Source: UNIVERSITY OF MISSOURI submitted to
PATHOGEN-RESPONSIVE MITOGEN-ACTIVATED PROTEIN KINASE CASCADE IN SIGNALING PLANT PHYTOALEXIN BIOSYNTHESIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0213541
Grant No.
(N/A)
Project No.
MO-BCSL0704
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Nov 5, 2006
Project End Date
Nov 4, 2011
Grant Year
(N/A)
Project Director
Zhang, S.
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
BIOCHEMISTRY
Non Technical Summary
Plant resistance to pathogens correlates with the production of antimicrobial phytoalexins. We found that phytoalexin induction, an integral part of the induced plant disease resistance, is regulated by MPK3/MPK6 cascade in Arabidopsis. In this project, we will use Arabidopsis as a model system to determine the underlying molecular mechanism. The knowledge gained can be used to investigate the signaling pathway in crops, which may eventually lead to crops with enhance disease resistance. The contribution of this research to basic science research includes: 1) identifying new signaling components in pathogen-induced phytoalexin biosynthesis; 2) identifying new MAPK substrate; and 3) revealing one of the mechanisms by which MPK3/MPK6 cascade regulates disease resistance. The biosynthetic pathways of a number of phytoalexins have been fully defined. However, the signaling pathway is largely unknown. Our research identified the Arabidopsis MPK3/MPK6 cascade as an important component in the signaling pathway leading to phytoalexin biosynthesis (Previous Work and Present Outlook). The proposed research will identify additional components in the pathway and reveal how phytoalexin biosynthesis is induced after recognition of pathogen-associated molecular patterns (PAMPs) and Avr factors.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2062499100010%
2062499101020%
2062499116020%
2122499100010%
2122499101020%
2122499116020%
Goals / Objectives
Our long-term goal is to identify components in plant defense signaling pathway(s) that can be manipulated to confer broad-spectrum resistance to plants. Plant disease has a major negative impact on crops. A number of strategies/methods have been proposed/used to fight against plant diseases. One of the attractive strategies is to enhance the plant's own defense system by engineering regulatory components in resistance signaling pathways. Such an approach will lead to the generation of crops with resistance against a broad spectrum of pathogens, because similar defense responses are elicited in plants by viral, bacterial and fungal pathogens, suggesting shared signaling pathway(s). The MAPK cascade that we have studied for almost ten years is a converging point in the signaling pathway, therefore, a suitable target for crop improvement. We found that camalexin induction, an important event in Arabidopsis disease resistance, is regulated by MPK3/MPK6 cascade. Figure 1 is our current model depicting how MEKK1/MAPKKK?-MKK4/MKK5-MPK3/MPK6 cascade regulates the biosynthesis of camalexin. It is likely that MPK3/MPK6 can activate a transcription factor or transcription factors that can coordinate the induction of all rate-limiting enzymes (PAD3 and others) in the biosynthetic pathway, which channels the metabolism to produce camalexin. In the next five years, we are going to identify additional components downstream of MPK3/MPK6 cascade using PAD3 gene as an entry point. PAD3 is a critical enzyme in the phytoalexin biosynthesis and is highly responsive to MAPK activation. The specific objectives of are: 1) To identify the cis-element involved in MAPK-induced PAD3 gene activation. 2) To identify the trans-acting factor(s) involved in MAPK-induced PAD3 gene activation. They are likely to coordinate the activation of other genes in the camalexin biosynthetic pathway. 3) To determine the MAPK substrate(s) involved in the induction of phytoalexin
Project Methods
1. Identification of cis-acting element in the MAPK signaling pathway that lead to PAD3 gene activation: Multi-gene activation is involved in the induction of camalexin and the up-regulation of these genes is coordinated by the same MAPK pathway. We are going to use PAD3 as an entry point to identify additional components in the signaling pathway. The dramatic activation of PAD3 gene (more than 1000-fold increase based on qRT-PCR) after MPK3/MPK6 activation should facilitate the identification of cis-element in the PAD3 promoter. 2. Identification of trans-acting DNA-binding protein(s) in the MAPK signaling pathway that interact with the cis-acting elements in PAD3 promoter, or PAD3-promoter binding protein (PBP) using following approaches: i) Affinity purification and mass spectrometry identification of PBP, and ii) Yeast one-hybrid cloning of PBP. 3. Functional analysis of the identified PBP trans-acting factor: Further studies will be carried out to determine the role of the PBP trans-acting factor in MPK3/MPK6-induced PAD3 transcriptional activation. Understanding how the activity of PBP factor is regulated by MAPK will fill the gap between MAPK activation and PAD3 gene activation. 4. To determine if the identified PBP trans-acting factor(s) are MPK3/MPK6 substrate(s): We will look into the possibility of post-translation modification, especially phosphorylation by MPK3 and MPK6. Putative phosphorylation can be identified by searching the sequence for Px(S/T)P and (S/T)P motifs. Sequence conservation across the species and the existence of MAPK docking domain will be used to narrow down the MAPK phosphorylation sites. The same set of experiments as we did to identify ACS as MPK6 substrate will be performed, including preparation of recombinant PBP protein, phosphorylation assay using active MPK3 and MPK6, site-directed mutagenesis to generate mutant PBP proteins to confirm the phosphorylation site(s), in vivo analysis of gain- and loss-of-function PBP mutants. To mimic the phosphorylated PBP protein, we will mutate the Ser/The to Asp to generate a potentially gain-of-function (GOF) mutant. Overexpression of such mutant may lead to PAD3 gene activation in the absence of MAPK phosphorylation. In addition, we will generate the loss-of-function (LOF) PBP mutant by mutating the Ser/Thr residue to Ala, which will render the mutant irresponsive to MAPK activation. 5. Coordinated up-regulation of multiple enzymes leads to the phytoalexin induction: Camalexin is synthesized from tryptophan via indole-3-acetaldoxime in a reaction catalyzed by CYP79B2 and CYP79B3. We found that genes encoding enzymes in tryptophan and camalexin biosynthetic pathways are highly induced after MPK3/MPK6 activation. To link the identified transcription factors with the genes in camalexin biosynthetic pathway, we will determine the expression of these genes in PBP mutant/RNAi, PBPGOF, and PBPLOF plants by qRT-PCR. Gene expression profiling of these plants may also allow us to identify other unknown genes under the control of PBP.

Progress 11/05/06 to 11/04/11

Outputs
OUTPUTS: Plant metabolism is highly adaptive and changes rapidly after the sensing of extracellular stimuli. One good example is the rapid induction of phytoalexins, small secondary metabolites with antimicrobial activity in response to pathogen invasion, which is an integral part of plant defense. Biosynthetic pathways of a number of phytoalexins have been elucidated, and it is known that the induction of phytoalexin is associated with the activation of biosynthetic genes. However, the signaling pathway(s), downstream transcription factor(s), and how these signaling event(s) regulate transcription factor(s) are largely unknown. In search of the transcription factors downstream of MPK3/MPK6, we found that WRKY33 is required for MPK3/MPK6-induced camalexin biosynthesis. In wrky33 mutants, both gain-of-function MPK3/MPK6- and pathogen-induced camalexin production are compromised, which is associated with the loss of camalexin biosynthetic gene activation. WRKY33 is a pathogen-inducible transcription factor, whose expression is regulated by the MPK3/MPK6 cascade. Chromatin-immunoprecipitation assays reveal that WRKY33 binds to its own promoter in vivo, suggesting a potential positive feedback regulatory loop. Furthermore, WRKY33 is a substrate of MPK3/MPK6. Mutation of MPK3/MPK6-phosphorylation sites in WRKY33 compromises its ability to complement the camalexin induction in wrky33 mutant. Using a phospho-protein mobility shift assay, we demonstrate that WRKY33 is phosphorylated by MPK3/MPK6 in vivo in response to B. cinerea infection. Based on these data, we conclude that WRKY33 functions downstream of MPK3/MPK6 in reprogramming the expression of camalexin biosynthetic genes, which drives the metabolic flow to camalexin production in Arabidopsis challenged by pathogens. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In animals and yeast, stress-responsive MAPKs regulate gene expression by direct phosphorylation of transcription factors. It has been speculated that plant stress/pathogen-responsive MAPKs have similar mechanism of action, although the direct evidence is scarce. WRKY transcription factors are unique to plants, and Arabidopsis has more than 70 members. Detailed characterization of WRKY33, a newly identified MPK3/MPK6 substrate, and its target genes will further the understanding of MAPK signaling in plants as well as the functional diversity and signaling specificity of MPK3 and MPK6. The tools generated in this project will have broad applications and will be shared with other groups. This project served as an excellent training environment for students/post-docs by using an integrative approach encompassing both experimental and computational biology to understand the roles of a MAPK cascade and its downstream transcription factor in reprogramming plant metabolism. Training of students and post-docs who can use a combinatory approach to study complex biological processes is critical to the advance of post-genome biology. In addition to scientific methodology, students/post-docs will also receive training in ethics and career development. Students (both graduate and undergraduate) and post-docs from under-represented groups will be actively recruited through institutional programs that reach out to minorities. Post-docs involved in the project will follow a career development plan supported by the research training in the lab, student mentoring experiences, and professional development training. Understanding the signal transduction pathways and the identification of important regulators of plant secondary metabolism could positively impact food/feed/biofuel production and/or quality, which is important to sustain the increasing world population.

Publications

  • Mao, G., Meng, X., Liu, Y., Zheng, Z., Chen, Z., and Zhang, S. (2011) Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell, 23: 1639-1653. (Highlighted in the Plant Cell In Brief)
  • Ren, D., Liu, Y., Yang, K.-Y., Han, L., Mao, G., Glazebrook, J., and Zhang, S. (2008) Fungal-responsive mitogen-activated protein kinase cascade in signaling phytoalexin biosynthesis in Arabidopsis. Proceedings of the National Academy Sciences USA. 105: 5638-5643


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Induction of anti-microbial phytoalexins is an integral part of plant defense response. Camalexin, the major phytoalexin in Arabidopsis, is essential for fungal resistance. We recently demonstrated MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs) play essential role in signaling the Botrytis cinerea-induced camalexin biosynthesis (Ren et al., 2008). Genetic analysis placed the MPK3/MPK6 cascade upstream of PHYTOALEXIN DEFICIENT 2 (PAD2) and PAD3, but independent or downstream of PAD1 and PAD4. Camalexin induction after MPK3/MPK6 activation was preceded by rapid and coordinated up-regulation of multiple genes encoding enzymes in the tryptophan (Trp) biosynthetic pathway, in the conversion of Trp to indole-3-acetaldoxime (IAOx, a branch point between primary and secondary metabolism), and in the camalexin biosynthetic pathway downstream of IAOx. In search of the transcription factors downstream of MPK3/MPK6 in regulating defense-related gene expression, we found that a WRKY transcription factor is required for MPK3/MPK6-induced camalexin biosynthesis. In wrky33 mutant background, both the gain-of-function MPK3/MPK6- and pathogen-induced camalexin production are compromised, which is associated with the loss of activation of camalexin biosynthetic genes. WRKY33 is a pathogen-inducible transcription factor, whose expression is regulated by MPK3/MPK6 cascade. In addition, WRKY33 protein is phosphorylated by MPK3/MPK6 in vivo in response to B. cinerea infection or the activation on MPK3/MPK6 in the gain-of-function transgenic plants. Mutation of MPK3/MPK6-phosphorylation sites in WRKY33 compromises the camalexin induction in response to B. cinerea attack and in conditional gain-of- function MPK3/MPK6 system. We further demonstrated that WRKY33 is phosphorylated by MPK3/MPK6 in vivo in response to B. cinerea. These results provide direct evidences that WRKY33 is a substrate of MPK3/MPK6 and is involved in regulating camalexin biosynthesis in Arabidopsis. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Activation of biosynthetic genes is known to be associated with the induction of phytoalexins in plants challenged by pathogens. However, the transcription factor(s) and regulatory pathways are largely unknown. Built on the finding of a specific MAPK cascade in signaling the pathogen-induced biosynthesis of camalexin, the major phytoalexin in Arabidopsis, we identified a WRKY transcription factor downstream of MPK3/MPK6 cascade. WRKY33 is regulated at two levels, transcriptional activation and post-translational phosphorylation by MPK3/MPK6. Understanding of the regulation of phytoalexin biosynthesis in plants may lead to the engineering of crops with enhanced resistance.

Publications