Screening And Resistance Response Mechanism Analysis Of Tomato Leaf Mould Resistant Candidate Gene Cf-12

As one of the most important vegetable crops in our country and the world,the tomato（Solanum lycopersicum）also plays an important role in the agricultural industry and people’s daily life.Susceptible to many pathogens infestation in tomato cultivation process,especially in the high temperature and high humidity cultivation area in the main tomato producing area in central China,it is very easy to cause large-scale occurrence of tomato leaf mold disease,caused a serious decline in fruit yield and quality in tomato cultivation,threatening the safety of local tomato production.The compatible interaction between C.fulvum and tomato plants is the main reason for the occurrence of tomato leaf mold.Therefore,the resistance Cf breeding work is the key point of our work to control leaf leaf mold.At present,Cf-1,Cf-2,Cf-4,Cf-4E,Cf-5,Cf-9,Cf-11,Cf-9DC and multiples Cf-ECPs resistance genes have been identified,and the Cf-4,Cf-5 and Cf-9 gene have been successfully applied in the production.The key of Cf gene-containing tomato can resist C.fulvum is that the Cf gene encoding a receptor-like protein can specifically identified the effector factors secreted by C.fulvum,which mediate plant production of HR（Hypersensitive response）.However,the new problem is that with the long-term and large-scale popularization of commercial tomato varieties with different Cf genes,C.fulvum races have also undergone a fierce evolutionary selective,among which the recently discovered C.fulvum race 2.4 overcomes the resistance of Cf-2 and Cf-4 genes,and C.fulvum race 2.5and 2.5.9 overcomes the resistance of Cf-5 and Cf-9 genes,which have broad spectrum or complex toxicity C.fulvum physiological races made disease resistant varieties lost resistance to C.fulvum,and also changed the trend of C.fulvum more polymorphic.Therefore,it is urgent for us to exploit the new resistance of Cf genes,and apply it to production.The Cf genes have been found to be derived from wild tomatoes.Therefore,mining new Cf resistance genes and introducing it into conventional tomato cultivars by transgenic or distant hybridization is the key of tomato leaf mould resistant breeding.CGN7495 has excellent C.fulvum resistance through the identification of disease resistance for many years and many cultivated areas.The Kanwar’s identificate that the material contains Cf-12 gene,which is derived from L.pimpinellifolium,There is no research basis for this gene.Therefore,the main focus of our study was to identify and screen the new candidate tomato leaf mould resistance gene Cf-12 and the resistance response mechanism as the main research focus.At the same time,the histological characteristics and inheritance of incompatible interaction between Cf-12 and C.fulvum were also analyzed.The main results are as follows:（1）The detailed microscopic observation of the incompatible interaction histological characteristics between Cf-12 resistant tomato materials and C.fulvum was clearly identified.For example,C.fulvum spores in the germination process and stomatal invasion on resistant and susceptible materials and did not show any difference.However,when C.fulvum hyphae invaded Cf-5,Cf-12,and other resistant material’s stomata,the hyphal swelling,bending and collapse,and host mesophyll cells also collapse,so that C.fulvum invaded mycelium was confined in a small confined space,can’t be infected more,and the necrotic spots generated gradually increased and expanded.The resistance characteristics of Cf-12 and Cf-5,Cf-9 and other genes to C.fulvum were compared and analyzed.The results showed that there were some differences in the quantity and area of necrotic spots in the resistance of different Cf genes to C.fulvum.（2）The resistant range of Cf gene on the C.fulvum species resistance survey results show that different Cf genes can resist C.fulvum races are significant differences.Tomato materials containing Cf-12 gene can resist the infection of C.fulvum physiological races such as 1.2,1.2.4,1.5,1.2.4.5,1.2.5and 1.2.4.9,but not resistant to physiological race 1.2.3.4.The range of resistance was clear,and it could provide the reference for the reasonable planting of Cf-12 gene tomato in different C.fulvum physiological races endemic areas.（3）Based on the six generations of S.pennellii LA0716 and CGN7495,the inheritance of resistance to Cf-12 gene in the population showed that the separation of Cf-12 gene in F2 and BC1P1 population accorded with the 3: 1 and 1:1 Mendel’s law of segregation,which showed that the resistance of Cf-12 gene to C.fulvum accorded with the dominant inheritance of single gene,and was consistent with the other Cf genes.（4）Based on the dominant inheritance of single gene Cf-12,we further performed the combined analysis of ED（Euclidean Distance）and SNP-index using parental re-sequencing and F2 generation of resistant population and susceptible population SLAF simplified sequencing.The Cf-12 candidate gene was successfully mapped to tomato chromosome 6,with the functional annotation and LRR-TM-sCT structure prediction by NR,NT,trEMBL,Swiss-prot,GO,KEGG and COG databases in the target region.We identified three candidate genes,with the structure,physicochemical properties,subcellular localization,secondary and tertiary structure,PPI network of the candidate genes were analyzed in detail by Proparam,pfam,SMART,SignalP,ProtComp,SOPMA,Swiss-Model,gene expression pattern and co-expression analysis.The Ccg2 gene,which is located at the Solyc06g008270.2 gene locus on the CGN7495 resistance gene,was inserted into the 72 base fragment,The Ccg2 gene is the Cf-12 gene we screened.In addition,we used the principle of reverse genetics to carry out the whole genome search of tomato genome with the function of binding activity and the eLRR-TM structure,and we found that all Hcr2 s,Hcr9s and Cf-Ecps gene were covered,this idea provides a new way for us to study other unknown Cf genes.（5）In order to study the mechanism of Cf-12 resistance to C.fulvum,high-throughput sequencing-based RNA-seq technique was used to perform of multi-time incompatible interaction samples.Through the K-mean cluster analysis of DEGs,we found a total of 3 types of expression patterns,the most worthy of our attention is 34 high expression DEGs of subcluster₄,and 41 DEGs with low expression first and high expression later of subcluster₃.GO analysis revealed that they were enriched in oxidation-reduction,nitrogen metabolism,phosphorus metabolism,binding activity,transferase activity,hormone response and defense response,ion transport and other categories.GO analysis plays an important role in the selection of genes specifically expressed in subsequent experiments.AS analysis showed that SE and MXE events mainly occurred in the process of incompatible interaction,and the total number of SE and MXE events increased with the prolongation of time,indicating that these significant differences AS events are undergoing dramatic changes,which regulation the complexity and diversity of the gene expression of Cf-12 gene resistant to C.fulvum at the gene expression level and transcriptional level.GO and Pathway analysis showed that the genes involved in the biological processes such as phenylalanine metabolism,plant-pathogen interaction,phenylpropane biosynthesis,glutathione metabolism and biosynthesis of secondary metabolites were significantly upregulated,Hormone signal transduction pathway analysis showed that SA and ET played an important role in the defense response of Cf-12,and JA might play a auxiliary role in Cf-12 resistance to C.fulvum.In addition,we also enrich a large number of oxidase,glutathione peroxidase genes,phytoalexin synthesis genes,and phenylpropanoid and flavonoid metabolic pathway associated phenylalanine ammonia lyase genes,4CL,TC4 M and F5 H genes,the flavonoids synthesis genes of CHSs,CHI,DFR and FLS,pectin and related to cell wall synthesis cellulose synthase gene and pectinase activity（a）also enriched to upregulated trend,and ubiquitin related E1,E2 and E3 genes were up-regulating,indicating that protein ubiquitination was positive response.In general,the SA,ET,PR protein,and the synthesis of phytoalexin,thionine,lignin,osmotic protein,peroxidase,flavonoids and cell wall enhanced the resistance of Cf-12 tomato to C.fulvum.In the analysis of TFs,we first discovered that several novel TFs such as ARF,MADS,G2-like,G3 H,C2H2,b HLH were involved in Cf-12 resistance to C.fulvum.And,we detected the 60 TFs family showed two kinds of expression patterns,the expressions of bHLH,GRAS,HB,SBP,MYB and WRKY showed high expression in the later stage,and the expression of b HIP,HBH,HSH,MADS and NAC were up-regulated in the early stage,and then down regulated later.Finally,through a comprehensive analysis of the C.fulvum incompatible interaction with the Cf-12,we also have a certain understanding of the mechanism of resistance to Cf-12 and C.fulvum.（6）Some of the genes and candidate genes in Cf-12 resistance to C.fulvum were verified by VIGS.After the silence of candidate genes Ccg2,NPR1（Solyc07g044980.2）and CDPK（Solyc03g113390.2）,some of the plants showed a sense of disease symptoms,the disease index showed that NPR1 and CDPK plants remained in the middle disease resistant,while the silent Ccg2 plants showed a susceptiblesymptom,suggesting that the Ccg2,NPR1（Solyc07g044980.2）and CDPK（Solyc03g113390.2）gene are related to the resistance response of Cf-12 tomato plants to C.fulvum.