Screening And Functional Analysis Of Resistant Response Gene Of Tomato Gray Leaf Spot

In this study,tomato resistant materials(Motelle)and susceptible materials(Moneymaker)were inoculated with S.lycopersici,then the transcriptome sequencing(RNA-seq)was carried on the uninfected and inoculated materials.The stable and significant differential expression genes SlNLC1,SlPKY1,and SlWYN1 were selected.Their expression patterns were analyzed by exogenous hormones methyl jasmonate(Me JA)and salicylic acid(SA).In order to further verify the functions of SlNLC1,SlPKY1,and SlWYN1,we used the virus-induced gene silencing method(VIGS)to silence three genes in the disease-resistant materials,the silenced plants were inoculated the pathogens and performed histopathological observation.The results indicated that the SlNLC1 plays an important role in the regulation of tomato gray leaf spot resistance.In order to analyze the differentially expressed genes,which was involved in the regulation of tomato resistance to gray leaf spot,bioinformatics analysis was used to predict the physicochemical properties and domains of the gene and so on.The main findings are as follows:(1)After RNA-seq and q RT-PCR analysis,we picked up three genes SlNLC1,SlPKY1 and SlWYN1,which were significantly and stably upregulated.(2)In order to analyze the expression patterns of three genes SlNLC1,SlPKY1 and SlWYN1 the resistant materials and susceptible materials under the SA and Me JA stress,we carried on the q RT-PCR analysis.The results showed that the expression levels of SlNLC1,SlPKY1 and SlWYN1 were increased by different multiple compared with untreated ones.These results indicated that the three genes SlNLC1,SlPKY1 and SlWYN1 could be successfully induced by SA and Me JA.(3)The three genes of SlNLC1,SlPKY1 and SlWYN1 were silenced by VIGS technique.The plants with successfully silenced were selected,after inoculation with pathogens,it was found that the silenced-SlNLC1 tomato plants showed susceptible symptoms,and it was speculated that SlNLC1 was resistant to the tomato gray leaf spot.(4)In order to further verify the relationship between SlNLC1 and disease resistance,the histopathological observation were determined on the silenced tomato plants.The results showed that on the third day of inoculation the pathogen,compared with the control,the HR on the silenced SlNLC1 plants was weakly and had a small amount of hyphae,which was observe d by microscopic observation.In addition,the accumulation of active oxygen H2O2 and O2-,lignin and callose were lower than the control.Based on these results,the SlNLC1 plays an important role in the resistance response to tomato gray leaf spot.(5)Some basic physical,chemical properties and structures of SlNLC1 were predicted,and sequence alignment and phylogenetic tree construction were also carried out.The SlNLC1 belongs to the "CC-NBS-LRR" in the NBS-LRR class.