| Tomato leaf mold, caused by the fungus Cladosporium fulvum, is a common disease in tomato cultivation. Breeding to obtain resistant cultivars is an efficient method to control leaf mold outbreaks, but use of Cf genes also has accelerated the differentiation of physiological races. To date, many Cf genes including Cf-2, Cf-4 and Cf-6 have been overcome by some physiological races. Therefore, it is vital to identify new Cf genes for breeding use. C. fulvum-tomato pathosystem is a model for the study of gene-for-gene interactions. Most studies about Cf/Avr interaction were focused on Cf-4/Avr4 and Cf-9/Avr9. The consistency between the amino acid sequence of Cf-9 and Cf-4 protein is 91%, while there is no significant homology between Avr9 and Avr4 protein, and the hypersensitivity reactions that mediated by Cf-9 and Cf-4 is very different. The mechanism that lead to the differences in hypersensitivity reactions between Cf-9 and Cf-4 is still unclear, so studies on the hypersensitivity reactions mediated by new Cf genes will help us to know more about Cf/Avr interaction mechanism, and even provide us new methods to breeding resistance cultivars.Tomato plants carrying Cf19 gene showed efficient resistance to tomato leaf mold in the field, and can be used as new candidate material for breeding. Cf-19 gene was firstly assigned to the long arm of chromosome 2 in 1980, and no more studies about this gene has been done according to our knowledge. The lack of understanding about this gene restricts its application. We carried out a comprehensive research including resistance valid range identification, resistance characteristic, inheritance of Cf19 gene, gene mapping, differencial expressed gene screening and expression pattern analysis to get a comprehensive understanding about the phenotypic resistance characteristics, genetic characteristics and even the mechanism of disease resistance response of Cf-19 gene. This study will provide bases for the application of Cf-19 gene in breeding, Cf-19 gene cloning and further study of Cf/Avr interaction mechanism.In this study, we applied many methods including trypan blue staining, high-throughput sequencing, quantitative real-time PCR and c DNA-AFLP on different parts of the experiment. The main results we obtained were shown as following:(1) Artificial inoculation results showed that Cf-19 gene resistant to C. fulvum physiological races 1.2.3.4.5 and 1.2.3.4.9, immune to 1.2.3 and 1.2.3.4. These physiological races of C. fulvum can stand for the C. fulvum differentiation level in China.(2) A remarkable hypersensitive response(HR) phenomenon was observed in trypan blue staining experiment, and the response intensity that Cf19 mediated is between Cf4 and Cf9. The necrotic spots are larger and distributed along the veins.(3) Inoculation results from two F2 segregating population showed that the resistance of tomato material CGN18423 was controlled by a single dominant gene(Cf-19) and that fits the monogenic Mendelian inheritance law completely.(4) Results of microscopic analysis revealed that penetration occurred at 4 days after inoculation(DAI), infected cells started to die was observed at 5 DAI, necrotic lesions formed between 5 and 7 DAI, Hyphae in necrotic lesions disappeared between 7 and 10 DAI, necrotic lesions increased and expanded in a restrict range between 10 and 21 DAI. Seven key time points were found out: 0, 4, 5, 7, 10, 15 and 21 DAI.(5) Cf-19 gene is mapped on Cf-4/9 locus on the short arm of chromosome 1, and 2 candidate genes Solyc01g005870.1.1 and Solyc01g006550.2.1 were screened out based on several methods including the parents resequencing, SLAF-seq analysis, gene annotation, marker developing and gene expression pattern verification. Solyc01g006550.2.1 is a Cf-4/9 locus gene as an allele of Cf-0 gene.(6) According to the amino acid sequence analysis of candidate genes, there were no significant difference of conserve domain in Solyc01g005870.1.1 locus between the resistant and susceptible plants. Solyc01g006550.2.1(KT845954)of CGN18423 is a new member of Cf-4/9 locus, a 60bp-insertion provided a signal peptide for the original polypeptide of Cf-0 and the changes of ORF lead to an increase of LRR domain number. DNA sequences multiple alignment of candidate genes and other Cf-4/9 locus genes showed that, Solyc01g006550.2.1 locus of CGN18423 had close evolution relationship with Cf-4 and Cf-9 genes. While Solyc01g005870.1.1 locus of CGN18423 showed a short evolution distance with Cf-0 gene, and the evolution level was very low.(7) A co-dominant SCAR marker P7 that cosegregated with the resistance trait was developed. A fragment which is 300 bp in size was amplified in CGN18423, a fragment which is 240 bp in size was amplified in Moneymaker, and both of these two fragments were amplified in the F1 samples. The marker was tested by 345 F2 plants and 3 F3 lines with different genotypes, the results showed that it can be used in MAS breeding.(8) The differencial expressed genes in the Cf-19 mediated resistance response were screened using c DNA-AFLP method. Tewety-six TDFs that successfully sequenced were obtained, the accession numbers are JZ717721~ JZ717746. Most of these TDFs showed the same types and expression patterns with genes from Cf-4 and Cf-9 mediated resistance response.(9) TDF35 and TDF49 were firstly detected in Cf/Avr interaction. TDF35 was homologous to Solanum lycopersicum S-Adenosylmethionine decarboxylase 1(SAMDC) gene. TDF49 showed similarity with the Solanum lycopersicum Eli3 protein. The expression patterns indicating that one or more pathways which SAMDC involved in played a key role in the Cf-19-C.fulvum interaction and the expression of Eli3 may dependent on the occurrence of R gene-Avr gene interaction. TDF1 was homologous to predicted Solanum lycopersicum non-specific lipid-transfer protein(ns LTP) 2-like(91%), and was up-regulated in response to infection by C. fulvum in both resistant and susceptible plants. TDF14 corresponds to the Solanum lycopersicum Bax inhibitor(BI-1) and was up-regulated in both CGN18423 and Moneymaker in our study. TDF47 was homologous to Calcium-dependent protein kinase gene(CDPK) and was up-regulated remarkably in Cf-19-mediated defense response.(10) An efficient infect method was found, and the recombinant vectors( VIGS-TRV2) of candidate genes(Solyc01g005870.1.1 and Solyc01g006550.2.1) and several resistance response genes were constructed successfully. |
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