Functions And Molecular Mechanisms Of CAMTA3 In Regulating Plant Resistance To Sclerotinia Sclerotiorum And Xanthomonas Oryzae Pv.oryzae

Stem rot disease caused by Sclerotinia sclerotiorum and bacter:ial blight disease caused by Xanthomonas oryzae pv.oryzae(Xoo)are important diseases of oil and vegetable crops and rice,respectively.Dissection of molecular mechanisms underlying resistance to these pathogens is beneficial to propose new strategy and provide theoretical guidance to control these diseases.Calcium signaling pathway is involved in regulating plant growth and development and stress responses.CAMTA(Calmodulin-binding transcription activator)family transcription factors are CaM(Ca2+ receptor)-binding components of calcium signaling pathway.Among the family is CAMTA3,which is known to play roles in plant basal resistance.However,roles and molecular mechanisms of CAMTA3 in other types of resistance such as induced resistance and nonhost resistance remain unclear.Additionally,calcium suppliers as fertilizers are applied widely in production.However,their role in induced resistance is seldom reported and the molecular mechanisms are unknown.In this study,selecting S.sclerotiorum and Xoo as pathogens of interest,and basal resistance and induced resistance to S.sclerotiorum and nonhost resistance to Xoo as resistance of interest,roles and molecular mechanisms underlying a calcium fertilizer-and CAMTA3-mediated resistance were analyzed.The main results are as follows:(1)Broad-spectrum regulatory roles of CAMTA3 in basal resistance and induced resistance to S.sclerotiorum and nonhost resistance to Xoo were elucidated and new salicylic acid(SA)biosynthesis pathway based on the CAMTA3-CBP60g/SARD1 module and its role in regulating the above resistances was revealed.Inoculation assays in Arabidopsis loss-of-function mutants cbp60g-1 and sardl-1 single and double mutants and sidl,sid2/icsl and nprl-1 single and double mutants demonstrated that CBP60g/SARD1-ICS 1-mediated SA biosynthesis and signaling pathway positively regulated these resistances.Inoculation analyses in Arabidopsis loss-of-function mutant camta3 and gain-of-function mutant camta3-3D showed that CAMTA3 negatively regulated these resistances.Expression of CBP60g and SARD1 genes was significantly upregulated and downregulated in plants of camta3 and camta3-3Dmutants,respectively compared to wild type.EMSA analysis indicated that CAMTA3 CG-1 domain specifically and strongly bound to CGCG boxes in promoter of CBP60g and SARD1 genes.Promoter activity LUC reporter assay showed that CAMTA3 reduced expression of CBP60g and SARD1 genes in planta and Pep1 and Xoo alleviated this negative regulatory role of CAMTA3.These results revealed that CAMTA3 directly and negatively regulated expression of CBP60g while probably indirectly down-regulated expression of SARD1,and Pep1 and Xoo suppressed this function of CAMTA3.Collectively,these results unveiled that CAMTA3 directly down-regulated CBP60g while indirectly down-regulated SARD1 to suppress SA biosynthesis and thereby negatively regulated resistances,and Pep1 and Xoo disabled CAMTA3 to induce resistances.(2)Role of calcium fertilizer in inducing resistance to S.sclerotiorum and the molecular mechanisms underlying this induced resistance were revealed.Leaf spray with calcium fertilizer(Effective component:CaCl2,0.3 mg/ml)significantly induced resistance to S.sclerotiorum and the resistance could last at least 5 days.The identical treatment of calcium fertilizer could not induce resistance to S.sclerotiorum in cbp60g-1 and sardl-1 mutants.LUC reporter assay showed that supply of calcium fertilizer repressed CAMTA3's suppression of CBP60g and SARD1 genes in planta while did not obviously altered expression of CAMTA3.These results uncovered that calcium fertilizer released CBP60g and SARD1 by inhibiting CAMTA3's suppression of these genes,thereby activated resistances.(3)The CAMTA3 target genes of jasmonic acid(JA),reactive oxygen species(ROS)and RNA intefering(RNAi)pathways were identified and thereby the involvement of CAMTA3 in regulating these pathways was revealed.Promoter cis-element analyses predicted that JA pathway genes JAZ1,JAZ2,JAZ5,JAZ7,JAZ8 and MYC2;ROS pathway NADPH oxidase gene RbohF and RNAi pathway genes AG04,AG05,AG06,RDR1,RDR3 and RDR4 contained CGCG boxes in their promoters.EMSA analysis indicated that Arabidopsis CAMTA3 CG-1 domain directly bound to CGCG boxes in promoters of 7 genes including AG04,AGO5,AGo6,RDR3,RBOHF,JAZ1 and MYC2.LUC reporter assay demonstrated that CAMTA3 increased expression of AGO5 and JAZ1 genes in planta.Collectively,this study revealed broad-spectrum regulatory roles of CAMTA3 in basal resistance and induced resistance to S.sclerotiorum and nonhost resistance to Xoo,uncovered a new SA biosynthesis regulatory pathway dependent on CAMTA3's suppression of CBP60g and SARD1,and elucidated the mechanisms underlying broad-spectrum resistance based on the above pathway.Moreover,the CAMTA3 target genes of JA,ROS and RNAi pathways were identified and thereby the involvement of CAMTA3 in regulating these pathways was revealed.Furthermore,role of calcium fertilizer in inducing resistance to S.sclerotiorum and the molecular mechanisms underlying this induced resistance were clarified.This study provides some insights into molecular mechanisms underlying resistances,and offers new potential ways to control S.sclerotiorum stem rot disease using Pep1 and calcium fertilizers.