STEVEN D. HARRIS UNIVERSITY OF NEBRASKA THE LIFE HISTORY AND SYSTEMS BIOLOGY OF FUNGAL-ALGAL MUTUALISMS EXTREMOPHILES ARE WIDELY RECOGNIZED AS ORGANISMS THAT CAN LIVE IN ENVIRONMENTS THAT ARE INHOSPITABLE TO MOST FORMS OF LIFE. THESE ENVIRONMENTS OFTEN EXHIBIT EXTREMES OF TEMPERATURE AS WELL AS WATER AVAILABILITY AND MAY ALSO FEATURE UNUSUALLY HIGH LEVELS OF RADIATION METALS ACIDS AND OTHER TOXIC COMPOUNDS. ALTHOUGH THE BEST STUDIED EXAMPLES OF EXTREMOPHILES ARE PROKARYOTIC AN INCREASINGLY DIVERSE ARRAY OF EUKARYOTIC FUNGI HAVE BEEN FOUND THAT THRIVE IN EXTREME COLD DRY SALTY OR ACIDIC HABITATS. EXTREMOPHILIC FUNGI POSSESS A NUMBER OF ADAPTATIONS THAT ENABLE THE COLONIZATION OF HARSH ENVIRONMENTS INCLUDING THE ACCUMULATION OF PROTECTIVE PIGMENTS SIMPLIFIED CELLULAR MORPHOLOGIES AND VERY SLOW GROWTH. NEVERTHELESS THERE IS MUCH THAT REMAINS TO BE UNDERSTOOD ABOUT THE LIFE HISTORY AND SYSTEMS BIOLOGY OF THESE FUNGI. WE PROPOSE TO ADDRESS THIS PROBLEM BY TESTING THE HYPOTHESIS THAT EXTREMOPHILIC FUNGI ENGAGE IN MUTUALISTIC ASSOCIATIONS WITH ALGAE (OR CYANOBACTERIA) TO SUPPORT THEIR COLONIZATION OF EXTREME HABITATS. IN PARTICULAR THESE TRANSIENT SYMBIOSES PROVIDE AN ACCESSIBLE SOURCE OF NUTRITION AND MAY ALSO CONTRIBUTE TO STRESS TOLERANCE. WE WILL TEST THIS HYPOTHESIS BY PURSUING TWO SPECIFIC OBJECTIVES. FIRST WE WILL USE IMAGE ANALYSIS AND MICROSCOPY TO DETERMINE THE RELATIVE TIMING OF EVENTS UNDERLYING THE MUTUALISTIC ASSOCIATION OF THE EXTREMOPHILE FUNGUS EXOPHIALA DERMATITIDIS WITH THE ALGA CHLORELLA SOROKINIANA. TRANSCRIPTIONAL PROFILING AND BIOCHEMICAL ANALYSES WILL BE USED TO INVESTIGATE THE RESPONSES OF EACH PARTNER AS THEY INTERACT. SECOND WE WILL EMPLOY CULTURE-BASED AND META-GENOMIC APPROACHES TO ASSESS THE DIVERSITY OF FUNGI AND ALGAE THAT COLONIZE ROCK SURFACES IN A BOREAL BIOME (PRE-CAMBRIAN SHIELD). ISOLATED FUNGI AND ALGAE WILL BE SYSTEMATICALLY TESTED FOR THE CAPACITY TO ENGAGE IN MUTUALISTIC ASSOCIATIONS. OUR PROPOSED RESEARCH FALLS WITHIN THE SCOPE OF THE NASA EXOBIOLOGY PROGRAM EMPHASIS AREA EARLY EVOLUTION OF LIFE AND THE BIOSPHERE . FOR EXAMPLE OUR RESULTS WILL PROVIDE INSIGHT INTO THE PHYSIOLOGY OF EXTREMOPHILIC MICROORGANISMS THAT ARE ADAPTED TO ENVIRONMENTS THAT SHARE FEATURES IN COMMON WITH EARLY EARTH (AND PERHAPS OTHER PLANETS). NOTABLY WE EXPECT TO UNDERSTAND HOW THE CELLULAR SYSTEMS OF INTERACTING FUNGI AND ALGAE EVOLVE IN RESPONSE TO THESE ENVIRONMENTS. IN ADDITION OUR RESULTS WILL ALSO SHED LIGHT ON HOW THE COMPOSITION OF MICROBIAL COMMUNITIES AND THEIR CONSTITUENT INTERACTIONS IMPACTS THE GEOCHEMISTRY OF ROCK SURFACES. FOR OUR FIRST OBJECTIVE WE HAVE A DEVELOPED A PROTOCOL FOR THE SUCCESSFUL CO-CULTIVATION OF E. DERMATITIDIS WITH C. SOROKINIANA. RNA-SEQ WILL BE USED TO GENERATE TRANSCRIPTIONAL PROFILES AT MULTIPLE STAGES OF CO-CULTIVATION. THESE PROFILES WILL BE CORRELATED WITH MICROSCOPIC ANALYSIS OF FUNGAL MORPHOLOGY AS WELL AS BIOCHEMICAL ANALYSIS OF FUNGAL LIPIDS. IN TOTAL WE EXPECT THESE EXPERIMENTS TO PROVIDE IMPORTANT INSIGHT INTO THE STRATEGIES EMPLOYED BY EXTREMOPHILE FUNGI TO ACQUIRE NUTRIENTS IN ENVIRONMENTS THAT ARE REPRESENTATIVE OF EARLY EARTH. FOR OUR SECOND OBJECTIVE WE WILL SAMPLE EXPOSED ROCK OUTCROPS AT THREE SPECIFIC LOCATIONS IN THE PRE-CAMBRIAN SHIELD OF CANADA. A ROUTINE CULTURE-BASED SELECTION WILL BE USED TO RECOVER EXTREMOPHILIC FUNGI AND ALGAE (OR CYANOBACTERIA) FROM ROCK SURFACES AS WELL AS FROM PULVERIZED FRAGMENTS. DNA SEQUENCE ANALYSIS WILL BE USED TO ESTABLISH THE IDENTITY OF EACH ISOLATE. IN PARALLEL DNA EXTRACTED FROM PULVERIZED ROCKS WILL BE SEQUENCED TO PROVIDE A META-GENOMIC OVERVIEW OF FUNGAL AND ALGAL COMMUNITIES. UP TO 50 DISTINCT FUNGAL AND ALGAL (OR CYANOBACTERIAL) ISOLATES WILL BE CO-CULTIVATED USING A MICROTITER PLATE FORMAT TO ASSESS THEIR CAPACITY TO ENGAGE IN MUTUALISTIC INTERACTIONS BASED ON ACCUMULATION OF BIOMASS AND PHYSICAL ASSOCIATION.