SlWRKY3 Negatively Regulates The Resistance To Botrytis Cinerea Via TPK1b In Tomato

Tomato Solanum lycopersicum L.is the most widely grown vegetable crop in the world and is widely demanded by consumers,but as a plant that grows fixedly,it is susceptible to disease.Among them,Botrytis cinerea is a necrotic vegetative disease and is ranked the second largest fungal pathogen in the world.It can infect a wide range of open field and facility-grown tomatoes,causing serious economic losses to the tomato industry.Therefore,exploring the genes of tomato in response to Botrytis cinerea and analyzing the molecular mechanism of tomato resistance to Botrytis cinerea is helpful to accelerate the process of disease resistance breeding in molecular level.Tomato TPK1 b gene encodes a RLCK protein kinase located on the plasma membrane,which has been reported to positively regulate tomato resistance to Botrytis cinerea.In this study,a part of transcription factors were screened through the yeast one-hybrid assay using the TPK1 b promoter as bait,and then these transcription factors were genetically transformed in tomatoes,and the transgenic plants were inoculated with Botrytis cinerea.A WRKY protein Sl WRKY3 that can negatively regulate tomato resistance to Botrytis cinerea was identified.We conducted a preliminary analysis of the resistance regulation mechanism of Sl WRKY3.The research results showed that Sl WRKY3 can negatively regulate the expression of TPK1 b gene,which in turn affects SA,ROS and other components to finally achieve the regulation of resistance to tomato Botrytis cinerea.The main research results of this paper are as follows:1.According to the bioinformational analysis,there are many cis-acting elements in the part of the 3000 bp upstream of the TPK1 b gene,including response elements of light,hormones and other life activities.This part of the TPK1 b promoter was subjected to screen the library by yeast one-hybrid,and several transcription factors that could bind to it were identified.We selected some transcription factors for the genetic transformation by overexpression and RNAi technology in tomatoes,and then the resistence was checked by inoculation Botrytis cinerea on transgenic plants,and found that only the resistance of Sl WRKY3 transgenic plants to Botrytis cinerea changed,and the overexpression plants of Sl WRKY3 show susceptible to Botrytis cinerea,on the contrary,the RNAi plants are more resistant to Botrytis cinerea.2.A comparative analysis of the amino acid sequence of Sl WRKY3 reveals that it is an evolutionarily conserved WRKY family protein,contains two WRKYGQK conserved domains and two C2H2 type zinc finger motifs,it’s a typical type I WRKY protein.And through sequence alignment and evolutionary tree analysis,Sl WRKY3 has high sequence similarity and homology with other type I WRKY proteins.The website predicts that it is located in the nucleus and contains a section signal of nuclear localization.Tissue expression analysis found that Sl WRKY3 had the highest expression in flowers,followed by leaves and red ripe fruits,with the lowest expression in roots and stems.3.There are several W-box elements on the TPK1 b promoter.Through further yeast one-hybrid assays,it was found that Sl WRKY3 can be combined with two of these W-boxes.In order to investigate the regulation of the expression of TPK1 b by Sl WRKY3,we examined the relationship between the expression levels of Sl WRKY3 and TPK1 b in the transgenic lines of Sl WRKY3.It was found that the expression level of TPK1 b in Sl WRKY3 overexpression lines was significantly lower than that of the control A57,and the expression of TPK1 b in the Sl WRKY3 RNAi plants was higher,indicating that Sl WRKY3 can negatively regulate the expression of TPK1 b in tomato.The negative correlation between Sl WRKY3 and TPK1 b can also be seen from the expression change pattern of Sl WRKY3 and TPK1 b after inoculation with Botrytis cinerea.4.After A57 plants were inoculated with Botrytis cinerea,the expression of Sl WRKY3 could be quickly activated in a short time.We inoculated Sl WRKY3 transgenic plants with Botrytis cinerea,and tested the expression of signal pathway marker genes at different time points after inoculation.Among the detected genes,it was found that the expression levels of the SA pathway marker gene PR1 and the ROS pathway related genes CAT1 and CAT2 have opposite expression trends in Sl WRKY3 overexpression and RNAi plants.In order to further explore the relationship between Sl WRKY3 and these signaling pathways,we used DAB staining to detect the generation of ROS after Sl WRKY3 transgenic plants were inoculated with Botrytis cinerea,and found that the production of ROS in the overexpression lines of Sl WRKY3 was significantly higher than that of the RNAi lines.Totally,the experiments show that Sl WRKY3 can regulate the expression of TPK1 b and futher affect SA and ROS signals,thereby negatively regulating tomato resistance to Botrytis cinerea.