The Mechanisms Of CCT1 And BZIP59 Transcription Factors Regulating Plant Immunity In Arabidopsis Thaliana

Plant diseases cause serious losses to agricultural production.The most economical and effective control strategy is to breed and utilize disease-resistant varieties.The research on the regulation mechanism of plant immune signals can provide genetic resources for plant disease resistance breeding.The signaling regulatory network constructed by transcription factors can alleviate or eliminate the damage caused by pathogens when plants face pathogenic infection.At present,studies have shown that transcription factors are involved in the regulation of plant stomatal immunity,but the specific regulatory mechanism is still unclear.This study focuses on the BBX family transcription factor CCT1 and the b ZIP family transcription factor b ZIP59,and studies their roles in stomatal immunity and apoplastic immunity,respectively.The main results are as follows:1.CCT1 is involved in stomatal immunity and apoplastic immunity against Pst DC3000 in ArabidopsisCCT1 is constitutively expressed in guard cells of Arabidopsis and responds to the induction of Pseudomonas syringae pv.tomato strain(Pst)DC3000.When dipinoculation with Pst DC3000,CCT1 mutation attenuated the resistance of Arabidopsis to Pst DC3000,while CCT1 overexpression enhanced the resistance of Arabidopsis to Pst DC3000.Further studies found that CCT1 overexpression inhibited Pst DC3000 and COR-induced stomatal reopening,suggesting that CCT1 positively regulates Arabidopsis stomatal immunity to Pst DC3000 by inhibiting COR signaling.CCT1 mutation and overexpression also attenuated and enhanced Arabidopsis resistance to Pst DC3000,respectively,when infiltration inoculated with Pst DC3000.In apoplasts,CCT1 mutation suppressed ROS accumulation,MAPK kinase activity,and callose accumulation induced by the PAMP flg22 derived from Pst DC3000,whereas CCT1 overexpression enhanced these PTI responses,suggesting that CCT1 positively regulated Arabidopsis apoplastic immunity to Pst DC3000 through flg22-induced defense signals.In addition,Ch IP-seq was used to screen CCT1 binding targets across the genome,and the target genes were found to be related to "transmembrane transport or ion channel","ROS synthesis process","kinase activity" and other defense signaling pathways.These results suggest that CCT1 is involved in stomatal and apoplastic immunity against Pst DC3000 in Arabidopsis,which may be mediated by these signaling pathways.2.NAC53 participates in Arabidopsis resistance to Pst DC3000 by regulating CCT1The transcription factor NAC53 interacting with the CCT1 promoter was screened by the yeast one-hybrid technique.Bioinformatics analysis found that NAC53 was highly expressed in guard cells and was induced by Pst DC3000.Transcription activation activity and subcellular localization analysis showed that NAC53 was a nuclear protein with transactivation activity.The truncation analysis of the CCT1 promoter showed that the-1 to-264 positions of the promoter sequence were the key segments for the interaction between NAC53 and the CCT1 promoter.On this basis,the minimal characteristic sequence of NAC53 binding to the CCT1 promoter was determined by electrophoretic mobility shift assay(EMSA)as CCCAAGAAA.Dualluciferase experiments found that NAC53 positively regulated CCT1.In addition,a knockout mutant of NAC53 was obtained by CRISPR/Cas9 gene editing,and it was found that NAC53 mutation inhibited Pst DC3000-induced CCT1 expression and attenuated Arabidopsis resistance to Pst DC3000,indicating that NAC53 was involved in the resistance of Arabidopsis to Pst DC3000 by regulating CCT1.3.b ZIP59 regulates stomatal immunity of Arabidopsis to Pst DC3000,and apoplastic immunity to Tobacco mosaic virus(TMV)and Sclerotinia sclerotiorumOur previous study found that b ZIP59 was involved in Pst DC3000 and flg22-induced stomatal closure.Based on this,it was found that the b ZIP59 mutation attenuated the resistance of Arabidopsis to Pst DC3000 by dip-inoculation,while the b ZIP59 mutation did not affect the resistance of Arabidopsis to Pst DC3000 by infiltration inoculation,indicating that b ZIP59 positively regulates stomatal immunity of Arabidopsis to Pst DC3000.Furthermore,b ZIP59 mutation attenuated Arabidopsis resistance to the biotrophic pathogen TMV,while enhancing Arabidopsis resistance to the necrotrophic fungus Sclerotinia sclerotiorum,consistent with RNA-seq showing that b ZIP59 positively and negatively regulates defense-related gene expression.Ch IPseq and EMSA showed that b ZIP59 could bind not only the Atf1 and Atf7 motifs containing the ACGT core sequence,but also the novel KAN1 motif.Taken together,this study found that CCT1 and b ZIP59 positively regulate stomatal immunity to Pst DC3000 in Arabidopsis.Differently,CCT1 also positively regulated Arabidopsis apoplastic immunity to Pst DC3000 through flg22-induced defense signals,while b ZIP59 was not involved in Arabidopsis apoplastic immunity to Pst DC3000.In addition,b ZIP59 also regulates the opposite resistance of Arabidopsis to the biotrophic pathogen TMV and the necrotrophic fungus Sclerotinia sclerotiorum.The results provide a reference for transcription factors to play different roles in stomatal immunity and apoplast immunity,and also provide a theoretical basis for the cell-type specific strategies of plants against different pathogens.