Construção de vetor plasmidial para deleção gênica da ORF XAC3162 de Xanthomonas citri subsp.citri

Abstract

Citrus canker is a bacterial disease caused by Xanthomonas citri (XAC), which affects all citrus species and causes damage to fruit quality and productivity. In 2022, in Brazil, the world´s largest producer of oranges, the disease presented a high rate of dissemination. The production of β- lactamases by XAC may contribute to its defense against microorganisms, including endophytes, which can secrete β-lactam antibiotics. The gene XAC3162, which is annotated as a β-lactamase in XAC, is an interesting gene to investigate its possible contribution to XAC pathogenicity. This work aimed to obtain a recombinant vector for deletion of XAC3162 gene from XAC genome by double homologous recombination. The l kb upstream and downstream regions of the XAC3162 gene were cloned into the pJET1.2 vector for propagation and subcloned in tandem into the suicide vector pNPTS138, which encodes for kanamycin resistance. Vector construction was successfully performed, as indicated by enzymatic digestion followed by SDS-PAGE. Electroporation of XAC did not yield kanamycin-resistant transformants, despite of several attempts performed under different conditions. More investigations will be needed in the future to elucidate why XAC3162 gene is refractory to deletion by homologous recombination.