Identification and analysis of Arabidopsis WRKY33-interacting proteins in plant defense

Arabidopsis WRKY33 transcription factor positively regulates plant resistance to necrotrophic fungal pathogens. Mutation of WRKY33 enhances plant susceptibility to necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola. Ectopic overexpression of WRKY33 increases plant resistance to these two necrotrophic pathogens. It has been previously shown that WRKY33 interacts with MKS1, a MPK4 substrate that contains the conserved VQ motif. In the absence of pathogen infection, MPK4, MKS1 and WRKY33 are present as nuclear complexes. After infection with the bacterial pathogen Pseudomonas syringae or treatment with a functional analog of salicylic acid, activated MPK4 phosphorylates MKS1 and releases the MKS1/WRKY33 complexes, allowing WRKY33 to target the promoter of PAD3 and activate its expression. This MPK4-releasing-WRKY33 model is consistent with the opposite roles of MPK4 and WRKY33 in the regulation of PAD3. However, the mks1 mutants do not share the common phenotype of hyper-susceptibility to necrotrophic pathogens, suggesting existence of additional mechanisms in WRKY33-mediated gene expression. Using yeast-two hybrid screens, we have identified three additional WRKY33-interacting proteins: SIGMA FACTOR BINDING PROTEIN 1 (SIB1), SIGMA FACTOR BINDING PROTEIN 2 (SIB2), and AUTOPHAGY 18a (ATG18a). Biomolecular fluorescence complementation (BiFC) assays confirm that WRKY33 interacts with these three proteins in the nucleus. As with WRKY33, genes encoding the WRKY33-interacting proteins: SIB1, SIB2 and ATG18a are induced by B. cinerea. Like MKS1, SIB1 and SIB2 each contain a short VQ motif and analysis with SIB1 indicates that the VQ motif is required for its interacting with WRKY33. Pathogen inoculation assays show that T-DNA insertion mutants for SIB1 and SIB2 are hypersusceptible to B. cinerea. DNA-binding assays indicate that SIB1 and SIB2 can enhance the DNA-binding activity of WRKY33 but do not affect the transcription-regulatory activity of WRKY33 in plants. SIB1 and SIB2 were previously reported to have function in chloroplasts, but the function of SIB1 and SIB2 in chloroplasts does not appear to play a role in resistance to B. cinerea. These results strongly suggest that SIB1 and SIB2 are coactivators of WRKY33 in plant defense against B. cinerea through enhancing WRKY33 DNA-binding activity. ATG18a, which also interacts with WRKY33, is a critical autophagy protein in Arabidopsis. Transcripts of autophagy genes and formation of autophagosome are induced in Arabidopsis by the necrotrophic fungal pathogen Botrytis cinerea. Induction of ATG18a and autophagy by B. cinerea was compromised in the wrky33 mutant, which is highly susceptible to necrotrophic pathogens. Arabidopsis mutants defective in autophagy exhibit enhanced susceptibility to the necrotrophic fungal pathogens B. cinerea and Alternaria brassicicola based on increased pathogen growth in the mutants. The hypersusceptibility of the autophagy mutants was associated with reduced expression of the jasmmonate-regulated gene PFD1.2, accelerated development of senescence-like chlorotic symptoms and increased protein degradation in infected plant tissues. These results strongly suggest that autophagy cooperates with jasmonate- and WRKY33-mediated signaling pathways in the regulation of plant defense responses to necrotrophic pathogens