Source: MICHIGAN STATE UNIV submitted to
AFLATOXIN B1 BIOSYNTHESIS IN ASPERGILLUS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0176024
Grant No.
(N/A)
Project No.
MICL01856
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Nov 1, 2012
Project End Date
Oct 31, 2017
Grant Year
(N/A)
Project Director
Linz, J.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Food Science
Non Technical Summary
Aflatoxin is a toxin made by a mold that grows on food and feed crops. We are studying the role of oxidants and antioxidants in control of aflatoxin synthesis because we might be able to use these compounds to prevent aflatoxin synthesis on crops in the field or during storage.
Animal Health Component
20%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7121219104020%
7121510104020%
7121810104020%
7124020104020%
7126110104020%
Goals / Objectives
Goals - understand the role of aflatoxin synthesis in redox signaling and stress response in Aspergillus. Expected outputs include publications in nationally recognized journals and presentation of data at national and international meetins and symposia.
Project Methods
We will measure accumulation of reactive oxygen species in endosomes under aflatoxin inducing and noninducing growth conditions. These measurements will be made using fluorescent dyes and microscopy on whole cells, protoplasts, and cell extracts. We will conduct similar studies on mutants that are deficient in specific steps in aflatoxin synthesis as well as in biogenesis of endosomes.

Progress 11/01/12 to 10/31/17

Outputs
Target Audience:university faculty, farmers, producers of corn, cotton, peanuts, and treenuts, Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We trained over 20 graduate students, 19 postdocs, and 40 undergraduate lab assistants under project How have the results been disseminated to communities of interest?Publication in peer-reviewed scientific journals and presentation at local, national, and international conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Developed several novel approaches to prevent aflatoxin in crops mostly focused on natural products isolated from plants, fungi, and lichens.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Mmongoyo JA, Wu F, Linz JE, Nair MG, Mugula JK, Tempelman RJ, Strasburg GM. 2017. Aflatoxin levels in sunflower seeds and cakes collected from micro- and small scale sunflower oil processors in Tanzania. Plos One. Volume 12. e0175801.doi 10.137


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Scientists in secondary metabolism research, consumers, and the public via published journal articles. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?1 peer reviewed publication and 2 additional manuscripts under review. What do you plan to do during the next reporting period to accomplish the goals?Continue to develop additional natural products that block fungal virulence on plants and humans and move towards bringing safe and effecgtive compounds to market within 5 years.

Impacts
What was accomplished under these goals? We screened several biological sources of natural products and identified several which block Aspergillus growth, sporulation, spore germination, and/or mycotoxin biosynthesis. These pure compounds are being scaled up for field tests and safety testing in animal models.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wee, J., Day, D.M., and Linz, J.E. 2016. Effects of zinc chelators on aflatoxin production in Aspergillus parasiticus. Toxins 8:171.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Scientists in the area of secondary metabolism, stress, response, and fungal development. Federal agencies associated with food safety. Consumers concerned about human exposure to toxic fungal metabolites. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?My PhD student just finished her degree program and initiated a Post Doctoral program. How have the results been disseminated to communities of interest?One publication in a highly visisble journal. What do you plan to do during the next reporting period to accomplish the goals?We are shifting emphasis to continue to identify natural products from plants that block mycotoxiin biosynthesis on susceptible plants including corn and peanuts

Impacts
What was accomplished under these goals? We demonstrated that the bZIP transcription factor AtfB regulates a gene network that controls mycotoxin synthesis, stress response, and conidiospore development.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Roze LV, Lavenieks M, Hong SY, Wee J, Wong SS, Vanos B, Awad D, Ehrlich KC, Linz JE. 2015. Aflatoxin biosynthesis is a novel source of reactive oxygen species - a potential redox signal to initiate resistance to oxidative stress. Toxins 7, 1411-1430.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Our work on prevention of aflatoxin in the food supply is of interest to consumers, farmers, government, and students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We are currently training 1 PhD level student and 1 MS level student. How have the results been disseminated to communities of interest? Primiarly by publication but Josephine Wee also presented at a national meeting of the American Plant Pathology Society. What do you plan to do during the next reporting period to accomplish the goals? We continue to study genes involved in expression of aflatoxin biosynthesis but also genes that regulate storage and export of aflatoxin.

Impacts
What was accomplished under these goals? We published 3 peer reviewed manuscripts and 1 peer reviewed book chapter. We also made great strides in understanding the AtfB regulatory network that controls secondary metabolism, stress response, and development in Aspergillus parasiticus.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Spraker, J., Jewell, K., Roze, L.V., Scharf, J., Beaudry, R., Linz, J.E., Allen, C., Keller, N.P. 2014. A volatile relationship:profiling interkingdom dialogue betwenn two plant pathogens, Raltonia solanacearum and Aspergillus flavus. J. Chemical Ecology 40:502-513.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Linz, J.E., wee, J., Roze, L.V. 2014. Chapter 5. Pp. 89-110. Aflatoxin biosynthesis: regulation at the level of transcription and sub-cellular localization. IN. Biosynthesis and molecular genetic of fungal secondary metabolites. Martin, J.F. (Ed). Springer. NY, NY.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: BAidya, S., Doran, R., Lohmar, J.M., Cary, J.W., Hong, S.Y., Roze, L.V., Linz, J.E., Calvo, A.M. 2014. VeA is associated with response to oxidative stress in the aflatoxin producer, A. flavus. Eukaryotic Cell: 1095-1103
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Linz, JH.E., Wee., J., Roze, L.V. 2014. Aspergillus parasiticus genome sequence, predicted chromosome structure, and gene expression under aflatoxin inducing and non-inducing conditions: evidence that differential expression contributes to species phenotype. Eukaryotic Cell: 1113-1123.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Researchers in the aflatoxin community and consumers who eat products that can be contaiminated with aflatoxin Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? We have trained 21 graduate students, 25 postdoctoral researchers, and over 40 undergraduate students on this project which has spanned 27 years. How have the results been disseminated to communities of interest? Publications in peer reviewed journals, oral and poster presentations at local, national, and international conferences and symposia. What do you plan to do during the next reporting period to accomplish the goals? Continue work on AtfB and Vps34.

Impacts
What was accomplished under these goals? We demonstrated that the transcription factor AtfB regulates expression of genes involved in aflatoxin synthesis, other secondary metabolic pathways, stress response, and asexual sporulation. We demonstrated that the Class III PI3-kinase Vps34 regulates asexual sporulation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hong, S.Y., Roze, L.V., and Linz, J.E. 2013. Oxidative stress-related transcript factors in regulation of secondary metabolism. Toxins 5:683-702.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hong, S.Y., Roze, L.V., and Linz, J.E. 2013. Evidence that a transcription factor regulatory network coordinates oxidative stress response and secondary metabolism. MicrobiologyOpen 2:144-160.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Activities - we continue to conduct experiments in several major focus areas. 1) The role of the stress related transcription factor AtfB in regulation of stress response and secondary metabolism; 2) Utilization of plant and fungal volatiles to prevent aflatoxin biosynthesis in the laboratory and during crop storage; 3) The role of endosome biogenesis in stress response and secondary metabolism. Products - data generated from our experiments allowed us to expand our previously proposed model on the role of stress response in regulation of secondary metabolism. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Although many investigators provided evidence of a regulatory link between stress response and secondary metabolism, our work contributed to understanding the specific molecular mechanisms that link these cellular processes. Specifically our work on the AtfB regulatory network and the role of endosomes in stress response and secondary metabolism in A. parasiticus provides novel insights into these specific regulatory mechanisms.

Publications

  • Linz, J.E., Chanda, A., and Roze, L.V. (2012). Proteomic and biochemical evidence support of role for tranport vesicles and endosomes in stress response and secondary metabolism, J. Proteome Res., 11:767-775.
  • Roze, L.V., Chanda, A., and Linz, J.E.. (2012). Analysis of volatile compounds emitted by filametous fungi using solid pahse microextraction-gas chromatography/mass spectrometry (SPME-GC/MS), Methods Mol. Biol., 944:133-142.
  • Chanda., A., Roze, L.V., and Linz, J.E. (2012). Purification of a vesicle-vacuole fraction (V) from Aspergillus, Methods Mol. Biol., 944:259-266.
  • Roze, L.V., Hong, S.Y., and Linz, J.E. (2012). Aflatoxin synthesis: Current frontiers, Ann. Rev. Food Sci. Technol., (in press).


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Attended Fungal Genetics national meeting in April 2011. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Research data were published in 4 publications in 2011

Publications

  • Linz, J.E., Chanda, A., and Roze, L.V. 2011. Proteomic and biochemical evidence support a role for transport vesicles and endosomes in stress response and secondary metabolism. J Proteome Res. (in press).
  • Roze, L.V., Chanda,A., and Linz, J.E. 2011. Stress-related transcription factor AtfB integrates secondary metabolism with oxidative stress response in Aspergilli. J. Biol. Chem. 286:35137-35148.
  • Roze, L.V., Koptina, A.V., Laivenieks, M., Beaudry, R.M., Jones, A.D., Kanarsky, A.V., and Linz, J.E. 2011. Willow volatiles influence growth, development, and secondary metabolism in Aspergillus parasiticus. Applied Microbiology and Biotechnology 92:359-370.
  • Roze, LV, Chanda, A.and Linz,J.E. 2011. Compartmentalization and molecular traffic in secondary metabolism: novel functions for established cell machinery. Fungal Gen. and Biol. 48:35-48.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We summarized our research on the role of the aflatoxisome in aflatoxin synthesis at three invited talks over the past 18 months. I presented a talk at the Gordon Conference on Myctoxins and Phycotoxins in June, 2009. Ludmila Roze presented an invited talk (in my place) at the International Mycology Congress (Edinburgh, Scotland) in Aug. 2010, and I will present an invited talk at the Fungal Genetics conference in California in Mar. 2011. PARTICIPANTS: Albert Kanarsky and Anna Koptina (Russia) collaborated with our group on characterization of the Aspergillus volatilome. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our novel work on aflatoxisomes made a large impact on the field of fungal biology. We provided the first evidence that aflatoxin synthesis is compartmentalized and then exported from the cell via fusion of the aflatoxisome with the cytoplasmic membrane.

Publications

  • Chanda, A, Roze, LV, Linz, JE. (2010) Aflatoxin export in Aspergillus parasiticus: a possible role for exocytosis. Eukaryotic Cell 9: 1724-1727
  • Roze, LV, Chanda, A, Linz, JE. (2010). Comparmentalization and metabolic traffic in secondary metabolism: novel functions for established cellular machinery. Fungal Genetics and Biology (in press).
  • Roze, LV, Chanda, A, Laivenieks, M, Beaudry, RM, Jones, AD, Artymovich, KA, Koptina, AV, Awad, D, Valeeva, D, Linz, JE. (2010). Volatolome analysis reveals intracellular metabolic changes in Aspergilli: VeA regulates branched chain amino acid and ethanol metabolism. BMC Biochemistry (in press)


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The results were discussed during a presentation at the Gordon Conference on Mycotoxins and Phycotoxins at Colby Sawyer College, June 2009. PARTICIPANTS: We are currently collaborating with Ana Calvo at Northern Illinois University. TARGET AUDIENCES: Our work should be of general interest to cell biologists but also is relevant to the agricultural sector based on implications to control of aflatoxin contamination in food and feed crops. PROJECT MODIFICATIONS: No major changes

Impacts
We recently discovered that vesicles are involved in the synthesis, storage and export of aflatoxin. The structural and functional connection between vesicles and secondary metabolism demonstrates an important and novel role of vesicles in cell biology.

Publications

  • Chanda, A., Roze, L.V., Pastor, A., Frame, M. and Linz, J.E. 2009. A key role for vesicles in fungal secondary metabolism. PNAS 106:19533-19538.
  • Chanda, A., Roze, L.V., Pastor, A., Frame, M. and Linz, J.E. 2009. Purification of a vesicle-vacuole fraction functionally linked to aflatoxin synthesis in Aspergillus parasiticus. J. Microbiol. Methods. 76:28-33.
  • Hong, S-Y, and Linz, J.E. 2009. Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme, Nor-1, in Aspergillus parasiticus. Mycological Research 113: 591-601.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: We made significant progress on work focused on subcellular localization of aflatoxin synthesis. Two manuscripts were accepted for publication and 3 more are in preparation for 2009. One PhD candidate graduated in 2008 (Hong). PARTICIPANTS: Sung Yong Hong is currently employed as a PostDoctoral researcher at UC Berkeley. Anindya Chanda is now a PostDoctoral researcher in my laboratory at MSU TARGET AUDIENCES: Anyone interested in prevention of mycotoxins in food and feed. Includes farmers and consumers of affected crops and other scientists in the mycotoxin and secondary metabolism fields. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We clearly demonstrated for the first time that the late aflatoxin enzymes localize to vesicles and vacuoles and carry out the last two steps in aflatoxin synthesis at that location. We also demonstrated that vesicles and vacuoles compartmentalize aflatoxin from the rest of the cell and that aflatoxin is predominantly secreted to the growth environment by an unknown mechanism. The novel finding is that the aflatoxin enzymes are functional in the vacuole and vesicle.

Publications

  • Hong, S-Y, and Linz, J.E. 2008. Functional expression and sub-cellular localization of the early aflatoxin pathway enzyme, Nor-1, in Aspergillus parasiticus. Mycological Research (in press)
  • Hong, S-Y, and Linz, J.E. 2008. Functional expression and sub-cellular localization of the aflatoxin pathway enzyme Ver-1 fused to enhanced green fluorescent protein. Appl. Environ. Microbiol. 74:6385-6396


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Aflatoxin, synthesized by filamentous fungi in the Genus Aspergillus,is one of the most potent naturally occurring toxins known. Presence of aflatoxin is closely associated with liver cancer in animals that ingest contaminated feed. A close epidemiological association wih human liver cancer has also been proposed. Our research activities have focused on development of practical solutions to the aflatoxin problem. In recent work, we identified a series of natural products synthesized by Aspergillus and/or by plants that control aflatoxin synthesis in culture and on plant materials. Part of current focus is to determine the mode of action of these natural product inhibitors so they can be developed into effective products that can be used in agriculture to control aflatoxin on economically important products including tree nuts, grains, and groundnuts. PARTICIPANTS: Ludmila Roze is a postdoctoral research associate working on this project. She has been with the project for over 11 years and helps direct the work of two graduate students and two undergraduate students. Sung Yong Hong is recently earned his PhD on this project. He is seeking a postdoctoral position in a US lab. Anindya Chanda is nearing completion of his degree program. It is likely he will graduate in May or August, 2008. Eric Harper and Kathrine Artymovich are undergraduate research assistants who assisted Hong, Chanda, and Roze with current research projects. TARGET AUDIENCES: THe primary target audience for our work is US farmers who grow crops susceptible to aflatoxin contamination. Currently, a larger portion of affected crops must be destroyed each year due to toxin contamination. It is our goal to eliminate this economic burden on affected farmers.

Impacts
We believe that natural product inhibitors of aflatoxin may eventually used to effectively control development of this toxin on affected crops including corn, peanuts, cotton seed, and treenuts. If applied properly, this technology could save hundereds of million dollars in lost revenue annually to the food and feed industry.

Publications

  • Roze L.V., Arthur A.E., and Linz J.E. (2007). Initiation and pattern of spread of histone H4 acetylation parallel the order of transcriptional activation of genes in the aflatoxin cluster. Mol. Microbiol. 66:713-726.
  • Roze, L.V., Calvo, A., Arthur, A., Beaudry, R. and Linz, J.E. (2007). Aspergillus volatiles regulate aflatoxin synthesis and asexual sporulation in Aspergillus parasiticus. Appl. Environ. Microbiol.73: 7268-7276.
  • Lee, J.-W., Miller, M.J., Roze, L.V.and Linz, J.E. (2007). A wortmannin-sensitive signal transduction pathway inhibits aflatoxin synthesis. Mycologia 99:562-568.
  • Gunterus, A., Roze, L.V. , Beaudry, R., and Linz, J.E. (2007). Ethylene inhibits aflatoxin biosynthesis in Aspergillus parasiticus grown on peanuts. Food Microbiology 24:658-663.


Progress 01/01/06 to 12/31/06

Outputs
Aflatoxins are among the most carcinogenic naturally occurring compounds known. They are synthesized primarily by Aspergillus parasiticus and A. flavus on economically important crops such as peanuts, cottonseed, corn, and treenuts. Our goal is to reduce or eliminate aflatoxin contamination of food and feed. Current research is focused in two primary areas. 1) We are analyzing several key elements of the genetic switch that turns the aflatoxin genes on - these elements include signal transduction pathways, transcription factors, chromatin remodeling, and sub-cellular protein localization. 2) We are developing new inhibitors of aflatoxin biosynthesis and analyzing the mechanism of action of several inhibitors identified in past research including ethylene, CO2, wortmannin, and crotyl alcohol.

Impacts
These studies will assist in identification of targets that will allow development of novel and effective strategies for reduction or elimination of aflatoxin if food and feed crops. Cellular recepetors for CO2, ethylene, gllucose, and crotyl alcohol show promise as targets for this purpose preharvest and postharvest.

Publications

  • No publications reported this period


Progress 01/01/05 to 12/31/05

Outputs
Aflatoxin, a potent carcinogen, contaminates a variety of food and feed crops in the US including corn, cotton, peanuts, and treenuts. This results in large health and economic losses each year. This project is focused on development of novel strategies to reduce aflatoxin contamination in food and feed crops. We currently study the function of the aflatoxin genes, the activity of their promoters, the sub-cellular localization and physical interaction of aflatoxin enzymes. We hope to identify specific targets for inhibition of aflatoxin synthesis via completion of these studies.

Impacts
These studies will assist in identification of targets that will allow development of novel and effective strategies for reduction or elimination of aflatoxin if food and feed crops. Cellular recepetors for CO2, ethylene, gllucose, and crotyl alcohol show promise as targets for this purpose preharvest and postharvest.

Publications

  • Miller, M.J and Linz, J.E. 2005. Genetic mechanisms involved in regulation of mycotoxin biosynthesis. Chpt. 3-19. Food Biotechnology, Second Edition.
  • Miller, M.J. Rarick, M. Roze, L. Trail, F. and Linz, J.E. 2005. Cis-acting sites, NorL, TATA box, and AflR1 play important roles in nor-1 transcriptional activation in Aspergillus. Appl. Environ. Microbiol. 71:1539-1545.


Progress 01/01/04 to 12/31/04

Outputs
During the past year we continued studies in three areas: 1) cellular localization of aflatoxin enzymes; 2)purification and functional analysis of transcription factors NorL and Orf3; 3) signalling pathways that regulate aflatoxin synthesis. Major advances include: 1)we developed protocols for purifying vacuoles from fungal mycelia to analyze the protein contents and confirm that OmtA resides in these vacuoles; 2)nearly completed constuction of vectors for expression of functional GFP fusions with OmtA, Nor-1, Ver-1, and VBS; 3) developed protocols for affinity purification of NorL and Orf3 based on DNA binding sites.

Impacts
These studies will assist in identification of targets that will allow development of novel and effective strategies for reduction or elimination of aflatoxin if food and feed crops. Ethylene gas shows promise for this purpose preharvest and postharvest.

Publications

  • Miller, M.J. Rarick, M. Roze, L. Trail, F. and Linz, J.E. 2004. Cis-acting sites, NorL, TATA box, and AflR1 play important roles in nor-1 transcriptional activation in Aspergillus. Appl. Environ. Microbiol.(in press).
  • Roze, L. Keller, N. Beaudry, R. and Linz, J. E. 2004. Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus. Mycopathologia 158:219-232.
  • Chiou, C-H. Lee, L-W. and Linz, J.E. 2004. Distribution and sub-cellular localization of the aflatoxin enzyme versicolorin B synthase (VBS) in time-fractionated colonies of Aspergillus parasiticus. Archives of Microbiology 182:67-79.
  • Roze, L. Miller, M. Mahanti, N. and Linz, J.E. 2004. A novel cAMP response element, CRE1, modulates expression of the nor-1 promoter in Aspergillus parasiticus. J. Biol. Chem. 279: 27428-27439.
  • Roze, L. Calvo-Byrd, A. Beaudry, R. and Linz, J.E. 2004. Ethylene modulates development and toxin synthesis in Aspergillus likely via an ethylene sensor-mediated signaling pathway. J. Food Protection 67:438-447.
  • Lee, L-W. Chiou, C-H. Klomparens, K. Cary, J.W. and Linz, J.E. 2004. Sub-cellular localization of aflatoxin biosynthetic enzymes in time-dependent fractionated colonies of Aspergillus parasiticus. Archives of Microbiology 181:204-214.
  • Yu, J. Chang, P-K. Cary, J.W. Bhatnagar, D. Cleveland, T.E. Payne, G.A. Linz, J.E. Woloshuk, C.P. and Bennett, J.W. 2004. Clustered pathway genes in aflatoxin biosynthesis. Appl. Environ. Microbiol. 70:1253-1262.


Progress 01/01/03 to 12/31/03

Outputs
During the past year we continued studies in 3 areas: 1) identification of transcription factors that mediate regulation of aflatoxin synthesis; 2) sub-cellular localization of enzymes involved in aflatoxin synthesis; and 3) signalling pathways that regulate aflatoxin synthesis in response to environmental stimuli. We demonstrated the importance of 3 transcription factors (NorL, CRE1, and TBP) and their cis-acting sites in addition to AflR in regulation of nor-1 promoter activity in response to carbon source. We demonstrated that Nor-1, OmtA, Ver-1, and VBS are localized in the cytoplasm of cells in colonies that are actively synthesizing aflatoxin. OmtA also localizes in vacuole-like organelles in cells near the basal surface of the colony on solid media. We also demonstrated that cAMP, Protein kinase A, and PI3 Kinase play important roles in regulation of aflatoxin synthesis. Finally we demonstrated that ethylene and CO2, alone and in combination, inhibit aflatoxin synthesis on solid growth media and on peanuts.

Impacts
These studies will assist in identification of targets that will allow development of novel and effective strategies for reduction or elimination of aflatoxin if food and feed crops. Ethylene gas shows promise for this purpose preharvest and postharvest.

Publications

  • Lee, L-W., Chiou, C-H., Klomparens, K., Cary, J.W. and Linz, J.E. 2003. Sub-cellular localization of aflatoxin biosynthetic enzymes in time-dependent fractionated colonies of Aspergillus parasiticus. Archives of Microbiology (in press)
  • Miller, M.J. and Linz, J.E. 2003. Genetic mechanisms involved in regulation of mycotoxin biosynthesis. Invited Book Chapter, Food Biotechnology. (in press)
  • Yu, J., Chang, P-K., Cary, J.W., Bhatnagar, D., Cleveland, T.E., Payne, G.A., Linz, J.E., Woloshuk, C.P. and Bennett, J.W.2003. The clustered genes in aflatoxin biosynthesis. Appl. Environ. Microbiol. (in press)
  • Roze, L., Calvo-Byrd, A., Beaudry, R. and Linz, J.E. 2003. Ethylene modulates development and toxin synthesis in Aspergillus likely via an ethylene sensor-mediated signaling pathway. J. Food Protection. (in press)
  • Roze, L., Keller, N., Beaudry, R. and Linz, J.E. 2003. Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus. Mycopathologia. (in press)


Progress 01/01/02 to 12/31/02

Outputs
The filamentous fungus Aspergillus parasiticus produces aflatoxin contamination in food and feed crops in the US. Aflatoxins are potent toxins which generate acute and chronic disease symptoms in animal and humans. Our goal is to eliminate aflatoxins from the food chain. The short term goal is to understand the mechanisms which regulated aflatoxin symthesis and to use this knowledge to develop intervention strategiesin the field and during storage. We have now confirmed tnat two novel transcription factors, CREB and NLBP, bind to specific cis-acting sites and regulate expression of one gene in the aflatoxin pathway called nor-1. In addition we have shown that two aflatoxin proteins, OmtA and VBS, localize to specific organelles during aflatoxin synthesis on solid growth media.

Impacts
The identification of these regulators will allow the development of effective intervention strategies to block aflatoxin synthesis by the fungus in association with susceptible crops in the field and during storage.

Publications

  • Cary, J.W., Ehrlich, K.C, Wright, M.S., Liang, S-H., Linz, J.E. Bhatnagar, D. 2002. Molecular and functional characterization of a second copy of the aflatoxin pathway regulatory gene, aflR-2 from Aspergillus parasiticus. Biochim. Biophys. Acta. 1576:316-323.
  • Lee, L-W., Chiou, C-H, Cary, J. Linz, J.E. 2002. Function of native OmtA in vitro and expression/distribution of this protein in colonies of Aspergillus parasiticus. Appl. Environ. Microbiol 68:5718-5727.
  • Chiou, C-H, Miller, M. Wilson, D., Trail, F., Linz, J.E.. 2002. Chromosomal location affects expression of genes involved in aflatoxin biosynthesis in Aspergillus parasiticus. Appl. Environ. Microbiol. 68:306-315.


Progress 01/01/01 to 12/31/01

Outputs
The filamentous fungus Aspergillus parasiticus produces aflatoxin contamination in food and feed crops in the US. Aflatoxins are potent toxins which generate acute and chronic disease symptoms in animal and humans. Our goal is to eliminate aflatoxin from the food chain. The short-term goal is to understand the mechanisms which regulate aflatoxin synthesis and to use this knowledge to develop novel intervention strtegies in the field and during storage. We have tentatively identifed two novel transcription factors, one positive and one negative, that regulate expression of one aflatoxin pathway gene(nor-1). We also tentatively identified the DNA binding site for these factors and are working to confirm these results.

Impacts
The identification of regulators of aflatoxin synthesis will allow the development of effective intervention strategies to block aflatoxin synthesis by the fungus in association with succeptible crops in the field and during storage.

Publications

  • Chiou, C-H, Miller,M. Wilson, D. Trail, F. Linz, J.E. 2001. Chromosomal location affects expression of genes involved in aflatoxin biosynthesis in Aspergillus parasiticus. Appl. Environ. Microbiol. (in press).
  • Hitchman, T.S. Schmidt,E.W. Rarick, M.A., Trail,F. Linz, J.E. Townsend, and C.A. 2001. Cloning and nucleotide sequence analysis of the genes encoding hexanoate synthase, a specialized type I fatty acid synthase from aflatoxin B1 biosynthesis in Aspergillus parasiticus. Bioorganic Chemistry 29:293-307.


Progress 01/01/00 to 12/31/00

Outputs
The filamentous fungus Aspergillus parasiticus produces aflatoxin (AF) contamintation in food and feed crops in the US. AFs are potent toxins which generate acute and chronic disease symptoms in animals and humans. Our goal is to eliminate AF from the food chain. The short term goal is to understand the mechanisms which regulate AF synthesis and to use this knowledge to develop novel intervention strategies in the field and during storage. We have tentatively identified several novel positive and negative regulators of AF synthesis which bind specifically to promoters in one structural gene (nor-1) and one regulatory gene (aflR). We have also utilized gold labeled antibodies and transmission electron microscopy to tentatively localize one AF protein (VBS) to a large membrane bound subcellular organelle which appears in the toxin producing strain but is significantly reduced or absent in a aflR gene disruption mutant.

Impacts
The identification of regulators of AF sysnthesis will allow the development of effective intervention strategies to block AF synthesis by the fungus in association with susceptible crops in the field and during storage.

Publications

  • Zhou, R-Q. Rasooly,R. Linz, J.E. 2000. Isolation and analysis of fluP, a gene associated with hyphal growth and sporulation in Aspergillus parasiticus. Mol Gen Genet 264:514-520.
  • Annis, S. Velasquez,L. Xu, H. Hammerschmidt, R. Linz, J.E. Trail, F. 2000. A novel procedure for identification of compounds inhibitory to transcription of genes involved in mycotoxin biosynthesis. J. Food Ag. Chem. 48: 4656-4660.
  • Xu, H. Annis, S. Linz, J.E. Trail, F. 2000. Infection and colonization of peanut pods by Aspergillus parasiticus and the expression of aflatoxin biosynthetic genes in infection hyphae. Physiological and Molecular Plant Pathology 56:185-196.
  • Oatley, J.T. Rarick, M.D. Ji, G-E Linz, J.E. 2000. Binding of Aflatoxin B1 to Bifidobacteria in vitro. Journal of Food Protection. 63:1133-1136.


Progress 01/01/99 to 12/31/99

Outputs
The filamentous fungus A. parasiticus and related species frequently produce aflatoxin (AF) contamination in food and feed crops in the US. In animals, AF are hepatotoxic, mutagenic, immunotoxic, and are among the most potent naturally occurring carcinogens known. Our goal is to eliminate AF from the food chain. The short term goal is to understand the molecular mechanisms which regulate expression of key genes involved in AF biosynthesis. We developed polyclonal antibodies to 5 AF enzymes and one pathway regulator that are being used in immunolocalization experiments via laser scanning microscopy (LSM; FITC label) to identify the location of the AF proteins in fungal colonies. We modified procedures for transmission electron microscopy (TEM) and are using gold-labeled antibodies to localize AF proteins within individual cells. Promoter deletion constructs and gel mobility shift analysis have tentatively identified 2 "novel" DNA binding proteins which may regulate AF gene expression. One DNA binding protein appears to be a repressor. We have also initiated a project on the influence of signal transduction pathways (cAMP) on AF gene expression, developed a rapid assay for site of DNA intergration, and developed a rapid assay for extraction of chromosomal DNA for PCR analysis.

Impacts
Identification of a putative repressor of AF gene expression may eventually allow inhibition of toxin synthesis by the fungus on crops in the field or on seeds during storage. The ability to influence AF gene expression via signal transduction may allow inhibition of toxin synthesis without the need to get an inhibitor into fungal cells. The rapid assays are instrumental in molecular genetic analysis of AF pathway function.

Publications

  • Zhou, R. and J.E. Linz. 1999. Enzymatic function of the Nor-1 protein in aflatoxin biosynthesis in Aspergillus parasiticus. Appl. Environ. Microbiol. 65:5639-5641.
  • Cary, J., Bhatnagar, D., and Linz,and J.E. 1999. Aflatoxins: Biological Significance and Regulation of Biosynthesis. In:Microbial foodborne diseases: mechanism of pathogenesis and toxin synthesis. Pp. 317-362. Technomic Co. (USA).


Progress 01/01/98 to 12/31/98

Outputs
The filamentous fungi Aspergillus flavus, A. nomius and A. parasiticus frequently produce aflatoxin (AF) contamination in food and feed crops in the US. and in other areas of the world. In several animal species, AF are hepatotoxic, mutagenic, immunotoxic, and are among the most potent naturally occurring carcinogens known. The long term goal of this research is to eliminate AF from the food chain. The short term goal is to understand the molecular mechanisms which regulate the expression of key genes involved in the biosynthesis of AF. We have recently developed a series of polyclonal antibodies which we are using to identify the location of the AF proteins in colonies of the fungus growing on AF inducing growth media. We have also modified existing procedures to view fungal preparations via laser scanning microscopy (LSM) and transmission electron microscopy (TEM) for fluorescent or gold-labeled antibodies to aid in the immunolocalization project. We have also developed a series of plasmid vectors containing reporter constructs fused to AF promoters to study the effects of gene location on expression. Finally AF promoter deletion constructs have been developed to study the importance of specific nucleotide sequences in AF promoter function.

Impacts
(N/A)

Publications

  • No publications reported this period


Progress 01/01/97 to 12/31/97

Outputs
The filamentous fungi Aspergillus flavus, A. nomius and A. parasiticus frequently produce aflatoxin contamination in food and feed crops in the US and in other areas of the world. In several animal species, aflatoxins are hepatotoxic, mutagenic, immunotoxic, and is one of the most potent naturally occurring carcinogens known. The long term goal of this research is to eliminate aflatoxin B1 (AFB1) from the food chain. The short term goal is to understand the molecular mechanisms which regulate the expression of key genes involved in the biosynthesis of AFB1. The ver-1A gene is a gene of immediate of interest in this regard. Previous studies have shown that ver-1A encodes an enzyme which is directly involved in aflatoxin biosynthesis. In this study two different tools, Ver-1 polyclonal antibodies and a GUS (uidA; -glucuronidase) reporter system were utilized to study the regulation of ver-1A expression. The observed temporal pattern of expression of GUS transcript, GUS protein, and GUS activity in transformants suggested that transcriptional regulation plays a major role in regulating ver-1A expression. Integration of the ver-1A/GUS reporter construct at the niaD locus resulted in 500 fold lower levels of expression than when the construct integrated at the ver-1A locus. These data suggest that (a) the location of a gene within the aflatoxin gene cluster may play a role in determining the level of its expression; and (b) there may therefore be two separate components regulating ver-1A expression; one regulates the timing of expression and the other regulates the level of expression.

Impacts
(N/A)

Publications

  • Liang, S-H., T-S. Wu, R. Lee, F.S. Chu, and J.E. Linz. 1997. Analysis of the mechanisms regulating the expression of the ver-1 gene involved in aflatoxin biosynthesis. Appl. Environ. Microbiol. 63:1058-1065.