CONDITIONAL SELECTION AND SUBSTRATE-FREE TECHNOLOGIES FOR HIGH-THROUGHPUT VIRAL RESISTANCE AND GAIN-OF-FUNCTION SCREENS IN PLANTS

PLANT VIRUSES CAUSE SIGNIFICANT ECONOMIC LOSSES TO CROPS WORLDWIDE. ALTHOUGH VIRAL RESISTANCE CAN BE ENGINEERED BY PLANT TRANSGENESIS, THIS RAISES PUBLIC CONCERN AND THE EUROPEAN UNION LEGISLATION ON GENETICALLY MODIFIED ORGANISMS IS EXTREMELY RESTRICTIVE. CONVENTIONAL BREEDING SCHEMES REQUIRE IDENTIFICATION OF RESISTANCE SOURCES THAT WILL BE INTROGRESSED TO COMMERCIAL CROPS, WHICH INVOLVES MASSIVE SCREENING OF NATURAL OR MUTANT POPULATIONS. ALTHOUGH GENETIC IDENTIFICATION OF CAUSAL GENES IS GREATLY FACILITATED BY GENOTYPING-BY-SEQUENCING APPROACHES, WE CURRENTLY LACK HIGH-THROUGHPUT PHENOTYPING PROCEDURES FOR VIRAL RESISTANCE, ESPECIALLY WHEN INFECTION SYMPTOMS ARE NOT SEVERE ENOUGH FOR VISUAL DETECTION. OUR PROPOSED SOLUTION IS TO GENERATE ENGINEERED VIRUSES THAT EXPRESS SELECTABLE MARKER GENES. THUS, VIRUS-MEDIATED EXPRESSION OF A D-AMINO ACID OXIDASE GENE WILL ALLOW DUAL, CONDITIONAL NEGATIVE OR POSITIVE SELECTION. APPLICATION OF D-VALINE WILL PERMIT IDENTIFICATION OF INFECTION-RESISTANT PLANTS, WHEREAS IDENTIFICATION OF GAIN-OF-SUSCEPTIBILITY PHENOTYPES WILL BE POSSIBLE USING THE SAME ENGINEERED VIRUS WITH APPLICATION OF D-ALANINE. THE SECOND ALTERNATIVE, IS VERY INNOVATIVE SINCE ALLOWS THE IDENTIFICATION OF GAIN-OF-FUNCTION PHENOTYPES WHICH ARE VERY INTERESTING BUT DIFICULT TO FIND. IN ANOTHER STRATEGY, WE PROPOSE TO EXPRESS CLAVATA3-LIKE GENES BY ENGINEERED VIRUSES. IN ARABIDOPSIS THALIANA, CLAVATA3 OVEREXPRESSION CAUSES A STRONG PHENOTYPE WITH SHOOT MERISTEM ARREST AND IMPAIRED SEED PRODUCTION. WE ANTICIPATE THAT PLANTS SUSCEPTIBLE TO A CLAVATA3-EXPRESSING VIRUS WILL HAVE THE SAME PHENOTYPE, WHEREAS RESISTANT PLANTS WILL SHOW NO DEVELOPMENTAL DEFECTS. THIS SELECTION APPROACH IS COST/TIME-SAVING, SINCE IT DOES NOT REQUIRE SUBSTRATE SUPPLY; MOREOVER, DUE TO CONSERVATION OF CLAVATA3-LIKE GENES IN BOTH MONOCOT AND DICOT SPECIES, WE PREDICT IT COULD BE EASILY EXTENDED TO OTHER PLANT-VIRUS SYSTEMS, INCLUDING CEREAL CROPS.