Functional analysis of xanB and xylA genes potentially involved with Xanthomonas citri pathogenicity

Abstract

Citrus canker is a disease caused by the bacterium Xanthomonas citri subsp. citri (XAC), which affects citrus of importance for Brazil’s economy, whereas Xanthomonas fuscans subsp. aurantifolii (XauB) causes cancrosis, a softer form of the disease. In previous Young Researcher Project-FAPESP of our group (2009-2017), the enzymes phosphomannose isomerase (PMI, codified by xanB gene) and xylose isomerase (XI, codified by xylA gene) were identified by proteomic analysis as potentially involved in XAC pathogenicity, being that XAC has two genes of xylA in distinct genomic contexts, whereas Xau-B has only one gene. PMI and XI catalyze interconversion between mannose-6-phosphate/fructose-6-phosphate and xylose/xylullose, respectively. This work aims to characterize functionally the two genes of xylA and the xanB gene, especially for their relation with XAC pathogenicity. XAC deletion mutants for such genes, including a double mutant for xylA, will be obtained by double homologous recombination between the genomic DNA and flanking regions of the gene cloned in the suicide vector pNPTS138, and mutants will be tested for in planta pathogenicity relatively to XAC. For the gene whose deletion mutant is unable of causing disease, a homology modelling of the correspondent protein will be performed in order to proceed virtual screening and selection of potential novel inhibitors from suitable commercial compounds databases. The inhibitory capacity of the selected compound will be evaluated in vitro, upon the activity of the target protein (to be produced in the recombinant form), and in vivo, during infection. XAC will be comparatively characterized with XauB and with the deletion mutants in relation to expression of the xylA genes by qRT-PCR and Western Blot and/or ELISA. The recombinant proteins PMI and XI will be produced in E. coli, purified and assayed for their predicted enzymatic activities. Once confirmed the activity, their tridimensional structures and in in vitro interaction with the XAC proteome by "Pull-Down" will be performed. The effects on XAC metabolism of xanB and xylA deletions will be evaluated by differential proteomic analysis between each deletion mutants and XAC, with 2D and/or free-gel techniques, as well as by targeting proteomics (SRM/ SWATH-MS). Until now, several steps were already concluded, such as: the 4 deletion mutants of XAC were obtained (3 for xylA and 1 for xanB) and one of them was unable to produce disease in plant; the target-protein was utilized in modeling and one of the compounds selected by virtual screening was commercially acquired for inhibition tests; XylA was already obtained in recombinant form and presented the predicted activity. The present project already shows promising partial results from the biotechnological point of view, and could lead to a proposition of new agricultural inputs for citrus canker control and also a better understanding of the biological role of xanB and xylA genes in XAC pathogenicity. (AU)