RNA INTERFERENCE (RNAI) DIRECTED AGAINST PESTS AND PATHOGENS, OFTEN CALLED HOST INDUCED GENE SILENCING (HIGS), IS A NEW APPROACH FOR ENGINEERING RESISTANT PLANT VARIETIES. HIGS IS EXPECTED TO PRODUCE HIGHLY SPECIFIC FORMS OF RESISTANCE COMPARED TO PESTICIDES AND MOST OTHER RESISTANT VARIETIES; HOWEVER, THIS PREDICTION HAS NOT BEEN RIGOROUSLY TESTED. A BETTER UNDERSTANDING OF THE SPECIFICITY OF HIGS WILL INFORM THE DEVELOPMENT OF REGULATORY STANDARDS FOR NON-TARGET ORGANISM STUDIES. WE PROPOSE TO USE THE POPULUS TRICHOCARPA MODEL SYSTEM TO STUDY HIGS. POPULUS IS GENETICALLY TRANSFORMABLE, HAS A WELL CHARACTERIZED MICROBIOME, AND HAS EXCELLENT GENOMIC RESOURCES. WE WILL FOCUS ON THE SPHAERULINA CANKER AND LEAF SPOT, THE MOST ECONOMICALLY IMPORTANT DISEASE OF POPLAR TREES. OUR PROPOSED STUDY WILL USE A COMBINATION OF FIELD AND GREENHOUSE EXPERIMENTS TO EVALUATE THE ECOLOGICAL IMPACT OF HIGS BASED DISEASE RESISTANCE ON THE POPULUS MICROBIOME. WE HAVE ALREADY CREATED HIGS CONSTRUCTS THAT ARE EFFECTIVE AGAINST THE SPHAERULINA PATHOGENS. THESE CONSTRUCTS ARE CURRENTLY BEINGTRANSFORMED INTO POPULUS. WE WILL TEST THESE TRANSGENIC LINES IN RANDOMIZED GREENHOUSE AND FIELD TRIALS TO ASSESS DISEASE RESISTANCE AND NON-TARGET EFFECTS OF HIGS ON THE POPULUS MICROBIOME.THIS PROJECT WILL PROVIDE GUIDANCE TO REGULATORY AGENCIES, INFORM THE DESIGN OF HIGS APPROACHES, AND ESTABLISH WHETHER HIGS IS EFFECTIVE FOR PRODUCING RESISTANCE AGAINST A MAJOR PATHOGEN OF POPLARS. OUR OVERARCHING OBJECTIVE WAS TO EVALUATE THE EFFECTIVENESS OF HIGS FOR DISEASE CONTROL WHILE CONFIRMING WITH SMALL TO NEGLIGIBLE IMPACTS TO THE HEALTH OF THE GE ORGANISM(OFF-TARGET) AND ITS ASSOCIATED MICROBIOME (NON-TAGET). SPECIFICALLY WE WILL: 1) SCREEN TRANSGENIC LINES FOR RESISTANCE TO SPHAERULINA IN VITRO AND BY ANALYSIS OF SIRNA PRODUCTION; 2) EVALUATE SPHAERULINA CANKER AND LEAF SPOT RESISTANCE OF TRANSGENIC LINES AND CONTROLS IN THE GREENHOUSE; 3) TEST THE IMPACT OF RNAI TRANSGENIC LINES ON: A) THE COMPOSITION OF A SYNTHETIC, INOCULATED FUNGAL LEAF ENDOPHYTE COMMUNITY; AND B) THE ABUNDANCE OF PATHOGEN ANTAGONISTS WITHIN THE COMMUNITY; AND 4) EVALUATE MICROBIOME DEVELOPMENT AND DISEASE RESISTANCE OF RNAI TRANSGENIC LINES AT FIELD SITES NEAR CORVALLIS, OR AND IN AN AREA OF HIGH NATURAL INOCULUM LOAD IN MORGANTOWN, WV.