2a). Note that, apart from the wild-type RG7204 datasheet strain (white arrows), all mutant colonies were deficient in starch degradation, which suggested that pPM2a could be integrated into the amy locus of Xac. To establish the site of plasmid integration, we performed a diagnostic
Southern blot. Total DNA from two independently generated kanR mutants was digested with EcoRV and probed with the labeled amy gene (Fig. 2b and c). The wild-type strain generated a single signal of ∼6051 bp (Fig. 2b, band 1), which corresponds to the EcoRV fragment containing the amy gene (Fig. 2c, genome coordinates 946 596 … 952 647). Conversely, both mutants displayed two signals: ∼2249 and ∼9686 bp (Fig. 2b, lanes 2 and
3, bands 2 and 3), a result expected in the event of Regorafenib order the integration of pPM2a into the bacterial amy locus (Fig. 2c). Together, data demonstrate that the expression vector had recombined with the amy gene at the chromosome. Before addressing the functionality of our protein expression system, it was necessary to check whether the Xac amy∷pPM2a mutants could still produce disease symptoms in planta, i.e., to evaluate whether α-amylase could play a role as a colonization and/or a pathogenicity/virulence factor in this bacterium. We inoculated Xac amy∷pPM2a mutants in leaves of sweet orange and lime (natural hosts for Xac) alongside the wild-type strain. We observed the appearance of symptoms for a period of 3 weeks, starting from the day of the inoculation, and photos were taken on the 20th day (Fig. 3 shows a representative experiment). As a result, no variation from the wild-type phenotype was detected on our tests, meaning that all the mutants inoculated were as competent as the wild-type strain in producing lesions on leaves. In addition, we did not detect alterations in the pattern of disease development, where lesions were all detected
at the same time scale. We also measured the viability of the mutant strains by analyzing their relative doubling times during growth in liquid media along with colony counting on agar plates, and, again, no variations Phospholipase D1 were observed (data not shown). Taking together, these results show that the ability to cause disease is not affected in Xac amy∷pPM2a mutants and strengthen the value of our GFP expression vectors for the characterization of ORFs suspected to be involved in virulence and pathogenicity. The functionality of our GFP expression vectors was first analyzed by Western blotting. A Xac amy∷pPM2a mutant strain, harboring a single copy of the expression cassette integrated into the amy locus, was cultivated in NYG medium alongside a wild-type strain (negative control) and treated with xylose to induce the GFP production by the mutant.