Necrotrophic pathogens cause diseases by killing plant cells during early stages of infection to extract nutrients from dead plant cells for pathogen growth and proliferation. To induce plant cell death, necrotrophic pathogens release toxins, reactive oxygen species and hydrolytic enzymes into plant tissues. Plant immune responses against necrotrophic pathogens involve defense mechanisms such as production of antimicrobial compounds and suppression of pathogen-induced cell death. How distinct defense responses are coordinately regulated in plant cells is poorly understood. Arabidopsis WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens. Plants lacking a functional WRKY33 gene display greatly increased susceptibility to necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola. As a transcription factor, WRKY33 regulates plant nuclear genes involved in defense responses. In addition, WRKY33 physically interacts with proteins involved in chloroplast transcription and autophagy, a protein degradation pathway critical for nutrient recycling and cell survival. This research will analyze the regulation, functions and coordination of the WRKY33, chloroplast transcription and autophagy pathways in plant defense against necrotrophic pathogens. The investigators will continue functional analysis of WRKY33-interacting chloroplast transcription factors in plant resistance to necrotrophic pathogens. The investigators will examine activation, roles and action mechanisms of autophagy in promoting plant cell survival during responses to necrotrophic pathogens. In addition, the investigators will analyze functional interactions of the three pathways required for an integrated immune response to necrotrophic pathogens. Progress in these studies will help establish a comprehensive plant immune system against necrotrophic pathogens. This knowledge is necessary for development of new and novel strategies for controlling necrotrophic pathogens, which cause many devastating diseases in crop and forest plants. The project will provide training for both graduate and undergraduate students, including students underrepresented in sciences.

Project Report

Necrotrophic pathogens are an important group of plant pathogens notorious for their aggressiveness by actively killing plant cells before colonization and extracting nutrients from dead plant tissues. Plant resistance to necrotrophic pathogens is often controlled by many genes, making breeding difficult to generate disease-resistant cultivars and currently the major means for controlling necrotrophic pathogens is through fungicide application, which is costly and environmentally unsound. Because of the complex genetic nature, our understanding of the underlying molecular mechanisms for plant resistance to necrotrophic pathogens is also very limited. The overall goal of the funded project is to understand the molecular basis of plant immune system against necrotrophic pathogens through functional and mechanistic analysis of the plant WRKY33 transcription factor, which plays a critical role in plant resistance to this important group of pathogens. Major outcomes and findings of the project include: i) plant immune system against necrotrophic pathogens is elevated upon pathogen infection partly through increased production of WRKY33 proteins due to induced expression of the WRKY33 gene by activated WRKY33 as a result of phosphorylation of WRKY33 proteins by a pathogen-responsive protein kinase cascade, ii) WRKY33 induces plant immune system against necrotrophic pathogens by activating a wide range of plant defense genes. This action of WRKY33 is through collaboration with two other proteins SIB1 and SIB2. The SIB proteins promote the action of WRKY33 in plant immune system by physically binding to WRKY33 and promoting the recognition of targeted defense genes by WRKY33, iii) WRKY33 also promotes plant immune system against necrotrophic pathogens by promoting a protein-degrading pathway called autophagy that enhances plant resistance to necrotrophic pathogens by promoting plant survival through removal of damaged and toxic proteins generated under stress conditions. These findings provide important new insights into the complex molecular mechanisms of plant immune system against necrotrophic pathogens. The identified proteins and mechanisms can be exploited to develop novel strategies of crop protection. Autophagy is a conserved cellular process and impaired autophagy has been linked with aging, cancer and metabolic and neurodegenerative diseases and, therefore, our insights about these processes in plant cells will have important implications beyond the plant world. During the funding period, seven graduate students and more than ten undergraduate students also received training in a variety of techniques in molecular and cellular biology. By incorporating some of the research activities of the project into a lecture/lab course called 'Molecular approaches in plant biology' more than 50 graduate and undergraduate students not only learnt necessary molecular techniques but also experienced the excitement and thrill of real scientific discoveries.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Michael L. Mishkind
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Purdue University
West Lafayette
United States
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