A GFP-LABELLED HYPOTHETICAL PROTEIN SHOWS A DISCRETE SUBCELLULAR LOCALIZATION PATTERN IN THE BACTERIUM XANTHOMONAS AXONOPODIS PV. CITRI

Paula Maria Moreira Martins (UNESP); Ivy Freitas Lau (UNESP); Alexandre Morais do Amaral (Embrapa); Henrique Ferreira (UNESP)

The bacterium Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus canker, a disease that affects citrus worldwide. Although the genome sequence of Xac has been available for almost a decade, little is known about its biology, the onset of infection and the late stages of host colonization that culminate in the development of disease symptoms in citrus plants. To start a systematic characterization of proteins encoded by Xac, and mostly to identify hypothetical factors that could be involved in pathogenesis, we developed an integrative vector for protein expression and subcellular localization of GFP-labelled proteins within Xac using fluorescence microscopy. Transcription from our GFP vector (pPM2a) is governed by the xylose promoter, and integration into the chromosome of Xac is achieved by a single crossover event between a fragment of the amy gene from Xac, contained in the vector, and its correspondent chromosomal version. Here we show that pPM2a can be stably integrated into the amy locus of Xac without altering its pathogenicity. The functionality of the GFP vector was evaluated by expressing, in Xac, a hypothetical factor that shares substantial homology to the Bacillus subtilis (Bsu) ZapA protein. ZapA-Bsu stimulates the assembly of FtsZ polymers in Bsu and is known to localize to the division septum. The Xac ortholog of ZapA-Bsu, when fused to GFP (GFP-ZapA-Xac), generated a localization pattern similar to that produced by GFP-ZapA-Bsu: a bar/ring-like structure perpendicular to the longitudinal axis of the cell and located in the middle of the rod, where the division septum develops. Additionally, we labelled four different hypothetical proteins encoded by Xac that had their expression stimulated or inhibited in contact with the host plant, and found that at least one of them localized as a discrete focus inside the cell. The possible involvement of this hypothetical factor with chromosome replication and cell wall development was investigated by subjecting the Xac mutant strain producing such a fusion to different sets of antibiotics. Moreover, our results corroborate the use of GFP-tagging to carry out functional characterization of proteins in Xanthomonas species, and constitute a new and useful tool for in vivo studies regarding protein factors in this bacterium. Financial support: FAPESP grant 04/09173-6; PMMM 06/59494-9 Keywords: Xanthomonas axonopodis; citrus; GFP-labelling; Protein expression