Source: UNIVERSITY OF FLORIDA submitted to NRP
MOLECULAR MECHANISMS OF BEAUVERIA BASSIANA ENTOMOPATHOGENICITY
Sponsoring Institution
State Agricultural Experiment Station
Project Status
COMPLETE
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0202101
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2004
Project End Date
Sep 30, 2006
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
MICROBIOLOGY & CELL SCIENCE
Non Technical Summary
Beauveria bassiana is an opportunistic fungal pathogen that has evolved a spectrum of strategies for the successful infection and degradation of a broad range of arthropod hosts. Arthropods are by far the most numerous species within the animal kingdom and fungi are responsible for a major portion of the recycling of the carbon and nitrogen contained in their bodies. Entomopathogenic fungi are able to infect living insects, and scientists since Pasteur have suggested their use for controlling a variety of agricultural, invasive, and nuisance pests. Aside from their use in biocontrol regimes, Beauveria sp are the second most widely used whole cell eukaryotic biocatalysts, and are responsible for a range of biological transformations and remediations that remain elusive to chemical and synthetic methods. Nonetheless, the molecular mechanisms of fungal entomopathogenicity are largely unknown. The outcomes of this project will yield in depth transcriptsome analyses of B. bassiana, providing a substantial sequence database for molecular characterization and gene discovery. Information derived from this project has the potential for expanding the boundaries of our current knowledge concerning fungal physiology, biochemistry, and cell biology.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2154020100010%
2154020104015%
2154020108015%
7213120116020%
7214020100010%
7214020104015%
7214020108015%
Knowledge Area
215 - Biological Control of Pests Affecting Plants; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
3120 - Spiders, mites, ticks, and other arthropods; 4020 - Fungi;

Field Of Science
1040 - Molecular biology; 1160 - Pathology; 1000 - Biochemistry and biophysics; 1080 - Genetics;
Goals / Objectives
Beauveria bassiana is a widely distributed fungal arthropod pathogen that holds considerable potential for insect biocontrol. In addition, Beauveria sp. are the second most widely used whole cell fungal biocatalysts. These biotechnological applications cover a range of biochemical transformations and remediation reactions. Despite this interest, few of the genes (and their protein products) involved in these critical biological processes have been characterized. In this project, we propose a transcriptsome analysis of Beauveria bassiana. In order to maximize the diversity of the transcripts, and increase the likelihood of novel gene discovery, we have constructed three cDNA libraries from distinct cell phenotypes of B. bassiana generated under different physiological conditions. These include (1) sporulating mycelial cells (conidia harvested from agar plates), (2) B. bassiana cells germinating on insect cuticle, and (3) in vivo produced hyphal bodies isolated from the hemolymph of infected insects. The specific aims of this project are: (1) To generate a robust dataset (transcriptsome analyses) of B. bassiana cell phenotypes. To date, we have sequenced 96 and 192 clones from the insect cuticle induced and the in vivo generated cDNA libraries, respectively. These data (1) confirm the integrity and representative nature of the libraries and (2) will allow us to proceed with the sequencing immediately upon funding. We propose to perform approximately 20,000 total sequence reads covering the three cDNA libraries. (2) Analyze, annotate, and disseminate the data. A series of specialized tools have been developed for analysis, annotation, comparison, and presentation of the data. (3) To enhance the teaching curriculum by exposing students to genomics-bioinformatics. Data generated from the research mission will be used to provide a hands-on learning tool (using primary scientific data) in an advanced genetics class currently taught by the PI as well as a Workshop to be developed.
Project Methods
Construction of cDNA Libraries. We have constructed three B. bassiana cDNA libraries using mRNA from (1) condia harvested from Potato Dextrose agar, (2) fungal cells grown on insect cuticles, and (3) fungal cells isolated from the hemolymph of infected insects (S. exigua). Both in vitro libraries were constructed using the OrientExpress Oligo(dT) cDNA cloning system and the lambda-screen-1 cloning vector according to the manufacturer's recommendations. (Novagen, Madison, WI). The tobacco hornworm Manduca sexta, accessed from laboratory colonies maintained at North Carolina State University, were used as hosts to propagate the in vivo cell phenotype of B. bassiana. Larvae were reared on artificial diet and at 25oC until they reached the 5th larval instar. Cohorts of fifth instar larvae were immobilized on ice and challenged by injection of blastospores into the hemocoel, as previously described (Hung & Boucias, 1992).

Progress 10/01/04 to 09/30/06

Outputs
OUTPUTS: Our overall goal is to understand the underlying molecular mechanisms of fungal pathogenesis of arthropods. Our model system utilizes the entomopthogenic fungus Beauveria bassiana, an economically important fungal pathogen of arthropods, which holds tremendous potential as a biological control agent. Indeed, B bassiana-insect interactions were used to develop the "germ theory" of disease, representing one of the first modern models of microbial based pathogenesis. Our goal was to begin to establish the genetic basis of B. bassiana pathogenesis. The major ouputs dervied from the granting period include:(1) The establishment of a data annotation and dissemination pipeline that can readily be expanded to ongoing efforts to obtain and characterize the genomic sequence of Beauveria bassiana. (2) Construction of a WEB based data platform for use by the scientific community that includes the full EST dataset in a searchable format with gene ontology categorizations. (3) Facilitation (decreases in error rates) of gene prediction models. The establishment of the EST database has increased the accuracy of gene calls by providing transcript based information for comparisons. PARTICIPANTS: Dr. Nemat O. Keyhani (PI) Dr. W. Farmerie (UF ICBR) Dr. Eun-Min Cho (Postdoctoral student) Dr. L. Liu (UF ICBR) Dr. D. Boucias (UF, Dept. of Entomology and Nematology) Dr. S.W. Huang (Collaborating professor, UF Medical School) Diane Holder (PhD student) Greg Westwood (PhD student) Brett Kirkland (PhD student) A. Eisa (undergraduate student) R. Teng (undergraduate student) M. Chen (undergraduate student) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The project has led to our ongoing genome sequencing efforts by helping to develop the necessary bioinformatics platform for annotation analysis and information dissemination. In addition, infomration generated from the project, namely gene expression data has contributed to our understanding diverse fields such as gaining insights into the array of depolymerases including cell wall degrading enzymes, proteases, glycosidases, phosphatases, and DNAses produced by the fungus. Impacts on changes in knowledge include the development of technologies for genetic transformation, gene manipulation and targeting, and gene identification in B. bassiana. The project has formed the basis for novel future directions of research and is being used by other laboratories world-wide in characterizing important genes involved in fungal development and pathogenesis.

Publications

  • Cho, E.-M., Boucias, D., and Keyhani, N.O. (2006) EST analysis of cDNA libraries from the entomophthogenic fungus Beauveria (Cordyceps) bassiana: II: fungal cells sporulating on chitin and producing oosporein. Microbiology 152: 2855-2864
  • Cho, E.-M., Liu, L., Farmerie, W., and Keyhani, N.O. (2006) EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana: I: Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores, and submerged conidia. Microbiology 152: 2843-2855
  • Westwood, G.S, Huang, S-H, and Keyhani, N.O. (2006) Molecular and immunological characterization of allergens from the entomopathogenic fungus Beauveria bassiana, Clinical and Molecular Allergy 4:12
  • Westwood, G.S., Huang, S-W., and Keyhani, N.O. (2005) Allergens of the entomopathogenic fungus Beauveria bassiana, Clinical and Molecular Allergy, 3:1.
  • Kirkland, B.H., Eisa, A., and Keyhani, N.O. (2005) Oxalic acid as a fungal acaracide virulence factor. J. Med. Entomol. 42: 346-351.
  • Holder, D.H. and Keyhani, N.O. (2005) Adhesion of Beauveria (Cordyceps) bassiana to substrata. Applied and Environmental Microbiology 71:5260-5266.


Progress 10/01/05 to 09/30/06

Outputs
Beauveria bassiana is an opportunistic fungal pathogen that has evolved a spectrum of strategies for the successful infection and degradation of a broad range of arthropod hosts. Arthropods are by far the most numerous species within the animal kingdom and fungi are responsible for a major portion of the recycling of the carbon and nitrogen contained in their bodies. Entomopathogenic fungi are able to infect living insects, and scientists since Pasteur have suggested their use for controlling a variety of agricultural, invasive, and nuisance pests. Aside from their use in biocontrol regimes, Beauveria sp are the second most widely used whole cell eukaryotic biocatalysts, and are responsible for a range of biological transformations and remediations that remain elusive to chemical and synthetic methods. Nonetheless, the molecular mechanisms of fungal entomopathogenicity are largely unknown. Published results from this project has yielded an in depth transcriptsome analyses of B. bassiana, and has provided a substantial sequence database for molecular characterization and gene discovery. Information derived from this project has the potential for expanding the boundaries of our current knowledge concerning fungal physiology, biochemistry, and cell biology. Studies are progressing on genomic, natural products, and strain improvement dealing with B. bassiana and other entomopathogenic fungi.

Impacts
Results derived from our research can be expected to have an important impact on the wide variety of biological control applications of entomopathogenic fungi, provide a framework for comparative genomics and bioinformatics, and will contribute to deciphering the fundamental biology underlying growth, development, and pathogenesis of entomopathogenic fungi.

Publications

  • Cho, E.-M., Liu, L., Farmerie, W., and Keyhani, N.O. (2006) EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana: I: Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores, and submerged conidia. Microbiology 152: 2843-2855
  • Cho, E.-M., Boucias, D., and Keyhani, N.O. (2006) EST analysis of cDNA libraries from the entomophthogenic fungus Beauveria (Cordyceps) bassiana: II: fungal cells sporulating on chitin and producing oosporein. Microbiology 152: 2855-2864
  • Westwood, G.S, Huang, S-H, and Keyhani, N.O. (2006) Molecular and immunological characterization of allergens from the entomopathogenic fungus Beauveria bassiana, Clinical and Molecular Allergy 4:12


Progress 10/01/04 to 09/30/05

Outputs
Our research seeks to investigate the key biochemical and genetic determinants involved in attachment, signaling in response to surface cues, and toxin production in the entomopathogenic fungus Beauveria bassiana during the infection and degradation of several arthrod hosts as model systems. In the past year, the laboratory has studied the role of fungal metabolites, in particular oxalate, as a fungal pathogenicity factor towards ticks (Acari:Ixodidae). This work has recently been publsihed in the Journal of Medical Entomology. Two additional papers have also been published in the past dealing with antigen characterization and adhesion of Beauveria bassiana. Progress on the characterization of expressed sequence tags (ESTs) derived from discrete fungal developmental stages has also been made and over 12,000 clones from five different B. bassiana cDNA libraries have been sequenced and analyzed. The results from this effort are now being written for publication and it is anticipated that a B. bassiana EST database curated by the PI will be available to the scientific community in 2006.

Impacts
Results derived from our research can be expected to have an important impact on the wide variety of biological control applications of entomopathogenic fungi, provide a framework for comparative genomics and bioinformatics, and will contribute to deciphering the fundamental biology underlying growth, development, and pathogenesis of entomopathogenic fungi.

Publications

  • Westwood, G.S., Huang, S-W., and Keyhani, N.O. (2005) Allergens of the entomopathogenic fungus Beauveria bassiana, Clinical and Molecular Allergy, 3:1.
  • Kirkland, B.H., Eisa, A., and Keyhani, N.O. (2005) Oxalic acid as a fungal acaracide virulence factor. J. Med. Entomol. 42: 346-351.
  • Holder, D.H. and Keyhani, N.O. (2005) Adhesion of Beauveria (Cordyceps) bassiana to substrata. Applied and Environmental Microbiology 71:5260-5266.