Differential proteome analysis of Pseudomonas syringae maculicola M6 in response to infection into Arabidopsis thaliana

Abstract

Our understanding on the molecular responses of bacteria post-infection into plant remains limited. Because of the intrinsic problem of insufficient sample amount and the inadequacies of available techniques, few studies were performed to investigate the pathogen responses at the protein level post-infection. This study aimed to understand the bacterial responses upon infection of the host plant. An incompatible plant-pathogen interaction model using Arabidopsis thaliana (A. thaliana or AT) and Pseudomonas syringae pv. maculicola (Psm) M6 had been successfully established. Furthermore, an efficient differential centrifugation method was developed for the separation of bacterial pathogens from the infected A. thaliana culture cells for proteomic analyses. This method yields sufficient sample materials for subsequent proteomic analysis. Two dimensional gel electrophoretic (2-DE) analyses of Psm M6 collected at 12th hr post-infection into the AT suspension cells were performed. Compared with the proteome of Psm M6 grown in MS medium (M6-MS proteome), 24 proteins were found to be differentially expressed in the proteome of Psm M6 which responded to the plant host response (plant defense responsive proteome or M6-AT interactome). With the provision of the MALDI-TOF mass spectrometer, 23 proteins were identified. A majority of these differentially expressed proteins were up-regulated and had known functions that were related to transportation, pathogenesis, phosphate uptake as well as amino acid synthesis. In contrast, 4 proteins were down-regulated 12 hrs post-infection of the A. thaliana. Transcriptional expression profiles of these 24 proteins were also measured by quantitative RT-PCR method. On the other hand, antibodies against some of these proteins were produced. With these home-made antibodies, the temporal expression profiles of these selected bacterial proteins (outer membrane protein D; OrpD, betaine aldehyde dehydrogenase; BadH, phosphate-ABC transporter; PstS, phosphonates-ABC transporter; PhnD, TAT sequence domain protein; Tat-sp and glycerophosphoryl diester phosphodiesterase glyceroaldehyde; GlpQ) were measured in Psm M6 post-infection into AT suspension cultured cells. For in vivo validation purposes, protein expression level of BadH and PstS were also measured after infection of A. thaliana planta. It reinforced the idea that the results obtained using cultured plant cells are also repeatable in planta. The strategy developed in this study could serve as a showcase for subsequent studies on plant bacterial pathogenesis. Further, in order to put the plant responses and the bacterial responses in perspective, the bacterial responses in relation to a rapid and massive production of nitric oxide (NO) and reactive oxygen species (ROS) production in plants were studied upon bacterial attack. The reaction of NO and free superoxide (O2-) yields peroxynitrite (ONOO-), which in turn will convert tyrosine residues in the protein backbone into nitrotyrosine residues. This process is called protein nitration. It is one of the post-translational modifications known to bring about the down-stream signaling by alternation of the protein structures. Since the bacteria are surrounded by such hostile environment, it would be interesting to detect the extent of bacterial protein nitration in vivo. Using the proteomic approach combining with the use of anti-nitrotyrosine antibodies in 2DE-western blotting, 19 proteins were found to be nitrated upon infection of A. thaliana suspension culture cells. Some of these proteins are known to be related to apoptosis. These results suggested that NO and ROS produced by the host plants modulated bacterial protein nitration in vivo and this nitration process may participate in biochemical sequences leading to bacterial cell death. Findings obtained from this study clearly demonstrate the complexity of the molecular response of bacteria to the host environment.