The pseudoenzyme CcNCS2, a CcDREB1B-activated PR-10 protein, works together with Cc4’OMT to protect Coptis chinensis against Fusarium oxysporum

Pathogenesis-related protein 10 (PR-10) plays a crucial role in plant responses to abiotic and biotic stresses. However, functional studies on PR-10 genes in Coptis chinensis Franch., a traditional Chinese medicinal plant rich in benzylisoquinoline alkaloids (BIAs), remain limited. The growth of C. chinensis is severely threatened by Fusarium oxysporum . Infection by this pathogen induced the transcription of a PR-10 gene ( CcNCS2 ), a transcription factor gene ( CcDREB1B ), and a BIAs biosynthetic enzyme gene ( Cc4′OMT ) in C. chinensis . Moreover, F. oxysporum infection enhanced the accumulation of BIAs, abscisic acid (ABA), and H₂O₂. Exogenous application of either ABA or H₂O₂ also upregulated the transcript levels of CcNCS2 , CcDREB1B , and Cc4′OMT . The localization of CcNCS2 and Cc4′OMT was determined to be cytoplastic. Recombinant CcNCS2 exhibited in vitro antifungal activity against F. oxysporum with a minimum inhibitory concentration (MIC50) of 33.70 μM, leading to disruption of cell wall integrity and inhibition of conidial germination. Notably, CcNCS2ᴸ¹ ¹⁹ᴷ site-directed mutant regained NCS enzymatic activity, exhibited reduced RNase activity, and showed substantially attenuated antifungal activity compared with wild-type CcNCS2, suggesting that the defensive function of CcNCS2 was largely independent of its NCS catalytic activity and partially dependent on its RNase activity. Transient overexpression of CcNCS2 in Nicotiana benthamiana enhanced tolerance to F. oxysporum . Meanwhile, recombinant Cc4′OMT efficiently catalyzed the conversion of ( S )-norcoclaurine to ( S )-coclaurine. Transient overexpression of Cc4′OMT in C. chinensis markedly increased the accumulation of BIAs, which exhibited antifungal activity against F. oxysporum . Furthermore, CcDREB1B directly bound to the promoters of CcNCS2 and Cc4′OMT and activated their transcription. A potential defense mechanism of C. chinensis against F. oxysporum was proposed: accumulation of ABA and H₂O₂ induced by F. oxysporum infection activated the transcription of the CcDREB1B / CcNCS2 / Cc4′OMT module, thereby enhancing tolerance to the pathogen. Overall, these findings provided valuable candidate genes for molecular breeding of disease-tolerant C. chinensis .