Yellow Leaf Curl China Virus Research Articles

A highly efficient Agrobacterium-mediated infectious system for Sweet potato leaf curl virus and a deltasatellite-based VIGS vector

Plant virus disease is one of the major threats to the yield and quality of sweetpotato (Ipomoea batatas [L.] Lam.), the seventh most important food crop in the world in terms of production. Sweepoviruses are a group of monopartite geminiviruses (genus Begomovirus, family Geminiviridae) that infect Ipomoea plants and cause significant damage. However, investigations on the mechanisms of sweepovirus infection are scarce due to the low efficiency of the current infectious system. In this work, we used the genomic sequence of Sweet potato leaf curl virus (SPLCV) to construct an effective infectious clone, SPLCV-1.01. We optimized the inoculation procedure in Nicotiana benthamiana by improving the Agrobacterium virulence using an induction medium. Finally, we established a highly efficient agroinoculation protocol for SPLCV on sweetpotato. Furthermore, a simple and reliable “Agro-soaking” method was developed for SPLCV on sweetpotato and Ipomoea aquatica. The “Agro-soaking” method also worked well with Tomato yellow leaf curl China virus (TYLCCNV) and Beet severe curly top virus (BSCTV) on tomato. Based on the “Agro-soaking” method and a deltasatellite, the SBG51 VIGS vector worked with the SPLCV-1.01 infectious clone and silenced two endogenous genes IbCHL (magnesium-chelatase subunit) and IbPDS (phytoene desaturase) in two weeks. Moreover, the VIGS system identified IbADK1 (adenosine kinase 1) as a positive regulator to dampen the SPLCV accumulation in sweetpotato. Our work provides a practical method for studying the interaction between sweetpotato and SPLCV and the functional studies of genes in sweetpotato.

Viral infections inhibit saponin biosynthesis and photosynthesis in Panax notoginseng

Virus-infected Panax notoginseng plants with chlorotic, mosaic, and pitted leaves are ubiquitous in the primary P. notoginseng-producing region in Wenshan autonomous prefecture, Yunnan province, China. However, the viruses that infect P. notoginseng and the effects of viral infections on the biosynthesis of secondary metabolites and photosynthesis remain unknown. This study identified a variety of viruses infecting P. notoginseng plants via deep-sequencing of small RNA (sRNA). Of the 10 identified viruses, seven had not previously been detected in P. notoginseng, including Cauliflower mosaic virus and Soybean chlorotic mottle virus. In addition, the simultaneous infection of P. notoginseng by Panax notoginseng virus A (PnVA), Panax cryptic virus 4 (PCV4), and Tomato yellow leaf curl China virus (TYLCCNV) was confirmed by PCR. Moreover, a quantitative PCR analysis showed that the expression levels of key genes related to saponin biosynthesis were generally down-regulated in the virus-infected P. notoginseng. Additionally, high-performance liquid chromatography results indicated the saponin content decreased in the roots of virus-infected P. notoginseng plants. The activities of photosynthesis-related enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase, fructose 1,6-bisphosphatase, and fructose 1,6-biphosphate aldolase, decreased significantly in the virus-infected P. notoginseng plants. The viral infections also induced the expression of antioxidant genes and increased antioxidant enzyme activities. Furthermore, the expression levels of many resistance-related genes were up-regulated in P. notoginseng plants inoculated with a viral suspension. The study results provide the foundation for future research on P. notoginseng viral diseases, which may lead to the development of enhanced disease control measures.

N-terminal acetylation of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus is critical for its viral pathogenicity

N-terminal acetylation (N-acetylation) is one of the most common protein modifications and plays crucial roles in viability and stress responses in animals and plants. However, very little is known about N-acetylation of viral proteins. Here, we identified the Thr residue at position 2 (Thr-2) in the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-βC1) as a novel N-acetylation site. Furthermore, the effects of TYLCCNB-βC1 N-acetylation on its function as a pathogenicity factor were determined via N-acetylation mutants in Nicotiana benthamiana plants. We found that N-acetylation of TYLCCNB-βC1 is critical for its self-interaction in the nucleus and viral pathogenesis, and that removal of N-acetylation of TYLCCNB-βC1 attenuated tomato yellow leaf curl China virus-induced symptoms and led to accelerated degradation of TYLCCNB-βC1 through the ubiquitin-proteasome system. Our data reveal a protective effect of N-acetylation of TYLCCNB-βC1 on its pathogenesis and demonstrate an antagonistic crosstalk between N-acetylation and ubiquitination in this geminiviral protein.

Diverse Begomoviruses Evolutionarily Hijack Plant Terpenoid-Based Defense to Promote Whitefly Performance.

Arthropod-borne pathogens and parasites are major threats to human health and global agriculture. They may directly or indirectly manipulate behaviors of arthropod vector for rapid transmission between hosts. The largest genus of plant viruses, Begomovirus, is transmitted exclusively by whitefly (Bemisia tabaci), a complex of at least 34 morphologically indistinguishable species. We have previously shown that plants infected with the tomato yellowleaf curl China virus (TYLCCNV) and its associated betasatellite (TYLCCNB) attract their whitefly vectors by subverting plant MYC2-regulated terpenoid biosynthesis, therefore forming an indirect mutualism between virus and vector via plant. However, the evolutionary mechanism of interactions between begomoviruses and their whitefly vectors is still poorly understood. Here we present evidence to suggest that indirect mutualism may happen over a millennium ago and at present extensively prevails. Detailed bioinformatics and functional analysis identified the serine-33 as an evolutionary conserved phosphorylation site in 105 of 119 Betasatellite species-encoded βC1 proteins, which are responsible for suppressing plant terpenoid-based defense by interfering with MYC2 dimerization and are essential to promote whitefly performance. The substitution of serine-33 of βC1 proteins with either aspartate (phosphorylation mimic mutants) or cysteine, the amino acid in the non-functional sβC1 encoded by Siegesbeckia yellow vein betasatellite SiYVB) impaired the ability of βC1 functions on suppression of MYC2 dimerization, whitefly attraction and fitness. Moreover the gain of function mutation of cysteine-31 to serine in sβC1 protein of SiYVB restored these functions of βC1 protein. Thus, the dynamic phosphorylation of serine-33 in βC1 proteins helps the virus to evade host defense against insect vectors with an evolutionarily conserved manner. Our data provide a mechanistic explanation of how arboviruses evolutionarily modulate host defenses for rapid transmission.

Geminivirus satellite-encoded βC1 activates UPR, induces bZIP60 nuclear export, and manipulates the expression of bZIP60 downstream genes to benefit virus infection.

UPR is a conserved response in eukaryotes and can alleviate endoplasmic reticulum (ER) stresses induced by abiotic and biotic stresses. The interactions between UPR and plant RNA viruses have been documented, while the interplays between UPR and plant DNA viruses remain unknown. Using tomato yellow leaf curl China virus (TYLCCNV) and its associated betasatellite (TYLCCNB) as a model, we indicate that TYLCCNB βC1 is a major inducer of UPR and can upregulate the expression of bZIP60, a transcription factor in Nicotiana benthamiana plants. Treatment using ER stress inducers or overexpression of NbbZIP60 increases βC1 accumulation and benefits TYLCCNV/TYLCCNB infection in N. benthamiana plants, and vice versa. In the TYLCCNV/TYLCCNB-infected or the βC1-expressing cells, NbbZIP60 is exported from the nucleus to the nuclear periphery via the XPO1 pathway, and blocking the XPO1 pathway inhibited TYLCCNV/TYLCCNB infection. We have found that the NbbZIP60-regulated pro-survival genes could promote virus infection, and the pro-death gene plays a contrasting role in virus infection. This study reveals that geminivirus infection activates UPR and utilizes the up-regulated molecular chaperons to promote viral infection, and then induces the nuclear export of NbbZIP60 to evade plant defense response, which is a distinct virulence strategy exploited by plant pathogens.

Molecular characterization of a novel Conyza canadensis-infecting begomovirus in China

A novel monopartite begomovirus was found in naturally infected Conyza canadensis plants exhibiting typical begomovirus-induced yellow vein symptoms in Yunnan Province of China. Analysis of two obtained full-length viral genome sequences showed that they shared 99.8% nucleotide (nt) sequence similarity, and both consisted of 2733 nts (GenBank accession no. OK120264 and OK120266, respectively). Further analysis showed that these two sequences shared the highest nt sequence similarity (78.9%) with tomato yellow leaf curl Vietnam virus (TYLCVV) (EU189150). In the assayed C. canadensis plants, a betasatellite of 1,341 nts (Accession no. OK120265 and OK120267, respectively) was also found, and it shared 98.1% sequence similarity with malvastrum yellow vein betasatellite (MaYVB) (JX679254). Based on the current classification criteria for begomoviruses, we consider that the two obtained viral isolates are a novel begomovirus, and named it as conyza yellow vein virus (CoYVV). Our further analysis result showed that CoYVV is likely originated from a recombination event between tomato yellow leaf curl Yunnan virus (TYLCYnV) and tomato yellow leaf curl China virus (TYLCCNV). To investigate the effect of CoYVV infection in plant, we constructed two infectious clones (i.e., pCoYVV and pMaYVB), and inoculated them individually or together to Nicotiana benthamiana plants through agro-infiltration. The result showed that the plants co-inoculated with CoYVV and MaYVB developed yellow vein and downward leaf-curling symptoms, whereas the plants inoculated with CoYVV alone showed no clear virus-like symptoms. Virus infection in the inoculated N. benthamiana plants was confirmed through polymerase chain reaction (PCR). The result of quantitative PCR (qPCR) showed that in the presence of MaYVB, the accumulation level of CoYVV DNA was significantly increased compared to that in the plants infected with CoYVV alone.

Geminiviruses employ host DNA glycosylases to subvert DNA methylation-mediated defense

DNA methylation is an epigenetic mechanism that plays important roles in gene regulation and transposon silencing. Active DNA demethylation has evolved to counterbalance DNA methylation at many endogenous loci. Here, we report that active DNA demethylation also targets viral DNAs, tomato yellow leaf curl China virus (TYLCCNV) and its satellite tomato yellow leaf curl China betasatellite (TYLCCNB), to promote their virulence. We demonstrate that the βC1 protein, encoded by TYLCCNB, interacts with a ROS1-like DNA glycosylase in Nicotiana benthamiana and with the DEMETER (DME) DNA glycosylase in Arabidopsis thaliana. The interaction between βC1 and DME facilitates the DNA glycosylase activity to decrease viral DNA methylation and promote viral virulence. These findings reveal that active DNA demethylation can be regulated by a viral protein to subvert DNA methylation-mediated defense.

Selective autophagic receptor NbNBR1 prevents NbRFP1-mediated UPS-dependent degradation of βC1 to promote geminivirus infection.

Autophagy is an evolutionarily conserved, lysosomal/vacuolar degradation mechanism that targets cell organelles and macromolecules. Autophagy and autophagy-related genes have been studied for their antiviral and pro-viral roles in virus-infected plants. Here, we demonstrate the pro-viral role of a selective autophagic receptor NbNBR1 in geminivirus-infected Nicotiana benthamiana plants. The βC1 protein encoded by tomato yellow leaf curl China betasatellite (TYLCCNB) that is associated with tomato yellow leaf curl China virus (TYLCCNV) enhanced the expression level of NbNBR1. Then NbNBR1 interacted with βC1 to form cytoplasmic granules. Interaction of NbNBR1 with βC1 could prevent degradation of βC1 by the NbRFP1, an E3 ligase. Overexpression of NbNBR1 in N. benthamiana plants increased βC1 accumulation and promoted virus infection. In contrast, silencing or knocking out NbNBR1 expression in N. benthamiana suppressed βC1 accumulation and inhibited virus infection. A single amino acid substitution in βC1 (βC1K4A) abolished its interaction with NbNBR1, leading to a reduced level of βC1K4A. The TYLCCNV/TYLCCNBK4A mutant virus caused milder disease symptoms and accumulated much less viral genomic DNAs in the infected plants. Collectively, the results presented here show how a viral satellite-encoded protein hijacks host autophagic receptor NbNBR1 to form cytoplasmic granules to protect itself from NbRFP1-mediated degradation and facilitate viral infection.

Coinfection by Two Begomoviruses Aggravates Plant Symptoms But Does Not Influence the Performance and Preference of Insect Vector Bemisia tabaci (Hemiptera: Aleyrodidae).

In nature, a plant can be infected by multiple viruses simultaneously. However, the effects of coinfection on plant-vector interactions are less well studied. Two begomoviruses of the family Geminiviridae, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl China virus (TYLCCNV), occur sympatrically in China. Each of them is reported to increase the performance of whitefly vector via manipulation of plant traits. In this study, we examined the effects of coinfection by the two viruses TYLCV and TYLCCNV on plant-whitefly interactions, compared to that infected by a single virus. We found that plants infected by two viruses showed aggravated symptoms but the performance and preference of whiteflies were not altered significantly compared to singly-infected plants. Coinfection suppressed the transcription of genes involved in jasmonic acid (JA) signaling pathway in plants, but showed no significant difference to single-virus infected plants. These findings suggest that although TYLCV and TYLCCNV may synergistically induce plant symptoms, they did not manipulate synergistically plant-mediated responses to the insect vector.

The Detection of Plant Viruses in Korean Ginseng (Panax ginseng) through RNA Sequencing

Korean ginseng (Panax ginseng) is a dicotyledonous, medicinal, perennial plant belonging to the genus Panax of the family Araliaceae. We investigated the occurrence and incidence of plant viruses in Panax ginseng in Korea. A total of 656 leaf samples were combined into one and total RNA was extracted from the polled sample, using RNA sequencing (RNA-Seq), a metatranscriptome analysis of the plant virome was conducted. The virus present in Panax ginseng was confirmed by reverse transcription polymerase chain reaction (RT-PCR) assay using virus-specific primers. In RNA-Seq data analysis, the multiplication protein of four viral contigs including Aristotelia chilensis virus 1 (AcV1), Turnip mosaic virus (TuMV), Watermelon mosaic virus (WMV), and Tobamovirus multiplication protein were discovered. From our metatranscriptome analysis and RT-PCR assay, TuMV and WMV were detected, whereas the three viruses reported in China such as tomato yellow leaf curl China virus; panax notoginseng virus A; and panax virus Y were not found in this study. The distribution of domestic ginseng viruses seems different from that recorded in China. Overall, this is the first plant virome analysis of Panax ginseng in Korea.

Begomovirus-Associated Betasatellite Virulence Factor βC1 Attenuates Tobacco Defense to Whiteflies via Interacting With Plant SKP1.

Plant-mediated interactions between plant viruses and their vectors are important determinants of the population dynamics of both types of organisms in the field. The whitefly Bemisia tabaci can establish mutualism with begomoviruses via their shared host plants. This mutualism is achieved by the interaction between virulence factors and their host proteins. While the virulence factor βC1 encoded by tomato yellow leaf curl China betasatellite (TYLCCNB), a subviral agent associated to the begomovirus tomato yellow leaf curl China virus (TYLCCNV), may interact with plant protein MYC2, thereby establishing the indirect mutualism between TYLCCNV and whitefly, whether other mechanisms are involved remains unknown. Here, we found the in vitro and in vivo interactions between βC1 and tobacco protein S-phase kinase associated protein 1 (NtSKP1). Silencing the expression of NtSKP1 enhanced the survival rate and fecundity of whiteflies on tobacco plants. NtSKP1 could activate the transcription of genes in jasmonic acid (JA) pathways by impairing the stabilization of JAZ1 protein. Moreover, βC1-NtSKP1 interaction could interfere JAZ1 degradation and attenuate the plant JA defense responses. These results revealed a novel mechanism underlying the better performance of whiteflies on TYLCCNV/TYLCCNB-infected plants.

Dynamic Subcellular Localization, Accumulation, and Interactions of Proteins From Tomato Yellow Leaf Curl China Virus and Its Associated Betasatellite.

Geminiviruses contain the largest number of species of plant viruses, and cause devastating crop diseases worldwide. The development of resistance to these viruses will require a clear understanding of viral protein function and interactions. Tomato yellow leaf curl China virus (TYLCCNV) is a typical monopartite geminivirus, which is associated with a tomato yellow leaf curl China betasatellite (TYLCCNB) in the field; the complex infection of TYLCCNV/TYLCCNB leads to serious economic losses in solanaceous plants. The functions of each protein encoded by the TYLCCNV/TYLCCNB complex have not yet been examined in a targeted manner. Here, we show the dynamic subcellular localization and accumulation of six viral proteins encoded by TYLCCNV and the βC1 protein encoded by TYLCCNB in plants over time, and analyzed the effect of TYLCCNV or TYLCCNV/TYLCCNB infection on these parameters. The interaction among the seven viral proteins was also tested in this study: C2 acts as a central player in the viral protein interaction network, since it interacts with C3, C4, V2, and βC1. Self-interactions were also found for C1, C2, and V2. Together, the data presented here provide a template for investigating the function of viral proteins with or without viral infection over time, and points at C2 as a pivotal protein potentially playing a central role in the coordination of the viral life cycle.

Functional analysis of a novel βV1 gene identified in a geminivirus betasatellite.

Betasatellites (DNA β) are circular ssDNA molecules that are associated with monopartite geminiviruses and exert a positive effect on the viral infection. Betasatellites encode one protein, named βC1, on the complementary strand; βC1 functions as a pathogenicity factor and RNA silencing suppressor. In this report, we describe the identification of another betasatellite-encoded protein, βVl, which also contributes to symptom development. The βVl open reading frame can be found on the viral strand of approximately 40% of reported betasatellite sequences, and is conserved in position and sequence. The presence of the βVl transcript was observed in plants infected with Tomato yellow leaf curl China virus (TYTCCNV) along with its associated betasatellite Tomato yellow leaf curl China betasatellite (TYTCCNB). Mutant viruses unable to produce βVl showed reduced virulence and decreased viral load. Ectopic expression of the TYTCCNB-PV1 gene in Nicotiana benthamiana plants from a PVX-based vector resulted in leaf mosaic and chlorosis. We further demonstrated that the βVl protein could elicit hypersensitive response (HR)-type cell death in N. benthamiana leaves. Our results uncover a novel betasatellite-encoded protein that contributes to the virus infection, and this discover gives us a more complete view of the plant-geminivirus interaction landscape.

Whitefly HES1 binds to the intergenic region of Tomato yellow leaf curl China virus and promotes viral gene transcription

Intergenic region of begomovirus genome is vital to virus replication and viral gene transcription in plants. Previous studies have reported that Tomato yellow leaf curl China virus (TYLCCNV), a begomovirus, is able to accumulate and transcribe in its whitefly vector. However, the viral and host components that participate in begomovirus transcription in whiteflies are hitherto unknown. Using a yeast one-hybrid system, we identified >50 whitefly proteins that interacted with TYLCCNV intergenic region. Dual luciferase analysis revealed that one of the identified proteins, the hairy and enhancer of split homolog-1 (HES1), specifically bound to CACGTG motif in TYLCCNV intergenic region. Silencing HES1 decreased viral transcription, accumulation and transmission. These results demonstrate that the interactions between whitefly proteins and the intergenic region of TYLCCNV may contribute to viral transcription in the whitefly vector. Our findings offer valuable clues for the research and development of novel strategies to interfere with begomovirus transmission.

Tomato Yellow Leaf Curl China Virus Impairs Photosynthesis in the Infected Nicotiana benthamiana with βC1 as an Aggravating Factor.

Tomato yellow leaf curl China virus is a species of the widespread geminiviruses. The infection of Nicotiana benthamiana by Tomato yellow leaf curl China virus (TYLCCNV) causes a reduction in photosynthetic activity, which is part of the viral symptoms. βC1 is a viral factor encoded by the betasatellite DNA (DNAβ) accompanying TYLCCNV. It is a major viral pathogenicity factor of TYLCCNV. To elucidate the effect of βC1 on plants’ photosynthesis, we measured the relative chlorophyll (Chl) content and Chl fluorescence in TYLCCNV-infected and βC1 transgenic N. benthamiana plants. The results showed that Chl content is reduced in TYLCCNV A–infected, TYLCCNV A plus DNAβ (TYLCCNV A + β)–infected and βC1 transgenic plants. Further, changes in Chl fluorescence parameters, such as electron transport rate, Fv/Fm, NPQ, and qP, revealed that photosynthetic efficiency is compromised in the aforementioned N. benthamiana plants. The presense of βC1 aggravated the decrease of Chl content and photosynthetic efficiency during viral infection. Additionally, the real-time quantitative PCR analysis of oxygen evolving complex genes in photosystem II, such as PsbO, PsbP, PsbQ, and PsbR, showed a significant reduction of the relative expression of these genes at the late stage of TYLCCNV A + β infection and at the vegetative stage of βC1 transgenic N. benthamiana plants. In summary, this study revealed the pathogenicity of TYLCCNV in photosynthesis and disclosed the effect of βC1 in exacerbating the damage in photosynthesis efficiency by TYLCCNV infection.

Identification and Analysis of Potential Genes Regulated by an Alphasatellite (TYLCCNA) that Contribute to Host Resistance against Tomato Yellow Leaf Curl China Virus and Its Betasatellite (TYLCCNV/TYLCCNB) Infection in Nicotiana benthamiana.

Recently, begomovirus/betasatellite disease complexes were found to be associated with alphasatellites, and their presence modulated disease symptoms and/or viral DNA accumulation in infected plants. However, the biological functions of alphasatellites during begomovirus/betasatellite infections remain unclear. Tomato yellow leaf curl China virus (TYLCCNV) associated with a betasatellite (TYLCCNB) is a widespread monopartite begomovirus in China. In the Yunnan province of China, the TYLCCNV/TYLCCNB disease complex is found in association with an alphasatellite (TYLCCNA). In this study, in order to explain the mechanisms underlying TYLCCNV/TYLCCNB infection and reductions in viral DNA accumulation caused by TYLCCNA, we analyzed the transcriptome profiles of Nicotiana benthamiana seedlings challenged by TYLCCNV/TYLCCNB or TYLCCNV/TYLCCNB/TYLCCNA using RNA sequencing. In total, 2272 and 1207 differentially expressed genes (DEGs) were identified to respond to TYLCCNV/TYLCCNB and TYLCCNV/TYLCCNB/TYLCCNA infections, respectively. Compared with the DEGs in the TYLCCNV/TYLCCNB-infected N. benthamiana seedlings, the number of DEGs in plants co-infected with TYLCCNV/TYLCCNB + TYLCCNA was significantly reduced. Additionally, 36 DEGs were identified to be regulated by TYLCCNA, six of which were further analyzed using the virus-induced gene silencing (VIGS) approach. Silencing of these six TYLCCNA responsive DEGs caused more severe disease symptoms and higher viral DNA accumulation levels, suggesting that TYLCCNA responsive DEGs may attenuate TYLCCNV/TYLCCNB infection.

βC1 protein encoded in geminivirus satellite concertedly targets MKK2 and MPK4 to counter host defense.

Plant viruses have evolved multiple strategies to overcome host defense to establish an infection. Here, we identified two components of a host mitogen-activated protein kinase (MAPK) cascade, MKK2 and MPK4, as bona fide targets of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNV). βC1 interacts with the kinase domain of MKK2 and inhibits its activity. In vivo, βC1 suppresses flagellin-induced MAPK activation and downstream responses by targeting MKK2. Furthermore, βC1 also interacts with MPK4 and inhibits its kinase activity. TYLCCNV infection induces the activation of the MAPK cascade, mutation in MKK2 or MPK4 renders the plant more susceptible to TYLCCNV, and can complement the lack of βC1. This work shows for the first time that a plant virus both activates and suppresses a MAPK cascade, and the discovery of the ability of βC1 to selectively interfere with the host MAPK activation illustrates a novel virulence function and counter-host defense mechanism of geminiviruses.

First Report of Tobacco curly shoot virus Infecting Phaseolus vulgaris in China

Common bean (Phaseolus vulgaris) is one of the most economically important vegetable crops in China. It was reported that begomoviruses could infect P. vulgaris, including tomato yellow leaf curl virus (TYLCV), tomato yellow leaf curl China virus, bean dwarf mosaic virus, and cotton leaf crumple virus. In 2016, P. vulgaris with leaf crumple, thickening, and curling symptoms was found in Sichuan province, China. To identify potential begomoviruses infecting P. vulgaris, five samples of P. vulgaris were collected, and total DNA was extracted from symptomatic leaves. An approximately 500-bp DNA fragment was amplified from all the samples using the degenerate primer pair PA/PB detecting members of the genus Begomovirus (Deng et al. 1994). The fragment of isolate SC740 was cloned, and four clones were randomly chosen to be sequenced. Sequence alignments showed that three clones shared over 99% identity to tobacco curly shoot virus (TbCSV-[China:Yunnan Y98:03]), and one clone was most closely related to TYLCV-[China:Yunnan 3560:15] (98.5%). Based on the obtained sequences, two pairs of primers TbCSV-full-F (5′-AGACTTATTCGCCTGACAC-3′)/TbCSV-full-R (5′-TCTCTACTAACTGCAGATAT-3′) and TYLCV-full-F (5′-GATTTCGTTGTATGTTAGCT-3′)/TYLCV-full-R (5′-CGTGAACAGATTCAGGAAAT-3′) were designed for amplification of full-length DNA of TbCSV and TYLCV, respectively. The full length of TbCSV (accession no. MH165181) was 2,743 nt and shared 99.6% identity with TbCSV-[China:Yunnan 3560:15], and the complete DNA sequence of TYLCV (accession no. MH205950) was determined to be 2,781 nt and had 99.7% identity with TYLCV-[Australia: AU1923:06]. A fragment of approximately 600 bp was obtained from all samples using TYLCV-specific primers TYT-F/TYT-R (Ruan et al. 2013) by polymerase chain reaction, and an approximately 1.0-kb fragment was obtained from one (SC740) out of the five samples using TbCSV-specific primer pair Y35F1/Y35+10R (Qing et al. 2009). With the universal abutting primers (Briddon et al. 2002) for betasatellite, an amplicon of approximately 1,300 bp was obtained from sample SC740, but no amplification was obtained from the other four samples. Sequence alignment showed the betasatellite was 1,354 nt (accession no. MH165182) and shared the highest identity (99.8%) with tobacco curly shoot betasatellite (TbCSB-[China:Sichuan 776:16]). The infectious clones of TYLCV, TbCSV, and TbCSB were constructed and coinoculated to P. vulgaris. Typical leaf crumple, thickening, and curling symptoms were observed at 25 days postinoculation, which were indistinguishable from those observed on P. vulgaris in the field. In Sichuan, TbCSV has been reported infecting Malvastrum coromandelianum and caused severe leaf curl disease on pepper but has not been found on P. vulgaris (Qing et al. 2010). To our knowledge, this is the first report of TbCSV associated with its betasatellite infecting P. vulgaris in China, and coinfection of TYLCV and TbCSV/TbCSB.

Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase.

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.

FIRST REPORT OF TOBACCO CURLY SHOOT VIRUS (TbCSV) AND ITS ASSOCIATED SATELLITES FROM WATERMELON IN CHINA

Tobacco curly shoot virus (TbCSV), a monopartite begomovirus (genus Begomovirus, family Geminiviridae), is a serious threat to many crops and weeds in China and India. In this study, a begomovirus disease complex was identified from a watermelon sample displaying severe leaf curling and yellowing symptoms. The genome of this TbCSV isolate Y3560 contained 2,744 nucleotides (nt) and shared 97.9% identity with the TbCSV YN4519 isolate. The complete nucleotide sequence of the betasatellite DNA molecule associated to isolate Y3560 contained 1,342 nt and shared 91.0% sequence identity with the Tomato yellow leaf curl China virus betasatellite isolate TYLCCNB-Y319. The nucleotide sequence of the alphasatellite contained 1,372 nt and shared the highest levels of identity (94.0%) with Tobacco curly shoot virus associated DNA1 (TbCSA). This is the first report of a monopartite begomovirus and associated satellites from watermelon.