Molecular Genetic Analysis Of The Role Of Nitric Oxide In Pathogen Defense In Arabidopsis

Nitric oxide(NO) is one of the ten smallest chemical molecules found so far in nature and is a small highly diffusible bioactive molecule.NO has been most studied in protozoa,bacteria,yeast,animal and human.Further investigations led to the finding that NO is a multifunctional effector involved in numerous mammalian physiological processes,including the relaxation of smooth muscle,inhibition of platelet aggregation,neural communication and immune regulation.NO is also a novel regulation molecule in plant growth and development.Scientists considered that NO play an important role in plant defense.Recently,more studies have been done about NO pharmacological effect in plant defense.However,the underlying cellular and molecular mechanisms of NO and SA, JA/ET crosstalk in plant defense pathway remain largely unknown.Here we have use reverse genetics,molecular biology and pharmacology methods to elucidate NO signaling with SA and JA/ET in plant defense mechanisms.Three important findings were made.First,the cue1 plants were found to be constitutively resistant to virulent pathogen Pseudomonas syringae pv.maculicola ES4326;to have endogenous expression of the pathogensis-related gene 1(PR1) and plant defesin 1.2(PDF1.2) by Semi-Quantitative RT-PCR;and to have elevated level of nitric oxide(NO) and salicylic acid(SA).Homozygous lines for cue1 and either the SA-degarding bacterial gene nahG or the SA-insensitive mutation npr1 do not express PR1 or exhibit resistance to P.s.maculicola ES4326.Treatment WT with SNP induced PR1 and PDF1.2 expression in a dose-dependent manner.The double mutants of cue1npr1 and cue1nahG displayed an elevated level of endogenous NO.Therefore,we conclude that NO acts upstream of SA in inducing SAR.Second,treatment ein2 and ein3 plants with SNP could not induce PDF1.2 gene expression,however,treatment jar1 plants could induce PDF1.2 expression.The double mutant cue1jar1 blocked PDF1.2 expression,but cue1ein2 did not block PDF1.2 expression.Homozygous lines for cue1 and either the ethylene-insensitive mutant ein2 or the jasmonic acid-insensitive mutatant jar1 displayed an elevated level of endogenous NO or exhibit resistance to P.s.maculicola ES4326.It is surprise that the cue1ein2 plants delay flowering time,but cue1jar1 is early flowering as same as jar1.The result indicated that NO induced PDF1.2 expression by EIN2 not JAR1,but NO how to effect flowering time of ein2 and jar1,we do not know as far.Third,treatment salicylic acid-insensitive mutant eds1 and eds5 plants with SNP induced PR1 and PDF1.2 expression in a dose-dependent manner.Therefore,we conclude that NO does not act upstream of EDS1 and EDS5 in inducing SAR.NO play an important role in eds1 and eds5 leaf development.Treatment eds1 and eds5 with≥20μM concentrations of SNP completely induced rectilate pattern leaves compared with WT.We can conclude that NO is not upstream EDS1 and EDS5,but NO have more effection on leaves development in eds1 and eds5.In this thesis study,we primarily explained NO and SA,JA/ET defense pathway by genetics,molecular biology and pharmacology methods.We can conclude that NO acts upstream of SA in inducing SAR and NO induced PDF1.2 expression by EIN2 and EIN3 not by JAR1.This is first time using genetics to study NO in plant defense pathway.We also give new insights and evidence about NO and JA/ET crosstalk.