Dilip M. Shah, Ph.D.

Domain Associate Member

My research is focused on understanding the molecular mechanisms underlying plant's defense against pathogens. We are investigating the modes of action of antifungal plant defensin, mechanisms of fungal resistance to these proteins and their use in development of mycotoxin-free disease resistant crops.

Plant diseases caused by fungal pathogens are responsible for substantial losses of crop yield worldwide. Effective and sustainable control of fungal pathogens remains one of the most important challenges of modern agriculture. The innate immune system of plants provides the first line of defense against fungal pathogens. Small cysteine-rich antifungal proteins called defensins are ubiquitous plant proteins implicated in the first-line host defense against fungal pathogens. A better understanding of how these proteins inhibit the growth of fungal pathogens will lead to the development of novel strategies for control of fungal diseases in transgenic crops.

Modes of Action of Plant Defensins:  Plant defensins are a family of antifungal proteins with remarkable structural conservation and rich diversity of variants.  The constitutive expression of these proteins in transgenic crops affords strong protection from fungal attack. A critical issue that needs to be addressed for effective use of these proteins in transgenic crops is understanding their modes of action and the mechanisms by which fungal resistance to these proteins might emerge.  My lab has been using Fusarium graminearum, a devastating multicellular filamentous fungal pathogen of wheat and barley, for defensin's mode(s) of action studies because genetic and genomics tools are well developed in this organism.  MsDef1 and MtDef4 are structurally similar defensins that share only 41% amino acid identity and potently inhibit the growth of F. graminearum. MsDef1 inhibits the growth of this fungus by inducing strong hyperbranching effect, whereas MtDef4 does so by only limiting polar growth of fungal hyphae (Figure 1).  We have recently isolated several mutants that selectively exhibit hypersensitivity to MsDef1, but not to MtDef4.  The molecular characterization of two of these mutants has revealed that MAP kinase signaling cascades play a major role in regulating sensitivity of F. graminearum to MsDef1, but not to MtDef4.  The MAP kinase signaling cascades are essential for the fungus to protect itself from these defensin. We have recently found that MsDef1 binds to a fungal membrane sphingolipid glucosylceramide.  Preliminary evidence indicates that glucosylceramide is indeed a membrane receptor for MsDef1 whose absence results in resistance to MsDef1 and a significant loss of fungal pathogenicity in wheat.  We are in the process of characterizing structural determinants of glucosylceramide receptor that are required for interaction with MsDef1.  Our current evidence indicates that MsDef1 and MtDef4 have different modes of antifungal action.  Using newly isolated mutants of F. graminearum we plan to identify and subsequently characterize mutant genes conferring resistance to these defensins.  These studies will be complemented by microarray and proteomic analyses of defensin-treated F. graminearum conidial cells to determine global gene expression elicited by MsDef1 and MtDef4.  These studies will result in identification of unique cellular responses to each defensin challenge in this fungus.

Disease Resistant Mycotoxin-Free Corn:  In recent years, ear rot disease caused by a fungal pathogen F. verticillioides has emerged as a major disease of corn limiting yield.  In addition to its direct negative impact on corn yield, the pathogen produces mycotoxins known as fumonisins that have been linked to human and animal mycotoxicosis.  Fumonisins pose a severe health hazard and their contamination in corn constitutes a costly and challenging problem. An environmentally sound and economical approach to address this problem is to plant corn hybrids that are highly resistant to ear rot.  Genetically engineered ear rot resistant corn will allow producers to generate high quality mycotoxin-free seed during normal as well as disease-favoring growing seasons.  We have found that plant defensins, MsDef1 and MtDef4, inhibit the growth of F. verticillioides in vitro at micromolar concentrations.   We have already determined that high level expression of these proteins in transgenic model plant Arabidopsis thaliana confers strong resistance to F. graminearum, a pathogen of wheat and barley, which is closely related to F. verticillioides.  We have constructed chimeric genes encoding MsDef1 and MtDef4 for high level expression in transgenic corn.  Several transgenic corn lines expressing either MsDef1 or MtDef4 have been identified. These lines will be tested for resistance to fungus disease and control of mycotoxin contamination in the field in 2008.

Lab Members:

Holly Carrell, Summer Intern

Jagdeep Kaur, Ph. D., Postdoctoral Associate

Anita K. Snyder, M.S., Research Associate

Mercy Thokala, Ph. D., Postdoctoral Associate