Genome-wide characterization of ADF proteins in Nicotiana tabacum uncovers the critical role of NtADF22 in defense against black shank disease

Tobacco is a vital economic crop, and its cultivation is severely threatened by black shank disease, caused by Phytophthora nicotianae (P. nicotianae). Actin depolymerizing factor (ADF) proteins are key regulators of actin dynamics and play crucial roles in plant growth, intracellular transport, and stress responses. Although the ADF gene family has been extensively studied in various plant species, characterization in tobacco remains limited, particularly regarding its role in disease resistance. In this study, 24 NtADF genes were identified in the tobacco genome. Phylogenetic analysis classified them into six groups, with structural and motif compositions supporting functional conservation within groups. Evolutionary analysis revealed that whole-genome duplication (WGD) or segmental duplication (SDR) was the primary drivers of NtADFs duplication, with strong purification selection. Promoter analysis identified numerous stress- and hormone-related cis-elements, and the regulatory network predictions indicated potential interactions with transcription factors (e.g., ERF, MYB, WRKY) and microRNAs (miRNAs). Expression profiling demonstrated tissue-specific patterns and differential responses to abiotic and biotic stresses. Notably, functional characterization demonstrated that NtADF22 played a critical role in conferring resistance to black shank disease. This study provides comprehensive insights into the NtADF gene family, highlighting its important roles in plant development and stress responses. These findings offer both theoretical and practical significance for molecular breeding and genetic improvement of tobacco.