Dilip Shah

Dr. Dilip Shah is a Research Associate Member at the Danforth Center. His lab is involved in studying plant-fungal pathogen interactions and developing strategies for the development of fungal disease resistant mycotoxin-free transgenic crops. He has over 25 years of experience in plant molecular biology and agricultural biotechnology, has made substantial contributions to the development of herbicide and disease resistant crops during his tenure at Monsanto Company, and is listed as a co-inventor on a number of patents.

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Research

1. Modes of antifungal action of plant defensins
2. Genetic modification of crops for enhanced resistance to fungal pathogens using antifungal defensins

1. Modes of action of antifungal 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 antifungal 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 elucidation of the mode(s) of action of these proteins because the genetic and genomics tools are well developed in this organism. MsDef1 and MtDef4 both contain four disulfide bonds, but share only 41% amino acid identity and potently inhibit the growth of F. graminearum. Recently published studies from my lab indicate that these two defensins act independently on the fungus and have different modes of antifungal action. We have isolated several mutants of F. graminearum that exhibit hypersensitivity or resistance to these defensins. Biochemical and molecular characterization of these mutants has revealed that two MAP kinase signaling cascades play an important 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 MsDef1. 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 fungal resistance to this defensin. Using a high-affinity nucleic acid stain SYTOX green, we have found that MtDef4 compromises the plasma membrane of the fungus more effectively than MsDef1. Furthermore, MtDef4 enters the cytoplasm of fungal cells more efficiently than MsDef1. We are in the process of characterizing structural determinants of glucosylceramide receptor that are required for interaction with MsDef1. Current studies in the lab are focused on deciphering the 3-D structures of these defensins and on elucidating the structure-activity relationships of these proteins. These studies will be complemented by transcriptomic and phosphoproteomic analyses of defensin-treated fungal cells to determine global gene expression elicited by MsDef1 and MtDef4. We hope to understand more thoroughly fungal toxicity pathways of these antifungal proteins.

Figure above: Three-dimensional structure of a plant defensin. It consists of one α-helix (shown in red) and three anti-parallel β-sheets (shown in blue). Four disulphide bonds (shown in yellow) stabilize the structure of this protein.


2. Genetic modification of crops for enhanced resistance to fungal pathogens using antifungal defensins.

A. 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, and virally encoded Ustilago maydis antifungal protein KP4 inhibit the growth of F. verticillioides in vitro at micromolar concentrations. We have constructed chimeric genes encoding MsDef1 and MtDef4 for high level expression in transgenic corn. Several transgenic corn lines expressing each protein individually or in combination with another protein have been generated. The field tests conducted in 2009 have identified transgenic lines that exhibit strong resistance to ear rot and accumulate low levels of fumonisins. These lines will be further tested in the greenhouse as well as field to confirm ear rot resistance and low accumulation of mycotoxins.

Figure above: The in vitro antifungal activity of plant defensins, MsDef1 and MtDef4 against a fungal pathogen, Fusarium graminearum. Note the inhibition of fungal growth at concentrations of 3 µM MsDef1 and higher and at a concentration of 1.5µM MtDef4 and higher.

B. Engineering resistance to F. graminearum in a model plant Arabidopsis thaliana.
Fusarium head blight (FHB) disease of wheat and barley caused by a fungal pathogen F. graminearum has become a severe problem in North America as a result of increased implementation of reduced tillage wheat production. This pathogen is intimately associated with the production of corn which provides a major reservoir of inoculum when its cultivation precedes wheat. Infection of spikelets causes sterility, poor seed fill and poor seed quality, resulting in yield loss. This fungus also produces the mycotoxin deoxynivalenol (DON) which destroys the quality of grain and makes it unsuitable for livestock and human consumption. The epidemics of FHB in the United States and Canada in the 1990s were extraordinarily severe. These epidemics caused unprecedented economic losses and prolonged hardships for wheat producers and rural communities. It is estimated that the combined production losses and price effects of FHB epidemics during the years 1991 to 1997 resulted in direct losses totaling more than $1.3 billion in the United States. It has been reported in recent years that F. graminearum is capable of causing a disease in a model plant Arabidopsis thaliana. A. thaliana-F. graminearum is therefore a suitable pathosystem to test antifungal defensins for their ability to provide resistance to FHB. We have generated a number of transgenic Arabidopsis lines constitutively expressing MtDef4 either extracellularly or intracellularly using the vacuolar or endoplasmic reticulum retention signals. Transgenic lines expressing MtDef4 either extracellularly or intracellularly exhibit strong resistance to F. graminearum and also substantially reduce the accumulation of DON. The current research in the lab is focused on generating transgenic lines co-expressing MtDef4 extra- and intracellularly to determine if extra- and intracellularly targeted MtDef4 provides even stronger resistance to this pathogen and little or no accumulation of the mycotoxin. Future research will also test MsDef1 for its ability to provide resistance to this pathogen in this model plant.

Figure above:Figure 3. Fluorescently labeled plant defensins, MsDef1 and MtDef4, are taken up by the fungal cells indicating these proteins likely have intracellular targets.

Technologies available for license: