppression, and that cytosine methylation levels in BCTV and TYLCCNV genomes, as well as the host genome, are substantially reduced by TYLCCNB or βC1 expression. We also demonstrate that while TYLCCNB or βC1 expression can reverse TGS, TYLCCNV by itself is ineffective. Thus its AC2/AL2 protein, known to have suppression activity in other geminiviruses, is likely a natural mutant in this respect. A yeast two-hybrid screen of candidate proteins, followed by bimolecular fluorescence complementation analysis, revealed that βC1 interacts with S-adenosyl homocysteine hydrolase (SAHH), a methyl cycle enzyme required for TGS. We further demonstrate that βC1 protein inhibits SAHH activity in vitro. That βC1 and other geminivirus proteins target the methyl cycle suggests that limiting its product, S-adenosyl methionine, may be a common viral strategy for methylation interference. We propose that inhibition of methylation and TGS by βC1 stabilizes geminivirus/betasatellite complexes.

Many circulative plant viruses transmitted by insect vectors are devastating to agriculture worldwide. The midgut wall of vector insects represents a major barrier and at the same time the key gate a circulative plant virus must cross for productive transmission. However, how these viruses enter insect midgut cells remains poorly understood. Here, we identified an endocytic receptor complex for begomoviruses in the midgut cells of their whitefly vector. Our results show that two whitefly proteins, BtCUBN and BtAMN, compose a receptor complex BtCubam, for which BtCUBN contributes a viral-binding region and BtAMN contributes to membrane anchorage. Begomoviruses appear to be internalized together with BtCubam via its interaction with the 12–19 CUB domains of BtCUBN via clathrin-dependent endocytosis. Functional analysis indicates that interruption of BtCUBN and BtAMN lead to reduction of virus acquisition and transmission by whitefly. In contrast, CUBN-begomovirus interaction was not observed in two non-competent whitefly-begomovirus combinations. These observations suggest a major role of the specific endocytic receptor in facilitating viral entry into vector midgut cells.

Most plant viruses are vectored by insects and the interactions of virus-plant-vector have important ecological and evolutionary implications. Insect vectors often perform better on virus-infected plants. This indirect mutualism between plant viruses and insect vectors promotes the spread of virus and has significant agronomical effects. However, few studies have investigated how plant viruses manipulate plant defenses and promote vector performance. Begomoviruses are a prominent group of plant viruses in tropical and sub-tropical agro-ecosystems and are transmitted by whiteflies. Working with the whitefly Bemisia tabaci, begomoviruses and tobacco, we revealed that C2 protein of begomoviruses lacking DNA satellites was responsible for the suppression of plant defenses against whitefly vectors. We found that infection of plants by tomato yellow leaf curl virus (TYLCV), one of the most devastating begomoviruses worldwide, promoted the survival and reproduction of whitefly vectors. TYLCV C2 protein suppressed plant defenses by interacting with plant ubiquitin. This interaction compromised the degradation of JAZ1 protein, thus inhibiting jasmonic acid defense and the expression of MYC2-regulated terpene synthase genes. We further demonstrated that function of C2 protein among begomoviruses not associated with satellites is well conserved and ubiquitination is an evolutionarily conserved target of begomoviruses for the suppression of plant resistance to whitefly vectors. Taken together, these results demonstrate that ubiquitination inhibition by begomovirus C2 protein might be a general mechanism in begomovirus, whitefly and plant interactions.

The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSKη-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants.

Gene silencing is a natural antiviral defense mechanism in plants. For effective infection, plant viruses encode viral silencing suppressors to counter this plant antiviral response. The geminivirus-encoded C4 protein has been identified as a gene silencing suppressor, but the underlying mechanism of action has not been characterized. Here, we report that Cotton Leaf Curl Multan virus (CLCuMuV) C4 protein interacts with S-adenosyl methionine synthetase (SAMS), a core enzyme in the methyl cycle, and inhibits SAMS enzymatic activity. By contrast, an R13A mutation in C4 abolished its capacity to interact with SAMS and to suppress SAMS enzymatic activity. Overexpression of wild-type C4, but not mutant C4R13A, suppresses both transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). Plants infected with CLCuMuV carrying C4R13A show decreased levels of symptoms and viral DNA accumulation associated with enhanced viral DNA methylation. Furthermore, silencing of NbSAMS2 reduces both TGS and PTGS, but enhanced plant susceptibility to two geminiviruses CLCuMuV and Tomato yellow leaf curl China virus. These data suggest that CLCuMuV C4 suppresses both TGS and PTGS by inhibiting SAMS activity to enhance CLCuMuV infection in plants.

Geminiviruses are major plant pathogens that threaten food security globally. They have a unique architecture built from two incomplete icosahedral particles, fused to form a geminate capsid. However, despite their importance to agricultural economies and fundamental biological interest, the details of how this is realized in 3D remain unknown. Here we report the structure of A geratum yellow vein virus at 3.3 Å resolution, using single-particle cryo-electron microscopy, together with an atomic model that shows that the N-terminus of the single capsid protein (CP) adopts three different conformations essential for building the interface between geminate halves. Our map also contains density for ~7 bases of single-stranded DNA bound to each CP, and we show that the interactions between the genome and CPs are different at the interface than in the rest of the capsid. With additional mutagenesis data, this suggests a central role for DNA binding-induced conformational change in directing the assembly of geminate capsids. Geminiviruses are an important plant pathogen that causes large food crop losses globally. Here the authors describe a high resolution cryo-EM structure of the Ageratum yellow vein virus and reveal the molecular details of how a single capsid protein sequence can adopt the different conformations needed to build that geminate capsid.

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.

Tomato is one of the major vegetable crops consumed worldwide. Tomato yellow leaf curl virus (TYLCV) and fungal Oidium sp. are devastating pathogens causing yellow leaf curl disease and powdery mildew. Such viral and fungal pathogens reduce tomato crop yields and cause substantial economic losses every year. Several commercial tomato varieties include Ty-5 (SlPelo) and Mildew resistance locus o 1 (SlMlo1) locus that carries the susceptibility (S-gene) factors for TYLCV and powdery mildew, respectively. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is a valuable genome editing tool to develop disease-resistant crop varieties. In this regard, targeting susceptibility factors encoded by the host plant genome instead of the viral genome is a promising approach to achieve pathogen resistance without the need for stable inheritance of CRISPR components. In this study, the CRISPR/Cas9 system was employed to target the SlPelo and SlMlo1 for trait introgression in elite tomato cultivar BN-86 to confer host-mediated immunity against pathogens. SlPelo-knockout lines were successfully generated, carrying the biallelic indel mutations. The pathogen resistance assays in SlPelo mutant lines confirmed the suppressed accumulation of TYLCV and restricted the spread to non-inoculated plant parts. Generated knockout lines for the SlMlo1 showed complete resistance to powdery mildew fungus. Overall, our results demonstrate the efficiency of the CRISPR/Cas9 system to introduce targeted mutagenesis for the rapid development of pathogen-resistant varieties in tomato.

Tomato yellow leaf curl Guangdong virus (TYLCGdV), a monopartite begomovirus first identified in 2004, remains poorly characterised. In this study, we demonstrate that TYLCGdV associates with a betasatellite, TYLCGdB, and the βC1 protein encoded by TYLCGdB is essential for symptom development. We also explore the role of TYLCGdV C4 protein by generating a C4‐deficient infectious clone (TYLCGdVmC4), revealing a dynamic role for TYLCGdV C4. Specifically, viral accumulation in TYLCGdVmC4/TYLCGdB‐inoculated plants was significantly lower than that in TYLCGdV/TYLCGdB‐inoculated plants at 7 and 14 days post‐inoculation (dpi), but surpassed that of TYLCGdV/TYLCGdB‐inoculated plants by 25 dpi. Furthermore, although C4 proteins in other begomoviruses typically exhibit one or more of the following properties: (i) suppression of post‐transcriptional gene silencing (PTGS), (ii) suppression of transcriptional gene silencing (TGS), (iii) enhancement of pathogenicity in potato virus X (PVX) and (iv) symptom induction when transgenically expressed, TYLCGdV C4 did not exhibit any of these properties. However, the dynamic role of TYLCGdV C4 in viral infection appears to result from its effects on viral DNA methylation. At 7 dpi, the cytosine methylation level in the TYLCGdVmC4 genome was notably elevated compared to that of the wild‐type virus. However, this trend reversed by 14 dpi, with the wild‐type virus exhibiting a higher methylation level. By 25 dpi, the cytosine methylation levels of both TYLCGdVmC4 and TYLCGdV were comparable. These results indicate that TYLCGdV C4 modulates viral infection via an unconventional mechanism. This novel observation highlights the need for further investigation into the diverse roles of C4 proteins in begomoviruses. The C4 protein, encoded by tomato yellow leaf curl Guangdong virus (TYLCGdV), does not inhibit PTGS or TGS; the C4 protein modulates TYLCGdV infection by regulating the cytosine methylation level of viral genome at different infection stages.