Study of the complementation of Xylella fastidiosa causing CVC with a functional polygalacturonase enzyme and its impact on bacterial physiology

Xylella fastidiosa is a phytopathogen restricted to the xylem of crops such as citrus and grapevine, causing citrus variegated chlorosis (CVC) and Pierce's disease (PD). Comparative genomics studies between the subspecies fastidiosa (Xf-PD) and pauca (Xf-CVC), it was observed that the precursor gene of the enzyme polygalacturonase (pglA) in Xf-CVC presents a frameshift that generates a non-functional protein. PglA degrades the pectins on the cell wall, allowing the movement of the pathogen. It suggests that the mutation of pglA in Xf-CVC may lead to the slower colonization observed in citrus or prevent the triggering of the plant immune system by DAMPs. To verify whether the expression of pglA confers greater virulence or absence of symptoms, Xf-CVC was complemented with the functional gene of Xf-PD, which was cloned into vector pXF20 and the complementation was confirmed by conventional PCR and restriction enzymes. The complemented bacteria was inoculated in sweet orange and after seven months they still had no symptoms, suggesting that the presence of pglA does not accelerate symptoms in citrus, and probably, the degradation of pectin leads to the activation of the immune system. Due to its fastidious behavior, further evaluations are necessary. To prove in vitro complementation and in vivo expression, a specific antibody against pglA was produced. For this, pglA-PD was cloned into pET28a vector, transformed into E. coli Rosetta and induced in a heterologous system. The protein was expressed in the insoluble fraction of the protein extract, isolated via SDS-PAGE and the antibody was synthesized for validation.