AtNHR2A and AtNHR2B: Two Players of the Plant Secretory Pathway Functioning in Biotic Stress Responses

AtNHR2A (Arabidopsis thaliana nonhost resistance 2A) and AtNHR2B (nonhost resistance 2B) are immunity-associated proteins required for resistance to the nonadapted pathogen Pseudomonas syringae pv. tabaci (Pstab) that is unable to cause disease in wild-type Col-0 plants. The enhanced susceptibility of the Atnhr2b Atnhr2a double mutant suggests that these proteins function synergistically in plant defense. Both AtNHR2A and AtNHR2B localize to endomembrane compartments and interact with secreted proteins, implicating them in immunity-related secretory processes. The objective of this study was to test the hypothesis that AtNHR2A and AtNHR2B are involved in the secretion of proteins to the apoplast by comparing the apoplastic proteomes of wild-type Col-0 and the Atnhr2b Atnhr2a double mutant plants at 24 and 72 h after inoculation with Pstab. Comparative quantitative proteomics identified 35 and 23 differentially abundant proteins (DAPs) carrying N-terminal signal peptides at 24 and 72 h, respectively. These DAPs were enriched for functions in cell wall remodeling, lipid metabolism, redox regulation, and proteolysis, reflecting broad alterations in the conventional secretory pathway. Many defense-associated proteins, including PR2-type β-1,3-glucanases, peroxidases, and cell wall-modifying enzymes, accumulated at reduced levels in the double mutant apoplast, whereas others, such as aspartyl proteases and plasmodesmata-located protein 2 (PDLP2) were elevated, suggesting dysregulation of apoplastic homeostasis. Importantly, several DAPs showed no corresponding changes in the whole leaf proteome, confirming that protein secretion rather than protein synthesis was affected. Our findings establish AtNHR2A and AtNHR2B as key components of endomembrane trafficking that safeguard the apoplastic proteostasis response during pathogen challenge.