**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** ABOUT 80% OF ALL LAND PLANTS FORM TIGHT INTERACTIONS WITH BENEFICIAL GLOMEROMYCOTINA SOIL FUNGI. THIS SYMBIOSIS IS CALLED ARBUSCULAR MYCORRHIZAL (AM) SYMBIOSIS. AM FUNGI PROVIDE MANY BENEFITS TO THEIR PLANT HOST, FOR EXAMPLE INCREASED RESISTANCE TO PATHOGENS AND IMPROVED ACQUISITION OF ESSENTIAL MACRONUTRIENTS SUCH AS PHOSPHATE. MAJOR CROPS SUCH AS SOYBEAN, CORN, OR RICE ENGAGE IN AM SYMBIOSIS WITH SOIL FUNGI. THUS, AN EFFICIENT USE OF THESE MICROBES IN AGRICULTURE COULD DIMINISH THE NEED FOR CHEMICAL FERTILIZERS, WHICH ARE A FINITE RESOURCE AND HAVE SEVERE NEGATIVE IMPACTS ON OUR ENVIRONMENT. BECAUSE OF THEIR BENEFICIAL EFFECTS ON PLANT HEALTH AND NUTRITION, AM FUNGI ARE COMMONLY SOLD AS 'BIOFERTILIZERS'. HOWEVER, NOT ALL AM FUNGAL SPECIES ARE EQUALLY BENEFICIAL FOR A GIVEN PLANT, AND WHILE WE KNOW THAT PLANTS CAN DISTINGUISH BETWEEN DIFFERENT AM FUNGAL COLONIZERS AND PREFERENTIALLY INTERACT WITH THE ONES THAT PROVIDE THE MOST BENEFITS, THE GENETIC MECHANISM UNDERLYING THIS 'HOST-SYMBIONT PREFERENCE' IS UNKNOWN. MOST AGRO-ECOSYSTEMS ALREADY CONTAIN NUMEROUS NATIVE AM FUNGAL SPECIES AND INTRODUCING 'BIOFERTILIZER' AM FUNGI WILL RESULT IN COMPETITION OF NATIVE AND INTRODUCED AM FUNGI, MAKING THE OVERALL IMPACT OF THE SYMBIOSIS UNPREDICTABLE. IN ORDER TO EFFICIENTLY UTILIZE AM FUNGI IN A FUTURE SUSTAINABLE AGRICULTURE, WE MUST FIRST GAIN A DETAILED UNDERSTANDING HOW PLANTS DISCRIMINATE BETWEEN DIFFERENT COLONIZERS AND PROMOTE SYMBIOSIS ONLY WITH THE MOST BENEFICIAL ONES.AM FUNGI ENTER THE INNER TISSUES OF PLANT ROOTS AND AT THE SAME TIME FORM A DENSE NETWORK OF FUNGAL FILAMENTS SURROUNDING THE PLANT ROOT. THE SURROUNDING FILAMENTS TAKE UP PHOSPHATE FROM DISTANT AREAS IN THE SOIL THAT CANNOT BE REACHED BY THE ROOTS. IN THE INNER TISSUES OF THE ROOT, THE FUNGUS THEN TRADES THESE NUTRIENTS TO THE HOST PLANT IN EXCHANGE FOR CARBON IN THE FORM OF SUGARS OR FATTY ACIDS. THE PLANT TIGHTLY CONTROLS ROOT COLONIZATION BY AM FUNGI TO ENSURE THAT ONLY FUNGI THAT PROVIDE SUFFICIENT PHOSPHATE RECEIVE CARBON. USING THE MODEL LEGUME MEDICAGO TRUNCATULA (BARREL MEDIC), WE PREVIOUSLY CHARACTERIZED A SYSTEMIC SIGNALING PATHWAY INVOLVING A MOBILE PEPTIDE HORMONE (CLE53) AND ITS RECEPTOR (SUNN). THE PEPTIDE HORMONE IS PRODUCED IN COLONIZED ROOTS AND ALLOWS THE PLANT TO 'QUANTIFY' EXISTING SYMBIOSES IN ITS ROOT SYSTEM. CLE53 AND ITS RECEPTOR SUNN THEN RESTRICT FURTHER AM FUNGAL ROOT COLONIZATION ONCE A CERTAIN COLONIZATION THRESHOLD IS REACHED (QUANTITATIVE SYMBIOSIS CONTROL). IT IS HYPOTHESIZED THAT THE CLE53-SUNN SIGNALING PATHWAY PREVENTS ROOT OVER-COLONIZATION AND OVER-SEQUESTRATION OF CARBON BY THE FUNGAL SYMBIONT.BASED ON PRELIMINARY DATA, WE HYPOTHESIZE THAT THE CLE53-SUNN MODULE IS ALSO INVOLVED IN QUALITATIVE SYMBIOSIS CONTROL (I.E., SELECTION OF THE MOST BENEFICIAL SYMBIONT). WE WILL USE A COMBINATION OF MOLECULAR BIOLOGY, MOLECULAR GENETICS, TRANSCRIPTOME SEQUENCING, AND MICROSCOPY TO CHARACTERIZE THE GENETIC BASIS OF HOST-SYMB,IONT PREFERENCE IN THE MODEL LEGUME MEDICAGO TRUNCATULA. USING MUTANTS IMPAIRED IN QUANTITATIVE SYMBIOSIS CONTROL, WE WILL FIRST ASSESS THE PHYSIOLOGICAL IMPLICATIONS ON PLANT GROWTH AND PRODUCTIVITY WHEN SUCH PLANTS ARE INOCULATED WITH LOW- OR HIGH-BENEFIT AM FUNGI. WE HYPOTHESIZE THAT OVER-COLONIZATION OF THE ROOT SYSTEM BY A LOW-BENEFIT FUNGUS WILL RESULT IN OVER-SEQUESTRATION OF CARBON AND THUS NEGATIVELY IMPACT PLANT GROWTH, WHILE OVER-COLONIZATION BY A HIGH-BENEFIT FUNGUS MAY POTENTIALLY BE BENEFICIAL FOR THE PLANT. WE WILL THEN DETERMINE IF THE KNOWN REGULATORS OF QUANTITATIVE SYMBIOSIS CONTROL (CLE53 AND SUNN) ARE ALSO INVOLVED IN QUALITATIVE SYMBIOSIS CONTROL, I.E. IF THE CLE53-SUNN MODULE RESPONDS DIFFERENTLY TO AM FUNGI CONFERRING LOW OR HIGH DEGREES OF BENEFITS (PHOSPHATE) TO THE PLANTS. USING A YEAST-1-HYBRID SCREEN TO DETECT INTERACTIONS OF DNA AND PROTEINS SUCH AS TRANSCRIPTIONAL REGULATORS, WE WILL IDENTIFY SPECIFIC REGULATORY PROTEINS INVOLVED IN THE REGULATION OF CLE53 EXPRESSION. LASTLY, WE WILL DEVELOP NEW MICROSCOPIC TOOLS THAT WILL ALLOW US TO SIMULTANEOUSLY IMAGE TWO DIFFERENT AM FUNGAL SPECIES COLONIZING THE SAME ROOT SYSTEM AND USE THESE TOOLS TO INVESTIGATE IF THE CLE53-SUNN MODULE IS INVOLVED IN HOST-SYMBIONT PREFERENCE. IN ADDITION, WE WILL PERFORM TRANSCRIPTOME SEQUENCING OF MEDICAGO TRUNCATULA ROOT SYSTEMS INOCULATED WITH HIGH- AND/OR LOW-BENEFIT AM FUNGI TO IDENTIFY YET UNKNOWN ADDITIONAL REGULATORS OF HOST-SYMBIONT PREFERENCE.TO DATE, VIRTUALLY NOTHING IS KNOWN ABOUT THE GENETIC BASIS OF PLANT RESPONSES TO SIMULTANEOUS COLONIZATION BY HIGH- AND LOW-BENEFIT AM FUNGI. THIS PROJECT WILL MAKE AN IMPORTANT CONTRIBUTION TO FILL THIS KNOWLEDGE GAP. FURTHERMORE, UNDERSTANDING HOST-SYMBIONT PREFERENCE ON A GENETIC LEVEL IS AN IMPORTANT PREREQUISITE TO DEVELOP PLANT LINES WITH INCREASED MYCORRHIZAL CAPACITIES AND OPTIMIZED ABILITIES TO SELECT FOR THE BEST SYMBIONT IN AGRO-ECOSYSTEMS WHERE MULTIPLE AM FUNGAL SPECIES ARE PRESENT. THE PROJECT WILL IDENTIFY SEVERAL POTENTIAL TARGETS FOR FUTURE CROP IMPROVEMENT, INCLUDING THE PREVIOUSLY DESCRIBED GENES CLE53 AND SUNN, THE TRANSCRIPTIONAL REGULATORS TO BE IDENTIFIED IN THE YEAST-1-HYBRID SCREEN, AND NOVEL REGULATORS TO BE IDENTIFIED IN THE TRANSCRIPTOME SEQUENCING EXPERIMENT. THIS PROJECT WILL LAY THE GROUNDWORK FOR FUTURE BREEDING EFFORTS TO MAXIMIZE SYMBIOTIC CAPACITIES OF CROP PLANTS AND WILL SIGNIFICANTLY CONTRIBUTE TO OUR PATH TOWARDS SUSTAINABLE AGRICULTURE.