Novel antifungal symbiotic peptides: Modes of action and control of fungal pathogens

Fungal pathogens cause significant losses of crop yield and are a serious biological threat to food security. Effective management of fungal diseases in crops has become a major challenge due to development of fungicide resistance in pathogen populations and lack of single gene resistance. Thus, it is important to look for countermeasures.

Small cysteine-rich antifungal peptides that exhibit potent antifungal activity against economically important fungal pathogens offer peptide-based biofungicide alternatives to on-farm chemical fungicides. We have identified two small antifungal peptides from leguminous plants that offer significant potential for development as second-generation safe and sustainable biofungicides.

For effective use of these peptides for crop protection in agriculture, it is important to understand their structure-activity relationships and mechanisms of antifungal action. Preliminary studies have revealed that these peptides exhibit multi-faceted modes of action. In this proposal, active sites of these peptides governing their antifungal activity will be determined and extensive high-resolution microscopy will be used to investigate subcellular targets of these peptides in a fungal pathogen that causes gray mold disease in fruits and vegetables.

Further, biochemical tools will be employed to identify molecular targets of these peptides in the fungus. This project provides interdisciplinary training to a postdoctoral student and under-represented minority undergraduate students. In addition, it provides hands-on research experiences to teachers and students from low performing public schools in the St Louis area.

Two cationic sequence-divergent nodule-specific cysteine-rich peptides, NCR044 and NCR13, exhibit potent antifungal activity against plant fungal pathogens. We hypothesize that these two NCR peptides exhibit unique structural characteristics and are highly effective due to their multi-faceted modes of action. We further propose that NCR peptides have untapped potential for development as safe spray-on biofungicides.

In this project, 3D structure of NCR13 will be determined. Sequence motifs of NCR044 and NCR13 governing membrane disruption and antifungal activity against Botrytis cinerea will be determined. In particular, sequence motif(s) required for nucleolus-localization of NCR044 will be elucidated.

Biochemical experiments will establish if NCR044 and NCR13 inhibit protein translation by binding to ribosomal RNA and/or ribosomal proteins. The fully completed gapless and annotated 38.8 Mb genome sequence of the B. cinerea genome and its functional genomics tools will provide essential resources for the protein-protein interaction studies. Extensive high-resolution live cell imaging of fungal cells with each peptide will be performed to identify mechanisms of internalization, subcellular dynamics and mobility of each peptide using organelle-specific fluorescent proteins and vital dyes with confocal and super-resolution microscopy and correlative electron microscopy.

Finally, spray-application of NCR044, NCR13 and their variants will determine if they confer resistance to gray mold disease in tomato plants. Experiments are planned to elucidate peptide-imposed changes in “spore germination transcriptome” in planta.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.