Pokhrel, A.; Nath, V. S.; Godwin, J.; Kalunke, R.; TETORYA, M.; Czymmek, K. J.; Shah, D. M.

Frogeye Leaf Spot (FLS) disease, caused by a fungal pathogen Cercospora sojina, is a serious threat to soybean production globally. The control of FLS is facing a major challenge due to the rapid emergence of pathogen resistance to Quinone outside Inhibitor (QoI) fungicides. Effective long-term management of FLS in soybean calls for the discovery of antifungal compounds with new modes of action (MoA), durability and safety. Here, we showed that chickpea nodule-specific cysteine-rich peptide, NCR13_Peptide Folding Variant1 (NCR13_PFV1), exhibited antifungal activity against QoI-sensitive and -resistant field isolates of C. sojina at nanomolar concentrations representing the first antifungal NCR peptide reported effective against C. sojina. Spray-application of this peptide showed no phytotoxicity and effectively protected soybean against FLS. When combined with the QoI fungicide azoxystrobin, NCR13_PFV1 provided additive control of FLS. NCR13_PFV1 induced plasma membrane disruption and production of reactive oxygen species (ROS) in C. sojina. NCR13_PFV1 was rapidly internalized into fungal cells where it accumulated in the cytoplasm, localized inside nucleus, bound to fungal ribosomal RNA and inhibited protein translation in vivo. RNA-seq studies revealed the upregulation of several genes encoding heme binding proteins in peptide-challenged C. sojina. Notably, iron supplementation in the growth medium reduced the peptide-induced ROS and antifungal activity, revealing the importance of iron homeostasis in protection or recovery of C. sojina from oxidative stress. Overall, NCR13_PFV1 with multiple MoA holds potential as a bio-fungicide for FLS control complementing conventional QoI fungicides and overcoming fungicide resistance in C. sojina.