Performing Department
(N/A)
Non Technical Summary
This project fits well in the NIFA program area priority of Pests and Beneficial Species inAgricultural Production Systems and addresses the Hatch Act area of plant and animal production, protection, and health. This project will lead to advanced knowledge of an established plant pathogen and a better understanding of the broader umbrellas of vascular plant pathogens and Gram-positive bacteria. The project focus on Clavibacter, a genus of Gram-positive, xylem-colonizing bacteria containing eight important plant pathogens. C. michiganensis is the most well-known and destructive of these, causing severe disease outbreaks in processing and fresh-market tomatoes. Management of Clavibacter diseases has historically been attempted with seed sterilization, seed treatment, and quarantine. However, no control method provides a complete solution. Plant pathogens are generally most effectively managed via plant resistance, but no genes controlling resistance to Clavibacter have been cloned and no genetic resistance to C. michiganensis has been found in processing tomatoes.Completion of this project will result in a greater understanding of the interactions between Gram-positive bacterial pathogens and plants. Despite the importance of Gram-positive plant pathogens, no immune receptors have been identified. Aim 1 of the project will identify and characterize a receptor for a Clavibacter-secreted effector, marking the discovery of the first plant receptor for a Gram-positive bacterium. Aim 2 will contribute to the understanding of plant defense to vascular plant pathogens. Understanding the role of damage receptors in the restriction of vascular pathogens can inform breeding decisions for resistant traits. The identification of a Clavibacter receptor and its transfer into tomato will have significant implications for disease resistance breeding, as well as provide valuable insight into the mechanisms behind Gram-positive pathogenicity.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
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Developmental
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Goals / Objectives
The major goals of this project are to improve understanding and management of plant diseases caused by Clavibacter species. Within these major goals exist six specific objectives broken into two aims. Aim one concerns the identification of the first Clavibacter resistance gene. The objectives within this aim are (i) the identification of a Cm resistance gene, (ii) the transfer of the resistance gene to tomato, and (iii) the evaluation of the resistance gene in tomato. Aim two concerns Clavibacter movement, colonization patterns, and restriction by plant hosts. The objectives within aim two are (i) the development of fluorescent Clavibacter strains, (ii) the visualization of Clavibacter movement, and (iii) the visualization of Clavibacter restriction.
Project Methods
Aim 1: Identify and characterize Clavibacter receptor.In my first aim, I seek to identify and characterize a plant receptor capable of recognizing a Cm-secreted effector. I will use a combination of receptor mining and gene silencing to identify receptor candidates, verify candidates by transient expression in N. benthamiana, and finally transfer the receptor to tomato to determine if the receptor controls Cm infection.Aim 1.1 - Identify the receptor: Clavibacter cannot secrete proteins directly into plant cells; we hypothesize Clavibacter effectors are recognized by surface localized receptors. Two types of plant surface receptors recognize proteinaceous molecules - receptor-like kinases (RLKs) and receptor-like proteins (RLPs). For proper function, RLPs require the RLK SUPPRESSOR OF BIR1-1 (SOBIR1). To determine if the receptor is an RLK or RLP we will use virus-induced gene silencing (VIGS) to silence SOBIR1 and test for effector-elicited cell death. Once the receptor type is identified, we will mine-resistant plant genomes for receptors using published receptor prediction models. A stacked VIGS library will be made to silence all genes identified by mining. This stacked VIGS method was pioneered in N. benthamiana and can be adapted for other plant species. Rather than silencing one gene at a time, these stacked constructs silence multiple gene candidates at once, allowing for rapid testing of several genes. Silencing of the receptor should lead to a reduction in cell death after Cm infiltration. Three plants will be silenced with each stacked VIGS construct or GUS or ChlH (photobleaching) controls. After three weeks, the plants will be challenged with Cm, a Cm knockout of the studied effector, and buffer (n = 6). Leaves will be imaged with UV and cell death quantified in ImageJ. A non-parametric test will be used for statistical testing. If any of the constructs lead to a reduction of cell death, individual VIGS constructs will be made for each candidate and the assay will be repeated. To confirm candidate receptors, individual receptor candidates will be expressed in N.benthamiana using Agrobacterium-mediated transient expression. Four plants will be infiltrated with the receptor expression construct or empty vector. 24h after expression, leaves will be challenged with wild-type Cm, a Cm knockout of the studied effector, and a buffer. The experiment will be repeated for 16 total replicates. As N. benthamiana cannot recognize Cm, a cell death response should only be observed after expressing the receptor. Leaves will be imaged with UV excitation and cell death quantified in ImageJ. Cell death will also be quantified by electrolyte leakage and analyzed by parametric statistical analysis (ANOVA, etc.) while images will be analyzed with a non-parametric test.Aim 1.2 - Transfer of the receptor to tomato: To further validate the receptor, it will be transferred into tomato and evaluated for efficacity in Cm control. The receptor will transferred into tomato at the UC Davis Transformation facility. Two independent homozygous lines will be generated and validated for expression. Three-week-old transgenic tomato and wild-type sister lines will be inoculated with Cm, a Cm knockout of the studied effector, or water by stab inoculation of the stem. Bacterial titers will be quantified at 14 days and disease symptoms will be scored on a quantitative scale as previously described. Six tomatoes per genotype per treatment will be analyzed and all experiments will be repeated at least three times.Aim 2: Investigate the importance of damage receptors in restricting Clavibacter infection.In aim 2 I seek to investigate the function and importance of damage receptors in the restriction of Clavibacter infection. While both pathogenic and endophytic Clavibacter secrete CAZymes, only pathogenic Clavibacter cause wilt symptoms. I hypothesize pathogenic Cm can suppress defense induced against CAZyme effectors, enabling pathogen movement and xylem destruction. To test this hypothesis, I will inoculate tomato lines with knockouts of DAMP and danger receptors with pathogenic and non-pathogenic Clavibacter strains and measure bacterial growth and disease severity. I will complement this assay with confocal microscopy to visualize bacterial movement and cell wall breakdown.Aim 2.1 - Investigating DAMP receptor impact on disease severity and CFUs: To test the importance of DAMP receptors in the restriction of Cm, three three-week-old tomato plants from each genotype (wild-type Rio Grande, DAMP knockouts) will be stab inoculated with a syringe with pathogenic Cm, non-pathogenic Cm, or water. Each week, symptoms will be quantified, and statistics run with a non-parametric test. After two weeks, the plants will be removed to quantify bacterial colony-forming units (CFUs). Aim 2.2 - Visualizing DAMP receptor restriction of colonization: Next, I will investigateClavibacter colonization of wild-type and DAMP knockout tomato with confocal microscopy. 9 three week-old tomato plants of each genotype will be inoculated as described above with GFP-tagged Clavibacter strains. Three tomato plants will be removed from each treatment at three timepoints and observed with confocal microscopy. Stem sections will be taken, fixed, sectioned with a vibratome, stained with propidium iodide visualize cell walls, and viewed with a Leica SP8 TCS microscope. Multiple images will be taken of each sample. Maximum fluorescence intensity will be used to infer bacterial titer and fluorescence area will be used to quantify the area of metaxylem, protoxylem, and pith colonized. Both intensity and area will be quantified in ImageJ and tested with a non-parametricstatistical test (n = 9). Interesting findings will be repeated with cell wall antibodies to visualizechanges in cell wall integrity.Efforts: Efforts during the project include mentorship, extension, and outreach. I will mentor at least two undergraduate students. At least one of these students will complete a research practicum contributing to their degree. For extension and outreach, I will present my findings at commodity board meetings and a disease round table with extension members present. Finally, I will communicate my research to the public through presentations in K-12 STEM programs, outreach within the university, and social media (twitter, blogs).Evaluation: Primary evaluation of the project will occur through bimonthly meetings and six-month progress reviews with my major professor, Gitta Coaker, as well as yearly thesis committee meetings. Outside evaluation of project success will come from feedback at commodity board meetings, presentations at international scientific conferences, and publications in peer-reviewed journals. Major milestones of the project include receptor identification, transfer, and evaluation (aim 1); and visualization of DAMP restriction (aim 2).