The Mechanism Of RBOHD 919th Glutamate Regulating LPS-triggered ROS Burst

Plants have established complicated immune responses during co-evolution with pathogens.Reactive oxygen species(ROS)burst is one of the most important immune responses.ROS not only play crucial roles in inhibiting pathogen growth,but also act as a second messenger that mediates a series of signal transduction.Lipopolysaccharide(LPS),known as endotoxin,are major component of the gram-negative bacterial outer membrane.LPS are important MAMP(Microbe-associate Molecular Pattern)that trigger biphasic production of ROS in Arabidopsis thaliana.The first transient ROS burst triggered by LPS and lipid A moiety was similar to that induced by other typical MAMPs.Whereas the second long-lasting burst induced only by LPS.Biphasic ROS LPS-induced are novel immune responses which are triggered by MAMPs.However,the molecular mechanism and functions of biphasic ROS are still unclear.In this research,we performed a genetic mutant screen in mutagenized seedlings and identified delt1(Defective in LPS-triggered late-ROS 1)which were shown no biphasic ROS production,and then further study elucidated the molecular mechanism of DELT1 in manipulation of LPS-triggered biphasic ROS burst.The main results are as follows:(1)delt1-1 and delt1-2 are caused by a single recessive mutation.We performed a genetic screen to identify mutants that showed insensitive to LPS triggered ROS burst and two of these mutants were shown no biphasic ROS burst after LPS treatment.These two mutants were indistinguishable from wild type plants when grown in soil.We crossed the two mutants with wild type plants.All F1 progeny showed a wild-type phenotype.In the F2 progeny,the mutant phenotype segregated approximately in a 1:3 ratio.Complementation tests between these two mutants indicated that they were allelic to each other,so named delt1-1,delt1-2.(2)DELT1 encoding RBOHD protein.We roughly mapped DELT1 position to the chromosome ? between 18.32 Mb(Megabase pair)and 22.19 Mb which are 3.87 Mb;we narrowed down the position to a 2.05 Mb interval between 19.05 Mb and 21.10 Mb by whole genome sequencing approach(MutMap).We analyzed the six mutant genes in this interval and then speculated RBOHD was the candidate gene.Allelic analysis between deltl and rbohD T-DNA insertion mutant indicated that loss of ROS in delt1-1 and deltl-2 was due to the mutations in RBOHD.The delt1-1 mutant harbors a G-to-A mutation at the nucleotide 830 of RBOHD,which results in a premature stop codon.The deltl-2 mutant harbors a G-to-A mutation at the nucleotide 4137 of RBOHD,resulting in an amino acid change from the 919th glutamate(E)to lysine(K).(3)E919 mutation affects the activity of RBOHD.In order to clarify the function of E919 in RBOHD,we first analyzed the transcriptional level of the RBOHD gene in the deltl-2 mutant and the effects of the E919 mutation on subcellular localization,protein interaction and enzyme activity of RBOHD.The results demonstrated that the transcript level of RBOHD was not affected in the deltl-2 mutant and E919 mutation did not affect RBOHD subcellular localization,endogenous protein accumulation,self-interaction and the interaction with receptor-like cytoplasmic kinase BIK1(Botrytis-induced Kinase 1).Then,we analyzed the effect of the E919 mutation on NADPH oxidase activity.We found that the activity of NADPH oxidase in deltl-2 and rbohD mutants were significantly lower than that in wild type,indicating that E919 mutation affects the activity of RBOHD oxidase.(4)RBOHD E919 mutation affects lipid A induced stomatal closure.We found lipid A can induce stomatal closure in Arabidopsis.but deltl-2 and rbohD mutants showed deficiency in stomata closure after lipid A treatment,indicating that E919 plays a key role in RBOHD-mediated lipid A-induced stomatal closure.Taken together,in this study,we found that NADPH oxidase RBOHD mediated LPS-induced biphasic ROS burst.In deltl-2 mutant,the 919th glutamate mutation of the RBOHD protein results in reduction of RBOHD oxidase activity.Therefore,ROS burst was lower after treated with flg22 and lipid A.Meanwhile,likewise with rbohD mutant,deltl-2 failed to close stomata.Because of the largely conservation of LPS-induced ROS among different plant species,this study provides great potential for engineering durable and broad-spectrum resistance traits in crop plants.