Geminiviruses and their application in biotechnology

Being a major class of single-stranded DNA viruses, geminiviruses are mostly studied due to their catastrophic infectious effect on crops. These DNA viruses are characteristic for their ability in quickly multiplying viral genetic materials without integrating into the genome of plants, which makes them ideal for developing viral vectors for plants bioengineering. Geminivirus-derived vectors can be classified into expression vectors and virus-induced gene silencing（VIGS） vectors. Details of the design, construction, application and improvements of these geminivirus vectors are summarized and discussed.

Tobacco RING E3 Ligase NtRFP1 Mediates Ubiquitination and Proteasomal Degradation of a Geminivirus-Encoded βC1

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.

Development and application of an efficient virus-induced gene silencing system in Nicotiana tabacum using geminivirus alphasatellite

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.

Chinese tomato yellow leaf curl virus--a new species of geminivirus

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.

Occurrence and distribution of geminiviruses in China

Geminiviruses are a group of plant viruses that cause severe diseases in many economically important crops worldwide, leading to devastating losses to agricultural production. Here we summarize the occurrence and distribution of geminiviruses in China,which provides valuable information for further epidemiological studies and supports the development of effective disease management strategies.

Nucieocytoplasmic Shuttling of Geminivirus C4 Protein Mediated by Phosphorylation and Myristoylation Is Critical for Viral Pathogenicity

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.

Chinese squash leaf curl virus: a new whitefly-transmitted geminivirus

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).

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

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.

Preliminary Study on Molecular Evolution of Tobacco Leaf Curl Geminivirus

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

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 TYLCCNBβC1 is a major inducer of UPR and can upregulate the expression of b ZIP60,a transcription factor in Nicotiana benthamiana plants.Treatment using ER stress inducers or overexpression of Nbb ZIP60 increasesβC1 accumulation and benefits TYLCCNV/TYLCCNB infection in N.benthamiana plants,and vice versa.In the TYLCCNV/TYLCCNB-infected or theβC1-expressing cells,Nbb ZIP60 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 Nbb ZIP60-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 Nbb ZIP60 to evade plant defense response,which is a distinct virulence strategy exploited by plant pathogens.

The Ever-Expanding Role of C4/AC4 in Geminivirus Infection: Punching above Its Weight?

Geminiviruses (family Geminiviridae) are among the most devastating plant pathogens worldwide, causing heavy losses on food and cash crops and representing a threat to global food security and sustainability. These viruses have very compact genomes comprising one (monopartite) or two (bipartite) circular singlestranded DNA (ssDNA) molecules, each ranging from 2.6 to 2.8 kb.

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the geminiviral infection in Arabidopsis

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 C2proteins 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 C2proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8did 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.

粉虱传双生病毒的TAS-ELISA及PCR快速检测

利用粉虱传双生病毒 (WTGs)多克隆抗体及单克隆抗体 ,建立了三抗体夹心 EL ISA (TAS-EL ISA)检测 WTGs的方法 ,并发现了单克隆抗体 SCR18可广泛用于我国 WTGs的检测。利用根据WTGs基因组上基因间隔区及外壳蛋白基因保守序列设计的引物 ,建立了 PCR特异检测 WTGs的方法。对田间病样的 TAS- EL ISA和 PCR检测表明 ,粉虱传双生病毒在云南省烟草、番茄和南瓜上均存在 ,2种方法的测定结果是一致的。

烟粉虱传播双生病毒研究进展

综述了烟粉虱Bemisia tabaci对双生病毒的获取、传播及存留等方面的特性.烟粉虱最短的获毒和接种时间为15～30 min;双生病毒在烟粉虱体内可存留1至数周,有的终身存在.烟粉虱对双生病毒的传毒效率除了随其获毒及传毒时间的延长、传毒烟粉虱个体数量的增加以及病毒体浓度的增加而提高外,还与烟粉虱的龄期及性别有关.双生病毒除了在植物与粉虱之间直接传播外,还可通过烟粉虱交配及经卵携带的途径在烟粉虱个体和代别间进行传播.寄主植物、双生病毒的一些特殊蛋白以及烟粉虱内共生菌产生的GroEL蛋白,都可影响烟粉虱携带的双生病毒种类及传毒的可能性.双生病毒可对烟粉虱的发育、存活和生殖产生不利或有利的影响.雌成虫携带番茄黄化曲叶病毒（tomato yellow leaf curl virus,TYLCV）后,存活力和生殖力均下降;而携带番茄斑驳病毒（tomatomottle virus,ToMoV）后,生殖力提高.此外,植物感染双生病毒后,其对烟粉虱的适合性可能提高.

云南省烟草上双生病毒的发生与分布(英文)

已报道云南省烟草曲叶病（Tobacco leaf curl disease，TLCD）由3种菜豆金色花叶病毒属（Begomovirus）的双生病毒引起。它们是烟草曲茎病毒（Tobacco curly shoot virus，TbCSV）、云南烟草曲叶病毒（Tobacco leaf curl Yunnan virus，TbLCYNV）和中国番茄黄化曲叶病毒（Tomato yellow leaf curl China virus，TYLCCNV）。为了了解这3种病毒的分布，2000—2005年，从云南省4个市（州）采集了109个烟草曲叶病样品，利用3种病毒的特异引物对采集样品进行了PCR检测。从保山市的烟草曲叶病样品中检测到3种病毒，并且存在TbCSV＋TbLCYNV、TbCSV＋TYLCCNV和TbCSV＋TbLYNV＋TYLCCNV的复合侵染，从大理州、文山州和红河州的烟草曲叶病样品中仅检测到TYLCCNV。对39个分离物部分序列的系统进化分析结果表明，病毒分离物依据病毒种类和地理位置聚成几簇。对烟草曲叶病的流行和病害控制策略进行了讨论。

香料烟粉虱传双生病毒的分子检测

发生在保山香料烟生产区粉虱传双生病毒病,已严重影响到了香料烟的品质和产量。本研究利用PCR技术快速检测香料烟植株及田间其它植物如胜红蓟、赛葵及菜豆等、以及病毒介体的带毒情况。对部分样品的PCR检测及序列分析表明,感染香料烟双生病毒有中国番茄黄曲叶病毒、番茄黄曲叶病毒、云南烟草曲茎病毒、烟草曲茎病毒以及云南烟草曲叶病毒等,且一些样品存在复合侵染现象。本研究为掌握病害发生流行的基本规律,监控病害在香料烟上的发生流行提供了依据。

吉林番茄黄化曲叶病毒分离物的检测及其DNA-A全基因组序列分析

从吉林省松原市田间表现黄化曲叶症状的番茄上分离到病毒分离物JLSY,基因组全序列测定结果表明,基因组全长2781bp,共编码6个ORF。序列比对表明,JLSY基因组与番茄黄化曲叶病毒(TYLCV)安徽分离物(FN650807)相似性最高,为99.5%,而与其他双生病毒的序列相似性低于89%。经系统进化分析,该病毒分离物属于TYLCV以色列株系(TYLCV-IL)。这是首次在我国吉林省检测到番茄黄化曲叶病毒。

中国南瓜曲叶病毒DNAA的克隆及其全序列

:对引起我国南瓜曲叶病的病毒分离物DNA的克隆和序列分析表明 ,中国南瓜曲叶病毒DNAA由 2 741个核苷酸组成 ,共编码 6个开放阅读框 (ORF) ,其中病毒链有 2个ORF :AV1 (2 56aa)和AV2 (1 40aa) ,AV1为外壳蛋白基因 ;病毒链的互补链有 4个ORF ,AC1 (2 4 3aa)编码复制酶基因 ,AC2 (1 34aa)编码反式激活蛋白 ,AC3(1 36aa)和AC4(1 72aa) ,该病毒属于旧世界Begomoviruses,是一个粉虱传播的联体病毒。

海南岛番木瓜和扶桑上粉虱传双生病毒的检测及序列分析

Three virus-infected Carica papaya samples showing mosaic,leaf roll symptoms and three virus-infected Hibiscus rosa-sinensis samples showing chlorosis and distortion symptoms were collected in Danzhou of Hainan Province.A pair of degenerate primers was designed according to the conserved sequence of CP gene and intergenic spacer of Whitefly-transmittal geminiviruses(WTGV),PCR product of expected size was amplified from six samples.The specific fragments were cloned and sequenced,and sequence analysis showed that PCR product had a geminiviral gene origin,which was demonstrated by BLAST.Partial DNA-A between P1 and H2 shared only 66% nucleotide sequence identity.Polygenie analysis with DNA-A of P1 and H2 from other 16 WTG,P1 was most closely related to LAYVV,H2 differed from those of other geminiviruses and constitute a new evolutionary branch.

海南岛长蒴母草和金腰箭上存在粉虱传双生病毒的侵染

从海南儋州的长蒴母草(Linderniaanagallis)上采集了呈现叶脉黄化、叶背叶脉突起的3个病样,从金腰箭(Synedrellanodiflora)上采集到叶片不均匀褪绿、叶背叶脉突起的3个病样。利用根据粉虱传双生病毒基因组DNA基因间隔区及外壳蛋白基因保守序列设计的简并引物,对这6个样品进行了PCR检测,结果发现所有样品均能扩增到预期大小的DNA片段。PCR产物经克隆后进行序列测定,BLAST结果表明,该序列为双生病毒基因组DNA序列,该2种杂草上均存在双生病毒的侵染。