The Role And Mechanism Of Tomato PUBs In Plant Defense Against Botrytis Cinerea

Crops are exposed to pathogenic microbes such as bacteria,fungi and viruses,which make them highly susceptible to pests and diseases.The warm and humid environment in the protected area leads to more frequent diseases of crops.Botrytis cinerea is a widespread plant fungal pathogen with a necrotrophic nutritional mode.The fungus has caused serious damage to the yield and quality of tomato.At present,the production is highly dependent on chemical fungicides,posing a serious threat to the ecological environment and food safety.CRISPR/Cas9 can be used to achieve precise regulation and germplasm innovation of Botrytis cinerea.Therefore,it is urgent to explore the internal mechanism of plant defense against Botrytis cinerea.Protein ubiquitination is one of the important post-translational modifications in eukaryotes and plays a role in many signal transduction pathways.Recent studies have shown that ubiquitination is widely involved in plant immune regulation.The PUB family is a class of E3 ubiquitin ligases containing a U-box domain,which can participate in plant immune defense as a motif through ubiquitination cascade reaction.In this paper,through the bioinformatics analysis of the tomato PUB gene family,the members of the tomato PUB gene family that negatively regulated the resistance to Botrytis cinerea were screened out,which was of great significance to clarify the internal mechanism of plant defense against Botrytis cinerea.The main research results are as follows:1.The PUB gene family of tomato was analyzed by bioinformatics.Through the comparison with Arabidopsis PUB gene family,61 genes containing U-box domain were found in tomato genome.In addition to the U-box domain,there are ARM,Kinase and other structural domains.The 61 PUB genes of tomato were divided into seven subgroups for their different domains.Among them,the largest subgroup is the second class with ARM repeating structure,a total of 30.The ARM repeat structure regulates signal transduction through protein interaction,so we speculate that tomato PUB class Ⅱ is important in plant immune defense.2.Screening for members of the tomato PUB gene family that negatively regulate tomato Botrytis cinerea resistance.Through the bioinformatics analysis of tomato PUB gene family in the previous chapter,it is speculated that tomato PUB class Ⅱ gene is of great significance in plant immune defense.Based on qRT-PCR analysis of tomato PUB class Ⅱ genes,PUB15/24/25/29/33/41/43/56 were down-regulated in response to Botrytis cinerea infection.The tomato PUB gene members which negatively regulatesBotrytis cinerea resistance were further screened by the method of transient overexpression of tobacco and Botrytis cinerea secreting protein.The selected genes were silenced by VIGS.Experiments showed that after silencing PUB33,PUB41,PUB43 and PUB56,the resistance to Botrytis cinerea was increased.The pub41 mutant was constructed by CRISPR/Cas9,and the resistance of pub41 to Botrytis cinerea was also significantly improved.It shows that these genes are important for the defense of Botrytis cinerea.3.This chapter makes a preliminary study on the mechanism of tomato PUB family genes in tomato Botrytis cinerea defense.It has been reported that PSKR1 and TPK1b are key genes in tomato defense againstBotrytis cinerea.Thepskr1 andtpk1b mutants obtained by CRISPR/Cas9 technology are more sensitive to Botrytis cinerea comparing with control,indicating that PSKR1 and TPK1b positively regulates Botrytis cinerea resistance.PUB33 was located on the plasma membrane,PUB43 and PUB56 were located on the plasma membrane and cytoplasm.And PUB43 and PUB56 have the function of E3 ubiquitin ligase.BIFC experiments have proved that PUB43 interacts with PSKR1 and TPK1b.It is preliminarily believed that PUB43 may degrade PSKR1 and TPKlb proteins through ubiquitination,thereby causing downstream signal transmission.