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Abstract

Plants face constant threats from various microbial pathogens. Successful sensing and defending against infections are essential to their survival. Arabidopsis thaliana receptor like protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2) is of great importance to plant basal immunity. The transcription factors CALMODULIN-BINDING PROTEIN 60g (CBP60g) and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) define two parallel signaling pathways downstream of SNC2 to activate defense responses. In my PhD research, I aimed to identify the components that are involved in the CBP60g-dependent signaling pathway downstream of SNC2. From a forward genetic screen, mutants with suppressed autoimmunity of sard1-1 snc2-1D were isolated. Characterization of suppressor mutant 125-1 revealed that Epsin-like clathrin adaptor ECA4 is an inhibiting factor of SNC2-mediated immunity. A gain-of-function allele eca4-4D or overexpression of ECA4 leads to enhanced susceptibility against pathogens. Transient expression of ECA4 or eca4-4D in Nicotiana benthamiana reduces the protein levels of BDA1, a transmembrane protein required for SNC2-mediated immunity. Additionally, eca4-4D partially suppresses the autoimmunity of bda1-17D. Taken together, ECA4 contributes to SNC2-mediated immunity by negatively modulating the protein level of BDA1. 116-1 is a partial suppressor of sard1-1 snc2-1D autoimmunity, which carries a premature stop codon in ZINC-FINGER AND OCRE DOMAIN-CONTAINING PROTEIN 1 (ZOP1). ZOP1 encodes a pre-mRNA splicing factor that is involved in gene regulation. zop1 mutant exhibits enhanced susceptibility against pathogens. Moreover, many defense-related genes, including BDA1, were downregulated in the zop1 plants. Altogether, ZOP1 positively contributes to plant immunity through regulating the transcript levels of defense related genes. Studies of the suppressor mutant 123-1 uncovered the role of deubiquitinating enzyme ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 1 (AMSH1) in SNC2-mediated immunity. Loss-of-function mutation in AMSH1 caused partial suppression of sard1-1 snc2-1D’s autoimmunity. BDA1 might be a substrate of AMSH1, as silencing AMSH1 homologs in Nicotiana benthamiana results in lower BDA1 protein levels. Thus, AMSH1 likely contributes to immunity regulation through controlling the stability of BDA1. In summary, my Ph.D. research expands the understanding of regulators involved in the CBP60g-dependent signaling pathway downstream of SNC2 and emphasizes the importance of homeostasis control of key immune regulators, such as BDA1.