Identification and functional analysis of ERF family genes in Pinus massoniana during infection of Bursaphelenchus xylophilus

BACKGROUND: Pinus massoniana forests in China cover approximately 40 million hectares but are severely threatened by pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus. Identifying resistance mechanisms and genes in resistant genotypes, such as MK94, is critical for managing this destructive disease. Ethylene responsive factors (ERFs), belonging to the AP2/ERF transcription factor superfamily, mediate diverse plant defense responses. However, their roles in resistance to PWN in P. massoniana remain poorly understood. This study aimd to identify ERF genes from the resistant genotype MK94, characterize their expression profiles under PWN infection, and validate their potential roles in plant defense. RESULTS: From transcriptome data of the resistant genotype MK94, 85 ERF genes (PmERFs) were identified and classified into 11 subfamilies. Expression analyses showed significant upregulation of genes in subfamilies VII, IX, and X following PWN infection. Real-time quantitative polymerase chain reaction (RT-qPCR) validation confirmed notable induction of PmERF46, PmERF48, PmERF49, PmERF52, and PmERF75, particularly in resistant genotypes. Further functional analysis revealed that transient heterologous expression of PmERF49 and PmERF52 in Nicotiana benthamiana significantly enhanced resistance against the fungal pathogen Botrytis cinerea, reducing lesion areas by approximately 50% and markedly decreasing pathogen biomass. Subcellular localization assays confirmed nuclear localization for both ERFs, and transcriptional activation assays showed that PmERF49 had transcriptional activation activity, whereas PmERF52 did not. CONCLUSION: The study identified and characterized ERF family genes associated with responses to PWN in the resistant P. massoniana genotype MK94. Functional validation of PmERF49 and PmERF52 highlights their significant potential for enhancing resistance to pathogens. These findings provide critical insights into the molecular mechanisms underlying resistance to pine wilt disease and offer promising targets for resistance breeding in forest management. © 2025 Society of Chemical Industry.