Functions And Molecular Mechanisms Of RALF22 In Plant Immunity Against Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum（Lib.）de Bary（Ss）is a devastating necrotrophic plant pathogen with a worldwide distribution.White mold/stem rot disease caused by Ss is one of the most severe diseases in China,posing a significant threat to oil crops and vegetables.Nevertheless,the mechanism underlying plant resistance to Ss is still poorly understood.Plants recognize pathogen-associated molecular patterns（PAMPs）and damage-associated molecular patterns（DAMPs）by pattern recognition receptors（PRRs）localized on the cell surface to trigger PTI（PAMP-triggered immunity）and DTI（DAMP-triggered immunity）immune responses.Yet few studies have been conducted on plant immunity against necrotrophic pathogens.In this thesis,following the idea on the functions of RALFs（Rapid alkalinization factors）,a new type of DAMP,in plant immunity to Ss,the functions and molecular mechanisms of RALF22 in immunity against Ss in Brassica napus and Arabidopsis thaliana were analyzed.The main results obtained are as follows:（1）The role and mechanisms of RALF22 in the immunity against Ss in Arabidopsis were revealed.The functional analysis using the Arabidopsis RALF22 gene overexpression and mutant plants and the synthetic RALF22 mature peptide-treated plants demonstrated that RALF22 positively regulated the resistance to Ss.The synthetic RALF22 peptide induced resistance to Ss in a variety of cruciferous crops（oilseed rape,cabbage,Chinese cabbage）,and stimulated diverse PTI immune responses such as reactive oxygen species（ROS）production,cytoplasmic Ca²⁺elevation,MAPK（Mitogen-activated protein kinase）phosphorylation,callose deposition,and resistance marker gene expression.The immune activation and anti-Ss resistance induction function of RALF22 depended on FER（FERONIA）.RALF22-recognition co-receptor LLG1（LORELEI（LRE）-like GPI-anchored protein 1）,NADPH oxidase RBOHD（Respiratory burst oxidase homolog D）and phosphokinases that activate RBOHD,including BAK1（Brassinosteroid-insensitive 1（BRI1）associated receptor kinase 1）,BIK1（Botrytis-induced kinase 1）,RIPK（RPM1-induced protein kinase）,and CAT2（Catalase 2）played an important role in RALF22-elicited ROS burst and accumulation.The C-terminal conserved cysteines were essential to the RALF22-induced ROS production and anti-Ss immunity.The analysis on synergistic effect of RALF22 and At Pep3（Arabidopsis thaliana plant elicitor peptide 3）and transcriptional regulation revealed that RALF22 enhanced the At Pep3-elicited ROS accumulation and anti-Ss immunity,and RALF22 may amplified the At Pep3-triggered immune responses via strongly augmenting the At Pep3 gene transcription.（2）The mechanism that FER regulated RALF-stimulated plant immunity to Ss through direct interaction with ASN2（Asparagine synthase 2）was unveiled.ASN2 was identified to be a component downstream to and binding to FER by using immunoprecipitation coupled with mass spectrometry（IP-MS）,which was verified by adopting other assays including Y2H（Yeast two-hybrid）,Bi FC（Bimolecular fluorescence complementation）,LCA（Luciferase complementation assay）and Co-IP（Co-immunoprecipitation）.Inoculation analysis of Arabidopsis ASN2 gene overexpression and mutant plants showed that ASN2 positively regulated the resistance to Ss.（3）The RALF and Cr RLK1L（Catharanthus roseus receptor-like kinase）families were identified in B.napus at genome-wide level and the function and mechanisms of Bn RALF10 and Bn FER1 in resistance to Ss were revealed.Genome of B.napus possessed61 Bn RALF and 45 Cr RLK1L genes,which were divided into 4 and 5 groups,respectively.The expression profiles of 61 Bn RALF genes in response to Ss infection,Ssnlp1 and Bn Pep5 treatments were identified,and about 40%of the genes were responsive to these treatments.Bn RALF10 was the ortholog of Arabidopsis RALF22.The synthetic Bn RALF10 mature peptide stimulated PTI immune responses such as ROS production and cytoplasmic Ca²⁺elevation,and induced resistance to Ss.Protein-protein interaction assays including Y2H,Bi FC and LCA and genetic analysis proved that Bn FER1 was the receptor of Bn RALF10.Functional analysis by VIGS（Virus-induced gene silencing）and using rape transgenic materials demonstrated that Bn RALF10 and Bn FER1 genes played positive role in resistance to Ss in rape.Comparative proteomics analyses were performed for Bn RALF10 synthetic peptide-treated and dd H₂O-treated rape leaves,and 314 differentially expressed proteins were identified.Secretion system,redox homeostasis,Ca²⁺and other immune signals,MAPK signaling pathway might participate in regulation of Bn RALF10-stimulated resistance to Ss.（4）The important role of p H in the interaction between plants and Ss was discovered and its underlying molecular mechanism was probed.Analysis of the influence of p H on the growth and pathogenicity of Ss uncovered that p H was negatively correlated with the growth and pathogenicity of Ss.Acidic condition was beneficial to the growth and host infection of Ss.Inoculation analysis of rape plants cultivated with nutrient solutions at different p Hs revealed that p H was positively correlated with rape immunity to Ss.Alkaline environment was favorable to rape resistance to Ss.The steady-state accumulation of ROS in rape leaves and the Ss hyphae was negatively correlated with the environmental p H,and high p H inhibited the steady-state accumulation of ROS.RNA-seq analysis showed that cultivation of rape seedlings under nutrient solutions at different p Hs resulted in gene transcription reprogramming,and high p H enabled the transcription of genes involved in the MAPK signal pathway,hormone signal pathway,plant pathogen interaction,redox balance and other pathway in favor to scavenge ROS and improve disease resistance.Collectively,this study uncovered the functions and molecular mechanisms of RALF22 in immunity against Ss,revealed the important effects of p H on rape-Ss interactions,and unveiled novel mechanisms underlying plant immunity against Ss,thereby provided new strategies for green management of white mold disease.