Functional Analysis Of Disease Resistance Related Transcription Factors ERF And WRKY1 In Nicotiana Benthamiana

Transcription factors (TFs) are a kind of proteins that can specifically combine with DNA sequences, to ensure the target DNA expression with appointed temporal and spatial in some moderate strength. The main function of these factors is to activate or suppress gene transcription. According to the conservative functional domains of DBD, transcription factors can be divided into different families. In recent years, the study on plant-specific TF families shows that some families can directly or indirectly involve in plant defense responses, such as, bZIP family, ERF family, MYB family and WRKY family. These transcription factors can interact with cis-element of gene promoter to regulate target gene transcription. They maybe form homologous dipolymer, or heterologous dipolymer, or directly combine with other proteins to stimulate the related regulatory network. At present, to examine the transcriptional network and cascade involved becomes the most important issue. In this study, we preliminary screened transcription factor WRKY1 related disease resistance in Nicotiana benthamiana. The further study showed that WRKY1 can modulate the activity of pathogen-induced promoter GmaPPO12. This study maybe provides valuable resources in plant disease resistant engineering.Selection of transcription factors ERF and WRKY1 related disease resistance in Nicotiana benthamiana: On the basis of previous studies, we chose the known transcription factors related disease resistance in Nicotiana tabacum. By multiple sequences alignment, we sought thirty homologous transcription factors in Nicotiana benthamiana genome, including WRKY family, ERF family and MYC family. We firstly constructed the TRV expression vectors and used virus-induced gene silencing technology to silence the required gene. By detecting the efficiency of silence, we preliminary chose two transcription factors those efficiency of silence was up to 50%.To further study, we observed cell death and examined lesion size of silenced Nicotiana benthamiana which was infected by Phytophthora capsici in different times:Oh, 12h,24h and 36h. Compared with control group, specific value of lesion size was 1.952 and 1.322 in ERF silenced plants, respectively. Specific value of lesion size was 1.556 and 1.080 in WRKY1 silenced plants, respectively. Our results showed that N. benthamiana enhanced susceptibility to Phytophthora, indicating that ERF and WRKY1 gene may be involved in resistance process of N. benthamiana.ERF and WRKY1 can regulate the activity of pathogen-induced promoter GmaPPO12:Preliminary work has shown that GmaPPO12 gene promoter was rapidly up-regulated in early Phytophthora infection and worked as pathogen-induced promoter. Furthermore, it was related with disease resistance of Phytophthora in plant. In order to explore whether the gene ERF or WRKY1 can be involved in regulate the activity of pathogen-induced promoter GmaPPO127 We have fused pathogen-induced promoter GmaPPO12 with reporter gene GUS. By using VIGS technology and transient expression assays in N.benthamiana to check enzyme activity and transcriptional level of GUS, we deduced the ERF and WRKY1 can directly regulate the activity of pathogen-induced promoter GmaPPO12.Based on the above research, we have selected transcription factors ERF and WRKY1 gene in N.benthamiana that is related to plant disease resistance. Furthermore, it is clear that ERF and WRKY1 can directly regulate the activity of pathogen-induced promoter GmaPPO12. Our research will provide new resources for engineering soybean and other crops for Phytophthora resistance.