Integrative analysis of the transcriptome and metabolome reveals the response mechanism to tomato spotted wilt virus

Tomato spotted wilt virus(TSWV)is an important virus that has rapidly spread throughout the world.TSWV seriously hinders the production of tomato(Solanum lycopersicum)and other plants.In order to discover more new genes and metabolites related to TSWV resistance in tomato plants,the genes and metabolites related to the resistance of tomato plants inoculated with TSWV were identified and studied herein.The tomato TSWV-resistance line YNAU335(335)and TSWV-susceptible lines NO5 and 96172I(961)were used as the transcriptome and metabolome research materials.Transcriptomic and metabolomic techniques were used to analyze the gene and metabolite response mechanisms to TSWV inoculation.A total of 3566,2951,and 2674 differentially expressed genes(DEGs)were identified in lines 335,NO5,and961,respectively.Meanwhile,208,228,and 273 differentially accumulated metabolites(DAMs)were identified in lines 335,NO5,and 961,respectively.In line 335,the number of DEGs was the highest,but the number of DAMs was lowest.Furthermore,903 DEGs and 94 DAMs were common to the response to TSWV in the three inbred lines.The 903 DEGs and 94 DAMs were mainly enriched in the plant hormone signal transduction and flavonoid synthesis pathways.In addition,many nucleotide-binding site-leucine-rich repeat genes and transcription factors were found that might be involved in the TSWV response.These results provide new insights into TSWV resistance mechanisms.

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.

警惕新发病毒番茄褐色皱果病毒Tomato brown rugose fruit virus对我国番茄产业的危害

番茄褐色皱果病毒Tomato brown rugose fruit virus(ToBRFV)于2014年首次在以色列发现,随后传播到欧洲、美洲以及亚洲等地。ToBRFV在番茄叶片上引起花叶,更重要的是在番茄果实上引起褐色皱缩斑,导致番茄完全失去商品价值,是番茄安全生产的重大威胁。为遏制ToBRFV的传播,多个国家已经将该病毒列入检疫对象。2019年,我们在山东番茄上检测到该病毒。本文综述了ToBRFV发生与危害、寄主范围和症状、传播方式、基因组结构、检测方法,并提出了防治建议,希望有助于防范该病毒在我国的扩散。

警惕种子传带的南方番茄病毒Southern tomato virus对我国番茄产业的危害

南方番茄病毒Southern tomato virus(STV)是近年来新发现的一种侵染番茄的病毒,通常与多种病毒复合侵染,可能与番茄的褪绿、黄化、衰退、果实变小等症状相关。该病毒最早在1984年发现,并于2005年命名。我国首先于2015年在山东寿光发现。最近,我们在山东和北京市多地的番茄样品中均检测到了该病毒,并通过检测发现国内一些主要品种番茄种子的STV携带率达40%。STV基因组为一条双链RNA,隶属Amalgaviridae科Amalgavirus属,是严格种传病毒,不能通过汁液摩擦接种和嫁接传播。鉴于STV的潜在危害以及与其他病毒高度复合侵染造成的严重损失,本文介绍了STV检测方法,并提出应开展对主栽品种的种子检测和针对性防控。

Transmission characteristics of Tomato chlorosis virus (ToCV) by Bemisia tabaci MED and its effects on host preference of vector whitefly

The epidemiology of Tomato chlorosis virus(ToCV)in China is closely associated with its vector whitefly,Bemisia tabaci(Gennadius)MED.However,the transmission characteristics of ToCV by B.tabaci MED remain poorly understood.In this study,we analyzed:1)the horizontal and vertical transmission of ToCV by B.tabaci MED whiteflies;2)the acquisition of ToCV by male and female B.tabaci MED whiteflies after different feeding durations;3)the transmission efficacy of viruliferous male and female B.tabaci MED whiteflies after different inoculation access periods(IAPs);4)the retention of ToCV by viruliferous male and female B.tabaci MED whiteflies after a 48 h acquisition access period(AAP);and 5)the effects of ToCV on host choice of healthy or ToCV-infected tomato plant of viruliferous and non-viruliferous B.tabaci MED at different time points.Our results showed that:1)viruliferous males could not transfer ToCV to non-viruliferous females,and vice versa,viruliferous females could not pass on ToCV to non-viruliferous males.ToCV could not be detected in the F1 generation adults;2)ToCV could be detected within 4.0%of females or males after a 20 min AAP;3)ToCV could be detected in 33.3%of tomato plants inoculated by 10 viruliferous males or females with IAPs of 20 or 30 min;4)the maximum retention time in females was 7 and 5 days in males;and 5)non-viruliferous B.tabaci MED did not show a preference for ToCV-infected tomato plants or healthy tomato plants.However,viruliferous B.tabaci MED whiteflies did prefer to settle on healthy tomato plants over ToCV-infected tomato plants.These findings will be helpful to better understand the epidemiology of the recently emerged plant virus,ToCV,in tomato fields in China.

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.

Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection

Tomato brown rugose fruit virus(ToBRFV) is a novel tobamovirus firstly reported in 2015 and poses a severe threat to the tomato industry. So far, it has spread to 10 countries in America, Asia, and Europe. In 2019, ToBRFV was identified in Shandong Province(ToBRFV-SD), China. In this study, it was shown that ToBRFV-SD induced mild to severe mosaic and blistering on leaves, necrosis on sepals and pedicles, and deformation, yellow spots, and brown rugose necrotic lesions on fruits. ToBRFV-SD induced distinct symptoms on plants of tomato, Capsicum annumm, and Nicotiana benthamiana, and caused latent infection on plants of Solanum tuberosum, Solanum melongena, and N. tabacum cv. Zhongyan 102. All the 50 tomato cultivars tested were highly sensitive to ToBRFV-SD. The complete genomic sequence of ToBRFV-SD shared the highest nucleotide and amino acid identities with isolate IL from Israel. In the phylogenetic tree constructed with the complete genomic sequence, all the ToBRFV isolates were clustered together and formed a sister branch with tobacco mosaic virus(TMV). Furthermore, a quadruplex RT-PCR system was developed that could differentiate ToBRFV from other economically important viruses affecting tomatoes, such as TMV, tomato mosaic virus, and tomato spotted wilt virus. The findings of this study enhance our understanding of the biological and molecular characteristics of ToBRFV and provide an efficient and effective detection method for multiple infections, which is helpful in the management of ToBRFV.

Detection and characterization of an isolate of Tomato mottle mosaic virus infecting tomato in China

Tomato（Solanum lycopersicum） plants exhibiting severe leaf distortion, mottle and systemic crinkling symptoms were identified in Hainan province in China in 2016. To survey and control the disease, it is necessary to identify and characterize the pathogen causing the disease. Dot enzyme-linked immunosorbent assay showed that the crude saps of the infected tomato samples reacted positively with the monoclonal antibody against Tobacco mosaic virus which indicated that one or more tobamoviruses are likely associated with the disease. RT-p CR and DNA sequence analysis results further elucidated that Tomato mottle mosaic virus（To MMV） in Tobamovirus was the pathogen causing the mottle disease in tomato. We amplified and sequenced the full-length sequence of the genome which showed the highest nucleotide identity with To MMV YYMLJ and To MMV Ti Lha LJ isolates. The putative virus isolate was named To MMV Hainan. Biological indexing studies showed that To MMV Hainan can infect Nicotiana benthamiana, Capsicum annuum and Solanum lycopersicum showing serious symptoms. This was the first identification and characterization of To MMV infecting tomato in Hainan of China.

Molecular Markers for Tm-2 Alleles of Tomato Mosaic Virus Resistance in Tomato

Tomato mosaic virus (ToMV) is one of the most infectious virus diseases in tomato (Solanum lycopersicum L). The practical and effective method of controlling this disease is through genetic control by using major resistance genes. So far, three genes Tm-1, Tm-2 and Tm-22 conferring resistance to ToMV have been reported and utilized in tomato culti-var development. Marker assisted selection (MAS) has become very important and useful tool in selection of ToMV re-sistant tomato lines or hybrids. The objective of this research was to identify allele-specific PCR-based, cleaved ampli-fied polymorphic sequence (CAPS), and allele-derived single nucleotide polymorphism (SNP) markers for Tm-2 loci. Four allele-specific PCR-based markers were identified: one for Tm-2, one for Tm-22, and two for the susceptible allele tm-2. Three allele-derived CAPS markers were identified, which can identify and distinguish three alleles, tm-2, Tm-2 and Tm-22 in tomato germplasm. Three SNP markers were developed specific for Tm-2 locus. These markers will pro-vide breeders with a tool in selection of Tm-2 and Tm-22 resistance genes in tomato breeding program.

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.

Population structure and association mapping to detect QTL controlling tomato spotted wilt virus resistance in cultivated peanuts

Tomato spotted wilt(TSW)is a serious virus disease of peanut in the United States.Breeding for TSWV resistance would be facilitated by the implementation of marker-assisted selection in breeding programs;however,genes associated with resistance have not been identified.Association mapping is a type of genetic mapping that can exploit relationships between markers and traits in many lineages.The objectives of this study were to examine genetic diversity and population structure in the U.S.peanut mini-core collection using simple sequence repeat(SSR)markers,and to conduct association mapping between SSR markers and TSWV resistance in cultivated peanuts.One hundred and thirty-three SSR markers were used for genotyping 104 accessions.Four subpopulations,generally corresponding to botanical varieties,were classified by population structure analysis.Association mapping analysis indicated that five markers:pP GPseq5D5,GM1135,GM1991,TC23C08,and TC24C06,were consistently associated with TSW resistance by the Q,PCA,Q+K,and PCA+K models.These markers together explained 36.4%of the phenotypic variance.Moreover,pP GPseq5D5 and GM1991 were associated with both visual symptoms of TSWV and ELISA values with a high R^2.The potential of these markers for use in a marker-assisted selection program to breed peanut for resistance to TSWV is discussed.

P19 of Tomato Bushy Stunt Virus Suppresses RNA Silencing Induced by Short Hairpin RNA in Mammal Cells

To counteract the immune system in parasitic hosts,some viruses encode proteins to suppress the RNA interference(RNAi)effect.In this report,we established two RNAi systems to be easily observed with strong and obvious effect.The function of the P19 of tomato bushy stunt virus,which suppresses RNAi in mammal cells,was then studied using these two systems.Short hairpin RNAs targeting green fluorescence protein(pshRNA-GFP)and firefly luciferase(pshRNA-luc)were designed and inserted into a eukaryotic transcriptional vector pTZU6+1,respectively.The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19.The GFP expression level was assayed by fluorescence microscopy,Western blotting and RT-PCR.The luciferase expression level was analyzed by the dual-luciferase assay system.pshRNA designed in this study down-regulated the target gene specifically and efficiently,with a decrease of expression of both genes of about 70%,respectively.When P19 was introduced into the RNAi systems,the expression of both GFP and the luciferase were mostly recovered compared with the control groups.The RNAi systems of GFP and luciferase were constructed successfully,demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.

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.

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.

Influence of Symbiotic Interaction between Fungus, Virus, and Tomato Plant in Combating Drought Stress

The influence of the three-way interaction between the fungus (Curvularia protuberata), virus (CThTV), and tomato (Solanum lycopersicum) in combating drought stress was evaluated in this study. The plants in this greenhouse experiment were grown under conditions of 400 ± 150 μmol·m-2·s-1 photon flux density, 45% to 50% relative humidity (RH), and 30°C ± 2°C. Tomato seeds were germinated and inoculated with the combination of the fungus and virus at the seedling stage. The plants were allowed to grow for two weeks and randomly selected individuals were utilized. The selected plants were grown in one gallon pots containing organic potting soil. The treatments included non-symbiotic (NS), virus-free (VF), and symbiotic (An) plants. Each treatment received twelve samples and each sample was allowed to grow for an additional two weeks under drought stress. At that time, plants were exhibiting drought stress symptoms including visible wilting. Six samples from each treatment were utilized in determining selected physiological responses of tomato at pre-anthesis stage. The remaining six samples from each treatment were re-watered once and allowed to grow until they reached the anthesis stage. When they showed visible signs of wilting, the same physiological responses measured during pre-anthesis were conducted. The samples of each treatment were utilized at the end of each stage in determining photosynthetic rate, stomata conductance, photosynthetic pigments, water potential, and soluble sugar content. Plant growth, chlorophyll a, chlorophyll b, photosynthetic rate, stomata conductance, water potential, and soluble sugar content were similarly affected by the various treatments. However, carotenoids were significantly higher at pre-anthesis in the symbiotic plants in comparison to other treatments. Additionally, photosynthesis appeared to be significantly higher at anthesis compared to pre-anthesis for all treatments.

Tomato mottle mosaic virus: Characterization, resistance gene effectiveness, and quintuplex RT-PCR detection system

Tomato mottle mosaic virus(ToMMV), an economically important species of the genus Tobamovirus, causes significant loss in yield and quality of tomato fruits. Here, we identified the Shandong isolate of ToMMV(ToMMV-SD) collected from symptomatic tomato fruits in Weifang, Shandong Province of China. ToMMV-SD caused symptoms such as severe mosaic, mottling, and necrosis of tomato leaves, yellow spot and necrotic lesions on tomato fruits. The obtained full genome of ToMMV-SD was 6 399 nucleotides(accession number MW373515) and had the highest identity of 99.5% with that of isolate SC13-051 from the United States of America at the genomic level. The infectious clone of ToMMV-SD was constructed and induced clear mosaic and necrotic symptoms onto Nicotiana benthamiana leaves. Several commercial tomato cultivars, harboring Tm-2~2 resistance gene, and pepper cultivars, containing L resistance gene, were susceptible to ToMMV-SD. Plants of Solanum melongena(eggplant) and Brassica pekinensis(napa cabbage) showed mottling symptoms, while N. tabacum cv. Zhongyan 100 displayed latent infection. ToMMV-SD did not infect plants of N. tabacum cv. Xanthi NN, Brassica rapa ssp. chinensis(bok choy), Raphanus sativus(radish), Vigna unguiculata cv. Yuanzhong 28-2(cowpea), or Tm-2~2 transgenic N. benthamiana. A quintuplex RT-PCR system differentiated ToMMV from tomato mosaic virus, tomato brown rugose fruit virus, tobacco mosaic virus, and tomato spotted wilt virus, with the threshold amount of 0.02 pg. These results highlight the threat posed by ToMMV to tomato and pepper cultivation and offer an efficient detection system for the simultaneous detection of four tobamoviruses and tomato spotted wilt virus infecting tomato plants in the field.

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.

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）

果型和抗性基因协同对番茄黄化曲叶病毒带毒量的影响

以12份番茄种质为试材,其具有2类果实大小和3种不同抗性基因,通过检测番茄黄化曲叶病毒病田间抗性和带毒量指标,探索果实大小、抗病基因与田间抗性、带毒量之间的关系。结果显示,在自然传毒情况下,所有番茄材料都能检测到病毒感染。在番茄定植后28 d,小果型番茄M8(I)、M5(I)、M3(I)、M7(I)、M9(I)和M11(HR)带毒量均较低,大果型番茄M6(S)、M10(S)、M4(S)和M2(S)带毒量均较高,且小果型番茄田间发病率明显低于大果型番茄。除了Ty-1/ty-1单基因抗性不受番茄果实大小影响外,携带其他单基因或聚合基因抗性的番茄种质均与果实大小有关。在此节点上,带毒量越高抗性越弱,带毒量越低抗性越强。总体来说,在遭受番茄黄化曲叶病毒侵染时,小果型番茄田间抗性强于大果型番茄。