Plants constantly encounter pathogen attacks, especially from pathogenic fungi. As part of various defense strategies to protect themselves against fungal attack, plants produce small antifungal proteins known as defensins. Defensins are produced during the normal growth and development of plants and also in response to fungal attack. Some of these proteins are potent inhibitors of fungal pathogens. The PI and his research team have isolated two plant defensins, MsDef1 and MtDef4, from Medicago spp. that inhibit the growth of Fusarium graminearum, a fungal pathogen which causes devastating head blight disease in wheat and barley. The constitutive expression of these proteins in transgenic plants 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 structure-activity relationships, modes of action and the mechanisms by which fungal resistance to these proteins might emerge. Preliminary data suggest that MsDef1 and MtDef4 have different modes of antifungal action and act independently on F. graminearum. The planned research will identify the structural features and active sites that are essential for the unique antifungal activity of these proteins. In addition, the fungal toxicity pathway utilized by each protein will be elucidated.
Broader Impacts The planned research will shed light on the molecular mechanisms by which antifungal plant defensins inhibit the growth of fungi. This knowledge will enable an effective long-term use of these proteins for biological control of fungal pathogens in transgenic crops. The research will also help establish the foundation for crop and mammalian safety, thus providing a greater sense of security about the use of these proteins in transgenic crops. Moreover, knowledge gained from the planned studies will facilitate future discovery of environmentally safe fungicides. In addition, this project provides a training opportunity for a postdoc as well as minority undergraduates and high school students. The PIs will continue to coordinate with and provide research opportunities through existing programs for students at the Danforth Center. Participants will learn not only protein biochemistry and engineering, but also cutting-edge fungal genomics and proteomics technologies. To enhance the wider scientific impact, presentations at professional meetings and publications in peer-reviewed journals are planned.
Intellectual Merit: Plant immune mechanisms that restrict pathogen growth are poorly understood. Defensins are small sequence divergent cysteine-rich effector proteins of innate immunity expressed in all plants. They exhibit potent antifungal activity and efficacy against filamentous fungi. NSF award 0924124 "Antimicrobial Plant Defensins: Structure-Activity Relationships and Modes of Action" has allowed us to investigate the mechanisms of action of two antifungal defensins, MtDef4 and MsDef1, from Medicago spp. These antifungal proteins are secreted by plant cells and potently inhibit the growth of filamentous fungi including Fusarium graminearum, a causal agent of Fusarium head blight disease in cereals. These two defensins share only 41% amino acid sequence identity and exhibit strikingly different antifungal properties. Whereas MsDef1 induces pronounced hyperbranching effect on fungal hyphal growth, MtDef4 does not. Three dimensional structures of MtDef4 and MsDef1 have been determined. A signature structural motif, the so-called γ-core motif (GXCX3-9C where G is glycine, C is cysteine and X is any amino acid), has been identified in each defensin. The γ-core motif is a major determinant of the antifungal activity and properties of each defensin. Specific amino acids of the γ-core motif critical for the in vitro antifungal activity of MtDef4 have been identified. A significant discovery has been made that MtDef4 and MsDef1 are internalized by fungal cells. It is therefore likely that both defensins inhibit the growth of fungal pathogens by targeting intracellular processes critical for fungal growth and differentiation. It has also been determined that MtDef4 binds to a bioactive phospholipid phosphatidic acid (PA) in vitro and the γ-core motif (GRCRGFRRRC) is involved in the binding of MtDef4 to PA. When the γ-core motif of MsDef1 is replaced with that of MtDef4, MsDef1 is endowed with the ability to bind to PA and exhibits antifungal properties of MtDef4. Amino acid substitutions within the γ-core motif that abolish interaction of MtDef4 with PA also abolish its antifungal activity. MsDef1 binds to a sphingolipid glucosylceramide present in the fungal cell wall. The mutant of F. graminearum which lacks glucosylceramide exhibits strong resistance to MsDef1. In addition, MsDef1 also binds to the bioactive phospholipid phosphatidylinositol 3,5-bisphosphate. Future research will determine the role each phospholipid plays in facilitating the fungal cell entry and/or intracellular perturbation of fungal cell physiology by each defensin. It will also reveal molecular mechanisms of fungal cell entry of each defensin and identify fungal genes required for this process. The antifungal activity of MsDef1, MtDef4, and peptides derived from their γ-core motifs has been characterized during colony initiation in the fungal model Neurospora crassa. The peptides inhibited conidial germination and conidial anastomosis (CAT) fusion with differential potency. Both defensins and their cognate peptides disrupted calcium homeostasis and signaling, possibly by targeting specific components of the calcium signaling/homeostatic machinery. In summary, this NSF-funded research has revealed that MsDef1 and MtDef4 differ markedly in their antifungal properties and their modes of antifungal action. Broader Impacts: This NSF funded research has important implications for the use of antifungal defensins in engineering resistance to fungal diseases in transgenic crops and ensuring their crop and mammalian safety. It will also facilitate engineering of other secreted antifungal proteins for entry into fungal cells and inhibiting their growth. Eight peer-reviewed publications and two review articles have resulted from the two NSF awards. One patent on the use of antifungal defensin MtDef4 for conferring resistance to fungal pathogens has been issued to the Danforth Center. This NSF funding has allowed the principal investigator to provide training for two postdocs and several undergraduate students.
