Progress 08/01/23 to 07/31/24
Outputs
Target Audience:The target audiences reached during the current reporting period include undergraduate students through guest lecturing in Dr. Ronald's course (SAS20 Genetics and Society) at UC Davis. Under the advisement of Dr. Ronald, I designed and gave a 90-minute lecture on "The Genetics of Domestication in Agriculture",designated assigned readings, and developed an in-class discussion activity for the students. Additionally,through mentoring opportunities in the lab, I was able to train undergraduate and graduate students, as well as incoming postdoctoral scholars in the Ronald Lab at UC Davis. Furthermore, we reached other research groups and scientists interested in receptor-ligand interactions and molecular aspects of plant pathology, especially those interested in the role sulfated peptides play in plant-microbe interactions. I was given the opportunity to attend three research conferences (Plant Peptides and Receptors Meeting 2023, Plant Biology 2024, and Plant Health 2024), where I was able to present my research at two of the conferences (Plant Biology 2024 and Plant Health 2024). Additionally, at the Plant Health 2024 meeting I was able to network and discuss project impact writing and communicationwith other USDA-NIFA Project Directors in similar fields.Additional target audiences will be reached in the next reporting year through outreach events, research presentations, publications, and teaching efforts. Changes/Problems: Given the unexpected outcome of Objective 1, I will not be able to pursue the TurboID proximity labeling approach with the rice immune receptor XA21 to identify signaling partners important for XA21-mediated immune response.After several attempts to confirm biotinylation activity and TurboID function of XA21 lines, it became clear that addition of the TurboID biotin ligase to the XA21 receptor was non-functional. We hypothesize that addition of TurboID to XA21 may have led to misfolding that potentially led to TurboID not being accessible for biotin recognition, therefore inhibiting biotinylation activity. Furthermore, given that the PSY receptors were recently identified in Arabidopsis this has shifted the research schedule for Objective 2. This finding allowed me to accelerate my research timeline and has allowed me to focus on characterizing the rice PSY receptors (OsPSYR1 and OsPSYR2) to understand their role inXooinfection of rice. Although this finding led to a shift in the research schedule, it does not change the overall goal of my research project, which is to better understand how plants perceive plant (PSY) and microbe (RaxX) derived peptides for the end goal of improving rice disease resistant. Now that we know the rice orthologues for the PSY receptors, I can (1) validate recognition of the the rice PSY peptides and theXoo-secreted RaxX peptide and (2) characterize howXoois hijacking the PSY peptide hormone signaling pathway to cause disease. Overall, the results of this work will lead to improved breeding efforts to create disease resistant crops by providing new strategies to engineer plant receptors. What opportunities for training and professional development has the project provided?With funding provided by NIFA for this project, the PD has been able to gain various opportunities for training and professional development. For professional development, the PD has networked at various conferences and developed collaborations. The PD was able to attend three conferences over the last year, one as an attendee and two as a poster presenter. Additionally, the PD was able to establish a collaboration with the Innovative Genomics Institute Plant Genetics and Transformation Facility in Berkeley. Additionally, the PD guest lectured in Dr. Ronald'sUC Davis undergraduate-level course: Genetics and Society (SAS20). Under the advisement of Dr. Ronald, the PD gave a 90-minute guest lecture, helped optimize in-class activities, and led classroom discussions. For research training, the PD has learned how to genetically engineer rice with CRISPR-Cas9 (from designing guide RNAs to in-vitro gRNA validation to cloning and downstream analyses), has established a biotinylation protocol necessary for TurboID proximity labeling efforts in rice, and has improved her skills in rice genetics. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? In the next reporting year, I aim to finish characterizing the CRISPR-Cas9 edited knock-out OsPSY receptor lines (Ospsyr1, Ospsyr2, and Ospsyr1,2).For these lines, I aim to address if knocking out one or both receptors will leadto phenotypic changes in root system architecture (i.e., elongated roots), flowering time, seed yield, panicle development, leaf development, and/or changes in susceptibility toXooinfection. To determine if these receptor knockouts are important forXooinfection, I will inoculate 4- to 5-week-old rice plants withXooby the scissor clipping method. Changes in host resistance or susceptibility will be measured by bacterial counts and lesion length measurements at 10 dpi and 14 dpi. For all inoculation assays, wild-type Kitaake and KitaakeX (Kitaake expressing Xa21 resistance gene) plants will be used as controls. In addition, differentXoostrains including the wild-type PXO99 strain, deltaRaxX strain, and deltaRaxX(RaxX) strain will be used to confirm if the OsPSY receptor knockout lines are able to recognize the RaxX peptide or not. Furthermore, in the next reporting year, I aim to confirm that the OsPSY receptors bind the OsPSY peptides (OsPSY1-8) and theXoosecreted RaxX peptide. To test if these plant and microbe derived peptides bind the OsPSY receptors I will use microscale thermophoresis (MST).MST is a tool used to quantitatively analyze protein and small molecule interactions with little sample consumption. Previous studies in the Ronald lab used MST to validate the binding affinity of the XA21 rice immune receptor with theXoo-secreted peptide RaxX, thus I plan touse the well-established XA21-RaxX system when optimizing MST parameters as a reference.
Impacts
What was accomplished under these goals?
The main goal of Objective 1 was to utilize TurboID proximity labeling (a proteomics approach) to identify signaling components involved in XA21-mediated immune signaling following RaxX recognition. However, when characterizing the Turbo-ID tagged rice lines (XA21 and the TM-control), I found that tagging a large, transmembrane receptor (XA21) with Turbo-ID makes the Turbo-ID tag non-functional. Although we were able to confirm that XA21 tagged with Turbo-ID (XA21-TurboID) was functional by gene expression analyses after RaxX peptide application andXooinfection, I was unable to confirm biotinylation activity of the Turbo-ID tag for these lines. XA21-TurboID lines showed increased defense gene expression following RaxX peptide treatment and showed resistance toXooinfection (short lesionlengths at 10 days post inoculation (dpi) and 14 dpi). Throughout the process of characterizing the Turbo-ID tagged lines, I was able to develop and optimize a protocol for testing biotinylation activity of older rice leaves (~3-4 week old plants). Through this approach I was able to confirm that our control Turbo-ID tagged lines (TM-TurboID) had high biotinylation activity, but none of the XA21 lines tagged with Turbo-ID showed any biotinylation activity. At first, I hypothesized that negative interactors of the XA21-immune signaling pathway could be blocking TurboID, hence there not being any biotinylation activity. So, I tested how XA21-TurboID tagged lines responded to biotin after treatment with the RaxX peptide (which should activate XA21-immune signaling). However, even addition of RaxX peptide before biotin application did not result in any biotinylation activity. After several attempts involving various time-points and concentrations of biotin, it became clear that addition of the TurboID biotin ligase to the XA21 receptor was non-functional. We hypothesize that addition of TurboID to XA21 may have led to misfolding, potentially making TurboIDinaccessible for biotin recognition andtherefore inhibiting biotinylation activity. The PSY receptors were recently identified in Arabidopsis (AtPSYR1-At1g17230, AtPSYR2-At2g33170, AtPSYR3- At5g63930) and confirmed to bind the AtPSY peptides with high affinity through a competitive binding assay. This discovery has allowed me to accelerate my research timeline, as one of the main goals of Objective 2 was the identify the PSY receptors in rice (Objective 2A) and Arabidopsis (Objective 2B). Based on the published work in Arabidopsis, I was able to identify two orthologues for the PSY receptors in rice based on phylogenetic analyses: OsPSYR1 = LOC_Os07g05740 and OsPSYR2 = LOC_Os04g42700. To better understand how PSY signaling and perception may be contributing to increased susceptibility of rice toXooinfection, I decided to generate CRISPR-edited Kitaake lines with null mutations in OsPSYR1 and OsPSYR2 through an optimized approach for rice. I designed guide RNAs (gRNAs) to target each receptor using the online program: CRISPR-P 2.0. Given that the OsPSY receptors are Leucine-Rich Repeat Receptor-Like Kinases (LRR-RLKs), I chose to design gRNAsthat would target each major part of the receptors. I generated three gRNAs for each receptor, one targeting the LRR-Cap coding sequence, one targeting the LRR-domain coding sequence, and one targeting the kinase domain coding sequence. The YPQ CRISPR-Cas9 multiplexing toolbox system was used to construct CRISPR plasmids using Golden Gate and Gateway cloning methods. Each gRNA was driven by a OsU6 promoter and each gRNA was screened for effective cleavage of their targetsin vitro. To screen for gRNA cleavage of target sites, I used the Takara Bio Guide-it sgRNAin vitroTranscription and validated each gRNA before Agrobacterium transformation into Kitaake. For single receptor knockouts(Ospsyr1and Ospsyr2)three gRNAs were usedand for the double receptor knock-out (Ospsyr1,2) only one gRNA for each receptor was used. In collaboration, with the Innovative Genomics Institute (IGI) Plant Genomics and Transformation Facility all the Kitaake transformations were done at IGI. I received over100 independently edited lines for each of the OsPSYR knockouts (Ospsyr1, Ospsyr2, and Ospsyr1,2). I genotypedall of the independently edited T0 lines andidentified those that contained deletions of interest and were Cas9-positive.Deletions identifiedin T0 transformants by PCRwere validated by Sanger sequencing for each of the OsPSYR knock-out lines. T1 lines are in the process of being genotyped (i.e., lacksCas9 but contains edit of interest seen in T0) and characterized (i.e., root length).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Ercoli M, Shigenaga AM, Teixeira de Araujo A, Jain R, Ronald P. Tyrosine-sulfated peptide
hormone induces flavonol biosynthesis to control elongation and differentiation in Arabidopsis
primary root. bioRxiv [PrePrint]. 2024 Feb 3:2024.02.02.578681. doi: 10.1101/2024.02.02.578681.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Shigenaga AM, Ercoli MF, Ronald PC. 2024. Elucidating the Role of Small, Sulfated Peptides in Facilitating Bacterial Infection of Rice. Poster Presentation. Plant Biology 2024 Conference (Honolulu, Hawaii).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Shigenaga AM, Ercoli MF, Ronald PC. 2024. Elucidating the Role of Small, Sulfated Peptides in Facilitating Bacterial Infection of Rice. Poster Presentation. Plant Health 2024 Conference (Memphis, Tennessee).
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