A PpHSP21‐Centred Regulatory Network Integrates Brassinosteroid Signalling to Enhance Black Spot Disease Tolerance in Pear

Pear black spot disease (BSD) is mainly a fungal disease caused mainly by Alternaria alternata. Although extensive research has been conducted, the precise molecular mechanisms underlying BSD resistance in pears, as well as potential genetic defence strategies, remain incompletely understood. This study investigates the role of PpHSP21, a small heat shock protein, in conferring resistance to A. alternata in pear calli. Overexpression of PpHSP21 in pear calli significantly enhances A. alternata tolerance, whereas silencing the gene in pear leaves increases susceptibility. Regulation of PpHSP21 involves the synergistic action of calmodulin-binding protein 60B (PpCBP60B) and brassinazole-resistant 1.1 (PpBZR1.1), with PpBZR1.1 also acting as a positive regulator of PpCBP60B expression. Both PpCBP60B and PpBZR1.1 directly bind to the promoter of PpHSP21, activating its expression and further confirming their roles as key transcription factors in the resistance mechanism. Additionally, pear calli overexpressing either PpCBP60B or PpBZR1.1 exhibit enhanced resistance to A. alternata, while their silencing results in increased susceptibility. Moreover, we identified a functional interaction between PpHSP21 and the large ribosomal subunit protein PpRPL12, which facilitates proteostasis and increases brassinolide levels, thereby strengthening A. alternata resistance. Collectively, these findings unveil a novel regulatory module centred on PpHSP21 that possibly regulates brassinosteroid-mediated tolerance to A. alternata. This study not only enhances our understanding of the molecular basis of A. alternata resistance but also provides potential genetic targets for improving crop resilience against A. alternata and other plant diseases.