DISSECTION OF THE SALICYLIC ACID-MEDIATED PLANT DEFENSE RESPONSES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0213615
• Project Start Date: Jan 1, 2008
• Project End Date: Sep 30, 2013
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Microbiology and Cell Science

Non Technical Summary
Salicylic acid (SA) is one of the most important signal molecules that regulate host responses in plant-pathogen interactions. Following a primary infection, SA accumulates in adjacent as well as distant tissues to induce a defense mechanism known as systemic acquired resistance (SAR), which provides protection against subsequent attacks by a broad spectrum of pathogens. Genetic screens performed in several laboratories have identified NPR1 as a key positive regulator of SAR. Characterization of the npr1 mutants has revealed that NPR1 has three functions in the SA-mediated signaling pathway: (1) activation of SAR; (2) feedback inhibition of the biosynthesis of SA; and (3) inhibition of the toxicity of SA. Our previous work has shown that SA changes plant cellular redox state to activate the NPR1 protein. However, how SA changes cellular redox state is unknown. Furthermore, although numerous studies have been conducted to understand the molecular function of NPR1 in the SA-mediated signaling pathway, less is known about its function in inhibition of the toxicity of SA. Lack of such knowledge is an important problem because it significantly limits our ability to modulate SAR for increasing plant resistance to pathogens. Accomplishment of this project will help understand the activation mechanism of SAR, which will undoubtedly provide new strategies for making crop plants more resistant to microbial attack.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2420	1040	50%
203	2499	1040	50%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2420 - Noncrop plant research; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology;

Keywords

salicylic acid

npr1

sar

redox

disease resistance

Goals / Objectives
The objective of this project is to elucidate the mechanism through which SA changes redox state of plant cells and the mechanism through which NPR1 inhibits the toxicity of SA. The central hypotheses of this project are: (1) SA regulates the oxidative pentose phosphate pathway (oxPPP), which leads to redox changes in the plant cells; (2) NPR1 regulates the expression of genes involving in SA detoxification. The hypotheses have been formulated based on preliminary characterization of several Arabidopsis mutants. One mutant, pgl, has a genetic mutation in an oxPPP gene. The pgl mutation perturbs the redox equilibrium of plant cells, resulting in redox state changes, which leads to NPR1-dependent defense responses. The defense responses activated by the pgl mutation mimic those activated by SA treatment, which led us to the first part of the central hypotheses. Another mutant, a suppressor of npr1, exhibits increased disease resistance, leading us to the second part of the central hypotheses. The rationale for the proposed research is that, once how SA changes redox state of plant cells and how the toxicity of SA is regulated are clear, they will help understand the activation mechanism of SAR, which will undoubtedly provide new strategies for making crop plants more resistant to microbial attack.

Project Methods
We plan to accomplish the objective of this application by pursuing the following two specific aims: (1) Determine the function of the oxidative pentose phosphate pathway (oxPPP) in plant defense responses. We will further characterize the Arabidopsis oxPPP mutant pgl. Transgenic plants containing a PGL:PGL-GFP transgene will be generated to determine the subcellular localization of the PGL protein. Double mutants pglnpr1, pglsid2, pgleds1, pglpad4, and pglndr1 will be generated and used to further dissect the defense responses activated in the pgl mutant. We will determine whether SA alters the expression of the PGL gene. We will use the PGL:PGL-GFP transgenic plants to determine whether SA regulates the accumulation of the PGL protein. We will also define whether the enzymatic activity of the PGL protein will be altered by SA treatment. Additionally, other oxPPP mutant will be characterized. (2) Identification and characterization of suppressors of npr1. We will screen for npr1 suppressors using its lethal phenotype on MS plates supplemented with 0.5 mM SA. In a pilot experiment, we have identified several suppressors. We will characterize the suppressors and clone the mutated genes. Meanwhile, we will saturate the mutant screen.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One garduate student and one undergrduate student were trained. How have the results been disseminated to communities of interest? Three papers were published. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A genetic screen for altered SA accumulation in the npr1 mutant background was performed. One mutant, which accumulates higher levels of SA than npr1, was identified and analyzed. The mutation causing the mutant pehnotypes was identified through map-based cloing.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: 4. DeFraia, C.T., Wang, Y., Yao, J. and Mou, Z. (2013). Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains. BMC Plant Biology 13, 102. 5. Zhang, X., Yao, J., Zhang, Y., Sun, Y. and Mou, Z. (2013). The Arabidopsis Mediator complex subunit MED14/SWP/ and MED16/SFR6/IEN1 differentially regulate defense gene expression in plant immune responses. Plant Journal 75, 484-497. 3. An, C., Orbovic, V. and Mou, Z. (2013). An efficient intragenic vector for generating intragenic and cisgenic plants in citrus. American Journal of Plant Sciences 4, 2131-2137.

Progress 01/01/08 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A graduate student and an undergraduate student were trained. How have the results been disseminated to communities of interest? Three papers were published. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A genetic screen for mutants with altered SA accumulation was performed. A mutant that accumulates significantly higher levels of SA was identified and charaterized. The mutation responsible for the mutant pehnotypes was identified through map-based cloning.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: An, C., Orbovic, V. and Mou, Z. (2013). An efficient intragenic vector for generating intragenic and cisgenic plants in citrus. American Journal of Plant Sciences 4, 2131-2137. DeFraia, C.T., Wang, Y., Yao, J. and Mou, Z. (2013). Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains. BMC Plant Biology 13, 102. Zhang, X., Yao, J., Zhang, Y., Sun, Y. and Mou, Z. (2013). The Arabidopsis Mediator complex subunit MED14/SWP/ and MED16/SFR6/IEN1 differentially regulate defense gene expression in plant immune responses. Plant Journal 75, 484-497.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: A genetic screen for mutants insensitive to exogenous NAD+ (ien) has been carried out. Several ien mutants have been identified, and one mutation, ien1, has been cloned. Furthermore, the human NAD(P)-metabolizing ectoenzyme CD38 has been expressed in Arabidopsis and the transgenic plants have been characterized. In addition, an Arabidopsis-Xanthomonas citri subsp. citri nonhost pathosystem has been established. PARTICIPANTS: Xudong Zhang, Biological Scientist, worked on the genetic screen for ien mutants and characterization and cloning of the ien1 mutation. She also worked on generation and characterization of transgenic Arabidopsis plants expressing the human CD38 gene. Chuanfu An, postdoc, worked on establishment of the Arabidopsis-Xanthomonas citri subsp. citri Pathosystem. Chenggang Wang, postdoc, worked on characterization of the ien1 mutant. Two postdocs and one graduate student have been trained. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Characterization of the ien1 mutant revealed that the Mediator subunit MED16 is a key regulator of salicylic acid-mediated systemic acquired resistance and jasmonate/ethylene-mediated defense responses. Characterization of Arabidopsis transgenic plants expressing the human CD38 gene suggested that extracellular NAD(P) may function as a signal molecule for activation of systemic acquired resistance. The Arabidopsis-Xanthomonas citri subsp. citri nonhost pathosystem holds great potential for identifying genes regulating resistance against the citrus canker-causing bacterial pathogen.

Publications

• Zhang, X., Wang, C., Zhang, Y., Sun, Y. and Mou, Z. (2012). The Arabidopsis Mediator complex subunit16 positively regulates salicylate-mediated systemic acquired resistance and jasmonate/ethylene-induced defense pathways. Plant Cell 24, 4294-4309.
• Zhang, X. and Mou, Z. (2012). Expression of the human NAD(P)-metabolizing ectoenzyme CD38 compromises systemic acquired resistance in Arabidopsis. Molecular Plant-Microbe Interactions 25, 1209-1218.
• An, C. and Mou, Z. (2012). Non-host defense response in a novel Arabidopsis-Xanthomonas citri subsp. Citri pathosystem. PLoS ONE 7(1): e31130.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Several Arabiddopsis mutants accumulating less salicylic acid after pathogen infection have been isolated. We are currently cloning the mutated genes. We have characterized the salicylic acid signaling mutant elp2 and are continuing the research on other elp mutants. PARTICIPANTS: George Mareck and Ryan Carver were undergraduate students. Chuanfu An is a postdoctoral researcher. TARGET AUDIENCES: The salicylic acid analysis method has been used in multiple labs around the world, which makes the analysis much faster. The elp mutant provided a new gene for the plant immunity community to study. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The fast salicylic acid quantification method has been used in multiple labs around the world. We have established the function of the ELP2 gene in plant defense responses, which added a new component in the signaling pathway of plant immunity.

Publications

• Marek, G., Carver, R., Ding, Y., Sathyanarayan, D., Zhang, X. and Mou, Z. (2010). A high-throughput method for isolation of salicylic acid metabolic mutants. Plant Methods 6, 21.2.
• An, C. and Mou, Z. (2011). Salicylic acic and its function in plant immunity. Journal of Integrative Plant Biology 53, 412-428.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Arabidopsis elp2 mutant has been isolated. Characterization of this mutant has been published in the Plant Journal. Transgenic citrus plants overexpressing the Arabidopsis NPR1 gene have been generated. These transgenic plants were shared with several citrus research labs at the Citrus Research and Education Center of the University of Florida to test their resistance to citrus diseases. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
ELP2 is a subunit of the transcription regulatory complex Elongator. Characterization of the Arabidopsis elp2 mutant demonstrated that the Elongator is an accelerator of plant immune responses. This work provided the foundation for further characterizing the Elongator complex and its function in plant immunity. Transgenic citrus plants exhibited increased resistance to citrus canker. This work provided a successful example of transferring disease resistance technology from the model plant Arabidopsis to crop plants.

Publications

• Defraia, T.C., Zhang X. and Mou, Z. (2010). Elongator subunit 2 is an accelerator of im-mune responses in Arabidopsis thaliana. Plant Journal 64, 511-523.
• Zhang, X., Francis, M.I., Dawson, W.O., Graham, J.H., Orbovic, V., Triplett, E.W. and Mou, Z. (2010). Overexpression of the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker. European Journal of Plant Pathology 128, 91-100.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Mutants generated in the study were shared with three laboratories. The idea of changing the activity of the oxPPP pathway has been applied to citrus research in selecting mutants with increased disease resistance. PARTICIPANTS: My postdoc Dr. Yuqing Xiong was trained in this project and published two articles. My graduate student Chris DeFraia participated in the research and was trained in plant molecular biology. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Mutant analysis indicated that the oxPPP pathway may play an important role in plant immunity.

Publications

• Xiong, Y., DeFraia, C., Williams, D., Zhang, X. and Mou, Z. (2009). Deficiency in a cytosolic ribose-5-phosphate isomerase results in chloroplast dysfunction, late flowering and premature cell death in Arabidopsis. Physiologia Plantarum 137, 249-263
• Xiong, Y., DeFraia, C., Williams, D., Zhang, X. and Mou, Z. (2009). Characterization of Arabidopsis 6-phosphogluconolactonase T-DNA insertion mutants reveals an essential role of the oxidative section of the plastidic pentose phosphate pathway in plant growth and development. Plant & Cell Physiology 50, 1277-1291.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Mutants of the Arabidopsis PGL1, PGL2, PGL3, and PGL5 have been isolated and the defense responses in the pgl3 mutant have been characterized. Double mutants pgl3sid2 and pgl3npr1 have been generated and characterized. Mutants of the Arabidopsis RPI2 gene were also isolated and characterized. Results from these studies have been included in two manuscripts, which are currently under review. The results will also be reported as abstracts on the 20th international conference on Arabidosis research and the plant biology 2009 meeting. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The Arabidopsis PGL1, PGL2, PGL4 and PGL5 are predicted to encode cytosolic isoforms of 6-phosphogluconolactonase (6PGL), whereas PGL3 is predicted to encode a 6PGL that has been shown to localize in both plastids and peroxisomes. Therefore, 6PGL may exist in the cytosol, plastids, and peroxisomes. However, the function of 6PGL in the three subcellular locations has not been well defined. Here we show that PGL3 is essential, whereas PGL1, PGL2, and PGL5 are individually dispensable for plant growth and development. Knockdown of PGL3 in the pgl3 mutant leads to a dramatic decrease in plant size, a significant increase in total glucose-6-phosphate dehydrogenase activity, and a marked decrease in cellular redox potential. Interestingly, the pgl3 plants exhibit constitutive pathogenesis-related gene expression and enhanced resistance to Pseudomonas syringae pv. maculicola ES4326 and Hyaloperonospora parasitica Noco2. We found that, though pgl3 does not spontaneously accumulate elevated levels of free salicylic acid (SA), the constitutive defense responses in pgl3 plants are almost completely suppressed by the npr1 and sid2/eds16/ics1 mutations, suggesting that the pgl3 mutation activates NPR1- and SID2/EDS16/ICS1-dependent defense responses. We demonstrate that plastidic (not peroxisomal) localization and 6PGL activity of the PGL3 protein are essential for complementing all pgl3 phenotypes, indicating that the oxidative section of the plastidic pentose phosphate pathway (PPP) is required for plant normal growth and development. Thus, pgl3 provides a useful tool not only for defining the role of the PPP in different subcellular compartments, but also for dissecting the SA/NPR1-mediated signaling pathway. We also characterized Arabidopsis T-DNA knockout mutants of the RPI2 gene, which encodes a cytosolic ribose-5-phosphate isomerase (RPI) that catalyzes the reversible interconversion of ribulose-5-phosphate and ribose-5-phosphate in the nonoxidative stage of the oxPPP. Although recombinant Arabidopsis RPI2 protein exhibits marked RPI enzymatic activity, knockout of the RPI2 gene does not significantly change the total RPI activity in the mutant plants. Interestingly, knockout of RPI2 interferes with chloroplast structure and decreases chloroplast photosynthetic capacity. The rpi2 mutants accumulate less starch in the leaves and flower significantly later than wild type when grown under short-day conditions. Furthermore, the rpi2 mutants display premature cell death in the leaves when grown at an above-normal temperature. These results demonstrate that a deficiency in the nonoxidative stage of the oxPPP has pleiotropic effects on plant growth and development and causes premature cell death.

Publications

• No publications reported this period