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According to molecular biology, genomic and proteomic data, the phytopathogenic gamma-proteobacterium Pseudomonas syringae pv. tomato DC3000 produces a number of proteins that may promote infection and draw nutrients from plants. Remarkably, P. syringae DC3000 strain possesses three paralogous gap genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes with different predicted molecular sizes and metabolic functions. As GAPDH was shown to be a virulence factor in other microbial pathogens, in the current study, we analyzed the expression levels of each paralogous gap gene by realtime PCR to understand the actual impact of their protein products on P. syringae virulence. We found that all of them were strongly induced during the infection process. Nevertheless, proteomic analysis of culture supernatants revealed that only Class I GAPDH1 encoded by the gap1 gene was identified as an extracellular protein in infective cells. These results strongly suggest that this GAPDH should play a role in the infective process, including its well-know enzymatic function in the glycolytic metabolic pathway.