Comparative transcriptome analysis reveals EjWRKY33 acts as a positive regulator of resistance against Colletotrichum fructicola through salicylic acid signalling

Loquat (Eriobotrya japonica) anthracnose, caused by Colletotrichum fructicola, affects multiple loquat tissues and leads to serious economic losses. In particular, foliar infections compromise not only fruit yield but also the medicinal value of loquat leaves. Despite this, no defence genes have been identified for effective disease management. In this study, an integrated analysis of symptom progression, cellular defence responses, and pathogen colonisation identified 72 h post-inoculation (hpi) as a critical time point. At this juncture, a significant difference in response to C. fructicola was observed between the resistant cultivar Peluches (Pell) and susceptible cultivar Donghuzao (DHZ). Comparative transcriptome analysis at 72 hpi revealed that Pell was enriched in activated key defence-related pathways, including ‘plant–pathogen interactions’ and ‘sesquiterpene and triterpene biosynthesis’, whereas DHZ was predominantly enriched in primary plant metabolism. We further identified EjWRKY33, a transcription factor significantly up-regulated in response to C. fructicola, with higher expression levels in Pell compared to those in DHZ. Silencing EjWRKY33 in loquat leaves resulted in larger lesions and increased pathogen colonisation, with down regulation of salicylic acid (SA)-related gene EjNPR1, jasmonic acid (JA)/ethylene (ET)-related gene EjPR4, the terpenoid biosynthetic gene EjCYP71B37, and defensin gene EjDef1. In contrast, overexpression of EjWRKY33 in Arabidopsis thaliana exhibited enhanced resistance to C. fructicola. Furthermore, our investigation with exogenous hormone treatments revealed that while all three hormones (SA, MeJA, or ETH) induced EjWRKY33 expression, the gene functions as a critical component of the SA-dependent pathway, with limited involvement in the JA/ET pathway. Collectively, these findings provide the molecular basis for developing anthracnose-resistant loquat cultivars and disease management applications.