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…

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

New Mexico State University

Results

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)