dilip m. shah, ph.d domain associate member dshah@danforthcenter.org


My research is focused on understanding the molecular mechanisms underlying the plant’s innate defense against pathogens. We are investigating the modes of action of antifungal plant defensins, 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 substan- tial losses of crop yield worldwide. Effective and sustainable control of fungal pathogens remains one of the most important challenges of modern agriculture. Te 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. Te 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 in gaining an understanding of 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 studies of modes of action of defensins of because the genetic and genomics tools are well developed in this or- ganism. 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 by inducing strong hyper- branching, whereas MtDef4 does so by limiting polar growth of fungal hyphae (Figure 1). We have recently isolated several mutants that are hypersensitive to MsDef1, but not to MtDef4. Te molecular character- ization 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. Te 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 glucosylce- ramide is indeed a membrane receptor for MsDef1: in absence of glucosylceramide in the fungus is resistant to MsDef1 and exhibits a significant loss of pathogenicity in wheat. We are in the process of characterizing structural determinants of a 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 resis- tance to these defensins. Tese studies will be complemented by mi- croarray and proteomic analyses of defensin-treated F. graminearum conidial cells to determine global gene expression elicited by MsDef1


recent publications: Allen, A., Snyder A.K., Preuss, M., Nielsen, E.E., Shah, D.M. and Smith, T.J. (2008). Plant defensins and fungal killer toxin KP4 inhibit root growth. Planta 227, 331-339


page 18 2009 scientific report the donald danforth plant science center


Entry of MtDef4 into fungal Hyphae. Fusarium graminearum hyphae were in- cubated with fluorophore-tagged MtDef4 for 4 hrs and pictures were taken using a wide-field microscope at 60X magnification. Entry of MtDef4 is indicated by the emission of intracellular fluorescence as shown by a picture on the right.


and MtDef4. Tese studies will result in identification of unique cel- lular 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, and the antifungal protein kp4 (encoded by a vi- rus of Ustilago maydis) inhibit the growth of F. verticillioides in vitro at micromolar concentrations. We have determined that high level expression of these proteins in transgenic Arabidopsis thaliana confers strong resistance to F. graminearum, a pathogen of wheat and barley closely related to F. verticillioides. We have developed transgenic corn lines expressing either MsDef1 or MtDef4 and are currently testing them for resistance to ear rot and reduction of fumonisin contami- nation in greenhouse and field trials.


lab members: Christina Chai, Summer Intern / Khira Gabliani, 2008 Pfizer-Solutia stars intern / Jagdeep Kaur, Ph. D., Postdoctoral Associate Mercy Tokala, Ph. D., Postdoctoral Associate / Himanshu Makadia, Volunteer Scientist


Ramamoorthy, V., Cahoon, E.B., Li, J., Tokala, M. and Shah, D.M. (2009) Sphingolipid C9-methyltransferases are essential for fungal growth and virulence but not for sensitivity to antifungal plant defensins in Fusarium graminearum. Eukaryotic Cell 8, 217-229.


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