Screening Transcription Factors Induced By Phytophthora And Function Analysis Of Two Nicotiana Benthamiana WRKY In Disease Resistance

Transcription factors can specifically combine with promoter region of gene sequence,to ensure the target gene expression in particular time and space.According to DNA binding domain,the transcription factors are divided into different families,including AP2/ERF family,WRKY family,MYB family,NAC family and so on.The research shows that transcription factors play roles in diverse biological processes including plant growth and defense responses to pathogens.To study the defense-related transcription factors involved in plant disease resistance,gene engineering and acquired new defense genes.In this study,RNA-Seq technology was applied to investigate the dynamic changes of the N.benthamiana transcriptone in response to P.parasitica.Then we analyzed the eighteen transcription factors in N.benthamiana that play roles in defense responses to Phytophthora.by qRT-PCR.Through RNA-seq data and the method of gene homology comparison,we selected two transcription factors:NbWRKY40 and NbWRKY1 which participated in plant disease resistance in Nicotiana benthamiana.Screening transcription factors of N.benthamiana that are induced by Phytophthora:In order to find transcription factors that participated in the N.benthamiana resistance responses to Phytophthora,we analyzed the inoculated(6 h)and non-inoculated N.benthamiana leaves by RNA-Seq approach.Further we identify the differentially expressed transcriptional genes in these two samples.We identified that 235 transcription factors were up-regulated after Phytophthora parasitica inoculation.We selected 71 up-regulated expression of at least 4 times,which homologous genes is induced by pathogens in Arabidopsis thaliana and Nicotiana tabacum.Through qRT-PCR the expression pattern of them was verified in the N.bentharmiana.Finally,18 transcription factors were identified which were up-regulated in the early stage of the infection by the P.parasitica.Functional analysis of disease resistance related transcription factor WRKY40 in Nicotiana benthamiana:According to the reports about AtWRKY40 and NtWRKY40 which involved in disease resistance and many biological functions including abiotic and biotic stress.By using RNA-seq and the gene homology comparison method,we got the NbWRKY40.qRT-PCR revealed that NbWRKY40 was a P.parasitica induced gene.We performed the silencing ofNbWRKY40 via Virus-induced gene silencing approach to examine function of NbWRKY40 in disease resistance in N.benthamiana.Using the hemibioltrophic P.parasitica as the test strain,we found that silencing of NbWRKY40 reduced the disease resistance,with the increased lesion diameter,The H2O2 accumulation and callose deposition were reduced.Then we used the necrotrophic fungus Botrytis drierea as the test strain,and found that silencing of NbWRKY40 also reduced resistance of N.benthamiana to Botrytis cinerea.We choose four genes PRlb,PR2b,LOX,ERF1 to check the relative expression levels of genes involved in defense-associated signaling pathway.Experimental results showed that silencing of NbWRKY40 reduced the expression level of these genes induced by P.parasitica.The results revealed that resistance of NbWRKY40 is involved in SA mediated signaling pathway.Functional analysis of disease resistance related transcription factor WRKY1 in Nicotiana benthamiana:According to the reports and RNA-seq data,we selected another transcription factor named NbWRKY1.We did the silencing of NbWRKY1 via VIGS to its function in disease resistance in N.benthamiana.The results showed that silencing of NbWRKY1 reduced the resistance of P.parasitica and B.cinerea,then microscopic observation of tissues infected with P.parasitica showed that the lesion diameter was increased with the H2O2 accumulation,but callose deposition was not changed.We choose four genesPRlb,PR2b,LOX,ERF1 to check the relative expression levels of genes involved in defense-associated signaling pathway.The results showed that stance of NbPWRKY1 is mostly involved in SA and ETH mediated signaling pathway which are connected with PRlby PR2b and ERF1.