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.

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.

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.

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）

Markers for Rapid Evaluation of Virus Resistance for TYLCV in Tomato, ZYMV and PRSV-W in Zucchini and LMV in Lettuce and Hybrid Seeds in Pumpkin

Screening for the source of virus resistance in horticultural plants or specific characterization as hybridization, through symptoms, requires time and depends on the weather and knowledge of plant characteristics. So, it is important to develop specific gene markers to allow rapid diagnosis by PCR. Markers were developed based on sequences homology comparison of susceptible and resistant plants provided by HORTEC SEEDS in tomato for Tomato yellow leaf curl virus (TYLCV) by the resistance gene Ty-1, in zucchini for Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus estirpe watermelon (PRSV-W), and in lettuce for Lettuce mosaic virus (LMV). Fragments of 249 bp were amplified only by resistant plants to TYLCV as the hybrids 2648 and Aguamiel, and not for varieties as Santa Cruz or Carina. It were observed for ZYMV the amplification of 791 bp by the resistant hybrid Px7051 and not for the susceptible cultivar La Belle;for PRSV-W using the same zucchini plants the amplification of 650 bp for susceptible and 750 bp for resistant;for LMV the 421 bp amplification only for the resistant cultivar Brasil 303 and not for susceptible Babá de Verão. Finally, it was observed that primers PK47F/R were able to check the Cabotiá seed hybrids of pumpkin Jabras.

Tomato leaf curl Bangladesh病毒完整基因组上微卫星分布

为了展示Tomato leaf curl Bangladesh病毒完整基因组上的微卫星分布特性。借助MATLAB软件、应用最优完全子图算法,提取并展示NCBI数据库中Tomato leaf curl Bangladesh病毒完整基因组(AF_188481.1)上的微卫星分布特性,计算1-碱基组至6-碱基组在完整基因组序列上重复出现的次数和位置,展示它们的分布规律(指数函数)。结果表明:Tomato leaf curl Bangladesh病毒完整基因组(AF_188481.1)上各种N-碱基组(N取1至6)最大的重复出现次数,随N按指数函数数减少;笔者提取和展示Tomato leaf curl Bangladesh病毒完整基因组序列微卫星分布特性(特别是微卫星各种N-碱基组重复出现的位置和次数)的方法,可以系统地运用到其他病毒完整基因组序列微卫星分布特性的提取和展示中,从而为有效利用微卫星分布特性研究完整基因组的结构和功能、遗传和变异规律提供完备、仔细的数据支撑。

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.

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.

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.

Molecular characterization of Tomato yellow leaf curl China virus and its satellite DNA isolated from tobacco

Virus isolates, Y8, Y36 and Y38, were obtained from tobacco plants showing leaf curl symptoms in Honghe, Yunnan Province. In reactions with 14 monoclonal antibod-ies raised against Begomovirus particles, Y8, Y36 and Y38had similar antigenic reaction in TAS-ELISA as Tomato yel-low leaf curl China virus (TYLCCNV). The complete DNA-A nucleotide sequences of Y8, Y36 and Y38 were determinedand they contain 2727, 2730 and 2730 nucleotides, respec-tively. Each of the DNA-A sequences has a typical Bego-movirus genome organization encoding 6 ORFs with 2 ORFs [AV1(CP) and AV2] in virion-sense DNA and 4 ORFs (AC1to AC4) in complementary-sense DNA. Comparisons withtotal DNA-A, intergenic region and deduced amino acid se-quences of individual ORFs show that Y8, Y36 and Y38 are isolates of TYLCCNV. Satellite DNA molecules (DNAb) were found to be associated with Y8, Y36 and Y38, which consist of 1338, 1339 and 1338 nucleotides, respectively. Compari-sons show that these DNAb molecules share 98%—99% se-quence identities on nucleotide level and have a commonORF (designated C1) encoding 126 amino acids on the com-plementary strand.

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.

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.

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.

AC2 and AC4 proteins of Tomato yellow leaf curl China virus and Tobacco curly shoot virus mediate suppression of RNA silencing

Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) alone could systemically infect host plants such as Nicotiana benthamiana without symptoms. In con- trast, Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) alone induces leaf curl symptoms in N. benthamiana. When inoculated into transgenic N. benthamiana plants expressing GFP gene (line 16c), TYLCCNV-Y10 neither reverses the established GFP silencing nor blocks the onset of GFP si- lencing. In contrast, TbCSV-Y35 can partially reverse the established GFP silencing and block the onset of GFP silenc- ing in new leaves. In the patch co-infiltration assays, the AC2 and AC4 proteins of TYLCCNV-Y10 and TbCSV-Y35 could suppress local GFP silencing and delay systemic GFP silenc- ing, suggesting that they are suppressors of RNA silencing. Comparison of the accumulation levels of GFP mRNA in the co-infiltration patches showed that Y10 AC2 and Y35 AC2 proteins had similar efficiency for suppression of RNA si- lencing. However, Y35 AC4 protein functioned as a stronger suppressor of RNA silencing than Y10 AC4 protein. There- fore, the pathogenicity difference between TbCSV-Y35 and TYLCCNV-Y10 may be related to the functional difference in their AC4 proteins.

Function of A-Rich region in DNAβassociated with Tomato yellow leaf curl China virus

A novel type of circular single-stranded satellite DNA, known as DNAb, was recently characterized and demonstrated to be associated with monopartite begomovi-ruses. DNAb was essential for induction of characteristic symptoms in plants. DNAb has three structural features: an 115 bp highly conserved region, bC1 gene and A-Rich region. The in-frame ATG mutation of bC1 gene of Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-TY10) DNAb demonstrated that bC1 gene is required for leaf curl symptom. Here, the function of A-Rich region in TYLCCNV-Y10 DNAb was identified. When A-Rich region was deleted, the A-Rich deleted mutant was still capable of replication and systemic infection in plant, indicating that A-Rich region is not required for trans-replication of DNAb. The immunotrapping-PCR demonstrated that A-Rich de-leted mutant could be encapsidated in the coat protein en-coded by TYLCCNV-Y10 DNA-A, suggesting that A-Rich region is not related with DNAb encapsidation. However, the A-Rich region deleted mutant caused milder symptom.

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.

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。

烟粉虱取食感染TYLCV番茄对其解毒酶和保护酶活性的影响

为了明确感染番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的番茄对烟粉虱生理防御的影响,本文采用生化分析法研究了烟粉虱取食感染TYLCV的番茄植株后解毒酶和保护酶活性的变化。结果表明,B型烟粉虱在带毒番茄上取食72h及30d(长期饲养种群)后,其谷胱甘肽S-转移酶(GSTs)和羧酸酯酶(CarE)活性呈现先升高后下降的趋势,而P450酶活性则持续上升,取食30d种群是对照种群的1.99倍;三大保护酶系超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)则在取食72h后,酶活性高于对照,而在感染TYLCV番茄上取食30d后,保护酶活性则持续高于72h及对照处理,分别是对照种群的1.45倍、8.42倍和5.03倍,差异显著;Q型烟粉虱表现趋势与B型烟粉虱相似。研究结果为进一步明确入侵烟粉虱与双生病毒之间的互作、揭示烟粉虱的适应性机制等补充了资料。