Manipulating Flowering Time in Citrus Using a TAL-based Effector Switch

Citrus has a long juvenility period. It typically takes a tree planted from seed about 8-10 years to flower. This presents a problem for citrus breeders when trying to select for disease resistance or horticultural traits because it often takes decades to make the serial crosses necessary to produce a commercially suitable variety. Three flowering locus T (FT) genes homologous to Arabidopsis FT have been identified in citrus (CiFT1, CiFT2 and CiFT3). Previous work at the University of Florida transforming citrus and tobacco individually with any of the CiFT genes behind a constitutive promoter suggested that CiFT3 was the most active of these genes. In ‘Duncan’ grapefruit, ‘Carrizo’ citrange, and ‘Pineapple’ sweet orange explants were transformed with the genomic CiFT3 gene behind a constitutive figwort mosaic virus (FMV) promoter, flowering occurred in vitro, preventing shoot production. The same phenotype occurred when citrus was transformed with a CiFT3 derived from cDNA in an otherwise identical construct. In tobacco, used as a model plant in these experiments, was transformed with a CiFT3 construct the time from planting to flowering was reduced from 170 to 50 days. By utilizing a transcriptional activator-like (TAL) effector designed to bind specifically to the endogenous CiFT3 gene promoter fused to an ecdysone receptor (EcR) - based expression switch and the virus protein VP16 transcriptional activator domain it will be possible to chemically induce flowering in citrus. Only when the EcR portion is activated by the field-approved chemical methoxyfenozide the TAL-EcR-VP16 fusion protein will be translocated to the nucleus and subsequently activate transcription of the target gene. The efficacy of this chemically-inducible promoter system will be evaluated using the β-glucuronidase (GUS) gene under the expression control of the chemically induced CiFT3 promoter in a tobacco model system and in citrus with the endogenous CiFT3. If successful, this system will enable breeders and growers to strategically regulate the initiation of flowering when plants are young and promote blossoming at an advantageous time.