Functional Study Of MiR825/825~* In Bacillus Cereus AR156 Mediated Induced Systemic Resistance And OsAGO2 In Rice Defense Responses Against Magnaporthe Oryzae

Bacillus cereus AR156 is a plant growth-promoting rhizobacterium,which can induce plant resistance against a broad-spectrum of pathogens.Induced systemic resistance(ISR)is mediated by growth-promoting beneficial microbes,which does not directly kill or inhibit pathogens,but through the induced resistance response to achieve disease control objective.Small RNAs is involved in regulating the plant immune response,but its role in AR156-induced Arabidopsis thaliana resistance to Botrytis cinerea is not clear.For function,small RNAs must interact with Argonaute proteins(AGOs)to silence the expression of the target genes.In this paper,we investigated the mechanism of small RNAs in AR156-mediated ISR by examining the function of some endogenous small RNAs.We also studied the role of OsAGO2 in rice defense response against Magnaporthe oryzae,and screened and identified the interaction proteins of OsAGO2.The main research results are showed as follows:First,induced systemic resistance against Botrytis cinerea by Bacillus cereus AR156 in Arabidopsis.Induced resistance response is a potent and cost effective plant defense against pathogen attack.The effectiveness and underlying mechanisms of the suppressive ability by Bacillus cereus AR156 to Pseudomonas syringae pv.tomato DC3000(Pst DC3000)in Arabidopsis has been investigated previously.Botrytis cinerea,a necrotrophic fungus causing gray mold disease,is considered an important pathogen around the world.However,the strength of induced systemic resistance(ISR)activity against Botrytis cinerea remains unknown.Here,we show that root-drench application of AR156 significantly reduces disease incidence through activation of ISR.This protection is accompanied with multilayered ISR defense response activated via enhanced accumulation of PR1 protein expression in a timely manner,hydrogen peroxide accumulation and callose deposition,which is significantly more intense in plants with both AR156 pretreatment and B.cinerea inoculation than that in plants with pathogen inoculation only.Moreover,AR156 can trigger ISR in sid2-2 mutant and NahG OE(overexpression),but not injarl,ein2 and nprl mutant plants.Our results indicate that AR156-induced ISR depends on JA/ET-signaling pathway and NPR1,but not SA.Also,AR156-treated plants are able to rapidly activate MAPK signaling and FRK1/WRKY53 gene expression,both of which are involved in pathogen associated molecular pattern(PAMP)-triggered immunity(PTI).Our results indicate that AR156 can induce ISR by the JA/ET-signaling pathways in an NPR1-dependent manner and involves multiple PTI components.Second,the role of miR825/825*in AR156-induced resistance to Botrytis cinerea.In order to study the mechanism of plant small RNAs in the regulation of plant-induced resistance,it has found that miR825/825*play a negative role in AR156-induced resistance to Arabidopsis thaliana infected by Pst DC3000.However,the role of miR825/825*in AR156-mediated Arabidopsis thaliana-induced resistance to Botrytis cinerea is still unknown.In this study,we investigated the pathogenicity,the expression of defense related genes,ROS accumulation and callose deposition of Col-0(wild type),miR825/825*OE(over-expression),and miR825/825*STTM(function inhibition)transgenic lines.We found that miR825/825*STTM showed resistance to Botrytis cinerea;on the contrary,miR825/825*OE is more susceptible to Botrytis cinerea.In addition,the expression and protein level of MPK6 and MPK3,and the expression of defense-related genes were detected by Western blot and qRT-PCR.The results showed that miR825/825*was involved in the regulation of PTI activated by AR156.These results suggested that AR156 inhibited the expression of miR825 and miR825*in Arabidopsis thaliana,caused upregulation of its target genes,and thereby the plant defense response was activated,and induced plant-derived system resistance.Third,OsAGO2 are involved in regulating the immune response to rice blast in rice.The expression of OsAGO2 were significantly induced after Guy11 infection(compatible),and the expression of OsAGO2 was significantly suppressed after JS153 invasion(incompatible).osago2 mutant result in cell death phenotypes,and enhance resistance,ROS accumulation and callose depostion to Magnaporthe oryzae Guyll infection,but they are susceptible to JS153 infection in rice.The results indicated that OsAGO2 were involved in the immune response to rice blast in rice.To further explore the specific signal pathway in OsAGO2 regulated the immune response to M.oryzae The expression patterns of SA and JA related defense genes in the osago2 mutant were detected by qRT-PCR.We found that the expression of SA synthesis and signal pathway related genes were significantly induced by Guy 11 infection,and the opposite trend were observed after JS153 infection.However,the expression of JA synthesis and signal pathway related genes was not significant by M.oryzae infection.However,.SA is an important signaling molecule of the plant innate immune response,and induced by hemibiotrophic and necrotrophic pathogens infection.SA plays as an important role in PAMP-induced immunity(PTI),effector-induced immunization(ETI)and systemic resistance(SAR),In this study we further examined the levels of SA and SAG in Nipponbare(NPB)and osago2 mutant by Guy 11 and JS153 infection.Compared with NPB,the content of SA and SAG in osago2 mutant was significantly increased after Guy 11 infection.However,compared with NPB,the level of SA and SAG in osago2 mutant were significantly reduced in the condition of JS153 infection.These results indicated that OsAGO2 regulates resistance response to rice blast through the SA signaling pathway.Fourth,screening and identification of OsAGO2 interacting proteins in rice.In order to analyze RNA silencing signal pathway and molecular mechanism induced by OsAGO2,the interaction proteins of OsAGO2 were screened by Yeast two hybrid and affinity purification in the study.Firstly,OsAGO2 were used as bait proteins,and the library of Nipponbare cDNA were screened by Yeast two-hybrid system.Secondly,we found OsPAL1 interacted with OsAGO2,and the proteins of OsAGO2 were further screened by affinity purification.We identified several candidate proteins,such as eukaryotic initiation factor 4A-1(OsIF4A1),and further analysis structural features by MS(Mass Spectrometry).And then we expressed OsPAL1 and candidate proteins in eukaryotes,and veritified the interactions by Co-IP.We proved that OsPAL1 interacts with OsAGO2,OsIF4A1 interacts with OsAGO2.