Mechanism Of Bacillus Cereus AR156 And Plant Metacaspase AtMC1 Involved In Plant Immunity

Bacillus cereus AR156 is a plant growth-promoting rhizobacterium isolated from soil.In our previous studies,we found applying AR156 to the roots of Arabidopsis Col-0 plants could elicit induced systemic resistance(ISR)against Pst and Botrytis cinerea.In this study,leaves of 4-week-old Arabidopsis Col-0 plants were syringe-infiltrated a low-dose concentration suspension of AR156,and that enhanced the resistance to the subsequent inoculation of Pst,which is similar to the resistance induced by flg22 against Pst.To further investigate the mechanism of disease resistance induced by AR156,we performed a series of comparisons between AR156 treatment and flg22 treatment on the defense responses to Pst.Caspase-like protein AtMC1 is an Arabidopsis thaliana type I metacaspase that positively regulates hypersensitive cell death response(HR).To identify the function of AtMC1 in plant immunity,atmc1 plants were inoculated with Pst,the atmc1 mutant was found to be more resistant than Col-0.We screened the interaction protein of AtMC1 by using the Arabidopsis Interactions Viewer and verified the interaction between LSM4 and AtMC1,Like AtMC1,LSM4 negatively regulates plant immunity to Pst infection.Since AtMC1 is associated with LSM4,which is involved in pre-mRNA splicing in Arabidopsis.By RNA-seq,AtMC1 was shown to modulate the splicing of many pre-mRNAs.Furthermore,AtMC1 was shown to interact with some pri-miRNAs in vivo,which in turn affects the expression level of miRNAs in plants.1.Bacillus cereus AR156 activates PTI responses in Arabidopsis thaliana.In this study,leaves of 4-week-old Arabidopsis plants were syringe-infiltrated with AR156 at 5×107 CFU/ml.One day after pretreatment,the leaves were collected for detection of ROS burst,callose deposition and histochemical GUS detection.Callose deposition,H2O2 accumulation and PR1 protein were noticeable in the leaves.We also examined whether AR156 can trigger MAPK activation,an early event in PTI signaling.As a result,phosphorylated MPK3 and MPK6 were were detected quickly in the leaves of two-week old Arabidopsis Col-0 seedings post treatment with AR156 applied as a foliar spray.The transcript levels of PAMP-response genes FRK1 WRKY22 and WRKY29 also increased in the Arabidopsis Col-0 seedings post treatment with AR156.Leaves of 4-week-old Arabidopsis plants were syringe-infiltrated with AR156 at 5 × 107 CFU/ml,flg22 at 1 ?M,or water(the control).One day after pretreatment,the leaves were challenge-inoculated by syringe-infiltration with a Pst suspension at the concentration of 5 × 105 CFU/ml.Compared to the non-treated control,Pst population density significantly declinedin leaves pretreated with AR156 or flg22.2.Bacillus cereus AR156 activates defense responses to Pst similarly to f1g22 in Arabidopsis.AR156-induced resistance to Pst requires AGO1 and DCL,but not FLS2 nor SA-,JA-,and ET-pathways.AGO1 is a critical component of RNA silencing pathways that binds small RNAs and mediates gene silencing at their target sites.DCL1 is required for miRNA biogenesis.AR156 and flg22 differentially regulate AGO1-bound miRNAs expression in a similar manner.We examined the similarity in transcriptional reprogramming induced by AR156 and flg22 by comparing full-genome transcriptional changes in Arabidopsis Co1-0,and the results indicated that AR156 and flg22 activated similar transcriptional programs.3.AtMC1 interacts with LSM4 to mediate plant immunity.AtMC1 is a positive regulator of cell death and negatively regulates plant immunity.To characterize the molecular mechanism of AtMC1 in plant immunity,we searched proteins interacting with it by using the Arabidopsis Interactions Viewer.LSM4 was found to be potentially able to interact with AtMC1.To verify the interaction between AtMC1 and LSM4,3 5S::AtMC1-FLAG and 35S::LSM4-HA plasmids were constructed and then transferred into Agrobacterium tumefaciens strain GV3101 for transient expression.Co-immunoprecipitation(Co-IP)assay was performed to test the potantial interaction between AtMC1 and LSM4.A bimolecular fluorescence complementation(BiFC)assay was subsequently carried out to confirm the interaction between AtMC1 and LSM4.AtMC1 and LSM4 were fused to the N-terminal and the C-terminal fragment of LUC and introduced into Nicotiana benthamiana leaves by infiltration.The results demonstrating the occurrence of their interaction.Given the small size of lsm4 mutant leaves,it was not possible to perform a pathogen growth assay in homozygous mutant plants.Moreover,we constructed transgenic plants overexpressing LSM4 under the control of the cauliflower mosiac virus(CaMV)35S promoter,two transgenic lines with higher LSM4 levels(LSM4-OE7 and-OE8)were selected for the analysis.When infected with Pst,LSM4-OE plants showed faster bacterial growth and severer disease symptoms compared to Col-0.4.Mechanism of AtMCl in the regulating of plant immunity.In view of the function of LSM4 in pre-mRNA splicing in Arabidopsis,we performed high-throughput sequencing of atmcl mutant plants.Bioinformatic analysis and semi-qRT-PCR assays validation indicate that some genes involved in plant immunity were defective in alternative splicing.Among the selected genes whose splicing is regulated by AtMC1,At2g30350 encodes HYL1-interacting GIY-YIG DNA endonuclease(HIGLE),a processing factor in the miRNAs biosynthesis pathway.At1G48500 encodes JASMONATE ZIM-DOMAIN 4(JAZ4)proteins,and At1G65060 encodes 4-coumarate:CoA ligase 3(4CL3),which are involved in the regulation of plant immunity.The accumulation of some miRNAs were affected in atmc1 mutant plants,such as miR398b,miR399a,miR159a and miR165a.Furthermore,no significant difference in the Pol ?,DCL1,HYL1,SE,and AGO1 protein abundance was detected between wild type and atmc1 mutant plants,suggesting that the impact of AtMC1 on miRNAs accumulation does not involve directly modulating key components.We performed RNA immunoprecipitation assay and found that AtMC1 binds pri-miRNAs in vivo and positively regulates the accumulation of mature miRNAs in Arabidopsis.Based on the results above,in this study found for the first time that Bacillus cereus AR156 activates PTI responses in Arabidopsis thaliana,thereby enhancing the disease resistance to Pst and providing a theoretical basis for the industrialization of AR156 as a biocontrol bacteria.However,the shortcoming is that the use of syringe injection to enhance plant disease resistance has no advantage in practical agricultural production applications.As an Arabidopsis thaliana type ? metacaspase,AtMC1 was first discovered to be involved in the plant immunity.AtMC1 negatively regulates plant immunity probably by positively regulating the accumulation of miRNAs and pre-mRNA splicing.