Genome-wide characterization of the JrCHI family and functional validation of JrCHI6 and JrCHI24 conferring resistance to Alternaria tenuissima in walnut

Alternaria tenuissima leaf spot disease severely hinders sustainable walnut production in Xinjiang. To dissect the molecular basis of walnut disease resistance and mine resistance-associated genes, we investigated the physiological and transcriptomic responses of a resistant wild walnut and a susceptible cultivar, ‘Xinxin No.2’. We found that the wild walnut displayed slower lesion spread and markedly higher defense enzyme activity; its chitinase activity stayed 30%–45% above that of ‘Xinxin No.2’ throughout pathogen infection. Transcriptomic data revealed significant enrichment of the amino sugar and nucleotide sugar metabolism pathway, proving chitin hydrolysis is vital for host defense. Genome-wide screening obtained 24 JrCHI genes classified into GH18 and GH19 families, whose promoters carry numerous jasmonic acid (JA) and salicylic acid (SA) response elements. JrCHI6 and JrCHI24 were screened as key resistance genes with elevated expression post-infection. Exogenous hormone treatments verified methyl jasmonate alone induced the expression of the two genes, while SA barely altered their transcript abundance. As JA signaling governs plant defense against necrotrophic pathogens, JrCHI6 and JrCHI24 are inferred to drive JA-mediated immunity to A. tenuissima. Transient overexpression of JrCHI6 and JrCHI24 improved leaf spot resistance in ‘Xinxin No.2’ and increased expression of JA/ET defense markers JrPR3 and JrPR4; in contrast, silencing these genes greatly compromised wild walnut resistance. This work systematically identified the walnut JrCHI gene family and verified JrCHI6 and JrCHI24 as core functional genes mediating chitinase-dependent resistance against A. tenuissima. ⚈ The first genome-wide identification of the chitinase gene family in walnut complements existing research on Juglandaceae. ⚈ JrCHI6 and JrCHI24 are identified as core resistance genes markedly upregulated upon Alternaria tenuissima infection. ⚈ JrCHI6 and JrCHI24 confer enhanced resistance against Alternaria tenuissima. ⚈ Functional JrCHI genes characterized in this work provide elite genetic resources for molecular resistance breeding against walnut Alternaria leaf spot.