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Molecular Mechanism Of The Resistance To Tomato Leaf Mould Disease And The Establishment Of A Technique System To Isolate Resistance-related Genes

Posted on:2008-08-16Degree:DoctorType:Dissertation
Country:ChinaCandidate:W HongFull Text:PDF
GTID:1103360242494309Subject:Vegetable science
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The resistance of tomato(Lycopersicon esculentum Mill.)against the leaf mould fungal pathogen Cladosporium fulvum is initiated by the recognition of C.fulvum arivulence(Avr)elicitors by tomato Cf resistance gene products,which activates the downstream signaling,resulting in defense-related gene expression.Over 91%of the amino acids of Cf-9 and Cf-4 proteins are identical,whereas no significant sequence homology is found between Avr9 and Avr4 proteins.However Avr9/Cf-9-and Avr4/Cf-4-induced hypersensitive cell death in tomato seedlings is distinct in the time of initiation,the speed of development and the tissues that HR occurs.The HR in cotyledons of Avr9/Cf-9 seedlings initiates earlier,spreads slower,developing more scattered spots,less associated with veins,when compared with that of Avr4/Cf-4 seedlings.The different HR could be caused by the different expression of the Cf genes and/or downstream signal transduction.To test the hypothesis,this thesis research employed the Avr/Cf tomato seedlings to compare the Avr/Cf-dependent HR developed after the growth temperature shift from 33℃to 20℃,investigate the transcript profiles upon the HR development through cDNA-AFLP, and isolate and clone the TDFs(transcript derived fragments)differentially expressed in the HR~+ and the HR~- seedlings.Expression of some TDFs was assayed in detail.System of TRV-induced gene silencing based on the cDNA-AFLP TDFs to identify the role in resistance of the genes corresponding to the TDFs,was established.In addition,the effect of development,temperature and the cognate Avr on the expression of Cf-9 and Cf-4 was comparatively studied.In order to make deep insights into the molecular mechanism of Cf-9-and Cf-4-dependent HR and resistance,and make a good basis on the future studies on identification and isolation of plant resistance-related genes employing the strategy based on gene silencing using the recombinant silencing vector directly inserted with the cDNA-AFLP TDFs.The main results are as follows:(1)The HR in Avr9/Cf-9 and Avr4/Cf-4 seedlings was compared through microscopic analysis.It was found that high temperature(33℃)attenuates the HR development in the Avr/Cf tomato seedlings. Some cells were stained blue by the dye trypan blue 4 hr and 16 hr post temperature shift from 33℃to 20℃in the Avr9/Cf-9 and Avr4/Cf-4 seedlings,respectively.More and large stained cell patches were observed 12 hr and 48 hr post temperature shift in the Avr/Cf seedlings,respectively.This result indicates that the HR is initiated 4 hr and 16 hr post temperature shift,and typical HR spots develop 12 hr and 48 hr post temperature shift in Avr9/Cf-9 and Avr4/Cf-4 seedlings,respectively.(2)cDNA-AFLP analysis conducted for the seedlings carrying a single Avr gene or Cf gene or a matching Avr/Cf gene pair sampled both at 33℃and after a temperature shift to 20℃.Transcript profiles were compared in HR~+ and HR~- seedlings.TDFs differentially expressed in the HR~+ and the HR~- seedlings were cloned and sequenced.Over 279 480 bands for totally 12 samples appeared in the cDNA-AFLP assay.Among these,367 ACE((?)vr/(?)f-(?)licited,either induced or repressed)TDFs shown differential expression in HR~+ and HR~- seedlings,among which 249 shown induced expression whereas 118 shown reduced expression in both HR~+ Avr9/Cf-9 and Avr4/Cf-4 seedlings.189 TDFs showing most significant differential expression in HR~+ and HR~- seedlings,were cloned and sequenced.The sequences were deposited at GenBank nucleotide sequence database under the accession numbers CD579099-CD579164, CK348286-CK348390,and DN956215-DN956225.The genes corresponding to the 189 TDFs encodes a resistance protein and proteins involved in HR/cell death,signal transduction,defense response,transcription regulation,metabolism and protein synthesis.In addition,9 full-length cDNA sequences were obtained,and named ACI genes for(?)vr/(?)f-(?)nduced genes.These sequences were deposited at GenBank under the accession numbers DQ056433-DQ056442.Among them,ACIs 14 25, 29,36,37 and 39 encodes a calcineurin-like phosphoesterase family protein,a xyloglucan-specific fungal endoglucanase inhibitor protein,a 60S ribosomal protein L10,an oxoglutarate/malate translocator,a cytochrome b559 and a cytoplasmic ribosomal protein S13,respectively,whereas ACIs 13,19 and 112 codes for proteins with unknown function.Results of transcript profiling assay revealed that although all 367 ACE TDFs showed the same trend(either induced or repressed)of differential expression between HR~+ and HR~- seedlings(either Avr4/Cf-4-or Avr9/Cf-9-dependent),42.8%of the ACE TDFs(157/367 in total)showed quantitatively different expression in the two types of HR~+ seedlings.The majority of these(135/157,86.0%)displayed significantly greater differential expression (either induced or repressed)in Avr4/Cf-4 HR~+ seedlings than in Avr9/Cf-9 HR~+ seedlings.These results demonstrate that the spectrum of the genes involved in the Cf-9 and Cf-4 downstream signaling may be identical;however,the fine-tuned regulation of these genes might be different during development of Avr9/Cf-9-and Avr4/Cf-4-dependent HR and resistance.(3)The expression of ACI29 upon development of HR and under stress conditions was analyzed.It was found that in the seedlings carrying a single Avr or Cf gene,and thus showing no HR,AC129 was weakly expressed in the 3d-old and younger seedlings,but strongly expressed in the 4d-old and older seedlings.Expression was stronger in the seedlings carrying a matching Avr/Cf gene pair and thus able to develop HR,and differed in the time-course and profundity in Avr9/Cf-9 and Avr4/Cf-4 seedlings.It was expressed earlier but less strongly upon development of HR in Avr9/Cf-9 seedlings compared with that in Avr4/Cf-4 seedlings,indicating that development and HR affects the expression of ACI29.In addition,expression of ACI29 was weakly induced by heavy metal treatments,strongly repressed by salt and drought treatments,but not affected by wounding treatments.Taken together,these results reveal that the ribosomal protein L10 may be involved in regulation of development,HR and stress response in plants.(4)TDFs 14 and 29 and a tPDS fragment as a positive control were subcloned into TRV-derived gene silencing vector pYL156,respectively.Silencing analysis was conducted in Cf4 and Cf9 tomato plants.Photobleaching phenotype was observed in the plants infiltrated with Agrobacterium transformed with pYL156::tPDS,demonstrating that the endogenous PDS gene has been silenced in these plants.The plants in which the gene corresponding to TDF 29(supposed to be ACI29)was silenced shown severe development retardation and dwarfing,whereas the plants in which the gene corresponding to TDF 14 (supposed to be the ACI14)was silenced,did not shown any visible symptom.This result reveals that this gene silencing system works for cDNA-AFLP TDFs.In addition,ACI29,the ribosomal protein L10, may be involved in regulation of development in plants;while ACI14,a calcineurin-like phosphoesterase family protein,may be not.These results indicate that the strategy employing gene silencing using the recombinant silencing vector directly inserted with the cDNA-AFLP TDFs,to rapidly dentify and isolate the resistance-related genes of plants in large quantities,is feasible.(5)The seedlings,carrying a single Avr gene or Cf gene or a matching Avr/Cf gene pair,were grown at 33℃for 1d,3d and 5d,respectively,and then were grown at 20℃.Results of Northern analysis shown that in the Cf seedlings without carrying a cognate Avr gene expression of the gene Cf-9 was not detected,whereas expression of the gene Cf-4 was obvious.The expression of Cf-4 gradually enhanced with the increasing growing days maintained at 20℃and the longer growing hours after the temperature shift.In Avr/Cf seedlings,however,both Cf genes were expressed more strongly than in Cf seedlings.The expression of both Cf genes rapidly strengthened with the increasing growing days maintained at 20℃and the longer growing hours after the temperature shift.After temperature shift,the expression of Cf-9 was more rapidly but the peak level was lower compared with that of Cf-4.These results demonstrate that development,temperature and the cognate Aw affect the expression of both Cf genes,but to a different extent.
Keywords/Search Tags:Tomato, Lycopersicon esculentum, leaf mould, Cladosporium fulvum, resistance gene, Cf, avirulence gene, Avr, hypersensitive response, development, temperature, cDNA-AFLP, gene expression, Tobacco rattle virus (TRY), virus-induced gene silencing (VIGS)
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