Regulation of plant defense against biotrophic and necrotrophic pathogens

Continuously exposed to the challenges from various kinds of pathogens, plants have evolved multilayered and cooperative defense mechanisms to protect themselves, which are usually dependent on the small signaling molecules, such as salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). However, how plants manipulate these defense signaling pathways to lead to defense responses to pathogens is still poorly understood. The first part of my studies is the functional characterization of pathogen-induced Arabidopsis WRKY33 and stress-responsive WRKY25 in disease resistance. Disruption of WRKY33 resulted in enhanced susceptibility to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola concomitant with reduced expression of the JA/ET-regulated plant defensin PDF1.2 gene. Constitutive overexpression of WRKY33, on the other hand, increased resistance to the two necrotrophic fungal pathogens. Double mutant analysis indicated that the enhanced susceptibility of the wrky33 mutants to B. cinerea was SA-independent. In addition, some genes involved in resistance to necrotrophic pathogens were identified as WRKY33 target genes by microarray experiments. On the other hand, although constitutive overexpression of WRKY33 resulted in enhanced susceptibility to a virulent strain of the bacterial pathogen Pseudomonas syringae, the wrky33 mutants did not show altered responses to this pathogen. WRKY33 was localized to the nucleus of plant cells with strong transcriptional activation activities and recognized DNA molecules containing the TTGACC W-box sequence. Taken together, these results indicate that pathogen-induced WRKY33 is an important transcription factor that positively regulates plant defense responses to necrotrophic pathogens. On the other hand, WRKY25 is mainly involved in resistance to the bacterial pathogen P. syringae. The expression of WRKY25 was responsive to general environmental stresses and this stress-responsive expression was positively regulated by the SA signaling pathway and negatively regulated by the JA signaling pathway. Two independent T-DNA insertion mutants for WRKY25 supported normal growth of a virulent strain of P. syringae, but developed reduced disease symptoms after infection. By contrast, Arabidopsis constitutively overexpressing WRKY25 supported enhanced growth of P. syringae and displayed increased disease symptom severity as compared to wild-type plants. These WRKY25 -overexpressing plants also displayed reduced expression of the SA-regulated PR1 gene after the pathogen infection, despite normal levels of free SA. Thus, WRKY25 appears to function as a negative regulator of SA-mediated defense responses to P. syringae. In the second part of my studies, I identified and characterized the enhanced susceptibility to Pseudomonas 1 (esp1) mutant in Arabidopsis thaliana that exhibited enhanced susceptibility to both virulent and avirulent strains of P. syringae. The ESP1 gene was isolated through positional cloning and found to encode a novel member of the BAHD CoA-dependent acyl transferase superfamily. Pathogen-induced accumulation of SA and expression of pathogenesis-related (PR) genes were compromised in the esp1 mutant. Application of exogenous SA could rescue the impaired PR genes expression and disease resistance of the esp1 mutant, suggesting the ESP1 functions upstream of SA. In addition, several lines of evidence suggest that ESP1 functions together with the acyl-adenylate/thioester-forming enzyme PBS3 in the synthesis of a precursor or a regulatory molecule for SA biosynthesis. Thus, ESP1 plays a critical role in regulating pathogen-induced SA accumulation and resistance to P. syringae. In the No-0 background, disruption of the ESP1 gene resulted in enhanced resistance to necrotrophic fungal pathogens B. cinerea and A. brassicicola, but compromised tolerance to oxidative stress, high salinity, ABA and osmotic stress. These results suggest that ESP1 functions in modulating the crosstalk between SA- and JA-dependent pathways and in regulating the plant responses to abiotic stresses