Role Of Sphingosine-1-phosphate In Plant Defense Response And Functional Analysis Of Arbidopsis And Rice BIK1 Gene In Stress Responses

During their life span,plants always suffer pathogen attack and environmental stress.To survive,plants have developed complicated and precisely regulated system to defend themselves against pathogens and abiotic stresses.To understand the molecular biology of defense responses in plants,I cloned and characterized genes involved in metabolism of sphingosine-1-phosphate(S1P),including OsSPK1, OsSPK2 and OsSPL1,and a receptor-like protein kinase gene OsBIK1,and performed functional analysis in transgenic tobacco and rice.Meanwhile,I also performed studies to elucidate the involvement of AtBIK1 in phosphorus nutrition in Arabidopsis.Recent studies have showed that S1P plays important role in regulation of abiotic stress responses.However,little is known about the role of S1P in regulation of defense response against pathogen infection.RT-PCR analysis showed that BTH (benzothiadiazole),SA(salicylic acid),ACC(1-aminocyclopropane-1-carboxylic acid) or JA(jasmonic acid,JA) can activate the expression of OsSPK1 and OsSPK2, but partially suppressed expression of OsSPL1.The expression of OsSPK1 and OsSPK2 was up-regulated in an incompatible interaction between Magnaporthe grisea and a resistant genotype,and no significant induced expression was observed in a compatible interaction.In order to investigate the role of S1P in plant disease resistance,functional analysis of OsSPK1 and OsSPL1 in transgenic tobacco plants were performed.Disease resistance assays revealed that overexpression of OsSPK1 in transgenie tobacco plants enhanced disease resistance against tobacco mosaic virus (TMV) and Pseudomonas syringae pv.tabaci(Pst),and up-regulated expression levels of defense-related PR gene,e.g.PR-1,PR-2,PR-3 and PR-5.Moreover, OsSPK1-overexpressing transgenic tobacco plants showed a reduced ABA sensitivity, and enhanced tolerance to oxidative stress.Significant necrotic lesion was observed in OsSPL1-overexpressing but not OsSPK1-overexpressing tobacco plants after injection with fumonisin B1(FB1),as revealed by detection of cell death in leaves by TUNNEL and staining approaches. It was previously shown that AtBIK1 plays important roles in defense response against necrotrophic fungi.Root architecture of the bik1 mutant seedling grown on normal MS medium,it was found that the root system of bik1 mutant is very similar to other mutants with defined function in phosphorous nutrition.RT-PCR and PBIK1::GUS analysis in transgenic lines demonstrated that AtBIK1 was expressed in the root,shoot and leaves in plants under low phosphorous condition.When in the phosphate deficient condition,the number and length of lateral root,the number and length of root hair were much more than the wild type.In bik1 mutants,it resulted in higher concentration of anthocyanin.In addition,when the mutants were deprived of phosphate,the activity of the total acid phosphatase was reduced while the total P content increased.Disease phenotypes of the wild-type and mutant both in the P sufficient and P deficient conditions were compared.The bik1 mutant in the P deficient condition was most susceptible to Botrytis cinera and Alternaria brassicicola,while the wild type in the P sufficient has the strongest disease resistance.These results suggest that the P condition affect the disease resistance. Together,these results suggest that AtBIK1 is a modulator of Pi starvation responses, including root development and the disease resistance.To investigate the function of OsBIK1,a functional analysis of OsBIK1 in transgenic rice plants was performed.RT-PCR analysis showed that BTH,SA,ACC and JA can activate the expression of OsBIK1.In the incompatible interaction between Magnaporthe grisea and a resistant genotype,OsBIK1 was strongly induced,while in the compatible interaction,there was no significant induction.Disease resistance assays revealed that overexpression of OsBIK1 in transgenic rice plants enhanced disease resistance against Magnaporthe grisea.Moreover,OsBIK1 overexpressing transgenic rice plants showed enhanced tolerance to cold stress,salt stress and drought. These results indicate that OsBIK1 play important role in defense responses against biotic and abiotic.