NCR13 peptide protects soybean against <i>Cercospora sojina</i> using multi-faceted modes of action and additive interaction with chemical fungicides

Frogeye Leaf Spot disease, caused by a fungus Cercospora sojina, is a serious disease affecting soybean production. Its control is challenged by resistance to Quinone outside Inhibitor (QoI) fungicides, but plant antifungal peptides with multi-faceted modes of action (MoA) offer a promising alternative. 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 disrupted plasma membrane, induced reactive oxygen species (ROS) production, and was rapidly internalized, localizing in the nucleus. It bound to fungal ribosomal RNA and inhibited protein translation. 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 its multi-faceted MoA holds potential as a bio-fungicide for FLS control, complementing QoI fungicides and overcoming resistance in C. sojina.