The plant hosts used were Bahia sweet orange [Citrus sinensis (L.) Osbeck] and Rangpur lime (Citrus limonia Osbeck). Citrus plants were cultivated under greenhouse conditions at 25–35 °C. Cells were

cultivated in the appropriate medium until OD600 nm∼0.6 (108 CFU mL−1). Following growth, cell suspensions were used to inoculate leaves on the abaxial surface with the help of hypodermic syringes (1 mL). Symptoms were observed during the course of 3 weeks. Cells were cultivated in the appropriate medium until OD600 nm∼0.3. Drops of 20 μL of cell culture were placed on microscope slides covered previously with a thin layer of 1% agarose in 1 × phosphate-buffered saline and covered with a slide cover slip. Visualization of cells was performed using an Olympus BX-60 microscope equipped with a Panobinostat DP-71 refrigerated camera. Images

were captured and processed using imagepro-mc (version 6.0). Before we could initiate studies of controlled protein expression into Xac, we had to develop protein expression systems for this bacterium. The expression vectors built (pPM2a and pPM7g) are integrative, and carry the xylose promoter (pxyl), the xylose repressor Romidepsin ic50 (xylR), and a gfp-coding sequence (Fig. 1). The xylose promoter is known for its fine-tuned control of protein expression levels, and it has been used extensively in B. subtilis (Lewis & Marston, 1999; Gueiros-Filho GPX6 & Losick, 2002). The xylose promoter and the gfp gene are separated by a short synthetic dsDNA that contains a RBS based on a consensus for B. subtilis and E. coli (Rocha et al., 1999). Unique restriction sites are present at both termini of the gfp gene, which allows the ligation of genes and the subsequent production of either N- or C-terminal GFP–protein fusions. Both vectors have a pCR2.1-TOPO backbone, so that they carry a kanamycin cassette, a selectable marker for Xac, and a pUC-like origin of replication. Therefore, these vectors do not replicate in Xac, and can be used for site-directed mutagenesis, a

key strategy to study gene function. Finally, pPM2a/pPM7g harbor a fragment of the α-amylase gene of Xac (amy106–912), intended to mediate their integration into the chromosome. The integration of pPM2a/pPM7g into the chromosome is an essential condition for placing the expression cassette into the bacterium. Integration occurs by at least a single homologous recombination event aiming as targets either the ORF to be characterized plus its native chromosomal copy or the amy106–912 fragment present in the vectors and the chromosomal amy gene. Recombination between amy106–912 and the chromosomal amy locus should produce Xac mutants unable to degrade starch on agar medium. To test for this integration, we inserted pPM2a into Xac by electrotransformation and searched for mutant strains on kanamycin-containing NYG-agar plates.