MOLECULAR BIOLOGY OF PLANT PATHOGENIC BACTERIA AND NEW APPROACHES TO DISEASE CONTROL

<br>What was accomplished under these goals? A paper was published in PLoS One that summarized the characterization of the syrG gene and the related salA and syrF regulatory genes associated with lipopeptide toxin production and plant pathogenesis by Pseudomonas syringae pv. syringae. We published a paper in Green Chemistry that describes a systems biology approach using Pseudomonas putida to convert lignin to biofuels. Significant progress was made with Drs. Joshua Yuan and E. A, Pierson towards usingPseudomonas putida to express enzymes for lignin conversion to biofuel production. Funding from the DOE was obtained to further develop the P. putida syustem for lignin degradation. A paper describing functional studies of a putative seralysin gene from Ca. Liberibacter solanacearum has been drafted and will be submitted for publicaiton in early 2017. <br><br><b>Publications</b><br>

<br>What was accomplished under these goals? We completed the publication of a paper describing the functional genomics of two P. syringae pv. syringae strains (i.e., B301D and HS191) in the journal MicrobiologyOpen in May 2015. The sequences have been uploaded and released to both NCBI and JGI-IMG. Our genomes work represents the 4th and 5th complete genome sequences of P. syringae strains. We also published a chapter describing the LAMP technology in "Methods in Molecular Biology -- Plant Pathology: Techniques and Protocols". This was published by Springer in 2015. A paper describing the characterization of the salA, syrF, and syrG regulatory networks of P. syringaewas prepared and submitted for publication with a target for publication in early 2016. This study shows that syrG and syrF are important transcriptional regulators of syringomycin biosynthesis genes. Serratia liquefaciens FK01 produces two serralysin-like metalloproteases, ser1 and ser2 and the serralysin-deficient mutant FK04 lack both ser1 and ser2. The putative serralysin proteins of 'Ca. Liberibacter' species were compared to the serralysin sequences in Serratia spp. and the conserved motifs (zinc binding: HEXXHXUGUXH, Met-turn: SXMXY, glycine rich repeats: GGXGXD, and ABC exporter: DXX) were identified in the predicted serralysin-like metalloproteases. According to amino acid alignment and function prediction, we predicted that the conserved motif is present in C-terminal region of the serralysin protein is responsible for the protease activity in both Serratia spp. and 'Ca. Liberibacter' species. We determined that the Lso serralysin-like metalloprotease cannot be secreted from S. liquefaciens due to an incompatible Type I secretion system. Therefore, we worked on a hybrid protein construct, which has the Serratia liquefaciens N-terminal region of serralysin (Type I secretion) and the C-terminal portion of the Lso serralysin-like region predicted to harbor protease activity. We used two PCR steps to make a complete hybrid serralysin to show activity of the critical LSO region. First, the N-terminal region of serralysin was PCR amplified from Serratia liquefaciens FK01. Second, PCR amplification of the C-terminal region of the putative serralysin from Lso was done to join the N- and C-terminal regions together. To test for protease activity, the complete hybrid serralysin sequences were cloned into a broad host range expression vector (pBBR1MCS-4) and in an expression vector (pET-14b). This was then transformed into the serralysin-deficient mutant FK04 and E. coli DE3, respectively. A proteolytic enzyme assay was used to determine if the Lso serralysin-like protein expressed in S. liquefaciens exhibited enzymatic activity. The Lso serralysin activity was expressed in E. coli DE3 and was observed by the zymogram assay. These studies are designed to characterize the Lso serralysin-like proteolytic activity as we hypothesize it is a major virulence determinant for the ZC pathogen in potato. The Lso serralysin gene also is a very useful target for understanding ZC disease development and control. For studying the serralysin-like gene at the expression level, qPCR primers were designed for the serralysin-like gene and the recA housekeeping genes of Lso; the primers were evaluated using conventional PCR. Total RNA was extracted from leaves at week 1 and week 3 of Lso-infected tomato plants; visible symptoms were not yet expressed in the plant. Total RNA was converted to double stranded cDNA. The Lso population was increased by week 3 as compared to week 1 (measured by both conventional PCR and qPCR). Using qPCR, Lso serralysin-like gene expression was 2.5-fold more in leaf samples at week 3 as compared to week 1. The Ct values were normalized to the recA housekeeping gene. The analysis shows a 2.5-fold increase in expression of the Lso serralysin-like gene in infected potatoes. Proteomic and secretory analysis of A514 grown with lignin as the sole carbon source supported the hypothesis that these complementary enzymatic systems may promote lignin depolymerization via the integration of oxidases and peroxidases to generate and utilize peroxide respectively to promote lignin depolymerization. An effective expression module depends on the identification of an effective promoter, enzyme, and secretion signal peptide. Several types of promoters were screened (constitutive, inducible, quorum sensing (QS)).We also identified a useful arabinose inducible promoter. For the module, a strong constitutive promoter was selected based on proteomic analysis and screening using a GFP reporter. The engineered strain grew faster on lignin. P31 NMR analyses (as part of another project) suggested that some hydroxyl contents of lignin (e.g., β-5, 5-5 and guaiacyl phenolic OH) were degraded faster by the engineered strain however, the percentage of lignin degraded was not specifically measured. Proteomic analysis identified two key PHA biosynthesis genes (there are several gene paralogs). These were over-expressed to enhance the carbon flux from fatty acid oxidation to PHA biosynthesis.The engineered strain grown in 1% lignin under nitrogen limitation reached 71% cellular PHA content. We developed methods to stimulate biofilm formation resulting in significant increases (10 fold) in cell growth on lignin as a sole carbon source. Lignin amalgams encouragingly demonstrated the potential for desired properties such as forming diffusion limited spaces where growth and enzyme activity could be highly concentrated next to lignin particles. <br><br><b>Publications</b><br>

<br>What was accomplished under these goals? A polished draft of a paper describing the functional genomics of two P. syringae pv. syringae strains (i.e., B301D and HS191) that are closely related but exhibit distinct plant host specificities. The sequences have been uploaded to both NCBI and to JGI-IMG. Our genome work represents the 4th and 5th complete genome sequences of P. syringae strains. The goal is to publish the paper in 2015. We will soon publish a chapter describing LAMP methodology to “Methods in Molecular Biology – Plant Pathology: Techniques and Protocolsâ€