Functional Study Of MiR825/825* Target Genes In Bacillus Cereus AR156-Induced Plant Disease Resistance And Screening Of Proteins Interacting With OsAGO18 In Rice

Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that elicits efficient induced systemic resistance(ISR)against various diseases.Small RNAs are key regulators in plant defense responses.Our data demonstrated that miR825/825*.played a negative role in AR156 mediated Arabidopsis ISR.To further investigate the mechanism of miRNAs involved in ISR,we characterized the molecular function of the target genes of miR825(At5G44940)and miR825*(At5G38850,At3G04220 and At5G40910)in AR156-induced ISR.Small RNA and Argonaute(AGO)play important roles in the interaction between plant and pathogen.It has been proven that OsAG018 is involved in the resistance against virus.In this study,we used yeast two-hybrid and affinity purification to screen the interacting proteins with OsAG018,in order to further study the molecular mechanism of OsAG018.Functional study of the roles of miR825/825*targrt genes in B.cereus AR156-induced disease resistance in Arabidopsis.ISR is an effective defense response against pathogen infection in plants.However,the mechanism of plant endogenous small RNAs in regulating plant induced resistance is not clear.This study was set up by using AR156 as biological strain;Arabidopsis thaliana as a plant mode;Botrytis cinerea and Pseudomonas syringae pv.tomato DC3000 as pathogens to deeply understand the biocontrol mechanism of AR156.In this study,we show that root-drench application of AR156 and then with B.cinerea inoculation significantly reduce disease incidence through activation of ISR.The results show that AR156 induces resistance against various pathogens.Previous studies showed that AR156 primes ISR by suppressing miR825/825*and activating their target genes which invole in defense responses against bacteria in Arabidopsis.To further study the regulatory mechanism of miR825/825*in AR156-induced ISR,we selected the T-DNA insertion mutants for the target gene of miR825 At5G44940(ubiquitin-protein ligase)and miR825*target genes At5G38850(R gene,TIR-NBS-LRR),At3G04220(R gene,TIR-NBS-LRR),and At5G40910(R gene,TIR-NBS-LRR).We pretreated Col-0 and these four mutants with AR156 in a root-drench application manner,followed by Pst DC3000 infection,and then detected pathogen infection,defense gene expression,ROS accumulation and callose deposition in plants.Compared with Col-0,we found that the antibacterial activity against Pst DC3000 is abolished in these four mutants,respectively.And AR156 pretreatment can increase the resistance against Pst DC3000 in these four target mutants.Taken together,our data demonstrate that the targets of miR825/825*are involved in AR156-induced ISR against bacteria in arabidopsis.Screening and identification of interacting proteins with OsAGO18.In order to explore the mechanism of OsAGO18 involved in RNA signaling pathway and plant disease resistance,we constructed rice cDNA library and to screened for proteins interacting with OsAGO18 by yeast two-hybrid system.In this study,70 proteins were identified as potential interacting partners with OsAGO18.However,one-to-one validation revealed that the interactions between OsAGO18 and these proteins are not authentic.Then we screened the OsAGO18 interacting proteins by affinity purification method,and identified 257 candidate proteins by mass spectrometry.We analyzed the structure of eight candidate proteins:eukaryotic initiation factor OsIF4A1 and OsIF3G;elongation factor OsEF1D1;ascorbate peroxidase OsAPX2;14-3-3-like protein OsGF14F;superoxide dismutase OsSODAl;heat shock protein OsHAP81-3 and catalase OsCatA.We were able to validate the interaction between OsIF4A1 and OsAGO18 by Co-IP assay.This study provides the basis for further study the function of OsAGO18.