The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, ...The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquiti- nation assays showed that NtRFP1 is a functional E3 ubiquiUn ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system.展开更多
Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assay...Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18-32°C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum.展开更多
Chinese tomato yellow leaf curl virus (TYLCV CHI) and other geminiviruses were analysed with 20 monoclonal antibodies. It was shown that TYLCV CHI is serologically close to Chinese tabacco leaf curl virus (TbLCV CHI)....Chinese tomato yellow leaf curl virus (TYLCV CHI) and other geminiviruses were analysed with 20 monoclonal antibodies. It was shown that TYLCV CHI is serologically close to Chinese tabacco leaf curl virus (TbLCV CHI). The fragment of TYLCV CHI DNA including the common region (CR), N terminal of coat protein gene and AV1 gene was amplified by PCR and cloned, and its DNA sequence was determined. These results showed that TYLCV CHI is different from other known geminiviruses in the world, and is a new whitefly transmitted geminivirus.展开更多
Coat protein (CP) gene of the Chinese squash !eaf curl virus (SqLCV-C) was amplified through PC’R, cloned and completely sequenced. Based on the comparisons at the levels of both CP gene nucleotide and CP -deduced am...Coat protein (CP) gene of the Chinese squash !eaf curl virus (SqLCV-C) was amplified through PC’R, cloned and completely sequenced. Based on the comparisons at the levels of both CP gene nucleotide and CP -deduced amino acid sequences with other geminiviruses, SqLCV-C is confirmed to be distinct from the American squash leaf curl virus (SqLCV-E). It is a new geminivirus transmitted by whitefly Bemisia tabaci, which infects dicotyledonous plants and is more closely related to the Indian cassava mosaic virus (ICMV).展开更多
During the last decade, the leafhopper transmitted Wheat dwarf virus （WDV） has become a serious problem both in northwestern China and Hungary. In order to study the molecular diversity and population structure of W...During the last decade, the leafhopper transmitted Wheat dwarf virus （WDV） has become a serious problem both in northwestern China and Hungary. In order to study the molecular diversity and population structure of WDV in these two countries, 39 Chinese isolates and 16 Hungarian isolates were collected from different regions of China and Hungary, and their genomes were sequenced. All isolates belonged to the wheat strain of WDV and showed limited genetic diversity. The highest and lowest nucleotide sequence identities among isolates from China and Hungary were 99.9 and 90%, respectively. In all isolates, the lowest nucleotide sequence identity was 89.5% between MO10-1 and KP10-5, which were collected from Martonv^is^r and Kompolt, Hungary. Phylogenetic analyses showed the genome sequences of 55 WDV isolates belong to two big clades, but no clear correlation to geographical location. Population difference analyses indicated that the Chinese and Hungarian WDV populations have no significant difference. The regions in WDV genome with relatively low nucleotide diversities represented protein coding regions suggested that these regions evolved under negative selection, and might be one of the causes restricting the number of genetic variants.展开更多
Many geminivirus C4 proteins induce severe developmental abnormalities in plants.We previously demon- strated that Tomato leaf curl Yunnan virus (TLCYnV)C4 induces plant developmental abnormalities at least partically...Many geminivirus C4 proteins induce severe developmental abnormalities in plants.We previously demon- strated that Tomato leaf curl Yunnan virus (TLCYnV)C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKη,an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway,in the nucleus through directing it to the plasma membrane.However, the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKη in the host cell remains unclear.Here,we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein,and that C4 shuttling is accompanied by nuclear export of NbSKTI.TLCYnV C4 is phosphorylated by NbSKη in the nucleus,which promotes myristoylation of the viral protein.Myristoylation of phosphorylated C4 favors its interaction with exportin-α(XPO I);which in turn facilitates nuclear export of the C4/NbSKTI complex. Supporting this model,chemical inhibition of N-myristoyltransferases or exportin-α enhanced nuclear retention of C4,and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention ofrC4 and thus decreased severity of C4-induced developmental abnormalities. The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus,restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling.Taken together,our results suggest that nucleocytoplasmic shuttling of TLCYnV C4,enabled by phosphorylation by NbSKη,myristoylation,and interaction with exportin-α is critical for its function as a pathogenicity factor.展开更多
Leaf curl is a serious disease of tobacco in tropical and subropical regions. Itscausative agent is tobacco leaf curl geminivirus (TLCV)which can be transmitted bywhitefly (Bemisia tabaci) and infect many other plant ...Leaf curl is a serious disease of tobacco in tropical and subropical regions. Itscausative agent is tobacco leaf curl geminivirus (TLCV)which can be transmitted bywhitefly (Bemisia tabaci) and infect many other plant species of Solanaceae andCaricaceae.The occurrence, host range and insect vector of TbLCV as well as itsserological reaction with monoclonal antibody of African cassava mosaic virus havebeen reported in China, and more recently, tobacco leaf curl disease has becomeendemic in southern areas. Here we first present the nucleotide sequence ofTbLCV coat protein (CP) gene and molecular evolutionary relationship with展开更多
Betasatellites(DNAβ)are circular ssDNA molecules that are associated with monopartite geminiviruses and exert a positive effect on the viral infection.Betasatellites encode one protein,namedβC1,on the complementary ...Betasatellites(DNAβ)are circular ssDNA molecules that are associated with monopartite geminiviruses and exert a positive effect on the viral infection.Betasatellites encode one protein,namedβC1,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,βV1,which also contributes to symptom development.TheβV1 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 theβV1 transcript was observed in plants infected with Tomato yellow leaf curl China virus(TYLCCNV)along with its associated betasatellite Tomato yellow leaf curl China betasatellite(TYLCCNB).Mutant viruses unable to produceβV1 showed reduced virulence and decreased viral load.Ectopic expression of the TYLCCNB-βV1 gene in Nicotiana benthamiana plants from a PVX-based vector resulted in leaf mosaic and chlorosis.We further demonstrated that theβV1 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.展开更多
文摘The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquiti- nation assays showed that NtRFP1 is a functional E3 ubiquiUn ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system.
基金supported by the National Science and Technology Major Projects of China (No. 2009ZX08009-026B)the China Postdoctoral Science Foundation (No. 20090461375)the National Basic Research Program (973) of China (No. 2006CB101903)
文摘Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18-32°C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum.
文摘Chinese tomato yellow leaf curl virus (TYLCV CHI) and other geminiviruses were analysed with 20 monoclonal antibodies. It was shown that TYLCV CHI is serologically close to Chinese tabacco leaf curl virus (TbLCV CHI). The fragment of TYLCV CHI DNA including the common region (CR), N terminal of coat protein gene and AV1 gene was amplified by PCR and cloned, and its DNA sequence was determined. These results showed that TYLCV CHI is different from other known geminiviruses in the world, and is a new whitefly transmitted geminivirus.
文摘Coat protein (CP) gene of the Chinese squash !eaf curl virus (SqLCV-C) was amplified through PC’R, cloned and completely sequenced. Based on the comparisons at the levels of both CP gene nucleotide and CP -deduced amino acid sequences with other geminiviruses, SqLCV-C is confirmed to be distinct from the American squash leaf curl virus (SqLCV-E). It is a new geminivirus transmitted by whitefly Bemisia tabaci, which infects dicotyledonous plants and is more closely related to the Indian cassava mosaic virus (ICMV).
基金supported by the Inter-Governmental Agreement in Science and Technology(Sino-Hungary 333/2008 and CN-13/2007)the National Natural Science Foundation of China(30900938)
文摘During the last decade, the leafhopper transmitted Wheat dwarf virus （WDV） has become a serious problem both in northwestern China and Hungary. In order to study the molecular diversity and population structure of WDV in these two countries, 39 Chinese isolates and 16 Hungarian isolates were collected from different regions of China and Hungary, and their genomes were sequenced. All isolates belonged to the wheat strain of WDV and showed limited genetic diversity. The highest and lowest nucleotide sequence identities among isolates from China and Hungary were 99.9 and 90%, respectively. In all isolates, the lowest nucleotide sequence identity was 89.5% between MO10-1 and KP10-5, which were collected from Martonv^is^r and Kompolt, Hungary. Phylogenetic analyses showed the genome sequences of 55 WDV isolates belong to two big clades, but no clear correlation to geographical location. Population difference analyses indicated that the Chinese and Hungarian WDV populations have no significant difference. The regions in WDV genome with relatively low nucleotide diversities represented protein coding regions suggested that these regions evolved under negative selection, and might be one of the causes restricting the number of genetic variants.
基金grants from the National Natural Science Foundation of China (31720103914 and 31390422).
文摘Many geminivirus C4 proteins induce severe developmental abnormalities in plants.We previously demon- strated that Tomato leaf curl Yunnan virus (TLCYnV)C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKη,an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway,in the nucleus through directing it to the plasma membrane.However, the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKη in the host cell remains unclear.Here,we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein,and that C4 shuttling is accompanied by nuclear export of NbSKTI.TLCYnV C4 is phosphorylated by NbSKη in the nucleus,which promotes myristoylation of the viral protein.Myristoylation of phosphorylated C4 favors its interaction with exportin-α(XPO I);which in turn facilitates nuclear export of the C4/NbSKTI complex. Supporting this model,chemical inhibition of N-myristoyltransferases or exportin-α enhanced nuclear retention of C4,and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention ofrC4 and thus decreased severity of C4-induced developmental abnormalities. The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus,restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling.Taken together,our results suggest that nucleocytoplasmic shuttling of TLCYnV C4,enabled by phosphorylation by NbSKη,myristoylation,and interaction with exportin-α is critical for its function as a pathogenicity factor.
文摘Leaf curl is a serious disease of tobacco in tropical and subropical regions. Itscausative agent is tobacco leaf curl geminivirus (TLCV)which can be transmitted bywhitefly (Bemisia tabaci) and infect many other plant species of Solanaceae andCaricaceae.The occurrence, host range and insect vector of TbLCV as well as itsserological reaction with monoclonal antibody of African cassava mosaic virus havebeen reported in China, and more recently, tobacco leaf curl disease has becomeendemic in southern areas. Here we first present the nucleotide sequence ofTbLCV coat protein (CP) gene and molecular evolutionary relationship with
文摘从浙江温州田间表现曲叶症状的番茄叶中提取获得病毒分离物wzh,对wzh全基因组序列进行测定。结果表明:wzh全长2 740个核苷酸,具有菜豆金色花叶病毒属(Begomovirus)病毒基因组典型特征,共编码6个开放阅读框(open reading frame,ORF),病毒链编码AV2及AV1 2个ORFs,互补链编码AC1、AC2、AC3及AC4 4个ORFs。BLAST搜索结果显示,wzh与Begomovirus中来自亚洲的病毒同源性较高,而与美洲、非洲等地的相对较低,与台湾番茄曲叶病毒(Tomatoleaf curl Tai wan virus,ToLCTWV)DNA-A全序列同源性最高,为99%。全序列系统进化关系树显示,wzh与TolCTWV的亲缘关系最近,并形成一个独立的分支,而与其他19种双生病毒的亲缘关系均相对较远。因此,wzh是台湾番茄曲叶病毒的一个分离物。
基金supported by grants from the National Natural Science Foundation of China to Y.Q.W.(31972235)and to X.P.Z.(31720103914)。
文摘Betasatellites(DNAβ)are circular ssDNA molecules that are associated with monopartite geminiviruses and exert a positive effect on the viral infection.Betasatellites encode one protein,namedβC1,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,βV1,which also contributes to symptom development.TheβV1 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 theβV1 transcript was observed in plants infected with Tomato yellow leaf curl China virus(TYLCCNV)along with its associated betasatellite Tomato yellow leaf curl China betasatellite(TYLCCNB).Mutant viruses unable to produceβV1 showed reduced virulence and decreased viral load.Ectopic expression of the TYLCCNB-βV1 gene in Nicotiana benthamiana plants from a PVX-based vector resulted in leaf mosaic and chlorosis.We further demonstrated that theβV1 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.
