Source: EAST STROUDSBURG UNIVERSITY OF PENNSYLVANIA submitted to
SPATIAL AND TEMPORAL IMAGING OF INTRACELLULAR PHOSPHATE PROFILES DURING ARBUSCULAR MYCORRHIZAL SYMBIOSES USING GENETICALLY-ENCODED BIOSENSORS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030541
Grant No.
2021-67034-39677
Cumulative Award Amt.
$77,867.96
Proposal No.
2022-11646
Multistate No.
(N/A)
Project Start Date
Oct 15, 2022
Project End Date
Oct 14, 2024
Grant Year
2023
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Project Director
Zhang, S.
Recipient Organization
EAST STROUDSBURG UNIVERSITY OF PENNSYLVANIA
200 PROSPECT ST
EAST STROUDSBURG,PA 18301
Performing Department
(N/A)
Non Technical Summary
Sufficient phosphate supply is crucial for crop production, but extensive use of phosphate fertilizers has led to a depletion of this non-renewable resource and considerable environmental degradation. One of the strategies for sustainable crop production is to utilize arbuscular mycorrhizal (AM) symbiosis in agriculture, an ancient association that occurs to ~72% of the flowering plants. Roots of the plant host often take up substantial phosphate from the AM fungal symbiont through AM-specific H+/phosphate transporters. Although the mechanism of the mycorrhizal phosphate acquisition has been investigated, profiling of the intracellular phosphate levels during different AM symbioses has not been done. Using a plant-specific, genetically encoded phosphate biosensor and a highly effective experimental system to trace phosphate translocation from the fungi to the roots, I will visualize the intracellular phosphate profiles during different AM symbioses spatially and temporally and characterize the roles of the AM symbiosis-induced phosphate transporters during the symbiotic phosphate uptake. The outcome of this research will provide novel and fundamental knowledge of phosphate dynamics during different AM symbioses.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
10224201030100%
Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
2420 - Noncrop plant research;

Field Of Science
1030 - Cellular biology;
Goals / Objectives
Using a plant-specific, genetically-encoded phosphate biosensor, the overall goal of this project is to spatially and temporally visualize the intracellular Pi profiles during Brachypodium distachyon symbiosis with four individual AM fungi: Rhizophagus irregularis (formerly Glomus intraradices), Diversispora epigaea (formerly Glomus versiforme), Claroideoglomus candidum (formerly Glomus candidum) and Claroideoglomus claroideum; and to characterize the roles of the AM symbiosis-induced Pi transporters in the symbiotic Pi uptake during B. distachyon AM symbiosis. This project is expected to provide a comprehensive understanding of how intracellular Pi of the host plant is differentially influenced spatially and temporally by the association ofevolutionarily divergent arbuscular mycorrhizal (AM) fungi, how these AM fungi differ in the Pi transport to their host and what the roles of the AM symbiosis-induced Pi transporters are in the AM symbiosis. Objective 1, spatial mapping of the cytosolic and plastidic Pi distribution of plant roots during symbiosis with four AM fungi (almost finished); objective 2, temporal monitoring of Pi translocation from individual AM fungi to the host roots; objective 3, characterize the individual roles of the AM symbiosis-induced Pi transporters in the cytosolic Pi changes during AM symbiosis (almost finished).
Project Methods
Objective 1, spatial mapping of the cytosolic and plastidic Pi distribution of plant roots during symbiosis with four AM fungiProgress to date: Spatial Pi distributions have been compared in colonized cells in roots colonized with Rhizophagus irregularis, Diversispora epigaea and Gigaspora gigantea. Such comparisons have shown that colonized cells in R. irregularis-colonized roots contained higher relative cytosolic Pi content 0.5 h after Pi application relative to cells in D. epigaea-colonized roots. Root cells associated with D. epigaea contained higher relative cytosolic Pi content relative to cells associated with G. gigantea same time after the application. Also, differences were found in the plastidic Pi levels in either cells containing young or mature arbuscules between D. epigaea- and R. irregularis-associated roots.Work to be completed: Currently propagating Claroideoglomus claroideum using Brachypodium plants as nursing plants. A few more experiments to compare the Pi distributions across plant roots colonized with R. irregularis, D. epigaea, C. claroideum and G. gigantea, respectively.Objective 2, temporal monitoring of Pi translocation from individual AM fungi to the host rootsWork to be completed: Tracing of 32Pi translocation from extraradical hyphae (ERH) to the host roots; monitoring the relative cytosolic Pi levels in the host root following Pi application to the ERH.Objective 3, characterize the individual roles of the AM symbiosis-induced Pi transporters in the cytosolic Pi changes during AM symbiosis?Progress to date: Crosses have been made between pt7 mutant and FLIPPi sensors, now genotyping their F1 generation. For the current mutants expressing biosensors in AM symbiosis-specific cells, comparisons in cytosolic Pi have been made between WT and pt7/pt12 mutants, between WT and pt3/pt7/pt12 mutants, and between WT and pt3/pt7/pt12/pt13 mutants. Unfortunately, it was found that these mutant lines have very weak signals of the Pi sensors. We are trying to select and compare the following mutants with WT: pt3/pt12, pt3/pt13, pt3/pt12/pt13.Work to be completed: Mapping the relative cytosolic Pi distribution in the pt7 mutant

Progress 10/15/22 to 10/14/24

Outputs
Target Audience:During the time of the project, the PD collaborated with over 10 undergraduate students and a master's student from East Stroudsburg University to establish a research lab and develop techniques using limited resources in ESU for the ongoing project. Additionally, the PD has published 2 first-authored open-access papers in peer-reviewed journals. Changes/Problems:To achieve Objective 2, which involves the use of a radioactive phosphorus isotope, I have had logistical constraints. My present institution does not hold an active permit for handling radioactive materials. On the other hand, Cornell University, while possessing the necessary permit, does not permit visiting scholars to work with radioactive isotopes. Unfortunately, these circumstances prevent me from completing this aspect of the objective. What opportunities for training and professional development has the project provided?During this period, the mentor of the PDclosely supervised the project, providing valuable guidance and feedback on research manuscripts and presentations. This mentorship played a crucial role in refining my research outputs and enhancing my professional development. During the funding period, I had the privilege of presenting my research at both the annual conference of the American Society of Plant Biologists (ASPB) and the regional ASPB meeting. These conferences provided valuable opportunities to share my findings with a broad audience, engage in meaningful discussions, and receive constructive feedback from experts in the field. In addition to presenting my work, I participated in various workshops at the ASPB annual conference. These included sessions on grant writing, which offered practical strategies for securing funding, and discussions on publishing in the era of artificial intelligence, which provided insights into navigating the evolving landscape of academic publishing. These workshops enhanced my professional skills and prepared me to address the challenges faced by early-career scientists. I also attended the Connect UR meeting, which significantly expanded my perspective on promoting and integrating undergraduate research into my academic activities. This experience emphasized the importance of mentoring undergraduate students and incorporating research into their learning experiences, aligning well with my role at a primarily undergraduate institution (PUI). Through these conferences and meetings, I built an extensive professional network that extends beyond my specific field and institution. This network introduced me to numerous resources and opportunities for early-career scientists, including collaborations, funding avenues, and mentorship opportunities. All these experiences contributed to my success in securing a tenure-track position at a PUI and helped me establish a strong foundation in this role. How have the results been disseminated to communities of interest?Results have been disseminated in two open-access peer-reviewed publications: one research article and one protocol paper. Unpublished dataare being prepared for submission to a peer-reviewed journal. Additionally, the results are disseminated via research presentationsat both the annual conference of the American Society of Plant Biologists (ASPB) and the regional ASPB meeting. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the project period, intracellular phosphate (Pi) profiles during arbuscular mycorrhizal (AM) symbiosis were analyzed, focusing primarily on three proposed AM fungi: Rhizophagus irregularis, Diversispora epigaea, and Gigaspora gigantea. The role of BdPT7, a phosphate transporter, in regulating intracellular Pi profiles was also characterized. The collected data revealed several key findings: Arbuscule-containing cells associated with Gigaspora gigantea showed lower cytosolic Pi levels compared to cells associated with Rhizophagus irregularis and Diversispora epigaea. The absence of BdPT7 during AM symbiosis significantly impacted cytosolic Pi levels, with a notable and potentially intriguing change0 in Pi levels compared to wild-type plants. The outcomes of this project have provided critical insights into Pi dynamics during AM symbiosis. Results have been disseminated in two open-access peer-reviewed publications: one research article and one protocol paper. Additionally, unpublished data related to the third objective are being prepared for submission to a peer-reviewed journal.

Publications

  • Type: Peer Reviewed Journal Articles Status: Awaiting Publication Year Published: 2025 Citation: Zhang, S., Jurgensen, L. and Harrison, M. J. (2025). Utilizing FRET-based Biosensors to Measure Cellular Phosphate Levels in Mycorrhizal Roots of Brachypodium distachyon. Bio-protocol 15(2): e5158. DOI: 10.21769/BioProtoc.5158.


Progress 10/15/22 to 10/14/23

Outputs
Target Audience:Over the past year, the PD has collaborated with several undergraduate students and a master's studentfrom East Stroudsburg University to establish a research lab and develop techniques using limited resources inESU for the ongoing project. Changes/Problems:To achieve Objective 2, which involves the use of a radioactive phosphorus isotope, I am currently facing logistical constraints. My present institution does not hold an active permit for handling radioactive materials. On the other hand, Cornell University, while possessing the necessary permit, does not permit visiting scholars to work with radioactive isotopes. Consequently, these circumstances prevent me from completing this aspect of the objective. What opportunities for training and professional development has the project provided?I had the privilege of presenting my previous findings at the annual conference of the American Society of Plant Biologists. During the conference, I had the opportunity to network with many colleagues and participate in various workshops, including those focused on grant writing and navigating the challenges of publishing in the era of artificial intelligence. How have the results been disseminated to communities of interest?Not yet. The results need further investigation. What do you plan to do during the next reporting period to accomplish the goals?Continue working on the objective to confirm the results we've gotten and possibly try again with another AM fungal species.

Impacts
What was accomplished under these goals? During the past year, my primary focus has been on Objective 3, involving the monitoring of cytosolic Pi levels in the pt7 mutant with FLIPPi sensors under various conditions: standard, high Pi, as well as low nitrogen and high Pi. This work has yielded intriguing results that warrant further exploration.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Zhang, S., Daniels, D. A., Ivanov, S., Jurgensen, L., M�ller, L. M., Versaw, W. K., & Harrison, M. J. (2022). A genetically encoded biosensor reveals spatiotemporal variation in cellular phosphate content in Brachypodium distachyon mycorrhizal roots. New Phytologist, 234(5), 1817-1831.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Title: Real-Time Imaging of Intracellular Phosphate Dynamics in Brachypodium Distachyon Mycorrhizal Roots Using FRET-Based Biosensors
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Talk at the annual conference of American Society of Plant Biologist. Title: Imaging phosphate (Pi) in mycorrhizal root cells using biosensors