Roles of Arabidopsis WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens

<p>Abstract</p> <p>Background</p> <p>Plant WRKY DNA-binding transcription factors are involved in plant responses to biotic and abiotic responses. It has been previously shown that <it>Arabidopsis WRKY3 </it>and <it>WRKY4</it>, which encode two structurally similar WRKY transcription factors, are induced by pathogen infection and salicylic acid (SA). However, the role of the two WRKY transcription factors in plant disease resistance has not been directly analyzed.</p> <p>Results</p> <p>Both WRKY3 and WRKY4 are nuclear-localized and specifically recognize the TTGACC W-box sequences <it>in vitro</it>. Expression of <it>WRKY3 </it>and <it>WRKY4 </it>was induced rapidly by stress conditions generated by liquid infiltration or spraying. Stress-induced expression of <it>WRKY4 </it>was further elevated by pathogen infection and SA treatment. To determine directly their role in plant disease resistance, we have isolated T-DNA insertion mutants and generated transgenic overexpression lines for <it>WRKY3 </it>and <it>WRKY4</it>. Both the loss-of-function mutants and transgenic overexpression lines were examined for responses to the biotrophic bacterial pathogen <it>Pseudomonas syringae </it>and the necrotrophic fungal pathogen <it>Botrytis cinerea</it>. The <it>wrky3 </it>and <it>wrky4 </it>single and double mutants exhibited more severe disease symptoms and support higher fungal growth than wild-type plants after <it>Botrytis </it>infection. Although disruption of <it>WRKY3 </it>and <it>WRKY4 </it>did not have a major effect on plant response to <it>P. syringae</it>, overexpression of <it>WRKY4 </it>greatly enhanced plant susceptibility to the bacterial pathogen and suppressed pathogen-induced <it>PR1 </it>gene expression.</p> <p>Conclusion</p> <p>The nuclear localization and sequence-specific DNA-binding activity support that WRKY3 and WRKY4 function as transcription factors. Functional analysis based on T-DNA insertion mutants and transgenic overexpression lines indicates that WRKY3 and WRKY4 have a positive role in plant resistance to necrotrophic pathogens and WRKY4 has a negative effect on plant resistance to biotrophic pathogens.</p

Functional analysis of Arabidopsis WRKY25 transcription factor in plant defense against Pseudomonas syringae

BACKGROUND: A common feature of plant defense responses is the transcriptional regulation of a large number of genes upon pathogen infection or treatment with pathogen elicitors. A large body of evidence suggests that plant WRKY transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. However, there is only limited information about the roles of specific WRKY DNA-binding transcription factors in plant defense. RESULTS: We analyzed the role of the WRKY25 transcription factor from Arabidopsis in plant defense against the bacterial pathogen Pseudomonas syringae. WRKY25 protein recognizes the TTGACC W-box sequences and its translational fusion with green fluorescent protein is localized to the nucleus. WRKY25 expression is responsive to general environmental stress. Analysis of stress-induced WRKY25 in the defense signaling mutants npr1, sid2, ein2 and coi1 further indicated that this gene is positively regulated by the salicylic acid (SA) signaling pathway and negatively regulated by the jasmonic acid 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. CONCLUSION: The nuclear localization and sequence-specific DNA-binding activity support that WRKY25 functions as a transcription factor. Based on analysis of both T-DNA insertion mutants and transgenic overexpression lines, stress-induced WRKY25 functions as a negative regulator of SA-mediated defense responses to P. syringae. This proposed role is consistent with the recent finding that WRKY25 is a substrate of Arabidopsis MAP kinase 4, a repressor of SA-dependent defense responses

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

Phosphorylation of a WRKY Transcription Factor by Two Pathogen-Responsive MAPKs Drives Phytoalexin Biosynthesis in Arabidopsis[C][W]

WRKY33 functions downstream of pathogen-responsive MPK3 and MPK6 in reprogramming the expression of camalexin biosynthetic genes; this drives the metabolic flow to camalexin production in Arabidopsis challenged by pathogens. Biochemical and genetic analyses demonstrate that the phosphorylation of WRKY33 by MPK3/MPK6 plays an important role in the process

Roles of WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens-8

Xes, while in the mPchn0 probe, the TTGACC sequences are mutated to TTGAAC. The wild-type and mutated W-box sequences are underlined. . Electrophoretic mobility shift assay (EMSA) of DNA binding of the WRKY3 and WRKY4 recombinant proteins.<p><b>Copyright information:</b></p><p>Taken from "Roles of WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens"</p><p>http://www.biomedcentral.com/1471-2229/8/68</p><p>BMC Plant Biology 2008;8():68-68.</p><p>Published online 20 Jun 2008</p><p>PMCID:PMC2464603.</p><p></p

Roles of WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens-6

-overexpressing lines 7 and 13 (and ) were infiltrated with a suspension of DC3000 (OD= 0.0001 in 10 mM MgCl). Samples were taken at 0 (open bars) or 3 days (closed bars) post inoculation (dpi) to determine the growth of the bacterial pathogen. The means and standard errors colony-forming units (cfu) were calculated from 10 plants for each treatment. Disease symptom development. Pathogen inoculation of wild type, mutants and overexpression lines was performed as in . Pictures of representative inoculated leaves taken at 4 dpi. Pathogen-induced expression. Wild type and -overexpressing plants were infiltrated with a suspension of DC3000 (OD= 0.0001 in 10 mM MgCl). Inoculated leaves were collected at indicated dpi for RNA isolation. RNA gel blot analysis was performed with P-labeled . These experiments were repeated three times with similar results.<p><b>Copyright information:</b></p><p>Taken from "Roles of WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens"</p><p>http://www.biomedcentral.com/1471-2229/8/68</p><p>BMC Plant Biology 2008;8():68-68.</p><p>Published online 20 Jun 2008</p><p>PMCID:PMC2464603.</p><p></p

Roles of WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens-1

Xes, while in the mPchn0 probe, the TTGACC sequences are mutated to TTGAAC. The wild-type and mutated W-box sequences are underlined. . Electrophoretic mobility shift assay (EMSA) of DNA binding of the WRKY3 and WRKY4 recombinant proteins.<p><b>Copyright information:</b></p><p>Taken from "Roles of WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens"</p><p>http://www.biomedcentral.com/1471-2229/8/68</p><p>BMC Plant Biology 2008;8():68-68.</p><p>Published online 20 Jun 2008</p><p>PMCID:PMC2464603.</p><p></p

Roles of WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens-4

Nd mutants. Wild-type and mutant plants were sprayed with SA (1 mM). The leaves were harvested 4 hours after treatment for total RNA isolation. After separation on the gels and blotting to nylon membranes, the blots were probed with corresponding gene-specific DNA fragments. and expression in transgenic plants. RNA samples were prepared from leaves of 5-week-old wild-type (Col-0) and transgenic plants and probed with a - or -specific probe. Transgenic lines 3 and 8 and transgenic lines 7 and 13 contained a single T-DNA insertion in the genome and exhibited stable expression of their respective transgenes. Their F3 homozygous progeny plants were used in all the experiments in the study.<p><b>Copyright information:</b></p><p>Taken from "Roles of WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens"</p><p>http://www.biomedcentral.com/1471-2229/8/68</p><p>BMC Plant Biology 2008;8():68-68.</p><p>Published online 20 Jun 2008</p><p>PMCID:PMC2464603.</p><p></p