Diverse Regulations of Viral and Host Genes in Tomato Germplasms Responding to Tomato Yellow Leaf Curl Virus Inoculation

Tomato yellow leaf curl virus(TYLCV)is the dominating pathogen of tomato yellow leaf curl disease that caused severe loss to tomato production in China.In this study,we found that a TYLCV-resistant tomato line drastically reduced the accumulation of viral complementary-sense strand mRNAs but just moderately inhibited that of viral DNA and virion-sense strand mRNAs.However,two other resistant lines did not have such virus inhibition pattern.Analysis of differential expressed genes showed that the potential host defense-relevant processes varied in different resistant tomatoes,as compared to the susceptible line,suggesting a diversity of tomato TYLCV-resistance mechanisms.

Monoclonal Antibodies Against the Whitefly-Transmitted Tomato Yellow Leaf Curl Virus and Their Application in Virus Detection

Tomato yellow leaf curl virus（TYLCV）is a species of the family Geminiviridae,causing serious yield losses in tomato production.The coat protein（CP）gene of TYLCV isolate SH2 was expressed in Escherichia coli BL21（DE3）using pET-32a as the expression vector.The recombinant protein was purified through Ni＋-NTA affinity column and used to immunize BALB/c mice.Three hybridoma cell lines（2B2,2E3 and 3E10）secreting monoclonal antibodies（MAbs）against TYLCV CP were obtained by fusing mouse myeloma cells（Sp 2/0）with spleen cells from the immunized BALB/c mouse.The titers of ascitic fluids of three MAbs ranged from 10-6 to 10-7 in indirect-ELISA.Isotypes and subclasses of all the MAbs belonged to IgG1,κ light chain.Triple antibody sandwich enzyme-linked immunosorbent assay（TAS-ELISA）showed that the MAb 3E10 could react with five begomoviruses infecting tomato,while the other two（2B2 and 2E3）mainly reacted with TYLCV.TAS-ELISA was set up using the MAb 3E10,and the established method could successfully detect virus in plant sap at 1：2 560（w/v,g mL-1）.Detection of field samples showed that begomoviruses were common in tomato crops in Zhejiang Province,China.

Development of high yield and tomato yellow leaf curl virus(TYLCV)resistance using conventional and molecular approaches:A review

Tomato(Solanum lycopersicum L.)belonging to the family Solanaceae is the second most consumed and cultivated vegetable globally.Since the ancient time of its domestication,thousands of cultivated tomato varieties have been developed targeting an array of aspects.Among which breeding for yield and yield-related traits are mostly focused.Cultivated tomato is extremely genetically poor and hence it is a victim for several biotic and abiotic stresses.Among the biotic stresses,the impact of viral diseases is critical all over tomato cultivating areas.Improvement of tomato still largely rely on conventional methods worldwide while molecular approaches,particularly Marker Assisted Selection(MAS)has become popular across the globe as a fast,low cost and precise tool which is essential in present day plant breeding.In this review paper,breeding tomato for high yield and viral disease resistance,particularly to tomato yellow leaf curl virus disease(TYLCVD)using conventional and molecular approaches will be discussed.Lining up of this set of information will be useful to those who are interested in tomato variety development with high yielding and TYLCVD resistance.

Mapping and Candidate Gene Screening of Tomato Yellow Leaf Curl Virus Resistant Gene ty-5

To identify the inheritance pattern and perform fne mapping of ty-5 gene, P1, P2, F1, BC1 and F2 generations were obtained through a cross between CLN32120a-23 （containing ty-5 gene, P1） and S. lycopersicum Moneymaker （fully susceptible, P2）. The results showed that resistance of ty-5 gene was determined by a recessive effect. Meanwhile, it was presumed that another resistance gene might be involved in mediating the resistance to tomato yellow leaf curl virus （TYLCV）. In this study, fne mapping was used to map TYLCV resistance locus to an interval between NAC1 and TES2461 on the short arm of chromosome 4 with genetic distances of 0.5 and 0.8 cM, respectively. qRT-PCR results showed that four candidate genes, SlNAC1; LOC104229164; LOC101260925 and LOC101261508 having resistance-related expression patterns, were the likely target genes of ty-5. In addition, it was found that the codominant marker TES2461 could be used in marker-assisted selection （MAS） breeding. The fndings of this research provided the basis for future cloning of ty-5 gene as well as MAS breeding and plant resistance mechanism studies.

Characterization and function of Tomato yellow leaf curl virus-derived small RNAs generated in tolerant and susceptible tomato varieties

supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China （20134320120013）;the Natural Science Foundation of Hunan Province, China （14JJ3095）

Tomato yellow leaf curl virus infection of tomato does not affect the performance of the Q and ZHJ2 biotypes of the viral vector Bemisia tabaci

To better understand the etiology of begomovirus epidemics in regions under invasion we need to know how indigenous and invasive whitefly vectors respond to virus infection. We investigated both direct and indirect effects of infection with Tomato yellow leaf curl virus （TYLCV） on the performance of the invasive Q biotype and the indigenous Asian ZHJ2 biotype of whitefly Bemisia tabaci. The Q biotype performed better than the ZHJ2 biotype on either uninfected or virus-infected tomato plants. However, virus-infection of host plants did not, or only marginally affected, the performance of either biotype of whiteflies in terms of fecundity, longevity, survival, development and population increase. Likewise, association of the vectors with TYLCV did not affect fecundity and longevity of the Q or ZHJ2 biotypes on cotton, a non-host of TYLCV. These results indicate that the alien Q biotype whitefly, but not the indigenous ZHJ2 biotype, is likely to become the major vector of TYLCV in the field and facilitate virus epidemics.

The novel C5 protein from tomato yellow leaf curl virus is a virulence factor and suppressor of gene silencing

Tomato yellow leaf curl virus(TYLCV)is known to encode 6 canonical viral proteins.Our recent study revealed that TYLCV also encodes some additional small proteins with potential virulence functions.The fifth ORF of TYLCV in the complementary sense,which we name C5,is evolutionarily conserved,but little is known about its expression and function during viral infection.Here,we confirmed the expression of the TYLCV C5 by analyzing the promoter activity of its upstream sequences and by detecting the C5 protein in infected cells by using a specific custom-made antibody.Ectopic expression of C5 using a potato virus X(PVX)vector resulted in severe mosaic symptoms and higher virus accumulation levels followed by a burst of reactive oxygen species(ROS)in Nicotiana benthamiana plants.C5 was able to effectively suppress local and systemic post-transcriptional gene silencing(PTGS)induced by single-stranded GFP but not double-stranded GFP,and reversed the transcriptional gene silencing(TGS)of GFP.Furthermore,the mutation of C5 in TYLCV inhibited viral replication and the development of disease symptoms in infected plants.Transgenic overexpression of C5 could complement the virulence of a TYLCV infectious clone encoding a dysfunctional C5.Collectively,this study reveals that TYLCV C5 is a pathogenicity determinant and RNA silencing suppressor,hence expanding our knowledge of the functional repertoire of the TYLCV proteome.

Molecular variability and evolution of a natural population of tomato yellow leaf curl virus in Shanghai,China

Tomato yellow leaf curl virus （TYLCV）, belonging to the genus Begomovirus of the family Geminiviridae, is emerging as the most destructive pathogen of tomato plants. Since the first report of TYLCV in Shanghai, China in 2006, TYLCV has spread rapidly to 13 provinces or autonomous regions of China. In this study, the molecular varia- bility and evolution of TYLCV were monitored in Shanghai from its first upsurge in 2006 until 2010. Full-length genomic sequences of 26 isolates were obtained by rolling circle amplification. Sequence analysis showed that the intergenic region was the most variable, with a mean mutation rate of 4.81×10-3 nucleotide substitutions per site per year. Ge- netic differentiation was found within isolates obtained from 2006, 2009, and 2010, though a linear increase in genetic diversity over time was not evident. Whilst significant parts of TYLCV genes were under negative selection, the C4 gene embedded entirely within the C1 gene had a tendency to undergo positive selection. Our results indicate that a mechanism of independent evolution of overlapping regions could apply to the natural population of TYLCV in Shanghai, China.

Molecular and biological characterization of melon-infecting squash leaf curl China virus in China

It has been reported that squash leaf curl China virus(SLCCNV)infects some Cucurbitaceae crops except for melon(Cucumis melo L.).A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan Province of China.In this study,the pathogen was identified as SLCCNV through biological and molecular characterization.The isolate(SLCCNV-HN)possess a bipartite genome,DNA-A(HM566112.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062251.1)pumpkin and SLCCNV-Hn61(AM260205.1)squash isolates from China,whereas DNA-B(HM566113.1)with the highest nucleotide identity(99%)to SLCCNV-Hn(MF062252.1).Phylogenetic analyses based on the full-length SLCCNV-HN DNA-A and-B sequences indicated that SLCCNV-HN melon isolate is clustered with SLCCNV-Hn pumpkin,SLCCNV-Hn61 and SLCCNV-SY squash isolates from southern China,forming an independent cluster.Infectious clone of SLCCNV-HN was constructed and the melon plants were inoculated and the infection rate is 100%,the systemic symptoms in melon showed identical to those of melon plants infected in fields.Additionally,melon plants transmission of this virus by Bemisia tabaci with a transmission rate of 95%(19/20)showed leaf curl and dwarf symptoms 15 days post transmission,thereby fulfilling Koch’s postulates.Analysis of genomic organization and phylogenetic trees indicated that SLCCNV-HN melon isolate belongs to the Begomovirus genus.To the best of our knowledge,this is the first characterization of meloninfecting SLCCNV through its genome,infectious clone and transmission.

Discovery and demonstration of small circular DNA molecules derived from Chinese tomato yellow leaf curl virus

Tomato yellow leaf curl viruses belong to Begomoviruses of geminiviruses. In this work, we first found and demonstrated that the small circular DNA molecules were derived from Chinese tomato yellow leaf curl viruses (TYLCV-CHI). These small circular DNA molecules are about 1.3 kb, which are half the full-length of TYLCV-CHI DNA A. It was shown by sequence determination and analysis that there was unknown-origin sequence insertion in the middle of the small molecules. These sequences of unknown-origin were neither homologous to DNA A nor to DNA B, and were formed by recombination of virus DNA and plant DNA. Although various defective molecules contained different unknown-origin sequence insertion, all the molecules contained the intergenic region and part of the AC1 (Rep) gene. But they did not contain full ORF.

Isolation and Identification of Squash Leaf Curl China Virus in Zucchini

[Objectives]In 2019,virus diseases occurred widely on zucchini planted in Shandong Province.The disease symptom was different from previous reports.This study aimed to identify the pathogen causing the zucchini virus disease.[Methods]Ten diseased zucchini leaves were collected in the field and used as materials for PCR and sequencing.[Results]PCR detection and sequencing showed that the nucleotide sequence of the amplified fragment had the highest identity with the squash isolate of squash leaf curl China virus(SLCCNV)(MW389919.1)in Guangdong Province.Primers were further designed for amplifying the full-length SLCCNV.The full-length DNA-A was 2730 bp(OM692270.1),and the full-length DNA-B was 2711 bp(OM692269.1).Through sequence alignment,it was found that the DNA-A sequence shared identity of 89.65%-99.42%with registered SLCCNV,and the identity with the SLCCNV-GDHY pumpkin isolate(MW389919.1)in Guangdong was the highest,at 99.42%.The DNA-B sequence was identical with registered SLCCNV in the range of 81.82%-97.29%,and the identity with the SLCCNV-GDHY pumpkin isolate(MW389918.1)in Guangdong,was the highest,at 97.29%.Therefore,it was speculated that SLCCNV is the pathogen of zucchini virus disease.Since the virus was first found on zucchini in Shandong,it was named SLCCNV-SD.[Conclusions]This study provides materials for the research on the spread of SLCCNV in China and the analysis of population genetic characteristics,as well as a reference for the prevention and control of the virus in zucchini.

Socio-Economic and Scientific Impact Created by Whitefly-Transmitted,Plant-Virus Disease Resistant Tomato Varieties in Southern India

Research carried out to assess the impact of open-pollinated Tomato leaf curl virus（ToLCV）-resistant tomatoes and hybrids on the livelihoods of resource-poor farmers in Southern India is described and discussed.Three high-yielding ToLCV-resistant tomato varieties were developed initially using conventional breeding and screening techniques involving inoculation by ToLCV-viruliferous whitefly,Bemisia tabaci.In 2003 and 2004,respectively,these varieties were released officially by the Karnataka State Seed Committee and the Indian Ministry of Agriculture through notification in the Gazette of India.From 2003 to 2005,eleven seed companies bought breeder seed of the ToLCV-resistant varieties and used them to begin breeding F1 hybrids from them.Socio-economic studies carried out to assess the benefits obtained from growing the ToLCV-resistant varieties found that farmers could gain up to 10 times the profit by growing the ToLCV-resistant varieties compared to the pre-existing ToLCV-susceptible varieties.Adoption of ToLCV-resistant tomatoes was also associated with reduced pesticide use.Extra income from tomato sales was prioritised by farmers to pay for children＇s education,better nutrition and medicines.In a joint effort with the commercial seed sector in India,a promotional field day was organised in 2007.As well as the three ToLCV-resistant varieties,62 ToLCV-resistant hybrid tomatoes were exhibited during a farmer-field day by 17 commercial seed companies and several public institutes.Tomatoes with ToLCV-resistance are now grown widely in South India and seeds of the three open-pollinated varieties have been distributed to more than 12 countries.In 2007,a conservative estimate of the financial-benefit to cost of the research ratio was already more than 837：1.

Tomato leaf curl New Delhi virus:an emerging plant begomovirus threatening cucurbit production

Tomato leaf curl New Delhi virus(ToLCNDV),a bipartite begomovirus,was first reported to infect tomato and has recently spread rapidly as an emerging disease to Cucurbitaceae crops.To date,the virus has been reported to infect more than 11 cucurbit crops,in 16 countries and regions,causing severe yield losses.In autumn 2022,ToLCNDV was first isolated from cucurbit plants in Southeastern coastal areas of China.Phylogenetic analysis established that these isolates belong to the Asian ToLCNDV clade,and shared high nucleotide identity and closest genetic relationship with the DNA-A sequence from the Chinese tomato-infecting ToLCNDV isolate(Accession no.OP356207)and the tomato New Delhi ToLCNDV-Severe isolate(Accession no.HM159454).In this review,we summarize the occurrence and distribution,host range,detection and diagnosis,control strategies,and genetic resistance of ToLCNDV in the Cucurbitaceae.We then summarize pathways that could be undertaken to improve our understanding of this emerging disease,with the objective to develop ToLCNDV-resistant cucurbit cultivars.

Molecular characterization and pathogenicity of an infectious clone of tomato leaf curl New Delhi virus isolate infecting Cucumis melo

Tomato leaf curl New Delhi virus(ToLCNDV)is a member of the genus Begomovirus,and causes devastating disease in the world.In recent years,ToLCNDV was rapidly spreading in China and induces severe economic losses in agriculture.In this study,we sequenced and characterized the complete genome of ToLCNDV isolates from melon plants showing leaf curling and stunting symptoms in Jiangsu Province of China.We constructed a full-length infectious cDNA clone of ToLCNDV,which could induce systemic infection with typical symptoms in Nicotiana benthamiana,Cit-rullus melo,and Citrullus lanatus plants through agrobacterium-mediated inoculation.Further experimental evidence demonstrated that the virions produced in plants infected with the infectious clone of ToLCNDV are biologically active and sap-transmissible.We also evaluated the resistance of commercial melon cultivars to ToLCNDV and found all testing melon cultivars were susceptible to ToLCNDV.Collectively,the reverse genetic system developed herein will facilitate further research on biological functions of proteins encoded by ToLCNDV and plant-ToLCNDV interactions,which might provide new insights into breeding resistance germplasm in crops.

Molecular characterization and infectivity of Papaya leaf curl China virus infecting tomato in China

Papaya leaf curl China virus (PaLCuCNV) was previously reported as a distinct begomovirus infecting papaya in southern China. Based on molecular diagnostic survey, 13 PaLCuCNV isolates were obtained from tomato plants showing leaf curl symptoms in Henan and Guangxi Provinces of China. Complete nucleotide sequences of 5 representative isolates (AJ558116, AJ558117, AJ704604, FN256260, and FN297834) were determined to be 2738–2751 nucleotides, which share 91.7%–97.9% sequence identities with PaLCuCNV isolate G2 (AJ558123). DNA-β was not found to be associated with PaLCuCNV isolates. To investigate the infectivity of PaLCuCNV, an in-fectious clone of PaLCuCNV-[CN:HeNZM1] was constructed and agro-inoculated into Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants, which induced severe leaf curling and crinkling symptoms in these plants. Southern blot analysis and polymerase chain reaction (PCR) indicated a systemic infection of test plants by the agro-infectious clone.

Gene expression profiling of the whitefly （Bemisia tabaci） Middle East- Asia Minor 1 feeding on healthy and Tomato yellow leaf curl China virus-infected tobacco

Begomoviruses are exclusively transmitted by whitefly （Bemisia tabaci） in a circulative, non-propagative manner. The influences of begomoviruses on whitefly vector are complex with both direct and indirect effects. However, the molecular mechanisms underlying these effects are poorly understood and the transcriptional profiles of whitefly on healthy and virus-infected plants have not yet been studied. Using suppression subtractive hybridization, we investigated the differentially expressed genes in whitefly Middle East - Asia Minor 1 feeding on healthy and Tomato yellow leaf curl China virus （TYLCCNV） infected tobacco. From the forward cDNA library, 124 differentially expressed expression sequence tags （ESTs） were obtained which represent up-regulated genes in the whiteflies feeding on the infected tobacco. From the reverse library, 112 ESTs were isolated which represent down-regulated genes. Among the up-regulated genes, we identified several genes that are probably involved in direct interaction between whitefly and TYLCCN~, including a 26/29-kDa proteinase that appears to participate in the elimination of foreign proteins, heparan sulfate proteoglycan which mediates the entry of several viruses into host cells and two genes of Rickettsia-a secondary endosymbiotic bacterium of whitefly. In addition, we identified a number of genes involved in metabolism, transcription and translation which might be the result of indirect effects of TYLCCNV on the whitefly via host plants. Collectively, our results suggest that TYLCCNV-infected tobacco changes the gene expression profiles of whitefly via both direct and indirect interactions. This study revealed a number of genes involved in whitefly-TYLCCNV-tobacco interactions and provided useful information for future study on this complex system.

番茄黄化卷叶病毒病(TYLCV)的研究进展

综述了国内外番茄黄化卷叶病毒病的研究现状,包括TYLCV的发病症状与鉴定、病毒的发生与流行及病原物的生物学特征、抗病遗传资源、抗病基因分子标记和抗病育种等方面的研究进展。

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。

侵染番茄的曲叶病毒检测及遗传变异分析

2017—2019年,从甘肃、广西、海南、河北和云南5个省份番茄主产区采集疑似感染曲叶病毒的番茄病叶,进行曲叶病毒检出率分析及种类鉴定。特异性PCR检测结果表明,5个省份曲叶病毒平均检出率为25.00%,其中广西最高,达到50.91%。PCR扩增序列同源性比对结果表明,侵染5个省份番茄的曲叶病毒为中国番木瓜曲叶病毒(papaya leaf curl China virus,PaLCuCNV)、广东番茄曲叶病毒(tomato leaf curl Guangdong virus,ToLCGDV)和广西番茄曲叶病毒(tomato leaf curl Guangxi virus,ToLCGXV)。序列系统发育分析表明,5个省份的曲叶病毒分离物可分为2个亚簇,无明显的地域特性和寄主依赖性。遗传进化分析表明,5个省份的曲叶病毒出现5个重组事件,序列中C/T替换率最高(18.73)。

Science Letters:Expression of a begomoviral DNAβ gene in transgenic Nicotiana plants induced abnormal cell division

An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAβ) is required to induce typical symptoms in host plants. DNAβ encodes a single gene (termed βC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing theβC1 gene of a DNAβ associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing βC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the βC1 protein showed that the βC1 protein accumulated in the nuclei of cells. The possible biological function of the βC1 protein was discussed.