Identification of Aerotaxis Receptor Proteins Involved in Host Plant Infection by Pseudomonas syringae pv. tabaci 6605.

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a foliar plant pathogen that causes wildfire disease on tobacco plants. It requires chemotaxis to enter plants and establish infection. While chemotactic signals appear to be the main mechanism by which Pta6605 performs directional movement, the involvement of aerotaxis or energy taxis by this foliar pathogen is currently unknown. Based on domain structures and similarity with more than 50 previously identified putative methyl-accepting chemotaxis proteins (MCPs), the genome of Pta6605 encodes three potential aerotaxis transducers. We identified AerA as the main aerotaxis transducer and found that it possesses a taxis-to-serine-and-repellent (Tsr)-like domain structure that supports a periplasmic 4HB-type ligand-binding domain (LBD). The secondary aerotaxis transducer, AerB, possesses a cytosolic PAS-type LBD, similar to the Aer of Escherichia coli and Pseudomonas aeruginosa. Aerotaxis ability by single and double mutant strains of aerA and aerB was weaker than that by wild-type Pta6605. On the other hand, another cytosolic PAS-type LBD containing MCP did not make a major contribution to Pta6605 aerotaxis in our assay system. Furthermore, mutations in aerotaxis transducer genes did not affect surface motility or chemotactic attraction to yeast extract. Single and double mutant strains of aerA and aerB showed less colonization in the early stage of host plant infection and lower biofilm production than wild-type Pta6605. These results demonstrate the presence of aerotaxis transducers and their contribution to host plant infection by Pta6605.

Identification of chemoreceptor proteins for amino acids involved in host plant infection in Pseudomonas syringae pv. tabaci 6605.

Chemotaxis is crucial for Pseudomonas syringae pv. tabaci (Pta) 6605 to evoke disease in tobacco plants. Pta6605 harbors more than fifty genes for methyl-accepting chemotaxis proteins (mcp), but almost all are functionally uncharacterized. Previously we identified a dCache_1 type MCP in Pta6605 that mediates chemotaxis to γ-aminobutyric acid, called McpG. In this study, we characterized four more dCache_1 type MCPs, three of which, PscA, PscB, and PscC2, are responsible for sensing amino acids. Using a capillary chemotaxis assay, we observed that PscA, PscB, and PscC2 mutant strains had reduced chemotaxis to most amino acids, indicating that PscA and PscB mediate chemotaxis to 14 amino acids, while PscC2 has a slightly narrower ligand recognition, mediating chemotaxis to 12 amino acids. Other cellular functions were also affected in ΔpscB and ΔpscC2: swarming motility was reduced, and biofilm formation was increased. Furthermore, ΔpscB and ΔpscC2 but not ΔpscA had reduced virulence in the host tobacco plant. On the other hand, ΔpscC1 was defective in motility and did not even respond to yeast extract and was unable to cause disease. These findings supported the idea that the chemosensory pathway correlated with virulence-related phenotypes. Amino acids are abundant in tobacco apoplast; having multiple MCPs appears to support the invasion of Pta6605 into the plant.

Cluster II che genes of Pseudomonas syringae pv. tabaci 6605, orthologs of cluster I in Pseudomonas aeruginosa, are required for chemotaxis and virulence.

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent of wildfire disease in host tobacco plants and is highly motile. Pta6605 has multiple clusters of chemotaxis genes including cheA, a gene encoding a histidine kinase, cheY, a gene encoding a response regulator, mcp, a gene for a methyl-accepting chemotaxis protein, as well as flagellar and pili biogenesis genes. However, only two major chemotaxis gene clusters, cluster I and cluster II, possess cheA and cheY. Deletion mutants of cheA or cheY were constructed to evaluate their possible role in Pta6605 chemotaxis and virulence. Motility tests and a chemotaxis assay to known attractant demonstrated that cheA2 and cheY2 mutants were unable to swarm and to perform chemotaxis, whereas cheA1 and cheY1 mutants retained chemotaxis ability almost equal to that of the wild-type (WT) strain. Although WT and cheY1 mutants of Pta6605 caused severe disease symptoms on host tobacco leaves, the cheA2 and cheY2 mutants did not, and symptom development with cheA1 depended on the inoculation method. These results indicate that chemotaxis genes located in cluster II are required for optimal chemotaxis and host plant infection by Pta6605 and that cluster I may partially contribute to these phenotypes.

Requirement of γ-Aminobutyric Acid Chemotaxis for Virulence of Pseudomonas syringae pv. tabaci 6605.

γ-Aminobutyric acid (GABA) is a widely distributed non-proteinogenic amino acid that accumulates in plants under biotic and abiotic stress conditions. Recent studies suggested that GABA also functions as an intracellular signaling molecule in plants and in signals mediating interactions between plants and phytopathogenic bacteria. However, the molecular mechanisms underlying GABA responses to bacterial pathogens remain unknown. In the present study, a GABA receptor, named McpG, was conserved in the highly motile plant-pathogenic bacteria Pseudomonas syringae pv. tabaci 6605 (Pta6605). We generated a deletion mutant of McpG to further investigate its involvement in GABA chemotaxis using quantitative capillary and qualitative plate assays. The wild-type strain of Pta6605 was attracted to GABA, while the ΔmcpG mutant abolished chemotaxis to 10| |mM GABA. However, ΔmcpG retained chemotaxis to proteinogenic amino acids and succinic semialdehyde, a structural analog of GABA. Furthermore, ΔmcpG was unable to effectively induce disease on host tobacco plants in three plant inoculation assays: flood, dip, and infiltration inoculations. These results revealed that the GABA sensing of Pta6605 is important for the interaction of Pta6605 with its host tobacco plant.