Study Of Isoflavonoids Synthesis Pathway Involved In Resistance To Rhizoctonia Solani AG-8 In Medicago Trunctula And Function Of Small Secreted Protein Of AG-1IA

Rhicotonia solani（R.solani）is a broad host,soilborne necrotrophic fungal pathogen which could infect more than 100 plant species,include many important food crops such as rice,maize,soybean,wheat etc,and may cause yield loss in food production.According to the fusion state of hyphae cell wall and similarity of ITS sequence,R.solani can be divided into 14 anastomosis groups（AGs）,from AG1 to AG13 and the bridging group AG-BI.No highly R.solani resistant germplasm can be used in breeding system has been reported until so far.Phytohormone are small molecules produced by plant,which has important roles in regulating development and response to biotic or abiotic stress of plant.It has been reported that the mutant of medicago truncatula A17 in homologous gene of Arabidopsis thaliana EIN2---sickle is more susceptible to AG-8 but the underlying mechanism of changes in resistance of A17 to AG-8 is still unclear.Effectors are proteins or molecules produced by pathogens which could modified structure and function of host cell.Pathogenicity of plant pathogen is strongly related to the function of effectors,but there is few report about the function of R.solani effectors.Study the pathogenicity of R.solani and the resistant mechanism of plant could lay ground for improving crop breeding system.In thesis,through various omics anlaysis of medicago truncatula A17 and its mutant sickle,the author hope to gain insight into the resistance of A17 to AG-8,and meanwhile cloned the small secreted proteins of AG-1IA,then did functional study in tobacco.Main results as below:1.Through transcriptome analysis of A17 and sickle before and after inoculation of AG-8,the author found that genes associated with ethylene signaling pathway,ROS burst,biotic stress response and isoflavonoid metabolism pathway were specially up-regulated in A17,but were not seen in sickle.By q RT-PCR of isoflavonoid metabolism pathway genes,we further confirmed that isoflavonoid metabolism pathway genes were up-regulated in A17 upon infection of AG-8.2.By HPLC analysis,the author found that isoflavonoid related compounds especially precursors of phytoalexin---medicarpin were more accumulated in A17 and the final compounds medicarpin was not accumulated in sickle which suggest that sickle might have defect in isoflavonoid metabolism.3.Over-expression of an isoflavone synthase gene in M.truncatula roots by hairy root transformation found that over-expression of IFS leads to accumulation of formononetin,one of the precursors of medicarpin and improved resistance to AG-8 in A17.Together,these results suggest that maybe the accumulation of phytoalexin---medicarpin and its synthesis related compounds of isoflavonoid metabolism pathway,is the reason that A17 is more resistance to AG-8.And whether isoflavonoid metabolism pathway is the downstream pathway which is regulated by ethylene signaling still needs investigation.4.32 of small secreted protein genes were cloned Rhizoctoani solani AG1-IA.By transient expression in tobacco,the author discovered that Rs IA＿SSP6 could induce HR-like cell death in tobacco.Level of ROS burst and expression of defense-relaed gene by q RT-PCR suggested that transient expression of Rs IA＿SSP6 could induce immune response in tobacco.5.By analysis of amino sequence of Rs IA＿SSP6 of 25 AG1-IA isolates from diseased leaves of different rice breeding area and reference AG-1IA,found that there are 2 amino acid sites changes among the Rs IA＿SSP6s amino sequence,the 37^thamino changed from S to A in 18 isolates and the 117^thamino changed from K to E in 2 isolates respectively.No changes in function were observed when mutate the corresponding amino sites in reference Rs IA＿SSP6 sequence.6.Through VIGS we silenced several tobacco genes involved in PTI and ETI signal pathway and then transient expression Rs IA＿SSP6,showed that SERK3B,HSP90,SGT1 but not RAR1 were required for Rs IA＿SSP6 induced cell death。7.By Y2H system,a protein SP6IP1（Rs IA＿SSP6 Interacting protein 1）from rice was found to interact with Rs IA＿SSP6 in vivo and in vitro.SP6IP1 encoded a putative FHA domain and 74aa chloroplast signal peptide and sub-cell location in tobacco confirmed that SP6IP1were located in chloroplast,which might suggest that chloroplast may involved in the interaction of Rs IA＿SSP6 and rice.