Transcriptomic Analysis Of Brassinosteroids-induced Resistance Of Cucumber(Cucumis Sativus L.)Seedlings To Phytophthora Melonis

Cucumber is one of the most important vegetable crops in the world.The cucumber blight which mainly caused by the infection of Phytophthora melonis is a devastating soil-borne disease that damages the production of cucumber in South China.At present,there are no fully immunized or highly resistant varieties in production,and there are also lack of effective agricultural measures and fungicides.Brassinosteroids(BRs)are novel hormones that have important effects on the growth and development of plants,and can increase plant resistance to various pathogens.Previous studies have shown that pretreatment with 1 ?M epibrassinolide(EBR)can induce disease resistance in cucumber seedlings.In this study,the RNA-seq technique was used to further analyze and compare the transcriptome data of EBR pretreatment and single inoculation with Phytophthora melonis for screening potential disease-resistance genes,and discussing the possibly molecular mechanism of BRs-induced resistance to cucumber plant diseases.The main conclusions are as follows:1 The cucumber susceptible variety ‘Zhongnong 20' was used as the material.The experiment was conducted with sterile water blank control(CK),single EBR pretreatment(BR),single inoculation with P.melonis(P),and inoculation of P.melonis after pretreatment with EBR(BR-P).Treatment was performed at the time of two leaves and the condition index was investigated 7 days after inoculation.The results showed that EBR pretreatment significantly reduced the incidence of cucumber plants.The disease index of BR-P treatment was 29.13%,and P treatment plant disease index was 55.77%.2 After 24 hours of inoculation with P.melonis,the stems of four treatment were subjected to RNA-seq sequencing.After comparing the sequencing results with the cucumber genome and annotating the gene function,the gene expression changes between the treatments were analyzed.The results are as follows:(1)The differential expression analysis evidenced that 196 genes were significantly differently expressed in BR-P treatment compared with P treatment,of which 85 genes were up-regulated and 111 genes were down-regulated.535 genes were significantly different in BR treatment and CK treatment,among which 152 genes were up-regulated,383 genes were down-regulated.2238 genes were significantly differently expressed in P treatment compared with CK treatment,of which 1322 were up-regulated and 1060 were down-regulated.There are 33 differentially expressed genes in the three comparison groups.(2)Through GO functional enrichment analysis,it was found that 196 differentially expressed genes in the comparison group of BR-P treatment and P treatment were enriched to 32 GO terms.535 differentially expressed genes of BR treatment compared to CK treatment were enriched in 43 GO terms.2382 differentially expressed genes were enriched to 46 GO terms in comparison group of P treatment and CK treatment.These GO terms are mainly involved to biological processes and catalytic activities.By KEGG enrichment analysis,it was found that the differentially expressed genes in the comparison group of BRP treatment and P treatment were significantly enriched in plant hormone signal transduction Pathway.The enriched Pathway of BR treatment compared to CK treatment was plant hormone signal transduction.The differentially expressed genes in comparison group of P treatment and CK treatment were: phenylpropanoid biosynthesis,phenylalanine metabolism,plant hormone signal transduction,photosynthesis-antenna proteins,plant-pathogen interactions.(3)RNA-seq analysis showed that EBR pretreatment and inoculation with P.melonis significantly affected the expression of resistance-related transcription factor genes.There were 2 TGA transcription factors,38 ERF transcription factors,21 WRKY transcription factors,24 MYB transcription factors,20 NAC transcription factors,and 9 HSF transcription factors in the comparison groups of P and CK.There were 16 ERF transcription factors,9 WRKY transcription factors,8 MYB transcription factors,3 NAC transcription factors and 4 HSF transcription factors in the BR compared with CK.There were 1 TGA transcription factor,10 ERF Transcription factors,6 WRKY transcription factors,2 MYB transcription factors,and 7 NAC transcription factors in BR-P treatment and P treatment comparison group,of which ERF transcription factor and NAC transcription factor were down-regulated.It can be seen that transcription factors play an important role in regulating EBR-induced cucumber disease.(4)Phytohormonal signal transduction may be involved in the response of cucumber plants to Phytophthora melonis.The genes BAK1,BKI1 and TCH4 related to BRs signal transduction in the comparison groups of P treatment and CK treatment all showed upregulated expression.EBR pretreatment down-regulated the expression of BKI1 gene.Significant enrichment of ethylene signaling pathways was observed in each of the comparison groups.Ethylene receptor(ETR),EIN3-binding F-box protein(EBF1/2)and ethylene-responsive transcription factor(ERF1/2)that associated with ET signaling transduction were both up-regulated in the P treatment and CK treatment comparison groups.EBR pretreatment reduced the expression of EBF1/2 and ERF1/2 in different degrees.It was suggested that EBR may be antagonistic to ethylene to regulate the resistance of cucumber.(5)18 ERFs genes of total DEGs were selected for validation through quantitative Real-Time PCR(qRT-PCR)and the results were consitant with RNA-seq,indicating that the results of RNA-seq were reliable.In conclusion,brassinosteroids induces a variety of different disease resistance pathways to participate in the resistance of cucumber to P.melonis,resulting in a complex regulatory network.Among them,transcription factors and ethylene signaling transduction play a major role in regulation.Brassinosteroids may be associated with ethylene antagonistic regulation of plant disease resistance.