SsPit2A/B Effectors from Sporisorium scitamineum Interact with the Sugarcane PLCP ScRD21A and Reduce ScRD21A-Associated Cysteine Protease Activity via a Conserved LXRR Motif

Papain-like cysteine proteases (PLCPs) are central immune hubs frequently targeted by pathogen effectors. Sugarcane smut, caused by Sporisorium scitamineum, threatens global sugarcane yield, yet effector manipulation of host PLCPs remains unclear. Genome-wide analysis of Saccharum spontaneum AP85-441 identified 61 PLCP-encoding genes, which were classified into nine conserved subfamilies. Among these, ScRD21A, a member of the RD21 subfamily, was prioritized for functional characterization. Two Pit2 homologs, SsPit2A and SsPit2B, were identified from S. scitamineum. Yeast two-hybrid, BiFC and pull-down assays demonstrated that both effectors interact with ScRD21A, and that this interaction depends on a conserved LXRR motif within their PID14-like region. In total protein extracts from Nicotiana benthamiana, co-expression of SsPit2A or SsPit2B reduced ScRD21A-associated cysteine protease activity. Transient expression of ScRD21A enhanced flg22-induced ROS production, attenuated Pst DC3000-induced hypersensitive response-associated necrosis, and increased resistance to Botrytis cinerea. Together, these results support a conserved PLCP-targeting strategy in smut fungi and identify the ScRD21A–SsPit2A/B module as a tractable framework for studying effector–protease interactions relevant to sugarcane smut.