Functional Study Of Small RNAs In AR156-mediated Induced Systemic Resistance(ISR) In Arabidopsis

Bacillus cereus strain AR156 can induce plant resistance against a broad-spectrum of pathogens.Induced systemic resistance(ISR)is mediated by nonpathogenic bacteria,such as plant growth promoting bacteria.ISR for the pathogen does not directly kill or inhibit the pathogens,but through the induced resistance response to achieve disease control objective.Small RNA plays an important role in the innate immunity of plants,but its role in ISR not clear.This paper mainly studies the small RNAs in Bacillus cereus AR156 on Arabidopsis thaliana mediated ISR mechanism.We found that AR156 pretreatment could effectively protect the leaves against Pst DC3000 infection.In order to study whether miRNAs are involved in AR156 induced ISR,we construct four small RNA libraries:AR156 pre-treated but without Pst DC3000 infection(AR156/none),control pre-treated but without Pst DC3000 infection(control/none),AR156 pre-treated but with Pst DC3000 infection(AR156/Pst),and control pre-treated but with Pst DC3000 infection(control/Pst).Through the control and AR156 treatments,as well as in the Pst DC3000 infected plants after the control and AR156 pretreatment,the results show that there is indeed a significant change in miRNA.Pst DC3000 infection in AR156 pretreatment,miR825 and miR825*expression significantly reduced,indicating that miR825 and miR825*targets are likely to be involved in the signal pathway of plant defense.In order to determine the function of miR825 and miR825*,we examine ISR on transgenic plants either the function of niR825/825*is blocked or is overexpressed.MiR825 and miR825*function is successfully blocked by short tandem target simulation strategy(STTM).After Pst DC3000 infection,miR825/825*-STTM plants showed similar phenotype to AR156 pretreated plants,and the disease resistance was significantly enhanced compared with the wild type plants.Over-expression lines were more susceptible to disease.The expression level of miR825*target gene At5G38850,At3G04220,At5G40910 and miR825 target gene At5G44940 were significantly increased by qRT-PCR detection,demonstrating the negative regulatory role of miR825 and miR825*in ISR.RDR6 is the key component of the secondary siRNA.Because miR825*is a 22 nucleotide miRNA,it is likely to play a role through the initiation of the 2nd siRNA as well as through a RDR6/DCL4 dependent manner.So we conducted a further study on rdr6 mutant in the ISR.The results of the study indicate that the ISR induced AR156 part depends on the RDR6.miR825*is likely to participate in the regulation of ISR through the RDR6 mediated pathway.AR156 induced more siRNA in control/Pst plants when compared to AR156/Pst pretreatment.4These siRNA are produced by the cleavage of At5G38850 by miR825*.The results showed that the secondary siRNA induced by miR825*at the At5G38850 site was significantly less than control/Pst in the AR156/Pst treatment.It showed that there was a correlation between the secondary siRNA and AR156/Pst treatment at the At5G38850 site,which promoted the expression of ISR,and it was beneficial to induce At5G38850 with other genes that have not yet been identified.The above results indicate that AR156 pretreatment prime plants aginst Pst DC3000 infection by inhibiting the activation of miR825 and miR825*and triggered ISR by activating their target genes with defense-related functions.In addition,we investigated the effect of AR156 on the induction of ISR against Botrytis cinerea infection,and the potential roles of miR825/825*in this process.Compared with the untreated wild type Arabidopsis thaliana,the AR156 pretreated plants showed obvious resistance to disease.The results showed that AR156 induced resistance to Botrytis cinerea in Arabidopsis thaliana.Since the involvment of miR825/825*in AR156 induced ISR against Pst DC3000 in Arabidopsis thaliana,we used Botrytis cinerea to infect miR825/825*-STTM plants,as well as transgenic plants expressing miR825 and miR825*.Compared with the wild type plants,the miR825/825*-STTM plants showed significant resistance to the wild type plants,and the transgenic plants over-expressing miR825 and miR825*showed significant disease phenotype.It indicates that miR825 and miR825*played a negative role in ISR against Botiytis cinereca.These results showed that AR156 can induced ISR against Botrytis cinerea infection in Arabidopsis thaliana,and miR825 and miR825*may play a role in this process.