Progress 06/15/21 to 01/11/24
Outputs
Target Audience:The primary target audience during this reporting period has been the plant biology scientific community, both at large as well as at the University of California, Davis. This includes professors and faculty across a variety of plant biology specializations, but chiefly those with active research in the field of host-microbe interaction. During the duration of the project, I communicated my research to the scientific community at large by presenting a poster at the American Society of Plant Biology annual meeting in Portland, OR. At the same meeting my project mentor, Dr. Dinesh-Kumar, gave a concurrent session using my research. Later, at the 2023 conference for the International Society of Molecular Plant-Microbe Interactions, I presented unpublished findings from this project at a concurrent session while Dr. Dinesh-Kumar presented our published findings from this project in a plenary session. Over the duration of the project, I participated and presented a seminar on my research at an international workshop between UC Davis, the Nara Institute of Science and Technology, and the Chinese Academy of Sciences on two separate occasions. I presented to the scientific community at UC Davis on several times throughout the duration of the project. First, I presented at the Plant Biology Graduate Student Recruitment, reaching members of the plant biology graduate group as well as students from other universities. Second, I gave an exit seminar at the cell biology seminar series at UC Davis, reaching faculty members outside of plant biology but still within the college of biological sciences. Further, our findings for KIS1, a kinesin which regulates immunity via chloroplast stromule formation were accepted and published in Science Advances. Going forward, we hope to submit further findings that were a part of this project. A secondary audience reached over the duration of the project was the undergraduate population at the University of California, Davis. Throughout, I was able to mentor three undergraduates who were interested in plant biology and wanted hands-on research experience. All of these undergraduates spent >10 hours a week working directly with me on this research project. The research that they were able to assist me with ranged from wet-lab work to data analysis. All of these undergraduates finished their degree programs and have since moved onto graduate school or state or industry positions. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Over the duration of this project, I was able to attend the 2022 ASPB annual meeting in Portland, OR and presented a poster on KIS1. I was also able to attend the International Student Workshop at UC Davis three separate times. Both of these presented great networking opportunities and will help guide me in my next career steps. I was also able to attend the 2023 IS-MPMI meeting in Providence, RI to present my work on effector-mediated stromule suppression. How have the results been disseminated to communities of interest?A manuscript of our findings on KIS1 has been published in Science Advances and is disseminated broadly in the scientific community. In addition, the project director has given three different seminars on KIS1 and effector-mediated stromule suppression to the scientific community at UC Davis as well as to the small international audience at the International Student Workshop at UC Davis. The Project Mentor presented our findings on KIS1 at the 2022 ASPB annual meeting during a concurrent session on Report organelle biology. He also presened our full results as a plenary speaker at the 2023 IS-MPMI meeting in July 2023. The project director gave a seminar on effector-mediated stromule suppression during a concurrent session on organelle biology at the 2023 IS-MPMI meeting in July 2023. A manuscript summarizing our results from Objective 1 is being prepared for submission. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Characterize mechanisms by which pathogen effectors regulate stromule formation As of now, we have completed a screen of all 28 effectors from Pseudomonas syringae pv tomato (Pst) DC3000 in the N-P50, Rx1-PVX-CP, and Sw5b-TSWV-NSm immune responses. We have identified one effector, HopR1, which is sufficient to suppress stromule formation induced by these three different immune responses as well as stromules that are induced constitutively by overexpression of chloroplast membrane proteins. Further, we have been able to show that HopR1 disrupts microtubule organization, potentially indicating a mechanism by which stromule formation is suppressed. We performed TurboID-based proximity labeling using HopR1 as the bait protein. Our results show that several interesting candidate interactors of HopR1. To test these candidate interactors, we have used virus-induced gene silencing (VIGS) to suppress these candidates in Nicotiana benthamiana and determined what happens to the macroscopic immune response as well as stromule formation and nuclear clustering. We have found one chloroplast membrane channel which is specifically required for immune stromule formation. Currently, we are characterizing the specific solute transported by this membrane channel. I presented these results at the IS-MPMI meeting in Providence, RI this July. Once submitted, this will represent a completion of Objective 1 and our findings will present a mechanism by which chloroplast stromules are initiated during the TNL-mediated immune response. Further, the other effectors identified from our screen warrant further investigation. Objective 2:Characterize proteins which interact with the stromule membrane during the immune response? On October 25, 2023, our manuscript titled "Calponin-homology domain containing kinesin, KIS1, regulates chloroplast stromule formation and immunity" was published in Science Advances. This represents a completion of Objective 2 of my original aims. In our publication, we show that a kinesin, KIS1, is required for chloroplast stromule formation during the immune response. Further, we conducted a structure-function study showing that the calponin homology (CH) domain of KIS1 is required for nuclear clustering, a required aspect of the immune response. Following this, we conducted a signaling study using knockout mutants of known players in the TNL-mediated immune response and found that KIS1-induced stromule formation is dependent on EDS1 and PAD4, but not NRG1. Taken together, these results identify KIS1 as the first known player in chloroplast stromule formation during defense. We have begun several follow-up experiments to further characterize KIS1 including attempts to purify KIS1 and generating transgenic lines in Arabidopsis to further study the CH domain. The transgenic and mutant lines generated from this study will be instrumental in identifying other components required for stomule biogenesis and function.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Nathan Meier, Yongliang Zhang, Jeffrey Caplan, and Savithramma P Dinesh-Kumar. July 16-20, 2023; Pathogen effectors target chloroplast stromules to interfere with immunity [Conference Presentation]. 2023 International Society of Molecular Plant-Microbe Interaction (IS-MPMI) Congress, Providence, RI.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Nathan D. Meier et al. ,Calponin homology domain containing kinesin, KIS1, regulates chloroplast stromule formation and immunity.Sci. Adv.9,eadi7407(2023).DOI:10.1126/sciadv.adi7407
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Progress 06/15/22 to 06/14/23
Outputs
Target Audience:The primary target audience during this reporting period has been the plant biology scientific community, both at large as well as at the University of California, Davis. This includes professors and faculty across a variety of plant biology specializations, but chiefly those with active research in the field of host-microbe interaction. During the reporting period, I communicated my research to the scientific community at large by presenting a poster at the American Society of Plant Biology annual meeting in Portland, OR. At the same meeting my project mentor, Dr. Dinesh-Kumar, gave a concurrent session using my research. In addition to communications at ASPB, I attended and presented a seminar on my research at an international workshop between UC Davis, the Nara Institute of Science and Technology, and the Chinese Academy of Sciences. I presented to the scientific community at UC Davis on two separate occasions this reporting period. First, I presented at the Plant Biology Graduate Student Recruitment, reaching members of the plant biology graduate group as well as students from other universities. Second, I gave an exit seminar at the cell biology seminar series at UC Davis, reaching faculty members outside of plant biology but still within the college of biological sciences. A secondary audience reached during this reporting period was the undergraduate population at the University of California, Davis. During this reporting period, I was able to mentor two undergraduates who were interested in plant biology and wanted hands-on research experience. Both undergraduates spent >10 hours a week working directly with me on this research project. The research that they were able to assist me with ranged from wet-lab work to data analysis. One of these undergraduates has finished her degree and will be working as a Junior Specialist at UC Davis and the other will be continuing to work with me throughout the next year. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, I was able to attend the 2022 ASPB annual meeting in Portland, OR and presented a poster on KIS1. I was also able to attend the International Student Workshop at UC Davis during this reporting period. Both of these presented great netwroking opportunities and will help guide me in my next steps. In July of this year, I will be attending the 2023 IS-MPMI meeting in Providence, RI to present my work on effector-mediatedstromule suppression. How have the results been disseminated to communities of interest?A manuscript of of our findings on KIS1has been submitted toScience Advances.If reviewed and accepted this will be disseminated broadly to the scientific community. In addition, the project director has given three different seminars on KIS1 and effector-mediated stromule suppression to the scientific community at UC Davis as well as to the small international audience at the International Student Workshop at UC Davis. The Project Mentor presented our findings on KIS1 at the 2022 ASPB annual meeting during a concurrent session on organelle biology. He will present our full results as a plenary speaker at the 2023 IS-MPMI meeting in July 2023. The project director will be giving a seminar on effector-mediated stromule suppression during a concurrent session on organelle biology at the 2023 IS-MPMI meeting in July 2023. What do you plan to do during the next reporting period to accomplish the goals?-Determine a mechanism of action for HopR1 mediated stromule suppression. -Attend IS-MPMI 2023 meeting in Providence, RI and speak during a concurrent session on organelle biology. -Complete the review process for KIS1 manuscript -Draft and submit a manuscript on HopR1 interactome and HopR1-mediated stromule suppression.
Impacts
What was accomplished under these goals?
Objective 1: Characterize mechanisms by which pathogen effectors regulate stromule formation As of now, we have completed a screen of all 28 effectors from Pseudomonas syringae pv tomato (Pst) DC3000 in the N-P50, Rx1-PVX-CP, and Sw5b-TSWV-NSm immune responses. We have identified one effector, HopR1, which is sufficient to suppress stromule formation induced by these three different immune responses as well as stromules that are induced constitutively by overexpression of chloroplast membrane proteins. Further, we have been able to show that HopR1 disrupts microtubule organization, potentially indicating a mechanism by which stromule formation is suppressed. We performed TurboID-based proximity labeling using HopR1 as the bait protein. Our results show that several known players of microtubule organization are likely candidate interactors of HopR1. To test these candidate interactors, we have used virus-induced gene silencing (VIGS) to suppress these candidates in Nicotiana benthamiana and determined what happens to the macroscopic immune response as well as stromule formation and nuclear clustering. As of now, we are determining the mechanism of action of these candidate proteins. I will be giving a presentation on these results at the IS-MPMI meeting in Providence, RI this July. Objective 2: Characterize proteins which interact with the stromule membrane during the immune response On May 15th, 2023, we submitted a manuscript titled "Calponin-homology domain containing kinesin, KIS1, regulates chloroplast stromule formation and immunity" to Science Advances. This represents a completion of Objective 2 of my original aims. In our manuscript, we show that a kinesin, KIS1, is required for chloroplast stromule formation during the immune response. Further, we conducted a structure-function study showing that the calponin homology (CH) domain of KIS1 is required for nuclear clustering, a required aspect of the immune response. Following this, we conducted a signaling study using knockout mutants of known players in the TNL-mediated immune response and found that KIS1-induced stromule formation is dependent on EDS1 and PAD4, but not NRG1. Taken Together, these results identify KIS1 as the first known player in chloroplast stromule formation during defense. We have begun several follow-up experiments to further characterize KIS1 including attempts to purify KIS1 and generating transgenic lines in Arabidopsis to further study the CH domain. Currently, these experiments are not complete and have not been included in the submitted manuscript.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Nagalakshmi, U., Meier, N., and Dinesh-Kumar, S. P. (2023) Virus-induced heritable gene editing in plants. Methods Mol. Biol. (in press).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Meier, N., Seward, K., Caplan, J., and Dinesh-Kumar, S. P. (2023) Calponin-homology domain containing kinesin, KIS1, regulates chloroplast stromule formation and immunity. Science Advances (Submitted)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Meier, N., Caplan, J., and Dinesh-Kumar, S. P. A kinesin is a positive regulator of chloroplast
stromule extension during the innate immune response. American Society of Plant Biology
(ASPB) Annual Meeting, July 9 13, 2022, Portland, OR.
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Progress 06/15/21 to 06/14/22
Outputs
Target Audience:The primary target audience during this reporting period has been the scientific community at the University of California, Davis. This includes professors and faculty across a variety of biological fields, but chiefly those with active research in the field of host-microbe interaction. During the reporting period, I communicated my research to the scientific community by presenting at two different seminars as well as presenting a poster during a departmental retreat. A secondary audience reached during this reporting period was the undergraduate population at the University of California, Davis. During this reporting period, I was able to mentor two undergraduates who were interested in biological science and wanted hands-on research experience. Both of these undergraduates spent 10 hours a week working directly with me on this research project. The research that they were able to assist me with ranged from wet-lab work to analysis of microscopy data. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In September 2021, the project director attended a workshop hosted by the UC Davis Genome Center on RNA-Seq Analysis. How have the results been disseminated to communities of interest?During the reporting period, the project director has presented results from this project at two different seminars as well as presented a poster at a Plan Biology Retreat. These presentations were all aimed at the scientific community at UC Davis. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period I plan to: -Publish a manuscript on the role of KSE2 in stromule formation. -Attend ASPB 2022 in Portland, OR where I will be presenting a poster on KSE2's role in stromule formation. -Finish work on EIP3 and draft a manuscript for the role of E8 suppressing EIP3-induced stromule formation. -Complete TurboID-based proximity labeling on another effector identifed from the suppressor screen. -Begin experiments to determine KSE2's role in plant immune signaling.
Impacts
What was accomplished under these goals?
Plant pathogens can lead to devastating crop losses and threaten food security both domestically and abroad. Toward this, great strides have been made in engineering plant resistance to pathogens. However, much of the current resistances to pathogens relies on gene-specific interactions between the pathogen and the plant host. Because of this, there is tremendous pressure for pathogens to overcome plant disease resistance; often this is referred to as an evolutionary arms race between the plant and pathogens. This arms race drives a need for continuous research into the plant immune system to cultivate new disease resistance mechanisms that can ultimately be deployed in key crop species. Recently, the Dinesh-Kumar lab has presented strong evidence that chloroplast stromules serve as positive regulators in plant immunity. Chloroplasts serve as a common battle ground for pathogens; as such, this project has been identifying components that are required for stromule formation during the plant immune response. To this end, we have made a significant change in knowledge by identifying the first known component that is required for stromule formation during the plant immune response. Our results show that a gene, KSE2, is required for stromule formation during the N-mediated immune response in Nicotiana benthamiana as well as the AvrRPS4-mediated immune response in Arabidopsis. Further study of this gene could allow for novel disease resistances to be developed and deployed into major crop plants as well as to improve our understanding of the role of chloroplast stromules during the plant immune response. We are also beginning to explore proteins that interact with pathogen effectors, these genes could be other significant components that are required for stromule formation. Objective 1: Characterize mechanisms by which pathogen effectors regulate stromule formation. We have completed initial stromule suppression screen for all of the effectors from Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in the Rx1-PVX-CP and Sw5b-TSWV-NSm induced immune responses. In order to reduce the number of potential false positives, we have begun secondary screening for any effectors that suppressed stromules in the first round of screening. One of the tested effectors, Effector 8 (E8), showed suppression in the Sw5b, Rx1, and N NLR systems. Due to this, we developed a Turbo-ID construct using this effector and proceeded with proximity labeling (PL). The PL data showed many interesting interacting partners. Based on our data from objective 2, we identified a kinesin, Effector-interacting protein 3 (EIP3), from the PL data as a potential stromule-regulating protein which is specifically suppressed by E8. To test this, we transiently expressed this kinesin in Nicotiana benthamiana and found that overexpressing EIP3 induces constitutive stromule formation. To verify our initial interaction observations, we found that if E8 is co-expressed with EIP3 then EIP3-induced stromule formation is suppressed. We are currently in the process of developing and testing TurboID constructs for other effectors which suppressed stromule formation in our initial screen. Objective 2:Characterize proteins which interact with the stromule membrane during the immune response. Using a candidate approach, we identified a kinesin, KSE2, which constitutively induces stromule formation when overexpressed in Nicotiana benthamiana. To quickly test what may happen with a loss-of-function of KSE2, we used Virus-induced Gene Silencing (VIGS) to knockdown KSE2 expression. We observed that when KSE2 is silenced, stromule induction is significantly suppressed during the N-mediated immune response. In order to verify our results from Nicotiana benthamiana, we isolated homozygous T-DNA mutants in Arabidopsis and transformed them with a stroma marker in order to observe stromule formation. We observed that during the immune response induced by Pst DC3000 expressing AvrRPS4 (Pst DC3000 (AvrRPS4)), kse2 mutants show significantly reduced stromule formation compared to Col-0. Not only are stomules suppressed in kse2, but trypan blue staining shows that kse2 mutants have a compromised cell death response to Pst DC3000 (AvrRPS4) when compared to Col-0. Finally, using a bacterial growth assay, we observed that kse2 mutants have significantly increased bacterial growth when compared to Col-0. Based on our data, KSE2 represents the first known component of stromule formation. We are preparing a manuscript to communicate our findings thus far for objective 2 tentatively titled "A kinesin, KSE2, is a positive regulator of stromule formation during the plant immune response".
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
High efficiency multiplex biallelic heritable editing in Arabidopsis using an RNA virus" by Ugrappa Nagalakshmi, Nathan Meier, Jau-Yi Liu, Daniel Voytas, and Savithramma Dinesh-Kumar
- Type:
Journal Articles
Status:
Accepted
Year Published:
2021
Citation:
Li Y, Meier N, Dinesh-Kumar SP (2021) Parasite effectors target helper NLRs in plants to suppress immunity-related cell death. PLoS Biol 19(9): e3001395
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Nathan Meier, Kody Seward , Jeffrey Caplan, and Savithramma P Dinesh-Kumar. A Kinesin is a Positive Regulator of Chloroplast Stromule Extension During the Innate Immune Response. Poster Presented at: Plant Biology Retreat; October 30, 2021; Davis, CA
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