The Bioinformatics Analysis Of Maize ZmBPMs Family And The Function Of ZmBPMl In Immune Response

Corn is one of the most important food crops in the world,and it is also used as feed and industrial materials and biofuels.Diverse maize diseases often lead to huge losses in maize yield.Therefore,it is very important to excavate maize disease resistance genes and investigate their molecular mechanisms.R proteins play an important role in the process of plant immunity,and the largest class of R proteins is called NLR(Nucleotide-binding,leucine-rich repeat).Rpl-D21 is an NLR protein,and it can produce autonomous HR in maize.Transient expression of the full-length or its N-terminal CC(Coiled-Coil)domain by Agrobacterium in tobacco can also induce autonomous HR.BPM(BTB/POZ-MATH)is the adaptor protein between CRL3(Cullin-RING ubiquitin ligases 3)E3 ligases and their substrates.Our previous RNA-seq analysis revealed that ZmBPM1 was significantly up-regulated in Rpl-D21 mutant compared to wild type.We analyzed the promoter regions of ZmBPMl and ZmBPM2,and found that both genes contained more stress response elements,including the binding sites of transcription factors related to disease resistance,suggesting that ZmBPMl and ZmBPM2 may play a role in maize disease resistance to different pathogens through transcriptional regulation.We further analyzed the position of ZmBPMs in chromosomes with the maize resistance QTL(Quantitative trait loci)to different pathogens,and found that several ZmBPMs were localized in a few maize resistance QTL,therefore ZmBPMs might be involved in the resistance to different pathogens.ZmBPM1 inhibits Rp1-D21 and CCD21 induced HR in tobacco by interacting with Rpl-D21 and CCD21,while ZmBPM2 does not.ZmBPM1 degrades Rpl-D21 and CCD21,while ZmBPM2 does not.To explore the molecular mechanism of ZmBPM1 in the degradation of Rpl-D21 and CCD21,ZmBPM1 was co-expressed with CCD21 and treated with the 26S proteasome inhibitor MG132 and the autophagy inhibitor 3-MA(3-Methyladenine),respectively,and the protein levels of CCD21 were restored when treated by 3-MA.These results suggested that ZmBPMl degraded CCD21 possibly through the autophagy pathway.ZmBPM1 mainly has a punctate subcellular localization,while ZmBPM2 is mainly localized in the cytoplasm and nucleus.We co-expressed ZmBPMl with CCD21 and found that ZmBPMl altered the subcellular localization of CCD21 from the cytoplasm and the nucleus to the dot structure.To further study its specific localization,ZmBPM1 was co-localized with the endoplasmic reticulum,golgi apparatus,peroxisome and other marker proteins in tobacco,and it was found that ZmBPMl did not co-localize with the above markers.Further localization in maize protoplasts showed that ZmBPM1 also showed a dot structure,which was consistent with the results in tobacco.We further transformed ZmBPMl with the dot-like localization markers such as autophagosome,early endosome and late endosome in maize protoplasts,and found that none of them were co-localized with the above markers.In conclusion,the specific localization of ZmBPM1 needs to be further investigated.In order to further study the mechanism of ZmBPMl,we used the MATH domain as a bait protein to conduct yeast two-hybrid screening of the maize cDNA library.A ring protein RF6028 and an E3 ubiquin ligase RGLG1 were screened.By co-expressing with Rpl-D21 and CCD21 in tobacco,neither of them could inhibit the HR produced by Rpl-D21 and CCD21 in tobacco.We obtained the UniforMu insert mutant of zmbpms from the maize mutant library,and constructed the maize over-expression lines of ZmBPM1,which will be inoculated with pathogens for disease resistance identification in the later stage.Taken together,maize ZmBPM1 may inhibit HR production by altering the subcellular localization of the Rpl-D21 and degrade it through the autophagy pathway.Therefore,ZmBPMs play important function in plant defense response,which provides a new genetic resource for maize disease resistance breeding.