Progress 12/15/13 to 12/14/17
Outputs
Target Audience:The primary users of the research in this project are other scientists engaged in research to improve disease management on small grain crops in both the private and public sector. We met with approximately 60 undergraduate students attending the America Society for Agronomy national meeting to inform them of NIFA sponsored research. Seminars on the funded research were given at major agricultural centers including China Agriculture University (Beijing), Northwest A&F University (Yangling); Chinese Academy of Sciences (Shanghai), Zhejiang University (Hangzhou), Cornell University, UC Davis, University of Maryland, BASF Crop Knowledge (Research Triangle Park) and the University of Minnesota. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoctoral and graduate student supported on the grant participated in the rich academic community at the University of Minnesota to the fullest extent. Opportunities included formal programs in Research Ethics (PlPa 8123; 0.5 credit), a Practicum for Future Faculty (GRAD 8102; 3 credits), and a course in Teaching in Higher Education (GRAD 8101; 3 credits). The courses sharpen rhetorical skills and provide thoughtful discussions of scientific communication and responsible scientific conduct that will serve STEM professionals in both the public and private sector. How have the results been disseminated to communities of interest?Theresearch generated gene expression data and functional analysis thatis freely available on the internet in databases and in open access journals. Another major customerhas beenmembers of the U.S. Wheat and Barley Scab Initiative consisting of scientists in 21 states conducting research on disease management, food safety, as well as chemical and biological control. Efforts from this workare available to wheat and barley breeders in public institutions and in private industry, and scientists conducting basic and applied research on genetics and genomics in wheat, barley and other crops. 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 finalyear of the project we made progress with respect to all objectives. Previously, enzymes catalyzing early and late steps in trichothecene biosynthesis, hydroxymethylglutaryl CoA reductase (Hmr1), trichodiene oxygenase (Tri4), and calonectrin oxygenase (Tri1), were shown to localize to spherical subcellular structures called "toxisomes" when grown in toxin inducing medium. We determined that these toxisomes are actually highly reorganized smooth endoplasmic reticulum (OSER) that form in response to toxin induction. The reorganization of the ER upon toxin induction was confirmed using ER-Tracker dye and super resolution microscopy as well as by co-florescence with other ER markers such as Sec22 and GFP linked to the ER-retrieval sequence -HDEL. Remarkably the cytosolic enzyme catalyzing the first step in trichothecene biosynthesis, trichodiene synthase (Tri5), also spatially associates with toxisomes and appears to be captured within the cytosolic domains within stacks of OSER membranes. We have recently found that tri5 deletion mutants do not form toxisomes but if the deletion is replace by the enzymatically inactive, but structurally similar tri5N225D/S229T allele, toxisome formation is restored. Remarkably, the synthesis culmorin, another sesquiterpene mycotoxin produced by F. graminearum, is dependent upon toxisome formation but not Tri5 activity. That is, culmorin is produced by wildtype and tri5N225D/S229T containing strains but not by Δtri5 strains. Unlike the ER-localized biosynthetic enzymes, the export of trichothecenes is linked to endosomes. The trichothecene transporter Tri12-GFP localizes to small (1 µm) motile vesicles, vacuoles and the plasma membrane, based on co-localization with fluorescent dyes CMAC and FM4-64. Motile Tri12-containing vesicles fuse with the vacuole or plasma membrane, suggesting that vesicular transport plays a role in export of trichothecenes. Mutants in four other potential trichothecene protein transporters also have been made and have been individually tested for their ability to facilitate toxin accumulation in the plant. Deletion of genes for two ABC transporters (Abc1 and Abc6), significantly reduce the ability of the fungus to accumulate DON in infected plants. The genes for Abc1 and Abc6 have been synthesized and codon optimized for yeast. The genes expressed in yeast will be tested for their ability to increase resistance to trichothecenes in culture in a prelude to expression of these genes in transgenic wheat. Also, mutants lacking the gene for the HOPS complex component Rab7, are unable to form late endosomes and do not accumulate detectable levels of toxin in plants. These results suggest a role for endosomal maturation in toxin export.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Tang, G., Chen, Y., Xu, J.-R., Kistler, H.C., and Ma, Z. 2018. The fungal myosin I is essential for Fusarium toxisome formation. PLoS Pathogens 14(1) e1006827.
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Progress 12/15/15 to 12/14/16
Outputs
Target Audience:The primary users of the research in this projectare other scientists engaged in research to improve disease management on small grain crops in both the private and public sector. During the reporting period we met with approximately 60 undergraduate students attending the America Society for Agronomy national meeting to inform them of NIFA sponsored research. Seminars on the funded research were given at major agriculturalcenters includingChina Agriculture University (Beijing), Northwest A&F University (Yangling); Chinese Academy of Sciences (Shanghai), Cornell University, UC Davis, BASF Crop Knowledge (Research Triangle Park) and the University of Minnesota. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The postdoctoral and graduate student supported on the grant participate in the rich academic community at the University of Minnesota to the fullest extent. Opportunities include formal programs in Research Ethics (PlPa 8123; 0.5 credit), a Practicum for Future Faculty (GRAD 8102; 3 credits), and a course in Teaching in Higher Education (GRAD 8101; 3 credits). The courses sharpen rhetorical skills and provide thoughtful discussions of scientific communication and responsible scientific conduct that will serve STEM professionals in both the public and private sector. How have the results been disseminated to communities of interest?The plan generates gene expression data and functional analysis thatis freely available on the internet in databases and in open access journals. Another major customeris the membership ofthe U.S. Wheat and Barley Scab Initiative consisting of scientists in 21 states conducting research on disease management, food safety, as well as chemical and biological control. Efforts from this work will be available to wheat and barley breeders in public institutions and in private industry, and scientists conducting basic and applied research on genetics and genomics in wheat, barley and other crops. What do you plan to do during the next reporting period to accomplish the goals?Current work is focused on generating double and triple mutants of F. graminearum, which lack multiple potential modes of DON transport. A Δabc1/Δsso1 mutant has been generated to assess whether Sso1-mediated exocytosis may function in parallel with the DON ABC1 transporter protein. In addition, Δtr12/Δabc1 and Δtri12/Δabc6 mutants are being generated to determine whether transporters may be functionally redundant. Another question is whether the presence of the trichothecenes themselves trigger OSER structure formation or whether the trichothecene proteins are the basis for formation. To address this question, we are currently constructing a Tri5 protein with a point mutation that retains the protein structure but which lacks enzymatic activity and thus lacks the ability to produce trichothecenes.
Impacts
What was accomplished under these goals?
During this year of the project we have made progress with respect to objective 1 and objective 2. Previously, enzymes catalyzing early and late steps in trichothecene biosynthesis, hydroxymethylglutaryl CoA reductase (Hmr1), trichodiene oxygenase (Tri4), and calonectrin oxygenase (Tri1), were shown to localize to spherical subcellular structures called "toxisomes" when grown in toxin inducing medium. We determined that these toxisomes are actually highly reorganized smooth endoplasmic reticulum (OSER) that form in response to toxin induction. The reorganization of the ER upon toxin induction was confirmed using ER-Tracker dye and super resolution microscopy as well as by co-florescence with other ER markers such as Sec22 and GFP linked to the ER-retrieval sequence HDEL. Remarkably the cytosolic enzyme catalyzing the first step in trichothecene biosynthesis, trichodiene synthase (Tri5), also spatially associates with toxisomes and appears to be captured within the cytosolic domains within stacks of OSER membranes. Unlike the ER-localized biosynthetic enzymes, the export of trichothecenes is linked to endosomes. The trichothecene transporter Tri12-GFP localizes to small (1 µm) motile vesicles, vacuoles and the plasma membrane, based on co-localization with fluorescent dyes CMAC and FM4-64. Motile Tri12-containing vesicles fuse with the vacuole or plasma membrane, suggesting that vesicular transport plays a role in export of trichothecenes. Mutants in four other potential trichothecene protein transporters also have been made and have been tested for their ability to facilitate toxin accumulation in the plant. Deletion of genes for two ABC transporters (ABC1 and ABC6), significantly reduce the ability of the fungus to accumulate DON in infected plants. Also, mutants lacking the gene for the HOPS complex component Rab7, are unable to form late endosomes and do not accumulate detectable levels of toxin in plants. These results suggest a role for endosomal maturation in toxin export.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Boenisch, M.J., Broz, K.L., Purvine, S.O., Chrisler, W.B., Nicora, C.D., Connolly, L.R., Freitag, M., Baker, S.E., Kistler, H.C. 2017. Structural reorganization of the fungal endoplasmic reticulum upon induction of mycotoxin biosynthesis. Scientific Reports doi: 10.1038/srep44296
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Progress 12/15/14 to 12/14/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctoral associate Marike Boenisch has expanded her skills and knowledge of confocal, super resolution and electron microscopy by one-on-one interactions with staff members at the University of Minnesota Imaging Center. She has learned molecular techniques for florescent labeling proteins for microscopy. She has increased her knowledge in her field of speciality by attending and presenting a talk at a national scientific meeting. In addition to course work at the University of Minnesota, graduate student Sean O'Mara has developed a research proposal for his Ph.D. project and gained experience in undergraduateteaching. He has also developed laboratory techniques for microbial genetics and molecular biology and plans to present the results of his preliminaryresearch at a scientific meeting in 2016. How have the results been disseminated to communities of interest?Results from the funded research have been presented to the scientific community at two scientific conferences: The American Phytopathological Society National Meeting and the Genetics Society of America sponsored Fungal Genetics Conference. More popular descriptions of the research were presentedto undergraduate attendees of the America Society for Agronomy national meeting, as well asattendees of the University of Minnesota Imaging Center Symposium and Nikon Center of Excellence Celebration. What do you plan to do during the next reporting period to accomplish the goals?Our goal is to further characterize the toxisome and to test at least six additional protein trasporters for their role in mycotoxin export and tolerance.
Impacts
What was accomplished under these goals?
In the second year of the project we have accomplished quite a bit, especially with respect to objective 1 and have made steady progress in objective 2. Previously, enzymes catalyzing early and late steps in trichothecene biosynthesis, hydroxymethylglutaryl CoA reductase (Hmr1p), trichodiene oxygenase (Tri4p), and calonectrin oxygenase (Tri1p), were shown to localize to spherical subcellular structures called "toxisomes" when grown in toxin inducing medium. Toxisomes co-localize with the endoplasmic reticulum (ER). Applying super resolution microscopy, we determined that the ER organization shifts from being highly reticulate under non-toxin inducing conditions to being tubular and exhibiting pronounced perinuclear ER upon toxin induction. The reorganization of the ER upon toxin induction was further confirmed by observing similar fluorescence patterns with the native ER resident protein Sec22p of F. graminearum tagged with GFP (green fluorescent protein), as well as with GFP containing the ER-retrieval sequence HDEL. High pressure freezing and freeze substitution for TEM (transmission electron microscopy) revealed the ultrastructure of ER membranes in toxin producing and non-toxin producing cells. Different types of proliferations of the smooth ER, including lamellar stacks of perinuclear ER membranes ("karmellae") as well as lamellar stacks of peripheral ER membranes ("stripes") and concentric stacks ("whorls") were observed in cells grown under toxin producing conditions. Visualizing nuclei of a histone H4p Tri4p::RFP (red fluorescent protein) doubly tagged strain under toxin induction supported that trichothecene biosynthesis is localized at both smooth perinuclear ER and peripheral ER. As a consequence, trichothecene biosynthesis seems to be localized at particular regions of the smooth ER. Subcellular changes which occur during trichothecene production may facilitate toxin biosynthesis and self-protection of the fungus. Unlike the ER-localized biosynthetic enzymes, the export of trichothecenes is linked to endosomes. The trichothecene transporter Tri12p::GFP localizes to small (1 µm) motile vesicles, vacuoles and the plasma membrane, based on co-localization with fluorescent dyes CMAC and FM4-64. Motile Tri12p vesicles fuse with the vacuole or plasma membrane, suggesting that vesicular transport plays a role in export of trichothecenes. Mutants in three other potential trichothecene protein transporters have been made and are now being tested for their ability to facilitate toxin accumulation in the plant. Mutants lacking the gene for SNARE protein Sso1, involved in subapical exocytosis, are significantly reduced in extracellular toxin accumulation in culture. These results suggest a role for vesicular trafficking and exocytosis in toxin export.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Kistler, H.C. and Broz, K. 2015. Cellular compartmentalization of secondary metabolism. Frontiers in Microbiology 6:68 doi:10.3389/fmicb.2015.00068.
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Progress 12/15/13 to 12/14/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The postdoctoral associate has expanded her skills and knowledge of confocal, super resolution and electron microscopy by one-on-one interactions with staff members at the University of Minnesota Imaging Center. She has increased her knowledge in her field of speciality by attending and presenting a talk at a national scientific meeting. How have the results been disseminated to communities of interest? Results from the funded research have been presented to the scientific community at two scientific conferences: The American Phytopathological Society National Meeting and the Gordon Conference on Cellular and Molecular Fungal Biology. Additionally portions of the results were presented to students from the minority serving institution, Minneapolis Community Technical College (MCTC) in a popular talk entitled "Understanding the USDA." What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Accumulation of trichothecene (TRI) mycotoxins by F. graminearum in wheat may require not only induction of enzymes for TRI biosynthesis but also may involve activation of cellular pathways to efficiently export these toxic metabolites and deliver them to the plant. Tri12 is a gene within the TRI biosynthetic gene cluster of F. graminearum that encodes a predicted drug resistance protein shown to play a role in export of TRI mycotoxins. Deletion mutants of Tri12 result in decreased TRI accumulation and reduced virulence when inoculated on wheat compared to the wild type strain. Mutants also are more prone to self-inhibition under toxin inducing conditions in vitro suggesting a role for Tri12 in self-protection, toxin export and virulence of the fungus in planta. Strains expressing Tri12p linked to green fluorescent protein show localization of the protein to the plasma membrane as well as to the vacuole and small (1 µm) motile vesicles. These vesicles are likely endosomes based on co-localization of Tri12::GFP with fluorescent dye FM4-64. Motile Tri12p linked vesicles may fuse with the vacuole or plasma membrane suggesting that vesicular transport of TRI may play a role in cellular sequestration and export of the toxin. The T-SNARE protein directing subapical exocytosis (SSO1) may be involved in toxin export. Deletion mutants (sso1) are significantly reduced in the ability to accumulate TRI in infected plants. Our results suggest a role for vesicular trafficking and exocytosis in export of TRI mycotoxins.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2015
Citation:
Kistler, H.C. and Broz, K. 201x. Cellular compartmentalization of secondary metabolism. Frontiers in Microbiology (submitted).
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