Modes of action of antifungal plant defensins
Genetic modification of crops for enhanced resistance to fungal pathogens using antifungal defenses
Modes of action of antifungal defensins
Plant defensins are naturally occurring, ribosomally synthesized cysteine-rich 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 for enhanced disease resistance is to unravel their modes of antifungal action and the mechanisms by which fungal resistance to these proteins might emerge. The Shah lab has been using Fusarium graminearum, a devastating multicellular filamentous fungal pathogen of wheat and barley, and a model filamentous fungus, Neurospora crassa, for elucidation of the mode(s) of action of these proteins. Genetic and genomics tools are well developed in these fungi for the mode of action studies.
Each plant contains a large gene family encoding defensin and defensin-like proteins. In the Shah lab, the research focus is on characterizing the antifungal properties and mechanism of antifungal action of three defensins, MsDef1, MtDef4 and MtDef5, each containing four disulfide bonds, but sharing only limited amino acid sequence homology. These proteins inhibit the growth of several filamentous fungi including Fusarium sp., Verticillium dahlia, Aspergillus sp. and N. crassa.
Research conducted by the Shah lab during the past few years has revealed that MsDef1 and MtDef4 exhibit different antifungal properties and different modes of antifungal action. MsDef1 is a morphogenic defensin which causes striking hyperbranching phenotype in the target fungus, whereas MtDef4 is a nonmorphogenic defensin which inhibits the growth of the target fungus without causing any significant morphological changes. MsDef1 binds to the fungal cell wall and plasma membrane localized sphingolipid glucosylceramide. The fungal signaling pathways operating downstream of this MsDef1/glucosylceramide interaction remain to be defined.
MtDef4 is a more potent antifungal protein and is internalized by fungal cells. It is very likely that its mechanism of action involves interaction with intracellular targets and processes. In preliminary studies, the Shah lab has found that MtDef4 binds to the phospholipid phosphatidic acid which is an important bioactive signaling lipid in all eukaryotic cells. In addition, they have determined that the γ-core motif (GRCRGFRRRC) of this protein plays a major role in facilitating fungal cell entry of MtDef4 into fungal cells. The current studies in the lab are focused on characterizing molecular processes involved in facilitating fungal cell entry of this defensin and identifying its intracellular targets.
The mode of antifungal action of the third antifungal defensin MtDef5 remains to be determined. Having antifungal defensins with different modes of action will pave the way for engineering more durable and broad-spectrum resistance to fungal pathogens in transgenic crops.
Genetic modification of crops for enhanced resistance to fungal pathogens using antifungal defensins
Because of their potent antifungal activity against economically important fungal pathogens, plants defensins have great potential to be used as antifungal agents in transgenic plants. For example, expression of apoplast-targeted MsDef1 in transgenic Russet Burbank potato provides strong resistance to Verticillium dahlia under the greenhouse and field conditions. More recent research in the Shah lab indicates that apoplast-targeted MtDef4 seems to be more effective in providing resistance to biotrophic fungal pathogens in transgenic model plant Arabidopsis thaliana and in wheat. The current research in the Shah lab is focused on generating fungal disease resistant transgenic wheat, soybean and corn expressing antifungal plant defensins. This research involves expressing these defensins in the target crop using constitutive, tissue-specific and pathogen-inducible promoters, and targeting these proteins to different subcellular compartments to effectively inhibit the growth of fungal pathogens with different lifestyles.