Progress 06/15/23 to 06/14/24

Outputs
Target Audience:During this project, broad scientific audiences were targeted by the PD. First, unpublished results were shared with scientific audiences within and beyond the PI's home institution (University of California Davis, and then University of Arizona). The scientific audiences reached during this project included researchers and scientists in the fields of microbial physiology, plant pathology, and microbial genomics. This project focuses on a fundamental understanding of Ralstonia solanacearum physiology, and a detailed understanding of this physiology could ultimately be leveraged to control this destructive pathogen. Ralstonia solanacearum is a persistent pest in tomato growing areas in SE US and there is an ongoing threat of introduction of select agent strains from asymptomatic ornamental imports. As such this work was also communicated through oral presentations with extension specialists interested in soil microbiomes and biocontrol research. This research was presented at 3 major conferences, at 5 different institutions departmental seminar series, and at several small colloquia in Davis and externally. The colloquia audiences were largely undergraduate, graduate, or postdoctoral audiences. During this project, several UCD undergraduates, and junior specialists participated in in-lab training and experimentation related to this project. At University of Arizona, undergraduates and graduate students are leveraging this data for further study as they join the Schomer Lab. Further, graduate trainees were targeted and educated about chemosensing in these plant pathogens in guest lectures and experimental demos at the Marine Biological Laboratory. As proposed a research-based course module was piloted in which students would screen chemoreceptors against an array of potential chemical ligands. This was piloted through the Microbial Diversity Course. Data was acquired by students in the course and this data is being leveraged in ongoing projects. This pilot provided valuable information about how this research technique can be leveraged into a successful CURE. Changes/Problems:As discussed in previous progress reports, the RB TnSeq experimental design ultimately had a significant bottleneck that limited the discovery that could have emerged from this powerful technique. This bottleneck contributed to the PDs and Sponsors ability to design improved experiments to bypass this bottleneck in the future, and exploit alternative selective pressures that will get at the same questions. For that reason, the work in the second and final progress periods focused on pursuing the functional screen (chimeric chemoreceptors in the fluorescent biosensors) and comparative genomics/metabolomics. Further, the pandemic limited the ability of the PD to initiate the CURE as planned originally. Rather than several iterations of the CURE, a pilot CURE was tested in 2022 and 2023 and a curriculum designed for future work. What opportunities for training and professional development has the project provided?The original proposal indicated that the PD would pursue professional development to further her career. These goals were accomplished, the PD established an online twitter presence and a professional website to communicate her research and build professional community. Further, the PD was able to develop a curriculum for a Course-based Undergraduate Research Experience and pilot that CURE through the Microbial Diversity Course at MBL. The PD mentored an undergraduate and two junior specialists during the project, two of which have moved on to a plant-focused research program, the other to a medically focused post graduate school. Finally, the PD has obtained a tenure-track position and transitioned into a leadership role at a new Univeristy.. How have the results been disseminated to communities of interest?The scientific progress of the project was presented at multiple conferences including APS (which targeted plant scientists, plant pathologists, and extension professionals), Lake Arrowhead Microbial Genomics (which targeted microbial phyiologist, geneticists, and genomicists), as well as several external institutions including NCSU, RIT, Virginia Tech, Purdue, UArizona and UC Davis. Importantly, this work has reached the broader international Ralstonia community, as the PD presented her work at an oral presentation at the International Bacterial Wilt Sympoisum in Uruguay. This symposium targets both extension specialists and basic/applied researchers in the field of Ralstonia. The work was presented also at symposia and workshops targeting graduate and postdoctoral researchers. Finally, the data described and analyzed above is currently being drafted into manuscripts that will be submitted for peer review and further reach communities of interest. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The goal of this project is to understand the role of chemotaxis (or the directional movement of bacteria towards specific chemicals) in the life cycle and pathogenesis of the plant pathogen, Ralstonia. Chemotactic behavior is a critical step in the infection of many nutritionally and economically relevant host plants by this particularly destructive pathogen. During this project period, root exudate samples were collected and metabolomic analyses were performed on the samples. Samples were taken over time and in replicates, using fabricated ecosystems, allowing for noninvasive and reproducible comparisons of multiple plants at different life stages. These exudates were found to be both growth substrate and attractants to the model Ralstonia strains. These exudate samples are currently under analysis. Targeted analyses revealed a small subset of target chemicals, while untargeted analyses generated a much larger pool of chemical data. The targeted chemicals have been incorporated into HTS and chemotaxis assays, while the untargeted analyses are being analyzed in greater detail to generate a subset of target chemicals for future studies. Further, chemotactic range of all three model Ralstonia strains was tested using individual chemical substrates. Chemicals were targeted based on their report as common plant metabolites. The ranges of the three model systems was variable. The broad-host range Ralstonia strain (IBSBF1503) was chosen as the first strain to proceed with a genomics-driven approach (Randomly Barcoded TransposonSequencing or RB TnSeq) to reveal fitness factors involved in sensing individual chemicals. RB TnSeq is a genome wide screen through which the fitness impact of every gene under variable selective pressures in the entire genome can be evaluated simultaneously Several dozen conditions across several exudates and rich media were tested and analyzed (>90 populations were collected, sequenced, and analyzed). Bottlenecking was discovered as a significant flaw for this experimental design. However, simultaneously, chimeric receptors of the chemoreceptors across all three strains were constructed and inserted into the E. coli reporting strains with lacZ and gfp readouts (44 unique fluorescent biosensors in total across three strains). A protocol was finessed to screen the chimeric receptors in 96-well plate format, using comparative analysis of the chimeras in the established betagalactasidase reporter to the newly designed fluorescent strain. Screens revealed several amino acid sensing chemoreceptors that were followed up on and studied in the original strains. Highly mobile versions of the three model strains were generated by classical enrichments. The whole genomes of these strains were sequenced to ensure any SNPs or mutants were not functionally altering core chemotaxis or flagellar genes. These enriched strains were established as wild-type strains and were shown to infect host plants similarly to the natural isolates by both cut petiole and soil soak inoculation. These strains were confirmed to have more significant chemotactic movement towards test attractants, but not variable chemotactic range from their natural isolate counterpart. The amino acid receptors were deleted from the genomes in all three model strains. Plants were challenged with wild-type and deletion strains to understand the role of each individual amino acid sensor, as well as amino acid sensing in general in plant discovery from the soil. Third, comparative genomics analyses were performed to determine across the RSSC which chemoreceptors are most conserved (core) or variable or cluster to specific phylotypes. This comparative data along with initial screen data has generated targets for downstream studies to continue to map the chemoreceptome of Ralstonia as it relates to host discovery.

Publications