Source: UNIV OF WISCONSIN submitted to NRP
FUNGAL PATHOGENS
Sponsoring Institution
State Agricultural Experiment Station
Project Status
COMPLETE
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0207402
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2005
Project End Date
Sep 30, 2013
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
PLANT PATHOLOGY
Non Technical Summary
Fungi are potent pathogens of plants, animals and humans. Many fungi produce toxins that injure or even kill the host. We are designing strategies to find means to inhibit toxin production by fungi.
Animal Health Component
10%
Research Effort Categories
Basic
75%
Applied
10%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21215991102100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1599 - Grain crops, general/other;

Field Of Science
1102 - Mycology;
Goals / Objectives
The work in my lab is aimed towards identifying and characterizing attributes of fungi that make them pathogens. I focus on identifying the genes and processes responsible for the production of fungal toxins and fungal spores. Toxins contribute to plant, animal and human disease. Fungal spores are the inoculum of fungi and start the disease process by landing and germination on a host. My laboratory research is aimed and understanding the molecular genetics behind both processes and, ultimately, designing control strategies based on our understanding of fungal biology.
Project Methods
A. Genetics. We identify genes involved in fungal toxin production, fungal development and disrupt or over express these genes to examine the role of the gene and its encoding protein. B. Physiological tests. We characterize above mutants with regard toxin production, sporulation, growth rate and virulence. C. Biochemistry. We often characterize activities of proteins important in the fungal virulence (e.g. enzymatic activities, substrate specificity, etc).

Progress 10/01/05 to 09/30/13

Outputs
OUTPUTS: Our laboratory addresses how fungi operate as pathogens and natural product producers. We have had three main thrusts this past year: 1). Understanding oxylipin signaling in the A. flavus/corn interaction and how this plays a role in pathogenicity and aflatoxin production, 2). How the protein LaeA regulates secondary metabolism in pathogenic fungi including Aspergillus and Fusarium species,and 3). How to activate silent secondary metabolite gene clusters in fungi and examine the potential of such metabolites as anti-microbials. Advances in these three areas have been reported as publications or in meetings as posters by my students and post-doctoral scientists. PARTICIPANTS: Training: students (undergrads, grads and post docs): Jon Palmer, Jeff Theisen, Mona Shabaan, Keat Shwab, Taylor Dagenais, Steve Giles; Collaborators: Clay Wang, Berl Oakley, Christina Hulll, Gary Payne, Natalie Fedorova, J Yu, Charlie Woloshuk, Bettina Tudzynski, Se el Habib TARGET AUDIENCES: The publications were directed towards the scientific community and as were the presentations. Some presentations were intended for a more lay community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Key outcomes from the above studies include our finding that: 1). THe laeA velvet complex is conserved in Fusarium species and regulates mycotoxins and sporulation in that genus as well as Aspergillus. 2). We have identified several gene clusters in A. nidulans including the orsellinic acid cluster, the F9775 cluster (a potential anti osteoporosis drug), the mondictyphenone cluster (with antifungal properties). 3). We have identified a BziP protein that remediates Velvet complex loss. 4). We have found that transposons contribute to secondary metabolite cluster regulation. 5). We have identified LaeA regulation of hydrophobins which impacts macrophage phagocytosis of aspergillus spores. 6). We have identified chromatin remodeling histone marks as important in secondary metabolite cluster regulation.

Publications

  • Reyes-Dominguez Y, Bok JW, Berger H, Shwab E, Gallmetzer A, Scazzocchio C, Keller NP, Strauss J. (2010) Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans.. Mol Microbiol 76(6):1376-86.
  • Wiemann P, Brown DW, Kleigrewe K, Bok JW, Keller NP, Humpf HU, Tudzynski B.(2010) FfVel1 and FfLae1, components of a velvet-like complex in Fusarium fujikuroi, affect differentiation, secondary metabolism and virulence. Mol Microbiol. 2010 Jun 21. [Epub ahead of print] Sanchez JF, Chiang YM, Szewczyk E, Davidson AD, Ahuja M, Elizabeth Oakley C, Woo Bok J, Keller N, Oakley BR, Wang CC 2010. Molecular genetic analysis of the orsellinic acid/F9775 gene cluster of Aspergillus nidulans.Mol Biosyst. 2010 Mar;6(3):587-93.
  • Shaaban MI, Bok JW, Lauer C, Keller NP (2010) Suppressor Mutagenesis Identifies a Velvet Complex Remediator of Aspergillus nidulans Secondary Metabolism. Eukaryot Cell. 2010 Dec;9(12):1816-24.
  • Shaaban M, Palmer JM, El-Naggar WA, El-Sokkary MA, Habib el-SE, Keller NP. (2010) Involvement of transposon-like elements in penicillin gene cluster regulation. Fungal Genet Biol. 2010 May;47(5):423-32.
  • Chiang YM, Szewczyk E, Davidson AD, Entwistle R, Keller NP, Wang CC, Oakley BR. 2010 Characterization of the Aspergillus nidulans monodictyphenone gene cluster. Appl Environ Microbiol. 2010 Apr;76(7):2067-74.
  • Palmer JM, Mallaredy S, Perry DW, Sanchez JF, Theisen JM, Szewczyk E, Oakley BR, Wang CC, Keller NP, Mirabito PM. (2010) Telomere position effect is regulated by heterochromatin-associated proteins and NkuA in Aspergillus nidulans.Microbiology. 2010 Dec;156(Pt 12):3522-31.
  • Georgianna DR, Fedorova ND, Burroughs JL, Dolezal AL, Bok JW, Horowitz-Brown S, Woloshuk CP, Yu J, Keller NP, Payne GA. 2010. Beyond aflatoxin: four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters. Mol Plant Pathol. 2010 Mar 1;11(2):213-26.


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

Outputs
OUTPUTS: We are starting an investigation of quorum signal interactions between Aspergillus and other pathogens found in same environments. We have found that LaeA mutant of the pathogen A. fumigatus is deficient in hydrophobin content leading to increased macrophage ingestion. We continue our work on the role of heterochromatin in secondary metabolite (toxins and pharmaceuticals) biosynthesis in Aspergillus nidulans and A. fumigatus. We have uncovered a cellular partnership between LaeA and VeA in the nucleus of Aspergillus. We collaborate with other researchers on identifying virulence factors in human pathogenic fungi. PARTICIPANTS: Graduate students trained: Kelsea Jewell, Jon Palmer, Ali Soukup, Yun Lim. Post docs trained: Graeme Garvey, Steve Giles, Taylor Dagenais. Senior scientist: JInWoo Bok. Technician: Carrie Lauer TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our research has spurred other researchers worldwide to collaborate with us on oxylipin and laeA research that have increased our outputs. We have developed new methodologies leading to outputs. We have a patent disclosure on various aspects of the laeA project.

Publications

  • Chiang Y-M, Lee K H, Sanchez JF, Keller NP, Wang CCC (2009) Unlocking fungal cryptic natural products. Nat Prod. Comm. 4: 1505-10.
  • Taylor R. T. Dagenais, Steve S. Giles, Vishukumar Aimanianda, Jean-Paul Latge, Christina M. Hull1, Nancy P. Keller(2009) Aspergillus fumigatus LaeA-mediated phagocytosis is associated with a decreased hydrophobin layer. Infect Immun. 2009 Nov 16. [Epub ahead of print]
  • Dagenais TR, Keller NP.Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis.Clin Microbiol Rev. 2009 Jul;22(3):447-65. Review.
  • Wortman JR, Gilsenan JM, Joardar V, Deegan J, Clutterbuck J, Andersen MR, Archer D, Bencina M, Braus G, Coutinho P, von Doehren H, Doonan J, Driessen AJ, Durek P, Espeso E, Fekete E, Flipphi M, Estrada CG, Geysens S, Goldman G, de Groot PW, Hansen K, Harris SD, Heinekamp T, Helmstaedt K, Henrissat B, Hofmann G, Homan T, Horio T, Horiuchi H, James S, Jones M, Karaffa L, Karanyi Z, Kato M, Keller N, Kelly DE, Kiel JA, Kim JM, van der Klei IJ, Klis FM, Kovalchuk A, Krasevec N, Kubicek CP, Liu B, Maccabe A, Meyer V, Mirabito P, Miskei M, Mos M, Mullins J, Nelson DR, Nielsen J, Oakley BR, Osmani SA, Pakula T, Paszewski A, Paulsen I, Pilsyk S, Pocsi I, Punt PJ, Ram AF, Ren Q, Robellet X, Robson G, Seiboth B, van Solingen P, Specht T, Sun J, Taheri-Talesh N, Takeshita N, Ussery D, vanKuyk PA, Visser H, van de Vondervoort PJ, de Vries RP, Walton J, Xiang X, Xiong Y, Zeng AP, Brandt BW, Cornell MJ, van den Hondel CA, Visser J, Oliver SG, Turner G (2009). The 2008 update of the Aspergillus nidulans genome annotation: a community effort. Fungal Genet Biol.46 Suppl 1:S2-13.
  • Bok JW, Keller NP, Tsitsigiannis DI. (2009) Real-time and semiquantitative RT-PCR methods to analyze gene expression patterns during Aspergillus-host interactions. Methods Mol Biol. 2009;470:151-67.
  • Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis. Dagenais TR, Keller NP. Clin Microbiol Rev. 2009 Jul;22(3):447-65.
  • Horowitz Brown S, Scott BJ, Bhaheetharan J, Sharpee WC, Milde L, Wilson RA, Keller NP. (2009) Oxygenase coordination is required for morphological transition and the host/fungal interaction of Aspergillus flavus. Mol Plant Microbe Interactions: 22:882-94
  • Giles SS, Dagenais TR, Botts MR, Keller NP, Hull CM. (2009) Elucidating the pathogenesis of spores from the human fungal pathogen Cryptococcus neoformans. Infect Immun. 77:3491-500
  • Chiang YM, Szewczyk E, Davidson AD, Keller N, Oakley BR, Wang CC. (2009) A gene cluster containing two fungal polyketide synthases encodes the biosynthetic pathway for a polyketide, asperfuranone, in Aspergillus nidulans. JACS 131:3148-3149
  • Bok JW, Chiang Y-M, Szewczyk E, Reyes-Domingez Y, Davidson AD, Sanchez JF, Lo H-C, Watanabe K, Strauss J, Oakley BR, Wang CCC, Keller NP. (2009) Chromatin- regulation of biosynthetic gene clusters. Nat Chem Biol 5:462-464
  • Palmer J, Perrin R, Dagenais T, Keller NP (2008) H3K9 methylation regulates growth and development in Aspergillus fumigatus Euk Cell 7:2052-60


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

Outputs
OUTPUTS: We have identified several oxygenase genes in Aspergillus flavus that are required for aflatoxin production and control the regulation of density dependent morphological development in this fungus. One paper has been published on this and one submitted. We have characterized an interaction between lipoxygenase mutants of maize and susceptibility to Aspergillus. We also have found that oxygenase mutants in A. fumigatus are abberant in macrophage uptake. We continue our work on the role of heterochromatin in secondary metabolite (toxins and pharmaceuticals) biosynthesis in Aspergillus nidulans and A. fumigatus. We have uncovered a cellular partnership between LaeA and VeA in the nucleus of Aspergillus and defined the role of RNAi machinery in Aspergillus/virus interactions. PARTICIPANTS: JinWoo Bok, Taylor Dagenais, Sigal Horowitz, Inhyung Lee, Marisa Trapp, Jeehwan Oh, Mona Shaaban, Jon Palmer. Jon is a PhD student trained during this period. Drs. Dagenais and Horowitz are post docs being trained. Dr. Lee is visiting scientist from Korea. Ms. Shaaban is a visiting PhD student from Egypt. Mr. Oh is a visiting PhD student from South Korea. We collaborate with groups in Denmark, Germany, Oklahoma, TIGR and Australia . TARGET AUDIENCES: Agricultural and health sectors of the country impacted by mycotoxin contamination. Health sectors impacted by aspergillosis. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our research has spurred other researchers worldwide to collaborate with us on oxylipin and laeA research that have increased our outputs. We have developed new methodologies leading to outputs. We have a patent disclosure on various aspects of the laeA project.

Publications

  • Gao X, Brodhagen M, Isakeit T, Horowitz Brown S, Goebel C, Betran J, Feussner I, Keller NP, Kolomiets MV (2008) Inactivation of the lipoxygenase ZmLOX3 increases susceptibility of maize to Aspergillus spp. Mol Plant Microbe Interactions 22:222-231
  • Palmer J, Perrin R, Dagenais T, Keller NP (2008) H3K9 methylation regulates growth and development in Aspergillus fumigatus Euk Cell in press.
  • Horowitz Brown S, Zarnowski R, Sharpee WC, Keller NP. (2008) Morphological transitions governed by density dependence and lipoxygenase activity in Aspergillus flavus. Appl Environ Microbiol. 74:5674-85.
  • Kale S, Milde L, Trapp M, Frisvad J, Keller NP, Bok JW. (2008) Requirement of LaeA for Secondary Metabolism and Sclerotial Production in Aspergillus flavus. Fungal Genetics Biology: 2008 Jul 11. [Epub ahead of print]
  • Dagenais T, Chung D, Giles S, Hull, C, Andes D, Keller NP. (2008) Abberancies in conidiophore development and conidial/macrophage interactions in a dioxygenase mutant of Aspergillus fumigatus. Infect Immun 76:3214-20.
  • Bayram O, Krappmann S, Ni M, Bok JW, Helmstaedt K, Valerius O, Braus-Stromeyer S, Kwon NJ, Keller NP, Yu JH, Braus GH. (2008) VelB/VeA/LaeA coordinate light information, fungal development and secondary metabolism. Science 320:1504-6
  • Hammond T M, Andrewski MD, Roossinck M, Keller NP. (2008) Aspergillus mycoviruses are targets and suppressors of RNA silencing. Euk Cell 2007 7:350-7
  • Hammond T M, Bok J-W, Andrewski MD, Reyes-Dominguez Y, Scazzocchio C, Keller NP. (2008) RNA silencing gene truncation in the filamentous fungus Aspergillus nidulans. Euk Cell 7:339-49 Brodhagen M, Tsitsigiannis D, Hornung E, Goebel C, Feussner I, Keller NP. (2008) Reciprocal oxylipin-mediated cross talk in the Aspergillus/seed pathosystem. Mol Microbiol 67:378-391


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

Outputs
OUTPUTS: We have previously cloned and characterized three Aspergillus nidulans oxygenases required for production of oxidized C18 fatty acids (e.g. oxylipins) that serve as hormone-like signals that modulate the timing and balance between sexual and asexual spore development as well as pathogenicity on seed. Recent work has identified similar genes in the pathogens A. flavus and A. fumigatus. We have also identified an A. flavus lipoxygenase important for sclerotial formation and aflatoxin production. We have shown that plant oxygenases remediate Aspergillus null mutants. We have preliminary biochemical data that LaeA methylates a nuclear protein that may be involved in chromatin re-organization in the nucleus. We have found that loss of histone deacetylases could remediate laeA null strains. This mechanism is preserved in A. fumigatus. We have started investigating the interaction of LaeA and VeA in A. flavus. We are looking at conserved mycotoxin regulation mechanisms in Fusarium spp. PARTICIPANTS: JinWoo Bok, Taylor Dagenais, Sigal Horowitz, Inhyung Lee, Marisa Trapp, Jon Palmer. Jon is PhD student trained during this period. Drs. Dagenais and Horowitz are post docs being trained. Dr. Lee is visiting scientist from Korea. We collaborate with groups in Sweden, Germany, New Orleans, TIGR and Australia as shown in our publications. TARGET AUDIENCES: Agricultural and health sectors of the country impacted by mycotoxin contamination. Health sectors impacted by aspergillosis.

Impacts
Our research has spurred other researchers worldwide to collaborate with us on oxylipin and laeA research that have increased our outputs. We have developed new methodologies leading to outputs. We have a patent disclosure on various aspects of the laeA project.

Publications

  • Brodhagen M, Tsitsigiannis D, Hornung E, Goebel C, Feussner I, Keller NP. (2008) Reciprocal oxylipin-mediated cross talk in the Aspergillus/seed pathosystem. Mol Microbiol 67:378-391
  • Garscha U, Jerneren F, Chung D, Keller NP, Hamberg M, Oliw EH. (2007) Effects of gene targeting on oxylipin biosynthesis by Aspergillus spp. and thei reaction mechanism of linoleate 5,8- and 8,00-diol synthases and 10R-dioxygenases. J Biol Chem 282:34707-18.
  • Fox E M, Gardiner DM, Keller NP, Howlett BJ (2008) A Zn(II)2Cys6 DNA binding protein regulates the sirodesmin PL biosynthetic gene cluster in Leptosphaeria maculans. Fungal Genetics Biology Oct 17; [Epub ahead of print]
  • Rohlfs M, Albert M, Keller NP, Kempken F. (2007) Secondary chemicals protect mould from fungivory. Biol Lett. 2007 3:523-5
  • Maggio-Hall L, Lyne P, Wolff J, Keller NP. (2007) A single acyl-CoA dehydrogenase is required for catabolism of isoleucine, valine and short-chain fatty acids in Aspergillus nidulans. Fungal Genet Biol Jun 21; [Epub ahead of print]
  • Perrin RM, Fedorova ND, Bok JW, Cramer RA, Wortman JR, Kim HS, Nierman WC, Keller NP. (2007) Transcriptional regulation of chemical diversity in Aspergillus fumigatus by LaeA. PloS Pathogens Apr;3(4):e50.
  • Stadler M, Keller NP (2008) Paradigm shifts in fungal secondary metabolite research. Mycologia in press


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

Outputs
1. Progress in identifying oxylipin signals for Aspergillus sporulation and secondary metabolism. We have previously cloned and characterized three Aspergillus nidulans oxygenases required for production of oxidized C18 fatty acids (e.g. oxylipins) that serve as hormone-like signals that modulate the timing and balance between sexual and asexual spore development as well as pathogenicity on seed. Recent work has identified similar genes in the pathogens A. flavus and A. fumigatus; these are being investigated for their role in fungal development and pathogenicity. These oxylipins are involved in a sophisticated lipid mediated signaling communication process operative in fungal:fungal communication and fungal:seed (or fungal:human) communication. 2. Mechanism and virulence of LaeA, a global regulator of toxin production. LaeA encodes a protein methyltransferase involved in global regulation of secondary metabolism gene clusters. This gene is conserved in all Aspergilli and is a virulence factor in the human pathogen A. fumgiatus and plant pathogen A. flavus. Recently Bok et al. showed that some, but not all, of the decrease of virulence in a laeA mutant is due to a decrease in production of the apoptotic factor, gliotoxin. Aside from understanding the mechanism by which LaeA works and its role in virulence, we have also shown that LaeA is an excellent tool for drug discovery as published in the Jan 2006 issue of Chem & Biol and reported by UW student news at http://www.news.wisc.edu/12053.html.

Impacts
Fungi are devastating pathogens of plants and animals including humans. Our lab focuses on understanding how fungal pathogens cause disease on both plants and animals. Results from our studies are expected to be applied towards strategies to minimize fungal diseases.

Publications

  • Bouhired S, Weber M, Kempf-Sontag A, Keller NP, Hoffmeister D.(2007) Accurate prediction of Aspergillus natural product gene cluster boundaries using the transcriptional regulator LaeA. Fung Genet Biology. In press
  • Keller NP, Bok JW, Chung DW, Perrin RM, Shwab K (2006) LaeA, a global regulator of Aspergillus toxins. Med Mycol 44:583-585.
  • Bok JW, Chung DW, Balajee S A, Marr K A, Andes D, Fog Nielsen K. Frisvad J C. Kirby K, Keller N P (2006) GliZ, a transcriptional regulator of gliotoxin biosynthesis, contributes to Aspergillus fumigatus virulence. Infect Immun 74:6761-6768
  • Bok JW, Noordermeer D, Kale S P, Keller NP (2006) Secondary metabolic gene cluster silencing in Aspergillus nidulans. Mol Microbiol 61:1636-1645
  • Tsitsigiannis D I, Keller N P (2006) Oxylipins act as determinants of natural product biosynthesis and seed colonization in Aspergillus nidulans. Mol Microbiol 59:882-892
  • Bok J-W, Hoffmeister D, Maggio-Hall L, Murillo R, Keller NP. (2006) Genomic mining for Aspergillus natural products. Chemistry & Biology 13:31-27.
  • Tsitsigiannis D I, Keller NP (2007) Oxylipins as developmental and host-fungal communication signals. Trends in Microbiology. In press.
  • Hoffmeister D, Keller NP (2007) Natural products of filamentous fungi: enzymes, genes, and their regulation. Natural Products Reports. In press.
  • Brodhagen M, Keller NP (2006) Signaling pathways connecting mycotoxin production and sporulation. Molecular Plant Pathology 7:285-301.