| The pathosystem of tomato (Solanum lycopersicum) and its leaf mould fungalpathogen Cladosporium fulvum conforms to the gene-for-gene hypothesis and is a modelsystem to study plant and pathogen interactions. Tomato Cf resistance gene productinitiates downstream signal tmnsduction by recognition of C.fulvum avirulence (Avr) geneproduct, resulting in expression of an array of defense related genes, thereby leads tomanifestation of resistance. Little is known about the signal transduction pathway thatdownstream the Cf genes. In this study, proteomic analysis in combination with virusinduced gene silencing (VIGS), a reverse genetic technology for rapid analysis of genefunction, were employed to screen and identify the genes involved in regulation ofresistance to the tomato leaf mold pathogen. The main results are as follows:(1) A two-dimensional electrophoresis (2-DE) system for separation analysis for totalprotein of tomato cotyledons was established. Through optimization of parameters of2-DE, a manipulation protocol for 2-DE with a high resolution and good reproducibilitywas generated: extract the total protein of tomato cotyledons by the method ofTCA/acetone precipitation, use the 24cm pH3-7NL IPG strips, adopt the rehydrationloading with 300μg protein, 8000v 56000～68000vhs for isoelectric focusingelectrophoresis.(2). Proteomic analysis for the resistance to C. fulvum was conducted. Proteomicprofiles for cotyledons of tomato seedling carrying Cf-4 (HR), Avr4/Cf-4 (HR+),Cf-9(HR-) or Avrg/Cf-9(HR+) respectively. 66 spots differently expressed in the Cf-4(HR-)and the Avr4/Cf-4(HR+) seedlings. 59 spots among them are up-regulated, while 7 aredown-regulated in the seedling containing Avr4/Cf-4 showing HR. 71 spots wreedifferently expressed between the Cf-9(HR-) and Avr9/Cf-9 (HR+) seedlings. Among them,59 were up-regulated whereas 12 were down-regulated in Avr9/Cf-9 HR+ seedlings. 43spots were differently expressed both between the Cf-4(HR-) and Avr4/Cf-4 (HR+)seedlings and between Cf-9 (HR-) and Avr9/Cf-9 (HR+) seedlings, with 39 up-regulatedand 4 down-regulated in the HR+ seedlings. 39 spots were differently expressed onlybetween the Cf-4(HR-) and Avr4/Cf-4 (HR+) seedlings, while 28 were differently expressed only between the Cf-9(HR-) and Avr9/Cf-9 (HR+) seedlings.65 spots were successfully identified by MALDI-TOF-MS andMALDI-TOF/TOF-MS analysis. These proteins belongs to several functional categories,including defense, signal transduction, metabolism, protein synthesis, transcriptregulation and photosynthesis.(3) Function in resistance of 19 differently expressed proteins were analyzed.Nicotiana benthamiana and N. tabacum homologs of the identified 65 differentlyexpressed proteins were selected in these Nicotiana sequence databases, and 19 wereretrieved with sequence identity rate higher than 68% in amino acid level or 80% innucleic acid level. These 19 genes were selected as targets for VIGS analysis in N.benthamiana. Fragments of the 19 N. benthamiana were amplicated by RT-PCR, thencloned into the TRV VIGS vector. The VIGS analysis was performed in N. benthamiana.Silencing of 7 genes, which encode a proteasome 20S beta1.1 subunit, a luminal-bindingprotein 5 precursor (BiP5), a cell division protease ftsH homolog, mitogen-activatedprotein kinase 1, an asparagine synthetase, ASR4 ((?)bscissic acid, (?)tress, (?)ipening), and achloroplast thiazole biosynthetic protein, resulted in necrosis, stunt, and abnormal leavesand floral organs in silenced plants. Silencing of genes encodingPIP1(Phytophthora-inhibited protease 1), mitogen-activated protein kinase 1, asparaginesynthetase and Lemir resulted in a significantly reduced Cf-4/Avr4 andCf-9/Avr9-dependent HR, indicating that these four genes may have essential roles in theCf/Avr dependent HR and disease resistance.(4) 56 ACE (Avr/Cf elicited) genes in Cf-4 and Cf-9-dependent HR were analyzedusing the approach of TRV-induced gene silencing. Silencing of eight genes, whichencode a lipid transfer protein, a pathogenesis-related family protein, cytochrome P45076A2, cytochrome P450 72A1, 60S ribosomal protein L10, bZIP transcription factor BZI-2, akinase interacting protein, and an unkown protein, significantly compromised HR,demonstrating that these 8 genes are important regulators of Cf/Avr-dependent HR.(5)The functions of eight genes related to ubiquitin-mediated protein degrationpathway and two genes related to Ca2+ signal transduction were analysed by VIGS. The results showed that one ubiquitin fusion degradation protein and two calcium-bindingproteins play roles in regulation of Cf-4 and Cf-9-dependent HR.Taken together, in this study, proteins differently expressed between seedlingsshowing and not showing Cf-dependent HR were identified through proteomic analysis.The role of genes differently expressed in nucleic acid or protein level in Cf-4/Avr4 andCf-9/Avr9-dependent HR was analysed. 15 genes potentially involved in regulation ofresistance to tomato leaf mould disease were obtained.Results of this study indicate that proteomic analysis in combination with VIGS is aneffective technique system for screening and identification of genes that regulate diseaseresistance. It may be also valuable in screening and identification of other functionalgenes. In addition, the results provide insights into the molecular mechanism of resistanceto C. fulvum, and provide beneficial gene resources for breeding leaf mold-resistanttomato cultivars.
|
PDF Full Text Request