Novel plasmid pCM3 harboring the <i>aph(3)</i> gene confers phosphorylation‐driven streptomycin resistance in <i>Clavibacter michiganensis</i>

Impact statement Antibiotic resistance in plant pathogens threatens sustainable crop protection, and yet, its molecular basis remains incompletely understood. We report that streptomycin resistance in Clavibacter michiganensis strain TX‐0702 is mediated by a plasmid‐borne aminoglycoside phosphotransferase, APH(3), encoded on an uncharacterized plasmid, pCM3. Functional and biochemical analyses demonstrate that APH(3) inactivates streptomycin through phosphorylation, establishing a phosphorylation‐driven resistance mechanism in Gram‐positive plant pathogens. Sequence analyses reveal that pCM3 carries mobile genetic elements, suggesting environmental dissemination of resistance genes in plant‐associated microbes. These findings expand understanding of phytopathogen antibiotic resistance and plant microbiomes as potential resistance gene sources for agricultural, food, and human health safety.