Functional Analysis Of ZmBPM1 In Maize Defense Response

In the long-term evolution,plants have evolved a multi-level immune defense system to prevent the pathogens infection.The first level is the physical barrier using cell wall to confer resistance to pathogens.The second level is called PTI(PAMPtriggered immunity)which is triggered by identifying the slowly evolved microbial-or pathogen-associated molecular patterns(MAMPS or PAMPs)recognized by transmembrane pattern recognition receptors(PRRs).The third level is the ETI(Effector-triggered immunity),which is induced by effectors secreted by pathogens that contribute to pathogenic virulence.These effectors are usually recognized by disease resistance(R)proteins and this process often trigger a rapid,localized cell death called the hypersensitive response(HR).The largest class of R proteins in plants is the nucleotide binding leucine rich repeat(NLR)protein.According to its N-terminal domain,it can be further divided into two categories:one contains the coiled-coil(CC)domain and the other contains the Toll/Interleukin-1-receptor(TIR)domain.A complex rust resistance locus Rpl locating on the chromosome 10 of maize contains 9 CNL genes:Rpl-dpl to Rpl-dp8 and RplD.An intragenic recombination between paralogs Rp1-D and Rpl-dp2 produces the chimeric gene Rp1-D21,which confers auto-active ’lesion mimic’ phenotype in the absence of pathogen infection.Using Rp1-D21 mutant as a tool combined with RNAseq,we identified a gene coding ZmBPM1 was significantly up-regulated in Rpl-D21 mutant.BPM1 contains a MATH domain and a BTB/POZ domain,and the complex formed by the interaction between BPM1 and E3 ubiquitin ligase CLRs is able to mediate the degradation of the target proteins and regulate various signaling pathways.ZmBPM1 and its homo log ZmBPM2 were transiently co-expressed with Rpl-D21 in tobacco leaves.We found that ZmBPM1,but not ZmBPM2 suppressed Rpl-D21mediated HR.ZmBPM1 and ZmBPM2 were also co-expressed with the CC domain(CCD21)the functional signaling domain of Rpl-D21,and the similar result was obtained.We further showed that ZmBPM1 interacted with Rpl-dp2 and CCD21,while ZmBPM2 had no or weak interaction with Rpl-dp2 and CCD21.Based on the western blot analysis,the protein accumulation of Rpl-D21,CCD21 and Rpl-dp2 was decreased when they were co-expressed with ZmBPM1,but not ZmBPM2,indicating that ZmBPM1 might regulate the degradation of Rpl proteins.The homologous proteins of ZmBPM1 in Arabidopsis thaliana degraded the interaction proteins through the ubiquitin pathway,thus the protease inhibitor MG132 was used to treat the samples to investigate whether ZmBPM1 is able to degrade Rpl proteins through the 26S proteasome pathway.When co-expressed with ZmBPMl,the protein accumulation of Rpl-D21 and CCD21 was not increased in MG132-treated samples compared to the control group.Therefore,it is less likely that ZmBPM1 degraded Rpl-D21 or CCD21 through the 26S proteasome pathway.Through subcellular localization studies,we found that ZmBPM1,but not ZmBPM2 formed some punctate aggregates similar to autophagosomes in the cytoplasm.Colocalization with the autophagy marker protein CFP:NbATG8f revealed that ZmBPM1 was partially colocalized with CFP:NbATG8f,which indicated that ZmBPM1 was partially localized in autophagosomes.Previous studies showed that the cytoplasmic and nuclear localizations of Rp1-D21 and CCD21 are very important for the HR induction.When co-expressed with ZmBPM1,Rpl-D21 or CCD21 was transferred to the punctate dot-like structure,suggesting that ZmBPM1 might degrade Rpl-D21 and CCD21 through autophagy.MATH and BTB/POZ,the two important domains of ZmBPM1,were examined on whether they have the regulation function on CCD21-mediated HR.The results showed that the MATH domain inhibits CCD21-mediated HR.The Co-IP examination also demonstrated that MATH had interaction with CCD21.Further studies also revealed the ability of MATH in increasing the dot-like structure of CCD21 in the cytoplasm.Based on the results above,it can be hypothesized that ZmBPM1 interacted with Rp1 proteins through its MATH domain,and it suppressed Rp1-D21-mediated HR by altering the subcellular localization of Rp1-D21 and degrading Rp1-D21 by autophagy.