Progress 10/01/14 to 09/30/18
Outputs
Target Audience: The target audiences served by the project are professional biologists and students with interests in the mechanisms by which plants resist pathogens and control development, especially traits related to disease resistance, or under circadian rhythm control. Because of its connections to disease resistance, the research may lead to insights relevant to the development of disease resistant agricultural crops. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project directly led to a student's completion of the Ph.D. and graduation. That individual is currently conducting research as a postdoctoral fellow in another state. During his graduate studies, he presented abstracts and delivered invited talks at several regional and national plant biology meetings, and presented an abstract and poster at an international meeting. He also participated in preparing his work for publication to several journals. Four undergraduate researchers have worked in this project. The graduate student and undergraduate students have learned methods for plant culture, plant genetics, molecular biology, use of informatics tools, pathogen culture, and biochemistry. How have the results been disseminated to communities of interest? This work has been communicated primarily through presentations (talks and posters) at scientific meetings, as well as through Dr. Prince Zogli's Ph.D. thesis at the University of Vermont. We submitted Dr. Zogli's work for publication in to several journals, but have not yet had it accepted for publication (a competing publication preempted some but not all of the novelty of our work, complicating its acceptance for publication). We are generating additional data that we will include in another version of the manuscript. 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 (Examine the role of Arabidopsis thaliana LNK genes in pathogen defense): We generated several resources required for these tests: The lnk1 and lnk2 mutants were identified, made homozygous, and crossed, to produce a double mutant line, which was thoroughly characterized. The double mutant line was stably transformed with wild- type LNK1 or LNK2 genes (fused with the GFP reporter gene), under control of the CaMV 35S promoter, to generate lines containing LNK1-GFP and LNK2-GFP fusion proteins. These lines were also later used in experiments under Objective 2 (below). We assessed whether the LNK-GFP fusion proteins were functional, by examining whether they could rescue the lnk1 lnk2 double mutant's long hypocotyl phenotype; both LNK1-GFP and LNK2-GFP restored hypocotyl length to wild-type, indicating that both transgenes were functional. We conducted pathogen growth assays using the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) in wild-type, lnk1 or lnk2 single mutant plants, lnk1 lnk2 double mutant plants, as well as double mutant plants transformed with wild- type LNK1-GFP or LNK2-GFP genes. We found that the double mutant was significantly less susceptible to Hpa. Double mutant plants transformed with wild-type LNK1 or LNK2 genes showed rescue, in that the transformants displayed higher susceptibility to Hpa, similar to WT plants. These data provide evidence for involvement of LNK gene products in pathogen susceptibility and/or resistance. Objective 2 (Assess whether LNK protein abundance is regulated by ubiquitin-mediated proteolysis): Test were begun to assess LNK1-GFP and LNK2-GFP stability in plant extracts, with the aim of determining whether ubiquitin- mediated proteolysis plays a role in controlling LNK1 or LNK2 protein accumulation. Total protein was extracted from 5-day- old leaves of lnk1 lnk2 double mutant plants transformed with wild-type LNK1-GFP or LNK2- GFP genes. Extracts were concentrated and placed in a degradation buffer, and combined with a bovine 26S proteasome- mediated protein degradation kit (UBP Bio, Aurora Colorado) following the manufacturer's instructions, and incubated over 2- hours. Some samples were incubated with inhibitors of the 26S proteasome (e.g. MG132, PSI, epoxomicin), as well as general protease inhibitor PMSF as control. Thirty-minute time points were stopped over the incubation period, and prepared for fractionation by SDS-PAGE, following western blotting and measurement of the GFP tag, using an anti-GFP primary monoclonal antibody, and secondary antibody conjugated to the IRDye 800CW fluorochrome. Blots were analyzed using an Odyssey CLx infrared imaging system (LiCor). Results showed degradation of LNK1-GFP or LNK2-GFP proteins over 120 minutes, which was reduced by the proteasome inhibitors MG132, PSI, or epoxomicin, but not by the general protease inhibitor PMSF. These preliminary data indicate that LNK1 and LNK2 are targeted for proteolysis by the ubiquitin- proteasome system. Additional experiments aer needed to determine whether the F-box protein SON1 participates in LNK2 degradation. We will usea heterologous expression system, by expressing a LNK2-GFP fusion protein, together with a SON1-myc fusion protein in Nicotiana benthamiana, using Agrobacterium-mediated gene introduction. LNK2 and SON1 proteins made in Agrobacterium-infiltrated N. benthamiana leaves will beanalyzed using western blot analysis, with anti-GFP and anti-myc monoclonal antibodies. By withholding SON1 in some infiltrated leaves, we can assess whether presence of SON1 increases LNK2-GFP degradation. By use of proteosome inhibitors, such as MG132, we can determine whether LNK2 degradation occurs form proteosomal activity. Together, we hope to establish the mechanism involved in LNK2 protein regulation, and in doing so, show how LNK2 and SON1 modulate development and disease resistance. Objective 3 (Evaluate circadian rhythmicity of LNK protein abundance): We intend to examine LNK2-GFP and SON1-myc plants across the circadian cycle, to assess LNK2 and SON! protein abundance, using western analysis.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Diversity of Gyroporus (Gyroporaceae, Boletales): rpb2 phylogeny and three new species (2020). N. Davoodian, K. Hosaka, O. Rasp�, O. A. Asher, A. R. Franck, A. De Kesel, T. P. Delaney, J. F. Ammirati, E. Nagasawa, B. Buyck, R. E. Halling. Phytotaxa 434: 208-218.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audiences served by the project are professional biologists and students with interests in the mechanisms by which plants resist pathogens and control development, especially traits related to disease resistance, or under circadian rhythm control. Because of its connections to disease resistance, the research may lead to insights relevant to the development of disease resistant agricultural crops. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has directly led to a student's completion of the Ph.D. and graduation. That individual is currently conducting research as a postdoctoral fellow in another state. During his graduate studies, the student presented abstracts and delivered invited talks at several regional and national plant biology meetings, and presented an abstract and poster at an international meeting. He also participated in preparing his work for publication to several journals. Four undergraduate researchers have worked in this project. The graduate student and undergraduate students have learned methods for plant culture, plant genetics, molecular biology, use of informatics tools, pathogen culture, and biochemistry. How have the results been disseminated to communities of interest?At this time, the work has been communicated primarily through presentations (talks and posters) at scientific meetings, as well as through Dr. Prince Zogli's Ph.D. thesis at the University of Vermont. We submitted Dr. Zogli's work for publication in to several journals, but have not yet had it accepted for publication. We are generating additional data that we will include in another version of the manuscript. What do you plan to do during the next reporting period to accomplish the goals?Our ongoing biochemistry experiments will add important details to explain the function and mechanism for LNK2 degradation, and the role of SON1 in that process. Those data will strengthen a revised publication that we will submit.
Impacts
What was accomplished under these goals?
Progress toward Objective 1 (Examine the role of Arabidopsis thaliana LNK genes in pathogen defense): We generated several resources required for these tests: The lnk1 and lnk2 mutants were identified, made homozygous, and crossed, to produce a double mutant line, which was thoroughly characterized. The double mutant line was stably transformed with wild-type LNK1 or LNK2 genes (fused with the GFP reporter gene), under control of the CaMV 35S promoter, to generate lines containing LNK1-GFP and LNK2-GFP fusion proteins. These lines were also later used in experiments under Objective 2 (below). We assessed whether the LNK-GFP fusion proteins were functional, by examining whether they could rescue the lnk1 lnk2 double mutant's long hypocotyl phenotype; both LNK1-GFP and LNK2-GFP restored hypocotyl length to wild-type, indicating that both transgenes were functional. We conducted pathogen growth assays using the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) in wild-type, lnk1 or lnk2 single mutant plants, lnk1 lnk2 double mutant plants, as well as double mutant plants transformed with wild- type LNK1-GFP or LNK2-GFP genes. We found that the double mutant was significantly less susceptible to Hpa. Double mutant plants transformed with wild-type LNK1 or LNK2 genes showed rescue, in that the transformants displayed higher susceptibility to Hpa, similar to WT plants. These data provide evidence for involvement of LNK gene products in pathogen susceptibility and/or resistance. Progress toward Objective 2 (Assess whether LNK protein abundance is regulated by ubiquitin-mediated proteolysis): Test were begun to assess LNK1-GFP and LNK2-GFP stability in plant extracts, with the aim of determining whether ubiquitin-mediated proteolysis plays a role in controlling LNK1 or LNK2 protein accumulation. Total protein was extracted from 5-day-old leaves of lnk1 lnk2 double mutant plants transformed with wild-type LNK1-GFP or LNK2- GFP genes. Extracts were concentrated and placed in a degradation buffer, and combined with a bovine 26S proteasome- mediated protein degradation kit (UBP Bio, Aurora Colorado) following the manufacturer's instructions, and incubated over 2- hours. Some samples were incubated with inhibitors of the 26S proteasome (e.g. MG132, PSI, epoxomicin), as well as general protease inhibitor PMSF as control. Thirty-minute time points were stopped over the incubation period, and prepared for fractionation by SDS-PAGE, following western blotting and measurement of the GFP tag, using an anti-GFP primary monoclonal antibody, and secondary antibody conjugated to the IRDye 800CW fluorochrome. Blots were analyzed using an Odyssey CLx infrared imaging system (LiCor). Results showed degradation of LNK1-GFP or LNK2-GFP proteins over 120 minutes, which was reduced by the proteasome inhibitors MG132, PSI, or epoxomicin, but not by the general protease inhibitor PMSF. These preliminary data indicate that LNK1 and LNK2 are targeted for proteolysis by the ubiquitin- proteasome system. We are currently performing experiments to determine whether the F-box protein SON1 participates in LNK2 degradation. We are using a heterologous expression system, by expressing a LNK2-GFP fusion protein, together with a SON1-myc fusion protein in Nicotiana benthamiana, using Agrobacterium-mediated gene introduction. LNK2 and SON1 proteins made in Agrobacterium-infiltrated N. benthamiana leaves are analyzed using western blot analysis, with anti-GFP and anti-myc monoclonal antibodies. By withholding SON1 in some infiltrated leaves, we can assess whether presence of SON1 increases LNK2-GFP degradation. By use of proteosome inhibitors, such as MG132, we can determine whether LNK2 degradation occurs form proteosomal activity. Together, we hope to establish the mechanism involved in LNK2 protein regulation, and in doing so, show how LNK2 and SON1 modulate development and disease resistance. These experiments are underway with two undergraduate researchers in the lab. Progress toward Objective 3 (Evaluate circadian rhythmicity of LNK protein abundance): We will examine LNK2-GFP and SON1-myc plants across the circadian cycle, to assess LNK2 and SON! protein abundance, using western analysis. These experiments will soon be underway.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audience served by the project are professional biologists and students with interests in the mechanisms by which plants resist pathogens and control development, especially as those traits are under circadian rhythm control. The research led to completion of a Ph.D. thesis and graduation by a student under supervision of the project manager. That student, Dr. Prince Zogli, reported his findings in seminars at our home university, at regional, national and international meetings, and in his Ph.D. thesis. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has directly led to the completion of a Ph.D. thesis and graduation by one student, who is currently conducting research elsewhere as a postdoctoral fellow. Four undergraduate researchers have worked in this project. The graduate student and undergraduate students have learned methods for plant culture, plant genetics, molecular biology, use of informatics tools, pathogen culture, and biochemistry. The graduate student presented invited talks at several regional and national plant biology meetings, and presented abstracts at those, and an international meeting. He also participated in preparing his work for publication to several journals. How have the results been disseminated to communities of interest?At this time, the work has been communicated primarily through presentations (talks and posters) at scientific meetings, as well as through Dr. Prince Zogli's Ph.D. thesis at the University of Vermont. We submitted Dr. Zogli's work for publication in to several journals, but have not yet had it accepted for publication. We are generating additional data that we will include in another version of the manuscript. What do you plan to do during the next reporting period to accomplish the goals?Our current biochemistry experiments will add important details to explain the function and mechanism for LNK2 degradation, and the role of SON1 in that process. Those data will strengthen a revised publication that we will submit.
Impacts
What was accomplished under these goals?
Progress toward Objective 1: We generated several resources required for these tests: The lnk1 and lnk2 mutants were crossed, and a double mutant line identified. This double mutant line was stably transformed with wild-type LNK1 or LNK2 genes, under control of the CaMV 35S promoter, and fused with the GFP reporter gene, to generate LNK-GFP fusion proteins. These lines were also later used in experiments under Objective 2 (below). We assessed whether the LNK-GFP fusion proteins were functional, by examining whether they could rescue the lnk1 lnk2 double mutant's long hypocotyl phenotype; both LNK1-GFP and LNK2-GFP restored hypocotyl length to wild-type, indicating that both transgenes were functional. We conducted pathogen growth assays using the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) in wild-type, lnk1 lnk2 double mutant plants, the single mutants, as well as double mutant plants transformed with wild- type LNK1-GFP or LNK2-GFP genes. We found that the double mutant was significantly less susceptible to Hpa, and that the double mutant plants transformed with wild-type LNK1 or LNK2 genes showed rescue, and wild-type levels of susceptibility to Hpa. These data provide evidence for involvement of LNK gene products in pathogen susceptibility and/or resistance. Progress toward Objective 2: Test were begun to assess LNK1-GFP and LNK2-GFP stability in plant extracts, with the aim of determining whether ubiquitin-mediated proteolysis plays a role in controlling LNK1 or LNK2 protein accumulation. Total protein was extracted from 5-day-old leaves of lnk1 lnk2 double mutant plants transformed with wild-type LNK1-GFP or LNK2- GFP genes. Extracts were concentrated and placed in a degradation buffer, and combined with a bovine 26S proteasome- mediated protein degradation kit (UBP Bio, Aurora Colorado) following the manufacturer's instructions, and incubated over 2- hours. Some samples were incubated with inhibitors of the 26S proteasome (e.g. MG132, PSI, epoxomicin), as well as general protease inhibitor PMSF as control. Thirty-minute time points were stopped over the incubation period, and prepared for fractionation by SDS-PAGE, following western blotting and measurement of the GFP tag, using an anti-GFP primary monoclonal antibody, and secondary antibody conjugated to the IRDye 800CW fluorochrome. Blots were analyzed using an Odyssey CLx infrared imaging system (LiCor). Results showed degradation of LNK1-GFP or LNK2-GFP proteins over 120 minutes, which was arrested by the proteasome inhibitors MG132, PSI, or epoxomicin, but not by the general protease inhibitor PMSF. Though preliminary, these data indicate that LNK1 and LNK2 are targeted for proteolysis by the ubiquitin- proteasome system. Our most recent work is aimed at determining whether the F-box protein SON1 participates in LNK2 degradation. We are using a heterologous expression system, where we express a LNK2-GFP fusion protein, together with a SON1-myc fusion protein in Nicotiana benthamiana, using Agrobacterium-mediated gene introduction. We are performing LNK2 and SON1 protein analysis from Agro-infiltrated N. benthamiana, using western blot analysis with anti-GFP and anti-myc monoclonal antibodies. By withholding SON1 in some infiltrated leaves, we can assess whether presence of SON1 increases LNK2-GFP degradation. By use of proteosome inhibitors, such as MG132, we can determine whether LNK2 degradation occurs form proteosomal activity. Together, we hope to establish the mechanism involved in LNK2 protein regulation, and how LNK2 and SON1 modulate development and disease resistance. These experiments are underway with two undergraduate researchers in the lab.
Publications
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience served by the project are professional biologists and students with interests in the mechanisms by which plants resist pathogens and control development, especially as those traits are under circadian rhythm control. A Ph.D. student finished his thesis during the reporting period, and reported his findings in seminars at our home university, and in his Ph.D. thesis. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A Ph.D. student finished his thesis research and wrote his thesis while supported by the project. We also wrote and submitted a manuscript that reports his findings, which has been through review but is currently in revision while we perform additional experiments. How have the results been disseminated to communities of interest?By the end of the reporting period, we submitted a manuscript based on this project which is currently under revision before publication. Results have also been shared in two seminars to the UVM community. What do you plan to do during the next reporting period to accomplish the goals?We hope to have the manuscript accepted for publication, and continue work on each of the three objectives. In addition, a graduate rotation student, and several undergraduate researchers have joined my lab, and I hope to engage them in work related to this project.
Impacts
What was accomplished under these goals?
Progress toward Objective 1: We generated several resources required for these tests: The lnk1 and lnk2 mutants were crossed, and a double mutant line identified. This double mutant line was stably transformed with wild-type LNK1 or LNK2 genes, under control of the CaMV 35S promoter, and fused with the GFP reporter gene, to generate LNK-GFP fusion proteins. These lines were aso later used in experiments under Objective 2 (below). We assessed whether the LNK-GFP fusion proteins were functional, by examining whether they could rescue the lnk1 lnk2 double mutant's long hypocotyl phenotype; both LNK1-GFP and LNK2-GFP restored hypocotyl length to wild-type, indicating that both transgenes were functional. We conducted pathogen growth assays using the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa)in wild-type, lnk1 lnk2 double mutant plants, the single mutants, as well as double mutant plants transformed with wild-type LNK1-GFP or LNK2-GFP genes. We found that the double mutant was significantly less susceptible to Hpa, and that the double mutant plants transformed with wild-type LNK1 or LNK2 genes showed rescue, and wild-type levels of susceptibility to Hpa. These data provide evidence for invovlement of LNK gene products in pathogen susceptibility and/or resistance. Progress toward Objective 2: Test were begun to assess LNK1-GFP and LNK2-GFP stability in plant extracts, with the aim of determining whether ubiquitin-mediated proteolysis plays a role in controlling LNK1 or LNK2 protein accumulation. Total protein was extracted from 5-day-old leaves oflnk1 lnk2 double mutant plants transformed with wild-type LNK1-GFP or LNK2-GFP genes. Extracts were concentrated and placed in a degradation buffer, and combined with a bovine 26S proteasome-mediated protein degradation kit (UBP Bio, Aurora Colorado) following the manufacturer's instructions, and incubated over 2-hours. Some samples were incubated with inhibitors of the 26S proteasome (e.g. MG132, PSI, epoxomicin), as well as general protease inhibitor PMSF as control. Thirty-minute time points were stopped over the incubation period, and prepared for fractionation by SDS-PAGE, following western blotting and measurement of the GFP tag, using an anti-GFP primary monoclonal antibody, and secondary antibody conjugated to the IRDye 800CW fluorochrome. Blots were analyzed using an Odyssey CLx infrared imaging system (LiCor). Results showed degradation of LNK1-GFP or LNK2-GFP proteins over 120 minutes, which was arrested by the proteasome inhibitors MG132, PSI, or epoxomicin, but not by the general protease inhibitor PMSF. Though preliminary, these data indicate that LNK1 and LNK2 are targeted for proteolysis by the ubiquitin-proteasome system. Work is continuing to expand upon these results, and to test for the requirement for SON1 in LNK2 degradation.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2015
Citation:
Prince K. Zogli (2015). Ph.D. Thesis at the University of Vermont.�Title:�Analysis of Function of the SON1-Interacting�Protein, LNK2 In�Arabidopsis thaliana.
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