Progress 09/01/20 to 08/31/23
Outputs
Target Audience:Scientists, virologists, plant pathology, plant science, mycology, horticulture, entomology Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The PI trained members of the Plant Pathology department to operate the confocal microscope: 19 scientists overall. Nagy lab: 7; Vaillancourt lab 2; Farman lab 2; P Kachroo lab 4; Schardl lab 1. Overall: 4 postdocs; 12 Ph. D students, 2 technicians, 1 undergraduate student; 1 professor and co-PI Kawashima has been training scientists how to use the confocal microscope: Dept. of Plant and Soil Sciences: 9 scientists overall. Kawashima lab: 8; Zhu lab 1. Overall 2 postdocs; 7 Ph. D students. Department of Horticulture: 1 scientist. Department of Entomology: 2 scientists. Ph. D students. Department of Veterinary Sciences: 1 scientist; a professor co-PI Kawashima taught two students (one undergrad and one PhD student) introductory confocal microscopy course ABT 480 in Fall 2021. co-PI Kawashima taught two students (one undergrad and one PhD student) advanced confocal microscopy course PLS 597 in Spring 2022. Altogether: 32 scientists have used the confocal microscope. How have the results been disseminated to communities of interest?Target Audience Scientists, virologists, plant pathology, plant science, mycology: 4 peer-reviewed publications from the PI lab were published in 2021, which included data using the new confocal microscope. 4 presentations on scientific meetings (Cold Spring Harbor meeting on autophagy and annual meeting of the American Society for Virology, all virtual-meeting presentations) were given by the PI lab members, which included data using the new confocal microscope in 2021. 4 peer-reviewed publications from the PI lab were published in 2022, which included data using the new confocal microscope. 3 presentations on scientific meetings (annual meeting of the American Society for Virology) were given by the PI lab members, 3 presentations by Vaillancourt Lab member in 2022 2 peer-reviewed publications from the Kawashima lab and 1 from Vaillancourt Lab were published in 2022, which included data using the new confocal microscope in 2023. 2 manuscript are under peer review from the Nagy lab 4 presentations on scientific meetings (annual meeting of the American Society for Virology,) were given by the PI lab members, 1 seminar at international meeting by the Nagy lab, 1 presentations by Vaillancourt Lab members, 6 presentations by Kawashima lab members, including seminars at international universities all of which included data using the new confocal microscope. all of which included data using the new confocal microscope. Undergraduate students (5 members) were taught how to use the confocal microscope. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
•We have successfully optimized the newly FV3000, Olympus, forlive-cell imaging and cellular studies. The following major discoveries were based partially or largely on the FV3000. • Aim 1:Understanding the Mechanism of VRO biogenesis, including the crucial roles of highly-conserved pro-viral host factors. Activities and results: The co-opted proteasomal Rpn11 protein interaction hub in cooperation with subverted actin filaments are targeted to deliver cytosolic host factors for viral replication. The PI lab demonstrated using the new confocal microscope that the co-opted proteasomal Rpn11 protein interaction is used for delivering cytosolic proteins, such as glycolytic and fermentation enzymes, which are readily subverted into viral replication organelles (VROs, site of viral replication) to produce ATP locally in support of VRO formation, replicase assembly and viral RNA replication. • Actin-assisted rapid biogenesis of replication organelles is used by TBSV to limit the recruitment of cellular restriction factors. • Allosteric inhibitors of the cytosolic Hsp70s interfered with tomato bushy stunt virus replication. interfering with the functions of the co-opted Hsp70s could be an effective antiviral approach against tombusviruses in plants. • Tombusviruses target a major crossroad in the endocytic and recycling pathways via co-opting Rab7 small GTPase. TBSV-driven usurping of Rab7 has pro-viral functions through facilitating the delivery of co-opted retromer complex, sorting nexin-BAR proteins and lipid enzymes into VROs to create optimal milieu for virus replication. • Multifunctional role of the co-opted Cdc48 AAA+ ATPase in tombusvirus replication. Based on pharmacological inhibition and genetic and biochemical approaches, we showed that Cdc48 AAA ATPase has critical pro-viral function in TBSV and CIRV replication inN. benthamianaand yeast. Pharmacological inhibition of Cdc48 also reduced the replication of turnip crinkle carmovirus inN. benthamianaprotoplasts. Surprisingly, thein vitroreplication experiments also showed that excess amount of Cdc48 facilitates the disassembly of the membrane-bound viral replicase-RNA template complex. • Key tethering function of Atg11 autophagy scaffold protein in formation of virus-induced membrane contact sites during tombusvirus replication. Deletion of ATG11 in yeast or knockdown of the homologous Atg11 in plants led to reduced tombusvirus replication, thus indicating pro-viral function for Atg11. • Subversion of selective autophagy for the biogenesis of tombusvirus replication organelles inhibits autophagy. Autophagy is a degradation pathway to recycle proteins, damaged organelles or destruct pathogens. However, TBSV recruited members of the autophagy pathway to VROs, sequestering them in biomolecular condensates, which were associated with VROs. This led to inhibition of autophagic flux, and protected TBSV from the antiviral autophagic degradation. • The centromeric histone CenH3 is recruited into the tombusvirus replication organelles. We found that over-expression of CenH3 greatly interferes with tombusvirus replication, whereas, mutation or knockdown of CenH3 enhances TBSV replication in yeast and plants. • Co-opted cytosolic proteins form condensate substructures within the membranous replication organelles of a positive-strand RNA virus. TBSV p33 and the CIRV p36 replication proteins sequester several co-opted cytosolic proteins, such as glycolytic and fermentation enzymes, in unique condensate substructures associated with membranous VROs. Wedemonstrated that subverted membranes and condensate substructures co-exist andare critical for VRO functions. • The PI lab used the plant virus, TBSV to dissect the role of a cellular proteasomal protein, called Rpn11 (POH1), as a protein interaction hub. They showed that knockdown of Rpn11 or retargeting Rpn11 into the nucleus and destruction of actin filaments diminishes TBSV replication in yeast and plant cells. This effect is due to diminished recruitment of pro-viral metabolic enzymes into VROs. Overall, their data support a novel viral recruitment strategy for cytosolic host factors. Key outcomes: Novel targets to develop new antiviral targets in plants have been identified. •Aim 2. Identification of cellular targets of human SARS-CoV-2 in yeast. Activities and results: This project included the expression of viral proteins in yeast by the PI lab. 5 SARS-CoV-2 proteins affected the replication of TBSV in yeast. The viral protein Nsp1 was found to bind to cellular targets in yeast, including Hsp70 and translation elongation factor 1A (eEF1A). Key outcomes: Novel targets to develop new antiviral targets have been identified. • Aims 4-5:Evaluation of the role of CPR1 in the secretion of fungal proteins in planta. Testing the hypothesis that developmental changes in the function of CPR1 in the WT are linked with changes in localization of the Cpr1 transcript. Activities and results: Confocal microscopy was used to study pathogenicity inColletotrichum graminicola, the fungal pathogen that causes anthracnose leaf blight and stalk rot of maize.demonstrated that the MT is not deficient in its ability to accumulate or secrete three different CWDE-mCherry fusions (two pectinases, and a cellulase). has been able to label the protein, which we named CPR1, with GFP and with mCherry, and he has shown that it localizes as expected to the fungal ER membrane, and that it seems to be particularly strongly associated with the nuclear envelope. The fluorescent protein complements the mutant phenotype, restoring pathogenicity. Key outcomes: Change in knowledge on the pathogenesis factors in a plant fungus. • We optimizedconfocal microscopy andlive-cell imaging of infection by Fungal Foliar Maize Pathogens • Aim 6:Characterize the cellular composition of equine organoid systems.Activities:Co-PI Shaffer has successfully optimized the live cell imaging and time-lapse monitoring of equine organoids. A special incubation chamber fitting into the FV3000 was purchased and was installed, which facilitated her studies. • Aims 7-10:Employing live cell, time-lapse confocal microscopy to monitor the biogenesis of rapidly proliferating equine gastrointestinal organoids; 8.Understanding how F-actin in the female gamete captures the sperm nucleus by visualizing the interaction between the F-actin and sperm nucleus. 9.Characterize the dynamics and biological significance of coenocytic endosperm F-actin in development by obtaining high resolution time-lapse Arabidopsis endosperm images. 10.Investigate the dynamics of the soybean coenocytic endosperm in development. It is critical to extend these studies of seed development to important crop plants such as soybean. Activities: Co-PI Kawashima has successfully optimized microscope conditions of the newly installed FV3000, Olympus, for Arabidopsis endosperm live-cell imaging. Results: By integrating confocal microscopy, live-cell imaging, and genetics, we have characterized the entire development of the coenocytic endosperm ofArabidopsis thaliana, including nuclear divisions, their timing intervals, nuclear movement, and cytoskeleton dynamics. Around each nucleus, microtubules organize into aster-shaped structures that drive F-actin organization. Microtubules promote nuclear movement after division, while F-actin restricts it. F-actin is also involved in controlling the size of both the coenocytic endosperm and mature seed. Additionally, Kawashima collaborated with a Japanese research group and quantitatively analyzed F-actin movement in the synergid cell for its function in attracting pollen tube for fertilization. Key outcomes: Change in knowledge on cytoskeleton dynamics on seed development and maturation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Feng Z, Inaba JI, Nagy PD. The retromer is co-opted to deliver lipid enzymes for the biogenesis of lipid-enriched tombusviral replication organelles. Proc Natl Acad Sci U S A. 2021;118(1). Epub 2020/12/31. doi: 10.1073/pnas.2016066118. PubMed PMID: 33376201; PubMed Central PMCID: PMCPMC7817191.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Feng Z, Inaba JI, Nagy PD. Tombusviruses Target a Major Crossroad in the Endocytic and Recycling Pathways via Co-opting Rab7 Small GTPase. J Virol. 2021;95(21):e0107621. Epub 20210818. doi: 10.1128/JVI.01076-21. PubMed PMID: 34406861; PubMed Central PMCID: PMCPMC8513485.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Feng Z, Kovalev N, Nagy PD. Key interplay between the co-opted sorting nexin-BAR proteins and PI3P phosphoinositide in the formation of the tombusvirus replicase. PLoS Pathog. 2020;16(12):e1009120. Epub 2020/12/29. doi: 10.1371/journal.ppat.1009120. PubMed PMID: 33370420; PubMed Central PMCID: PMCPMC7833164.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Nagy PD. Co-opted membranes, lipids, and host proteins: what have we learned from tombusviruses? Curr Opin Virol. 2022;56:101258. Epub 20220923. doi: 10.1016/j.coviro.2022.101258. PubMed PMID: 36166851.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
BIORXIV/2023/550743
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Nature Plants (https://www.nature.com/articles/s41477-022-01331-7)
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
The Plant Cell (https://academic.oup.com/plcell/article/35/4/1222/6958415?login=true)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Yuanrong Kang, Wenwu Lin, and Peter D. Nagy: Subversion of selective autophagy for the biogenesis of tombusvirus replication organelles inhibits autophagy
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Scientists, virologists, plant pathology, plant science, mycology: 3 peer-reviewed publications from the PI lab were published in 2022, which included data using the new confocal microscope. 3 presentations on scientific meetings (annual meeting of the American Society for Virology) were given by the PI lab members, 3 presentations by Vaillancourt Lab members, all of which included data using the new confocal microscope. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PI Nagy, technician Dr. Shifeng Wu, postdoc Dr. Zhike Feng and Wenwu Lin; graduate students Mr. Biao Sun, Mr. Y. Kang, Ms. Yuyan Liu and Chase Eastham undergraduate student are fully trained to use and operate the system. Additional researchers trained from the co-PIs lab: 3 co-PIs and from their labs 3 lab technicians and two graduate students are also trained. Two PhD students in the Vaillancourt lab (Caleb Mathias and Renata Belisario) have been trained in the use of the confocal microscope. In addition, 4 graduate students from the Kachroo's lab and Dr. Kachroo, and 1 postdoc from Dr. Schardl's lab and 1 student from Farman's lab from Department of Plant Pathology have also been trained to use the new confocal microscope. Co-PI Kawashima has been training scientists (1 technician, 2 postdocs and a graduate student) how to use the confocal microscope from the Dept. of Plant and Soil Sciences. Co-PI Kawashima has been training scientists how to use the confocal microscope (Dept. of Plant and Soil Sciences). Co-PI Kawashima taught two students (one undergrad and one PhD student) introductory confocal microscopy course ABT 480 in Fall 2021. Co-PI Kawashima taught two students (one undergrad and one PhD student) advanced confocal microscopy course PLS 597 in Spring 2022. How have the results been disseminated to communities of interest?3 peer-reviewed publications from the PI lab were published in 2022, which included data using the new confocal microscope. 3 presentations on scientific meetings (annual meeting of the American Society for Virology) were given by the PI lab members, 3 presentations by Vaillancourt Lab members, all of which included data using the new confocal microscope. What do you plan to do during the next reporting period to accomplish the goals?The PI plans to study the role of the co-opted ER meshwork and actin network in delivering various host factors into VROs to provide membranes, lipids and cargo proteins for tombusvirus replication. Co-PI Vaillancourt Lab will use the new confocal microscope to optimize the expression and visualization of Colletotrichum graminicolaeffector proteins. Co-PI Kawashima plans: To obtain time-lapse images to investigate F-actin and nuclear dynamics of Arabidopsis endosperm development. Based on the use of transgenic lines that alter F-actin dynamics in the endosperm, we will identify how F-actin regulates nuclear division and movement in the endosperm and their impact on endosperm development and final seed size. Co-PI Shaffer will continue optimizing the live cell imaging and time-lapse monitoring of equine organoids.
Impacts
What was accomplished under these goals?
The following research topics have been initiated with the progress described below: 1. The roles of host factors in the biogenesis of tombusvirus replication organelles. The PI lab demonstrated using the new confocal microscope that the critical roles of the cellular Cdc48 unfoldase/segregase protein in facilitating the replication of the peroxisome-associated tomato bushy stunt virus (TBSV) and the mitochondria-associated carnation Italian ringspot virus (CIRV). We show that TBSV and CIRV infections induce the expression of Cdc48 in Nicotiana benthamiana plants. Based on pharmacological inhibition and genetic and biochemical approaches, we show that Cdc48 AAA ATPase has critical pro-viral function in TBSV and CIRV replication in N. benthamiana and yeast. Pharmacological inhibition of Cdc48 also reduced the replication of turnip crinkle carmovirus in N. benthamiana protoplasts. Cdc48 binds to the TBSV and CIRV replication proteins through its N-terminal region. In vitro TBSV replicase reconstitution experiments demonstrated that Cdc48 is needed for efficient replicase assembly and activity. Surprisingly, the in vitro replication experiments also showed that excess amount of Cdc48 facilitates the disassembly of the membrane-bound viral replicase-RNA template complex. The multifunctional role of the co-opted Cdc48 is further supported by findings that Cdc48 facilitates the recruitment of the endosomal Vps34 PI3K to produce PI(3)P phosphoinositide within VROs. Cdc48 is also involved in the recruitment of the mitochondrial/peroxisomal Fis1 protein, and Sec13, which is component of the COPII vesicles coat. Because several human viruses, including flaviviruses, also utilize Cdc48, also called VCP/p97 for VRO biogenesis, we suggest that Cdc48 might be a common panviral host factor for plant and animal RNA viruses. In another project, the PI discovered that the nuclear centromeric CenH3 histone variant (Cse4p in yeast, CENP-A in human) plays a major role in tombusvirus replication in plants and in yeast model host. We find that over-expression of CenH3 greatly interferes with tombusvirus replication, whereas, mutation or knockdown of CenH3 enhances TBSV replication in yeast and plants. CenH3 binds to the viral RNA and acts as an RNA chaperone. Although, these data support a restriction role of CenH3 in tombusvirus replication, we demonstrate that by partially sequestering CenH3 into VROs, TBSV alters selective gene expression of the host, leading to more abundant protein pool. This in turn helps TBSV to subvert pro-viral host factors into replication. We show this through the example of hypoxia factors, glycolytic and fermentation enzymes, which are exploited more efficiently by tombusviruses to produce abundant ATP locally within the VROs in infected cells. Altogether, we propose that subversion of CenH3/Cse4p from the nucleus into cytosolic VROs facilitates transcriptional changes in the cells, which ultimately leads to more efficient ATP generation in situ within VROs by the co-opted glycolytic enzymes to support the energy requirement of virus replication. Altogether, CenH3 plays both pro-viral and restriction functions during tombusvirus replication. This is a surprising novel role for a nuclear histone variant in cytosolic RNA virus replication. Aim 2. Identification of cellular targets of human SARS-CoV-2 in yeast. This project includes the expression of viral proteins in yeast by the PI lab. 5 SARS-CoV-2 proteins affected the replication of TBSV in yeast. These proteins are now being investigated for cellular targets in yeast. Aim 4: The Co-PI Vaillancourt Lab is studying mechanisms of pathogenicity in Colletotrichum graminicola, the fungal pathogen that causes anthracnose leaf blight and stalk rot of maize. Several years ago we used insertional mutagenesis to produce a nonpathogenic strain of this fungus. The strain has an interruption in the 3'UTR of a gene encoding a protein similar to one component of the conserved endoplasmic-reticulum (ER) localized signal peptidase. Although this is an essential housekeeping protein, the mutation has created a conditional defect that only manifests during pathogenicity: growth in vitro is unaffected. Both students have been using the confocal microscope to visualize fluorescent protein fusions, in order to better understand the nature of this mutation and its role in pathogenicity. Mr. Mathias has been able to label the protein, which we named CPR1, with GFP and with mCherry, and he has shown that it localizes as expected to the fungal ER membrane, and that it seems to be particularly strongly associated with the nuclear envelope. The fluorescent protein complements the mutant phenotype, restoring pathogenicity, and so it seems likely that this the true locat ion of the endogenous protein. Our plan next is to use the confocal microscope to evaluate qualitative and quantitative changes in the protein and its localization in planta versus in vitro, to test our hypothesis that the protein is expressed at higher levels during infection of the living host tissues. Ms. Belisario has been testing the hypothesis that the mutant is deficient in secretion of proteins that are important for pathogenicity, including cell wall degrading enzymes (CWDE) and small protein effectors. She has been able to label several representatives of each of these protein classes and visualize them in the mutant and wild type fungi in planta. She has observed that the mutant seems to produce less of the effectors and more slowly, while the enzyme production is more similar between the two. The plan is to use confocal microscopy to quantify the production of these fluorescent fusion proteins in vitro and in vivo over a complete infection time course. Support for our hypothesis would be very exciting, as it would reveal a previously unsuspected specific activity for this housekeeping protein in the regulation of pathogenicity. Aims 8-9: Co-PI Kawashima has successfully optimized microscope conditions of the newly installed FV3000, Olympus, for Arabidopsis endosperm live-cell imaging. By integrating confocal microscopy live-cell imaging and genetics, we have characterized the entire development of the coenocytic endosperm ofArabidopsis including nuclear divisions, their timing intervals, nuclear movement, and cytoskeleton dynamics. Around each nucleus, microtubules organize into aster-shaped structures that drive F-actin organization. Microtubules promote nuclear movement after division while F-actin restricts it. F-actin is also involved in controlling the size of both the coenocytic endosperm and mature seed. The work has been published as a preprint in bioRxiv (https://www.biorxiv.org/content/10.1101/2022.04.01.485647v1) as well as is currently under revision in a peer-reviewed journal.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Gonzalez PA, Nagy PD. The centromeric histone CenH3 is recruited into the tombusvirus replication organelles. PLoS Pathog. 2022;18(6):e1010653. Epub 20220629. doi: 10.1371/journal.ppat.1010653. PubMed PMID: 35767596.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Kang Y, Lin W, Liu Y, Nagy PD. Key tethering function of Atg11 autophagy scaffold protein in formation of virus-induced membrane contact sites during tombusvirus replication. Virology. 2022;572:1-16. Epub 20220429. doi: 10.1016/j.virol.2022.04.007. PubMed PMID: 35533414.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Molho M, Zhu S, Nagy PD. Race against Time between the Virus and Host: Actin-Assisted Rapid Biogenesis of Replication Organelles is Used by TBSV to Limit the Recruitment of Cellular Restriction Factors. J Virol. 2022:e0016821. Epub 20220531. doi: 10.1128/jvi.00168-21. PubMed PMID: 35638821.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
preprint in bioRxiv (https://www.biorxiv.org/content/10.1101/2022.04.01.485647v1
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Scientists and bilogists via : 3 peer-reviewed publications from the PI lab were published in 2021, which included data using the new confocal microscope. Virologists and autophagy researchers via: 4 presentations on scientific meetings (Cold Spring Harbor meeting on autophagy and annual meeting of the American Society for Virology, all virtual-meeting presentations) were given by the PI lab members, which included data using the new confocal microscope. Changes/Problems:Unfortunately and tragically, co-PI Dr. Goodin passed away last year. Therefore, his expected contribution to objective 3 and running the facility was unfulfiled. PI Nagy took over the day-to-day operation and supervision of the facility and center CAFE-BI. Also, due to COVID-19 restrictions in using university facilities (many of us, we had to work from home for a longer period), and limited number of visits by the Olympus engineers to install the instrument and training activities, thus significantly slowing down the training of lab personnal of using and oprating the new confocal. In addition, undergraduate students were not allowed to work in our labs and facilities during this period. What opportunities for training and professional development has the project provided?In spite of COVID19-based restrictions, we were able to get the full-training on the using the new confocal microscope of important personnel described below. Undergraduates students were not allowed in our lab to do research due to COVID-19 restrictions. Training: PI Nagy, technician Dr. Shifeng Wu, postdoc Dr. Zhike Feng; graduate students Mr. Biao Sun, Mr. Y. Kang, Ms. M. Molho, Ms. P. Gonzales are fully trained to use and operate the system. Additional researchers trained from the co-PIs lab: 3 co-PIs and from their labs 3 lab technicians and two graduate students are also trained. In addition, 3 graduate students from the Kachroo's lab and Dr. Kachroo, and 1 postdoc from Dr. Schardl's lab from Department of Plant Pathology have also been trained to use the new confocal microscope. Co-PI Kawashima has been training scientists (1 technician, 2 postdocs and a graduate student) how to use the confocal microscope from the Dept. of Plant and Soil Sciences. How have the results been disseminated to communities of interest? 3 peer-reviewed publications from the PI lab were published in 2021, which included data using the new confocal microscope. 4 presentations on scientific meetings (Cold Spring Harbor meeting on autophagy and annual meeting of the American Society for Virology, all virtual-meeting presentations) were given by the PI lab members, which included data using the new confocal microscope. What do you plan to do during the next reporting period to accomplish the goals?The PI plans to study the role of the actin network in delivering Rab1 and Rab7-decorated vesicles into VROs to provide membranes, lipids and cargo proteins for tombusvirus replication. Co-PI Vaillancourt Lab will use the new confocal microscope to optimize the expression and visualization of Colletotrichum graminicolaeffector proteins. Co-PI Kawashima plans: Using our optimized confocal microscopy method, we plan to obtain time-lapse images to investigate F-actin and nuclear dynamics of Arabidopsis endosperm development. Using transgenic lines that alter F-actin dynamics in the endosperm, we will identify how F-actin regulates nuclear division and movement in the endosperm and their impact on endosperm development and final seed size. Co-PI Shaffer will optimize the live cell imaging and time-lapse monitoring of equine organoids. A special incubation chamber fitting into the FV3000 was purchased and was installed last month to facilitate her studies.
Impacts
What was accomplished under these goals?
Olympus FV3000 confocal laser microscope has been purchased, fully installed, and now housed in the Plant Pathology Department in the Plant Science Building. The system is fully operational. The following research topics have been initiated with the progress described below: 1. The roles of host factors in the biogenesis of tombusvirus replication organelles. The PI lab demonstrated using the new confocal microscope that the co-opted proteasomal Rpn11 protein interaction hub in cooperation with subverted actin filaments are targeted to deliver cytosolic host factors for viral replication. TBSV takes advantage of a noncanonical function of Rpn11 by exploiting Rpn11's interaction with highly abundant cytosolic proteins and the actin network. We provide supporting evidence that the co-opted Rpn11 in coordination with the subverted actin network is used for delivering cytosolic proteins, such as glycolytic and fermentation enzymes, which are readily subverted into viral replication organelles (VROs) to produce ATP locally in support of VRO formation, VRCs assembly and viral RNA replication. In another project, we have shown key tethering function of Atg11 autophagy scaffold protein in formation of virus-induced membrane contact sites during tombusvirus replication. Using the new confocal microscope, we have shown that the highly conserved Atg11 autophagy scaffold protein (FIP200 in mammals) is co-opted by both TBSV and CIRV via direct interactions with the viral replication proteins. Deletion of ATG11 in yeast or knockdown of the homologous Atg11 in plants led to reduced tombusvirus replication, thus indicating pro-viral function for Atg11. Based on co-purification, BiFC and proximity-labeling experiments, we find that Atg11 is exploited by tombusviruses to form and stabilize virus-induced membrane contact sites (vMCS) within VROs. We propose that the tethering and scaffold function of Atg11 is needed to glue together other co-opted host proteins, such as the ER resident VAP tethering proteins, Sac1 PI4P phosphatase and the cytosolic OSBP-like oxysterol-binding proteins, Fis1 mitochondrial fission protein and the viral replication protein in vMCSs. Aim 2. Identification of cellular targets of human SARS-CoV-2 in yeast. This project has been initiated by starting the expression of viral proteins in yeast by the PI lab. Aim 4: The Co-PI Vaillancourt Lab has begun to use the new confocal microscope for our NIFA-funded project to visualizeColletotrichum graminicolaeffector proteins that are labeled with fluorescent protein markers in fungal hyphae growingin planta. Preparation of the materials for assay is quite time consuming, so we have been able to engage only in one session per week. The past several months have been spent mostly in learning the operation and capabilities of the microscope. These are obviously considerably improved from the older model, allowing for much higher resolution of fungal structures and less autofluorescent background. Some of the effectors are expressed at very low levels, and so this new microscope will be really important for successful completion of our work. Aims 8-9: Co-PI Kawashima has successfully optimized microscope conditions of the newly installed FV3000, Olympus, for Arabidopsis endosperm live-cell imaging. The endosperm is deeply embedded in the seed coat layers, making it extremely difficult to obtain high resolution crisp images even utilizing confocal microscopy. Using the Olympus 30x silicon immersion objective lens, the endosperm actin filaments are clearly visualized (Figure 1). Furthermore, we have started dissecting out the interaction between actin filaments (F-actin) and nuclei in the endosperm (inset, Figure 1), which we were not able to resolve by our old confocal microscope (FV1200, Olympus). Further analyses will provide insight into how the coenocytic endosperm of Arabidopsis controls its nuclear division and movement as well as its dynamic development. Figure 1 (will be attached if requested): Z-projected confocal microscopy image of Arabidopsis endosperm. Arabidopsis endosperm is coenocytic and nuclei (magenta) and F-actin (green) are shown. Aster-shaped F-actin structure is on each nucleus, presumably controlling the position of the nucleus. Inset shows a single z-plane of the dashed box region of the endosperm to show the interaction between the F-actin and nucleus. Bar = 20 mm.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Feng, Z.Inaba, J. I.Nagy, P. D.Tombusviruses target a major crossroad in the endocytic and recycling pathways via co-opting Rab7 small GTPase. J. Virology, DOI: 10.1128/jvi.01076-21
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Molho, M.Chuang, C. Nagy, P. D. : Co-opting of nonATP-generating glycolytic enzymes for TBSV replication. Virology, Vol: 559, 15-29
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Molho, M. Lin, W. Nagy, P. D.: A novel viral strategy for host factor recruitment: The co-opted proteasomal Rpn11 protein interaction hub in cooperation with subverted actin filaments are targeted to deliver cytosolic host factors for viral replication. PLOS Pathogens, 17: e1009680
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