Bacillus Spp.improve Disease Resistance And Drought Tolerance Of Plants By Regulating Stomatal Closure

As people pay more and more close attention to environmental protection and food safety,the research on beneficial microbial pesticides,fertilizers and other agricultural inputs with no pollutions and damages is increasingly concerned.Beneficial bacteria are conducive to plant coping with biotic and abiotic stress and therefore play an important role in sustainable development of agriculture.Previously,we found that Bacillus cereus AR156 can induced systemic resistance to Pseudomonas syringae pv.tomato DC3000.In this study,we found that AR156 induced stomata closure through the salicylic acid(SA)and abscisic acid(ABA)signal pathways,thereby inhibiting Pst DC3000 invading plants through stomata.Further study showed that AR156 could inhibit the function of MYC2-MED25 transcription complex by enhancing the gene expression of EDS1(Enhanced Release Susceptibility1)and PAD4(Phytoalexin Defense 4),and then CYP70 7A 1(ABA 8'-hydroxylase)was suppressed,which destroyed coronatine(COR)secreted by Pst DC3000 to reopen stomata.In addition,the stomatal regulation not only participates in plant disease resistance,but also plays a vital role in plant drought tolerance.By screening drought tolerance phenotypes of plants induced by biocontrol strains,we found that Bacillus amyloliquefaciens JC65 can significantly improve the plants drought tolerance.Further study demonstrated that JC65 improves plants drought tolerance by inducing the stomata closure.Moreover,biofilm formation of JC65 contributed to induce stomatal closure and drought tolerance of plants.1.Bacillus cereus AR156 restricts foliar pathogen entry through stomataIn our study,it was found that,root inoculated with Bacillus cereus AR156 induced a momentary closure of stomata,and significantly reduced Pst DC3000 invasion,thus restrict Pst DC3000 colonization on the leaves.Although Escherichia coli DH5? and AR156 can induce stomata closure at 1h after inocultion,only AR156 can prevent Pst DC3000 from reopening the stomata at 3h,thereby inhibiting the Pst DC3000 invasion,indicating that AR156 can inhibit the ability of Pst DC3000 to reopen the stomata.Besides,AR156 can significantly increase the expression of ABA synthesis-related gene NCED3(9-cis-epoxycarotenoid dioxygenase 3)and the content of plant endogenous hormone ABA.Exogenous addition of ABA and SA induced plant stomatal closure and inhibited the colonization of Pst DC3000.Further study showed that the ability to induce stomatal closure of AR156 disappeared in mutants of the ABA and SA signal transduction pathways.Taken together,our data indicates AR156 could induce stomatal closure through ABA and SA signaling pathways,and then inhibit Pst DC3000 from entering the plant through stomata.2.Study on Mediate subunit MED25 involving in AR156 regulating of stomatal immunity in plantsThe mediator complex is a kind of multi-protein complex,which is composed of many subunits.It is an indispensable factor for eukaryotic transcription regulation whose main function is to link transcription factors and RNA polymerase II to regulate gene transcription.Previous study showed that AR156 increase resistance aganist Pst DC3000 by inducing stomatal closure,and AR156 can destroy the ability of Pst DC3000 to reopen stomata by secreting phytotoxin COR,but the mechanism is still not clear.In this study,we found that COR can significantly increase the gene expression of the transcription factor MYC2(Myelocytosis 2)and CYP707A1,the key enzyme of ABA metabolism pathway(ABA 8'hydroxylase),while AR156 can inhibit the expression of these genes.The activity of CYP707A1 promoter was significantly enhanced by the transcription factor MYC2.Further studies revealed that the DNA binding ability of MYC2 and the homodimer of MYC2 are critical for MYC2 to regulate the CYP707A1 promoter activity.Moreover,the G-box sequence on the CYP707A1 promoter is also required for MYC2 to regulate the activity of the CYP707A1 promoter.Meanwhile,MED25 is necessary for MYC2 to perform its transcription function.In addition,AR156 can significantly increase the gene expression of EDS1(Enhanced Release Sustainability 1)and PAD4(Phytoalexin Deficiency 4)genes.CoImmunoprecipitation(Co-IP)assasys demonstrated that EDS1,with its homologous protein PAD4 intreacted with transcription factor MYC2 and the mediator subunit MED25 and EDS1/PAD4 suppressed the expression of MYC2.It suggested that AR156 can inhibit the activity of MYC2-MED25 transcription complex by regulating the expression of EDS1 and PAD4,thereby inhibiting the expression of CYP707A1 and destroying the ability of COR to open stomata.3.Bacillus amyloliquefaciens JC65 induces drought tolerance in tomato plants through stomataIt has been reported that stomatal regulation not only participates in plant disease resistance,but also plays a vital role in plant drought tolerance.Here,we tried to explore the mechanism of Bacillus species to help plants resist drought stress by regulating stomata closure.In this study,the ability of three isolates,including Bacillus amyloliquefaciens JC65,from 30 potential PGPR to induce drought tolerance in tomato plants was examined via greenhouse screening.The results indicated that B.amyloliquefaciens JC65 induces stomatal closure of by regulating ABA biosynthesis.As the result,the relative water content(RWC)and root vigor were significantly improved by root inoculation of JC65.Coordinated changes were also observed in cellular defense responses,including decreased concentration of malondialdehyde and elevated concentration of antioxidant enzyme activities.Moreover,expression levels of stress-responsive genes,such as lea,tdi65,and ltpg2,increased in JC65treated plants.Furthermore,we constructed biofilm formation mutants and revealed the positive role of biofilm formation in JC65-induced stomatal closure and drought tolerance.The results showed that plants inoculated with hyper-robust biofilm(?abrB and AywcC)mutants were better able to induce stomatal closure and drought tolerance,while defective biofilm(?epsA-O and ?tasA)mutants were more vulnerable to induce stomatal closure and drought tolerance.Taken altogether,these results suggest that B.amyloliquefaciens JC65 induce drought tolerance by regulating the stomatal closure via ABA signaling pathway,and the biofilm formation of JC65 contribute to improve the drought tolerance of tomato plants.