Development of inhibitory ssDNA aptamers for the FtsZ cell division protein from citrus canker phytopathogen

• Recombinant FtsZ protein of X. axonopodis was cloned and purified.
• Activity of purified FtsZ was determined by polymerization assay.
• ssDNA apatmers with high binding affinity and specificity against FtsZ was identified.
• Three aptamers exhibited FtsZ inhibitory effects with nanomolar range of IC50 values.
• Candidate aptamers demonstrated high growth inhibitory activity at the cellular level.

Citrus canker caused by Xanthomonas axonopodis pv. citri (X. axonopodis) is a plant pathogenic bacterial disease infectious to citrus crops, resulting in reduced fruit quality and premature fruit drop. Many chemical substances to prevent citrus canker cannot cure the progressive disease caused by drug resistant pathogens. In this study, we identified the filamentous temperature-sensitive Z (FtsZ) protein of X. axonopodis, a GTPase essential for bacteria cell division, as a new target for anti-citrus canker agent. We found nine single-stranded DNA aptamers with 44–444 nM Kd values against recombinant FtsZ, using SELEX. Among these aptamers, three FtsZ binding aptamers (FBAs) exhibited potent inhibitory effects with IC50 values of 1–2 μM similar to berberine, a well-known commercial antibacterial agent. Furthermore, the FBAs also demonstrated high growth inhibitory activity at the cellular level with MIC50 values in the 100 μM range. Consequently, this is the first report of a biocompatible inhibitory aptamer as a drug against X. axonopodis FtsZ, and provides a novel strategy for the development of eco-friendly citrus canker prevention agents, thereby replacing the presently used chemical-based drug in near future.

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