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COLLABORATIVE RESEARCH: PLANTTRANSFORM: NOVEL METHODS OF PLANT GENOME EDITING ENABLED BY THE PARASITIC PLANT CUSCUTA CAMPESTRIS -A MAJOR BARRIER TO RESEARCH ON PLANT FUNCTION AND TO CROP IMPROVEMENT IS A LIMITATION IN METHODS AVAILABLE FOR GENETICALLY MANIPULATING PLANTS. TECHNIQUES CURRENTLY USED INCLUDE THE CULTURING OF PLANT TISSUES, THE INSERTION OR EDITING OF DNA, AND THE RECOVERY OF WHOLE PLANTS, BUT EACH OF THESE STEPS POSES CHALLENGES TO THE DEGREE THAT THEY WORK RELIABLY IN RELATIVELY FEW SPECIES, AND EVEN THEN MAY NOT WORK WELL IN ALL VARIETIES OF THE SPECIES. TO FILL THIS GAP, WE PROPOSE THAT THE PARASITIC PLANT DODDER (CUSCUTA SPECIES) CAN BE USED TO DELIVER GENE EDITING MOLECULES TO A WIDE RANGE OF PLANTS. DODDER PLANTS LIVE BY ATTACHING THEMSELVES TO THE STEMS OF HOST PLANTS AND FORMING CONNECTIONS TO WITHDRAW WATER AND NUTRIENTS. THE ORGANS THAT FORM THE CONNECTIONS ARE CALLED HAUSTORIA, AND FUNCTION SOMEWHAT SIMILAR TO THE WAY A MOSQUITO TAPS INTO A VEIN TO FEED, AND DODDER IS ABLE TO TRANSMIT A VARIETY OF LARGE MOLECULES, INCLUDING PROTEINS AND RNAS, TO THEIR HOSTS. ANOTHER KEY FEATURE OF DODDER IS ITS ABILITY TO CONNECT TO AN UNUSUALLY WIDE RANGE OF HOST SPECIES, INCLUDING THE MOST IMPORTANT BROADLEAF CROPS. WE WILL EVALUATE THE ABILITY OF DODDER TO MOBILIZE GENOME EDITING MOLECULES INTO ITS HOSTS, WITH THE GOAL OF PRODUCING GENE-EDITED SEEDS. SUCCESS IN THIS ACTIVITY WOULD ESTABLISH A NOVEL VEHICLE FOR GENETIC MODIFICATION OF PLANTS THAT IS RELATIVELY SIMPLE, RAPID, AND BROADLY APPLICABLE. THE PROJECT WILL EXPLORE MULTIPLE POSSIBILITIES FOR TRANSFERRING CAS9 AND SINGLE GUIDE RNA (SGRNA) BETWEEN DODDER AND HOSTS. AMONG THE POSSIBILITIES ARE THE MOVEMENT OF THESE MOLECULES FROM AN EASILY TRANSFORMED HOST, SUCH AS ARABIDOPSIS, TO RESULT IN TRANSFORMED DODDER, OR THE REVERSE FROM STABLY TRANSFORMED DODDER TO RESULT IN A TRANSFORMED HOST. GIVEN SUCCESS WITH THESE, WE WILL EXPLORE THE ABILITY FOR DODDER TO SERVE AS A BRIDGE BETWEEN A CAS9-SGRNA EXPRESSING DONOR HOST AND A TARGET HOST (E.G., TOMATO). THE PROJECT HAS THREE MAJOR AIMS TO ACHIEVE THESE OUTCOMES, INCLUDING: 1) THE STABLE TRANSFORMATION OF DODDER (C. CAMPESTRIS) TO BE USED IN HOST GENE EDITING, 2) THE DEVELOPMENT OF A DODDER PROTOPLAST SYSTEM FOR RAPID SCREENING OF GENE EDITING CONSTRUCTS, AND 3) THE DEVELOPMENT OF A DODDER-MEDIATED GENE EDITING SYSTEM. PRELIMINARY RESULTS INDICATE THAT AGROBACTERIUM-MEDIATED TRANSFORMATION OF DODDER IS POSSIBLE, AND THE PROCEDURE WILL BE OPTIMIZED TO ENABLE GENERATION OF MULTIPLE LINES BEARING VARIOUS TRANSGENE CONSTRUCTS. OTHER CONSIDERATIONS INCLUDE IDENTIFYING PROMOTORS FOR THE APPROPRIATE EXPRESSION OF GENE CONSTRUCTS AND OPTIMIZED TARGETING FOR SYSTEMIC TRAFFICKING OF CAS9/SGRNA IN THE PARASITE-HOST SYSTEM. FOR THE OUTREACH GOAL OF THIS PROJECT, PIS WILL DEVELOP A PROGRAM FOR REFUGEE STUDENTS TO HELP THEM PARTICIPATE IN PROJECT-RELATED ACTIVITIES AND DEVELOP THEIR UNDERSTANDING OF PLANT SCIENCE AT THE UNIVERSITY OF MISSOURI. IN SUMMARY, THE PROJECT WILL LEVERAGE THE INTRINSICALLY ENGAGING TOPICS OF PLANT PARASITISM, RNA MOBILITY, AND GENOME EDITING TO ATTRACT THESE STUDENTS TO PLANT SCIENCE. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.
$300.0k 6/17/24