The Expression And Function Analysis Of Tomato Response Factors After Tomato Yellow Leaf Curl Virus Infection

Originated from South America,tomato(Solanum lycopersicum L.)is one of the most important vegetables worldwide.Tomato fruits are rich in vitamins,lycopene,and essential amino acids,which playing an important role in balancing people's diets.However,tomato is susceptible to a series of pathogen infection(bacteria,fungi,and virus).Transmitted by the whitefly Bemisia tabaci,Tomato yellow leaf curl virus(TYLCV)brings devastating damages to tomato's development.Up to now,the different expression genes and metabolites patterns after TYLCV infection in tomato have been identified,but the function mechanism of TYLCV infection in tomato is not intact.In the text,transcription factors(TF)involved in TYLCV infection were identified according to the transcriptome database and the expression profiles of TFs after TYLCV infection in tomato were determined.The genes in response to TYLCV infection were transformed into Arabidopsis,,tomato,and tobacco plants.A comparative proteomic study of TYLCV infection in resistant and susceptible tomato cultivars was conducted to analyze the differently expressed proteins.The smHDP gene encoding single-stranded DNA-binding proteins(SSB)from M13 phage was overexpressed in tomato;the symptom and TYLCV DNA content between the transgenic and control tomato plants after TYLCV infection were identified.The major findings are as follows:1.Twenty-two AP2/ERF TFs in response to TYLCV infection were identified according to transcriptome database.Expression profiles of five ERF-B3 genes(Solyl9,Soly36,Soly66,Soly67,and Soly106)were detected by quantitative real-time polymerse chain reaction(RT-qPCR)after TYLCV infection in five tomato cultivars(highly resistant cultivar:'Hongbeibei',resistant cultivars:'Zheza-301' and 'Zhefen-702',susceptible cultivars:'Jinpeng-l' and 'Xianke-6').Interaction network indicated that tomato ERF TFs could interact with mitogen-activated protein kinase(MAPK).Yeast one-hybrid showed that the GCC-box binding ability of ERF-B3 TFs differed in resistant and susceptible tomato cultivars.2.Six WRKY? TFs(SolyWRKY41,SolyWRKY42,SolyWRKY53,SolyWRKY54,SolyWRKY80,and SolyWRKY81)were identified and these TFs responded to TYLCV infection.Subcellular localization analysis indicated that SolyWRKY41 and SolyWRKY54 were nuclear proteins.Many elements,including W-box,were found in the promoter region of WRKYIII TFs.Positive and negative expression patterns showed that WRKYIII genes could respond to TYLCV infection in tomato.The TYLCV DNA contents after SolyWRKY41 and SolyWRKY54-silencing resistant lines were lower than that in control tomato lines,indicating the negative roles played by SolyWRKY41 and SolyWRKY54 in the progress of tomato-TYLCV infection.3.Six NAC TFs were identified to respond to TYLCV infection in tomato.The transcripts of four NAC genes(SINAC20,SlNAC24,SlNAC47,and SlNAC61)were induced after TYLCV infection in resistant tomato cultivar.Virus-induced gene silencing analysis(VIGS)indicated that SINAC61 played positive roles in response to TYLCV infection.Tomato NAC TFs were not only involved in defense regulation but in development and stress progress.Some defense response TFs,such as WRKY,TGA,MYB,NAC,could interact with NAC proteins by binding cis-elements in promoter regions of NAC TFs.4.A TYLCV infection-related NAC gene SINAC20 was cloned from tomato.Expression levels of SINAC20 were increased after drought and salt treatments for 24 h.SINAC20 also responded to SA and MeJA treatments.The expression level of SINAC20 was increased about 60 times after SA treatment for 1 h.The sequences of SlNAC20 were split into three parts according to the subdomains to inhibit the self-activation.No interaction was identified between TYLCV Ren protein and three parts of SINAC20.The fresh water loss and activities of SOD and POD in SlNAC20-overexpression Arabidopsis plants were increased than in wild type plants.The SINAC20 gene were also transformed into tobacco and tomato plants and obtained transgenic plants with GUS blue stain for subsequent functional verification.5.A proteomic approach was used to investigate the molecular mechanisms involved in tomato leaf defense against TYLCV infection.Proteins extracted from leaves of resistant tomato cultivar 'Zheza-301' and susceptible tomato cultivar 'Jinpeng-1' after TYLCV infection were analyzed by two-dimensional polyacrylamide gel electrophoresis(2-DE).Eighty-six differentially expressed proteins were identified and classified into seven groups based on their functions.For several of the proteins,including PPO,CAB,and HSC70,expression patterns measured using RT-qPCR differed from the results of the proteomic analysis.A putative interaction network between tomato leaves and TYLCV infection provides us with important information about the cellular activities that are involved in the response to TYLCV infection.6.To identify the roles of single-stranded DNA-binding protein involved in TYLCV infection,we overexpressed the synthesized smHDP based on the HDP sequence of M13 phage in tomato.The symptoms of TYLCV infection in transgenic tomato lines were alleviated compared with those in the control lines,and the DNA content of TYLCV in the transgenic tomato lines was lower than that in the control lines.Some stress-related genes,including pathogenesis-related genes,HSC70,protease,APX,and CAT,were induced in the transgenic tomato lines,and their expression levels increased in certain infection stages.The expression patterns of two Ty loci(Ty-1 and Ty-5)also increased in the transgenic tomato lines.