Function And Mechanism Analysis Of Three Nicotiana Benthamiana Transcription Factors In Phytophthora Resistance

The genus Phytophthora contains many devastating plant pathogens,among which Phytophthora parasitica has a very wide host range and may damage many crops.Nicotiana benthamiana is an important model plant for studying the interaction between plants and Phytophthora pathogens because it is highly sensitive to Phytophthora,and is easy for virus-induced gene silencing and protein transient expression.Previous research shows that a large number of transcription factors are induced during P.parasitica infected N.benthamiana.However,there are still many questions to be addressed in the function and mechanism of these transcription factors during interaction.For example,which transcription factor plays important roles in the interaction process?What kinds of signals and metabolic pathways are involved in regulation and how they perform the roles?In this study,we used the interaction system between N.benthamiana and P.parasitica to screen and characterize a large number of transcription factors against Phytophthora.Three transcription factors were selected for further research.The main research contents are as follows:Bioinformatics Analysis during Interaction between Nicotiana benthamiana and Phytophthora.By analyzing RNA-Seq data of N.benthamiana infected with P.parasitica leaves,we identified 1,016 transcription factors that were differentially expressed during infection.We used fluorescence quantitative and semi-quantitative techniques to verify the expression levels of more than 300 transcription factors that were differentially expressed,and 32 transcription factors with significantly differential expression were further silenced by the VIGS.P.parasitica and Botrytis cinerea were inoculated to leaves with over 50%silencing efficiency.The disease resistance analysis was performed and 10 disease-resistance-related transcription factors were finally obtained.Bioinformatical analysis found that the bHLH(basic helix-loop-helix)family was differently significantly expressed during P.parasitica infecting N.benthamian.With the largest number,it was assumed that the transcription factor family must play important roles in the infection process of P.parasitica.Here,we identified N.benthamiana bHLH genes(NbbHLHs)using a whole-genome searching approach,and found that the NbbHLHs were highly enriched and some subfamilies were selectively expanded in N.benthamiana,resulting in high members in some subfamilies.Gene replication may be the main reason for the expansion of the bHLH family of N.benthamiana.In addition,we analyzed their expression profiles using P.parasitica infected N.benthamiana.Twenty-eight NbbHLHs were found to be significantly upregulated during P.parasitica infection,suggesting they may play an important role in phytophthora resistance.And,then cis-acting element analysis and protein-protein interaction networks prediction were performed for them.Functional characterizations of NbERF1 in the resistance of Nicotiana benthamiana.Analyzing RNA-Seq data for N.benthamiana infected with P.parasitica,we found that NbERF1 is one of the most strongly up-regulated genes.It contained a conserved 53-residue AP2/ERF DNA binding domain.Overexpression of NbERF1 enhanced resistance to P.parasitica while its silencing significantly reduced the resistance,suggesting that it may positively regulate host disease resistance.RNA-Seq analysis of NbERF1-silenced leaves upon P.parasitica infection showed that the upregulation of receptor-like protein kinase(RLK)family was strongly suppressed.Since previously reported that ERF1 can bind to the GCC-box in the promoter region of the target genes,we searched this element in the promoters of RLK genes and found that nine of them contained the element.These 9 RLK genes were selected for qRT-PCR validation,suggesting that RLK genes reduced sensitivity to P.parasitica when NbERF1 was silenced.In addition,resistance to P.parasitica was enhanced after the RLK genes were overexpressed in N.benthamiana.Therefore,NbERF1 enhances the resistance of N.benthamiana to P.parasitica by binding to the GCC-box in a RLK promoter and regulating their expressions.Functional characterizations of NbERF173 in the resistance of Nicotiana benthamiana.Based on the above results,we selected NbERF1 73,a transcription factor with significantly up-regulated during P.parasitica infection,and further studied its resistance to disease.The transcription factor was found to contain a conserved 52-residue AP2/ERF DNA binding domain.Overexpression of NbERF1 73 in N.benthamiana enhanced resistance to P.parasitica,while its silencing significantly reduced the resistance to both P.parasitica and Botrytis cinerea,indicating that it positively regulates host disease resistance.RNA-Seq analysis of NbERF173-silenced leaves upon P.parasitica infection showed that several defense-related genes were differentially regulated,especially two proteinase inhibitors that were strongly suppressed.Transient overexpression of these two protease inhibitor genes in N.benthamiana wild type enhanced resistance to P.parasitica,and overexpression of them in NbERF173-silenced N.benthamiana leaves,partially restored the resistance of N.benthamiana to P.parasitica.Functional characterizations of NbMYB57 in the resistance of Nicotiana benthamiana.By analyzing the RNA-Seq data of N.benthamiana infected with P.parasitica,NbMYB57 was found strongly induced to up-regulate expression and this gene contained two conserved MYB DNA binding domains.Silencing this transcription factor in N.benthamiana significantly reduced the resistance of N.benthamiana to P.parasitica and B.cinerea,indicating that NbMYB57 positively regulates the resistance of N.benthamiana.In combination with RNA-Seq analysis,ChIP-Seq analysis and yeast one-hybrid library screening,downstream target genes regulated byNbMYB57 were identified.It was found that JAZ3 genes in the jasmonic acid signaling pathway,WRKY and bHLH transcription factors may be target genes regulated by NbMYB57.