new mexico state university comparative analysis of fungal glyoxylate cycle promoters ian l....
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New Mexico State University
Comparative analysis of Fungal Glyoxylate cycle
Promoters Ian L. Gonzales, Jennifer C. Fleming, Jorge Gil C. Angeles,
Punsiri M. Colonne, and Peter J. Lammers
New Mexico State University
• Mycorrhizal fungi colonize over 85% of plants– G. Intraradices is an arbuscular mycorrhizal fungi
• Fungal mycelia form a network that connects plant roots with an extended volume of soil
• Fungi exchange water, N, P and other nutrients with plants for carbon in the form of hexose sugars
• Colonized plants have increased abiotic stress and pathogen resistance
• Fungi move carbon from roots to distal soil hyphae as triglyceride (fat) and then convert the fat back to carbohydrate via the glyoxylate cycle enzymes
• This study was designed to analyze regulation of glyoxylate cycle gene expression
Glomus Intraradices
New Mexico State University
The Glyoxylate Cycle…
• Photosynthetic sugars are converted into triacylglycerols (fats) then transported to extra-radical mycelia (ERM) and developing spores.
• The glyoxylate cycle utilizes the Isocitrate Lyase (ICL) and Malate Synthase (MS) for the conversion of fat back to carbohydrates in ERM and germinating spores
New Mexico State University
Difficulties in working with AM Fungi
• G. intraradices is the model AM fungus (genome project in progress at Joint Genome Institute)
• but….it is a difficult organism to work with– Grows slowly in laboratory (minimum 12 weeks from germinating spore to next
generation spores)– Obligate symbiont grown with Ri-transformed “hairy” carrot root cultures– Non-transformable (transient gene expression assays published in Febrary 2008)
• Therefore we used Laccaria bicolor
New Mexico State University
Laccaria Bicolor
• Ectomychorrhizal fungi• Shares similar sybiotic functionality as Glomus.
– i.e. Nutrient uptake, Glyoxylate cycle, stress tolerance, pathogen resistance…
• Model fungal system– Well studied fungal system compare to Glomus– Straightforward culturing procedures– Genome sequence available for bioinformatics analysis– Transformation is possible using Agrobacterium tumefaciens T-DNA
transfer
• This study was designed to analyze regulation of glyoxylate cycle gene expression in transgenic L. bicolor, an ectomycorrhizal fungus
New Mexico State University
Is Glyoxylate cycle regulation conserved in ECM and AM Fungi?
• Ectomycorrhizal fungi (40 MY) colonize perennial trees and shrubs and Arbuscular mycorhizal (400 MY) fungi are more widespread colonize most land plants
• Hypothesis: regulation of the glyoxylate cycle gene promoters is conserved in ECM and AM fungi
• Test: promoter::Green Fluorescence Protein Fusion constructs introduced in the ECM fungus Laccaria bicolor
New Mexico State University
How to study ICL and MS?
• ICL and MS genes are negatively regulated when glucose is abundant and upregulated when glucose is scarce but acetate or short chain fatty acids are present.
• Make promoter deletions of ICL and MS genes of Glomus and Laccaria bicolor fused with eGFP
• eGFP is our reporter protein used to account for ICL or MS up regulation during exposure to glucose or oleic acid– We should see higher fluoresce if genes are activated– Quantify with fluorimetry
New Mexico State University
Fusion protein Constructs
• Employing common PCR, endonuclease, and cloning techniques, we were able to clone our full length promoters for G. intradices ICL and MS into our pBGgHg vector, as well as our full length L. bicolor ICL and MS promoters.
New Mexico State University
The Promoters
279 bp
L. bicolor Isocitrate Lyase
1126 bp
L. bicolor Malate Synthase
538 bp
470 bp
G. Intraradices Isocitrate Lyase
G. Intraradices Malate Synthase
New Mexico State University
The Basic Setup
• Take our fusion protein constructs and use Agrobacterium mediated gene transfer to move our constructs into L. bicolor
HygR ICL or MS eGFPL/B R/B
New Mexico State University
The Basic Setup
Grow on cellophane in glucose media…select for Hygr, transfer to Glc or Oleate
Measure crude protein extract (A280) and eGFP fluorescence
Glc 0hrs Glc 12hrs Ole 12hrs
New Mexico State University
Results
• glucose repression and oleate stimulation was observed only for L. bicolor promoter fusions
• Variation in behavior of L. bicolor transformants indicates strong position effects on transcriptional regulation of transgenes.
• The experimental data do not support the hypothesis for conservation between glyoxylate cycle promoters in AM and ECM fungi
New Mexico State University
Future Directions• Kinetics study
– Measure growth rate of transformants vs. wild type
– If the insertion of DNA happened in a “non essential” region of the L. bicolor transformant, then rate of growth would be similar to wild type growth
New Mexico State University
Thanks!!!
• I would like to thank Dr. Lammers, Punsiri Colonne, and Jorge Angeles for their guidance throughout this project…and their patients
• I would also like to extend a great deal of gratitude to the MARC program for their constant support and giving me the opportunity to work in a lab setting
New Mexico State University
References • Orr-Weaver, Terry L., Jack W. Szostak, Rodney Rothstein. "Yeast Transformation: A model system for
the study of recombination." Genetics 78(1981): 6354-6358.
• Manivasakam, Palaniyandi, Shane C. Weber, John McElver, Robert Schiestl. "Micro-homology mediated PCR targeting in Saccharomyces cerevisiae." Nucleic Acids Research 23(1995): 2799-2800.
• Fang, J., W.X. Zhai, M. Wang, L.H. Zhu. "Amplification and analysis of T-DNA flanking sequences in transgenic rice lines." Rice Genetics Newsletter 17(2007):
• Wach, Achim, Arndt Brachat, Christina Alberti-Segui, Corinne Rebischung, Peter Philippsen. "Heterologous HIS3 Marker and GFP reporter modules for PCR-Targeting in Saccharomyces cerevisiae." Yeast 13(1997): 1065-1075.
• Balasubramanian, Sujata, Kim, Podila. "Differential expression of a malate synthase gene during the preinfection stage of symbiosis in the ectomycorrhizal fungus Laccaria bicolor." New Physiologist 154(2002): 517-527.
• Piers, Kevin L., Joe Heath, Xiaoyou Liang, Kathryn Stephens, Eugene Nester. "Agrobacterium tumefaciens-mediated transformation of Yest." Microbiology 93(1996): 1613-1618.
• Abubaker, Jehad. PhD. Dissertation. NMSU (2004)