Studies On The Resistance Response Process Mediated By Cf-19 And The Function Analysis Of The Important Response Genes In Tomato

Tomato?Solanum lycopersicum?belongs to the genus Tomato of the Solanaceae family.It is one of the important planting vegetables.Because of its unique flavor and rich nutrition,there are many types to choose from,which can be eaten fresh or processed.Tomato plants are highly adaptable to the environment,coupled with the promotion and application of greenhouse technology in recent years,the tomato planting area has continued to expand and has a high commercial value.The annual output in 2016 reached 177 million tons and the economic value reached 60 billion US dollars.Because of its strong adaptability to the environment and short growth cycle,it is a strict self-pollinating plant and has a small genome.It has become one of the model plants for genome research and the study of plant genetic evolution.In 2012,the tomato genome was sequenced for tomato.Genomic and gene function studies provide favorable conditions.Tomato leaf mildew is a common disease in tomato cultivation in protected areas.Infection with tomato leaf mildew will seriously affect the quality and yield of tomatoes.The tomato Cf gene product can be specifically identified with the Avr gene product of the tomato leaf mold pathogen,so that the tomato plants carrying the Cf gene have disease resistance,and the tomato varieties carrying the Cf-4,Cf-5 or Cf-9 genes can reflect well Field resistance,but as the physiological race of tomato leaf mildew continues to differentiate,the genes that have been used are constantly being overcome,so the problem of antigen shortage is becoming more and more serious.Tomato varieties that carry the tomato Cf-19 gene have shown good disease resistance in many years of tomato planting practice.Compared with other Cf genes,the Cf-19 gene has a stronger immune effect and a wider range of resistance.In our research,we cloned the gene and clarified the sequence and structure information of the gene,but the mechanism of its disease resistance response is unclear.In order to further clarify the disease resistance response mechanism of this gene,we inoculated the tomato leaf mold mildew physiological race 1.2.3.4 with the disease-resistant varieties?CGN18423?and susceptible varieties?Moneymaker?containing the Cf-19 gene,and collected Tomato leaves at 0,7 and 20 days were tested for salicylic acid?SA?and jasmonic acid?JA?content,and RNA-seq sequencing technology was used to analyze transcriptome data to screen important regulatory pathways and disease resistance Related genes,at the same time,VIGS gene silencing technology was used to perform preliminary functional verification on the selected important disease-related genes to clarify their role in tomato Cf-19gene-mediated disease response.The following conclusions are obtained through experiments:?1?The results of hormone content measurement showed that the SA content of CGN18423and Moneymaker plants reached the maximum value on the 7th day,and reached the minimum value on the 20th day and were lower than before inoculation,and showed an increase in the process of 0-20 days.After the downward trend.The JA content of CGN18423 plants reached the maximum on the 7th day,and the content on the 20th day decreased to the level before pre-inoculation.The overall trend is to increase first and then decrease.The JA content of Moneymaker plants increased slightly over the course of 0-20 days.?2?A total of 18 samples were tested in this experiment,and each sample produced an average of 4.40 Gb data.The predicted new genes are 1,513;the total number of expressed genes detected is 25,779,of which 24,295 are known genes,and the predicted new genes are 1,484;after recombination,a total of 20,395 new transcripts were obtained,of which 8,521 There are new alternative splicing subtypes of known protein-coding genes,1,513 transcripts belong to new protein-coding genes,and 10,361 belong to long-chain non-coding RNAs.?3?A total of 418 differentially expressed genes were identified during the CGN18423 plant response process.The differential genes were mainly annotated in GO classifications such as"plasma membrane","histidine decarboxylase activity",and"carboxylic acid metabolism process"by GO analysis.on.KEGG analysis showed that in the process of 0-7 days,"plant hormone signal transduction"is a unique differential metabolic pathway of CGN18423 plants compared with Moneymaker plants.?4?By analyzing the interaction pathways between plants and pathogens and phytohormone signaling pathways,a total of"SAUR"in the"tryptophan metabolism"pathway,"A-ARR"in the"zeatin biosynthesis"pathway,and"rapeseed rape"were obtained."BRI1"in the"steroid steroid biosynthesis"pathway,"JAZ"in the"?-linolenic acid metabolism"pathway,and"Ca MCML","WRKY25/22","WRKY28/33","CERK1","RRS1-R"and"WRKY1/2",including 10 key regulatory points,and excavated important differential genes of each regulatory point.?5?According to the results of this study and the existing knowledge system,a Cf-19gene-mediated disease resistance prediction model was established.When plants carrying the Cf-19 gene are infected with leaf mold,ETI and PTI are triggered simultaneously.Subsequently,Ca²⁺binds to different CAM/CML proteins and enters downstream signaling pathways.The increase in intracellular Ca²⁺concentration also creates conditions for the activation of CDPK and MAPK signaling components.Many TFs,especially WRKYs,accumulate and indirectly regulate the expression of defense-related genes.These changes ultimately lead to defense responses and HR phenomena.During the entire resistance reaction,both SA and JA play important regulatory roles.JAZ is likely to be the key factor in maintaining the balance of these two hormones.In addition,CKs,BRs,auxin and ET may play an important role in the resistance response by changing cell morphology or affecting other functions in the hormone regulatory network.?6?Using VIGS technology to conduct a functional study on the important differential response gene SAMDC,it was found that silencing the gene in disease-resistant materials,the PA content in the leaves of the plants decreased,and some hyphae were restricted in some allergic necrotic spots on the blade edges after inoculation Growth indicates that the plant's disease resistance has decreased.