Identification And Function Analysis Of Genes Regulated Resistance To Sclerotinia Sclerotiorum In Arabidopsis Thaliana

The plant disease caused by Sclerotinia sclerotiorum has been a serious threat to the high yield and good quality of the world economic crops especially oilrape and Sunflower,caused huge economic losses.The pathogenic mechanism of Sclerotinia sclerotiorum and its interactions with plants have drawn many researchers' attention.Once pathogen comes to infect,plants can take some measures to resist to pathogens.The measures plants taken include base defense response,resistance gene,inducible defense signal pathway(JA,SA,ET signaling pathways),plant programmed cell death and active oxygen species regulation.They are regulated by some regulator genes in defense process.We used Arabidopsis thaliana-Sclerotinia sclerotiorum pathosystem to investigate the genes regulating resistance to Sclerotinia sclerotiorum in Arabidopsis.Some genes differentially expressed response to Sclerotinia sclerotiorum were screened by qRT-PCR.Then their over-expression vectors were constructed and were transformed into wild-type of Arabidopsis mediated by Agrobacterium and the transgenic over-expressed plants(OE)were screened.Whilst,we confirmed the homozygotes of T-DNA insertion mutants SALK by three-primers method and RT-PCR.The detached leaves or alive plants of OE,Co10 and SALK were inoculated with S.sclerotiorum,then lesions were measured,the inoculated leaves were stained with DAB or Trypan blue and the expression change of defense signaling related genes after inoculation of S.sclerotiorum were analyzed by qRT-PCR.Upon these analysis,we preliminary identified and analyzed five genes participating in the regulation of resistance to S.sclerotiorum in A.thaliana.The results of Phenotypic analysis showed that:At4g13180-OE,At2g30870-OE,At3g52400-OE,At4g24220-OE,At4g12 720-OE could enhance resistance to Sclerotinia sclerotiorum,but some genes T-DNA instertion mutants were sensitive than Co10.The results of DAB staining showed that:The H2O2 level in At4g13180-OE,At2g30870-OE and At3g52400-OE inoculated leaves were smaller than that of Co10 or T-DNA insertion mutants.But The H2O2 level in At4g24220-OE,At4g12720-OE were not obviously different from that of Co10 or SALK lines.The results of trypan blue staining showed that hyphal growth were restrained in OE,while they were well-grown in the Co10 and SALK lines.The analysis of defense related gene expression change shows that:At3g52400 plays some roles in plant defense responses to S.sclerotiorum.It may regulate JA signaling pathway through the positive regulation of LOX2 and PDF1.2 expression and may also regulate SA Signaling pathway through the negative regulation PAL1 and positive regulation of ICSl and PRl gene expression.When plants were challenged by S.sclerotiorum,At4g24220 can also plays some roles in plant defense responses to Sclerotinia sclerotiorum,but this role is a little different from that of At3g52400.At4g24220 may regulate JA signaling pathway also through the positive regulation of LOX2 and PDF 12 expression,but it regulate SA Signaling pathway through the negative regulation of ICS1,PAL1 and PR1 expression and the SA signaling pathway are totally supressed.