Nodule-specific cystatin GmCYS18 promotes soybean nodulation by inhibiting expression of the cysteine protease gene GmCYP17

Phytocystatins (plant cystatins) are a type of protease inhibitor widely studied for their specific and reversible inhibitory effects on cysteine proteases. The equilibrium between phytocystatins and the cysteine proteases which they inhibit plays key roles in biotic and abiotic stresses, plant immunity, and so on. However, the roles of this balanced relationship in legume-rhizobium symbiosis remain poorly characterized. In the present study, we identified a nodule-specific cystatin gene GmCYS18 as a positive regulator of nodulation and nodule development in soybean. Overexpression of GmCYS18 increased the chlorophyll SPAD value, nodule number, plant height, weights of shoot, root, and nodule, and the expression of nodulation marker genes, particularly in the stable transgenic line GmCYS18-OX-1. Surprisingly, we found that GmCYS18 suppressed the expression of six root nodule symbiosis-related papain-like cysteine proteases (PLCPs) in nodules. Furthermore, the GmPLCP gene, GmCYP17, which shows high homology to GmCYS9 that plays a negative regulatory role in soybean nodulation, was silenced by RNAi. The results showed that GmCYP17 inhibits nodulation, nodule development, and the expression of nodulation marker genes in soybean. Our findings enriched knowledge on the function of phytocystatins and provided insights into the correlation between cystatin and cysteine protease in nodule symbiosis.