Study On Molecular Mechanism Of SmNAC Transcription Factor Regulated To Resistance Of Bacterial Wilt In Eggplant

Eggplant(Solanum melongena L.)is an annual crop of Solanaceae and is one of the most important vegetables in China.However,eggplant is susceptible to bacterial wilt,especially in southern China.Bacterial wilt is a soil-borne disease caused by Ralstonia solanacearum.In severe cases,the loss can reach 50 % or more.It has become an important factor limiting the production of eggplant.Previous studies have shown that NAC transcription factors play an important role in the growth and development of plants and in actively coping with environmental stresses such as high temperature,cold,salinity and disease.In the previous study,our group screened a NAC transcription factor with significant expression in susceptible(E-32)eggplants and resistance(E-31)eggplants,SmNAC(Gen Bank: KM435267).R.solanacearum could induce SmNAC expression.Functional identification studies showed that SmNAC negatively regulates the resistance of bacterial wilt.But how SmNAC regulates the resistance of bacterial wilt,the molecular regulation mechanism is still unclear.In this study,we deeply analyzed the mechanism of SmNAC transcription factors on resistance to bacterial wilt.The main results are as follows:1.The transcriptome sequencing analysis of the susceptible(E-32)eggplants and resistance(E-31)eggplants before and after inoculation with R.solanacearum by RNA-Seq technique was carried out,and 125,852 contigs were obtained by de novo assembly using multiple programs and steps.A total of 122,508 transcripts and 68,792 unigenes were obtained,with 51,165 non-redundant unigenes annotated.The gene expression changes after R.solanacearum inoculation were identified by RNA-Seq technology,the expression levels of different types of genes were analyzed.The analysis showed that there were 217 genes up-regulated and 738 genes down-regulated through the resistance materials compared with the susceptible materials before inoculation.There were 5,832 genes were up-regulated and 6087 genes were down-regulated before inoculation compared with after inoculation in susceptible materials.There were 9,048 genes were up-regulated and 1,137 genes were down-regulated before inoculation compared with after inoculation in resistance materials.After inoculation,12,523 genes were up-regulated and 4,712 genes were down-regulated through the resistance materials compared with susceptible materials.The function of the gene was annotated by GO classification and KEGG metabolic pathway.2.Previous studies have shown that SmNAC negatively regulate the resistance to bacterial wilt of eggplant.The SmNAC-RNAi transgenic plants were further silenced by RNAi technology,and the transgenic plants were inoculated with R.solanacearum.It suggests that non-transgenic plants‘ leaves showed no wilting symptoms compared with transgenic plants.However,the leaves of the transgenic plants showed symptoms of wilting.The experimental results show that silencing SmNAC can enhance the resistance to bacterial wilt of eggplants.3.The expression levels of related signal genes in JA and SA signaling pathways in SmNAC-overexpressing plants and SmNAC-RNAi transgenic plants were determined,and the expression levels of JA and its signaling pathway genes(JAR1,Pin2,Lox A)were found increased in SmNAC-overexpression plants,but decreased in SmNAC-RNAi plants;conversely,the expression levels of SA and its signaling pathways(EDS1,Glu A,NPR1,TGA,SGT1,PAD4,PR-1a)is decreased in SmNAC-overexpression plants,while increased in SmNAC-RNAi plants.At the same time,SmNAC also affected the expression of ICS1,a gene related to SA synthesis.In SmNAC-overexpressing plants,the expression level of ICS1 decreased,while in SmNAC-RNAi plants,the expression level of ICS1 increased.However,the expression of the other three SA metabolism-related genes(PBS3,SAGT1,BSMT1)had little effect.Application of 0.2 m M exogenous SA can increase the resistance to bacterial wilt of eggplants.4.The ICS1 promoter was cloned from eggplant and analyzed by software analysis.The promoter of ICS1 has six NAC binding sites and is detected by promoter activity,result indicates that it has priming activity.Yeast one-hybrid results confirmed that SmNAC can bind to the ICS1 promoter through the two binding sites(-1 to-370 and-550 to-750)of ICS1 promoter.The detection of dual luciferase transcriptional activity revealed that SmNAC reduced the expression of ICS1 by inhibiting the activity of the ICS1 promoter,thereby affecting the synthesis of SA.We infer that SmNAC reduces the accumulation of SA by inhibiting the expression of ICS1,thereby reducing the resistance of plants to bacterial wilt through above results.5.Primers was designed according to the NCBI database,the five genes Hrp B,Prh J(hrp G),Pop P1,Prh A and Pop P2 were successfully cloned from R.solanacearum‘s DNA as template,and the yeast two-hybrid vectors were constructed.Experiments show that there are no interaction between the five genes and the SmNAC.