Better together: the use of virus-induced gene silencing technique to repress the expression of two endogenous citrus genes simultaneously.

Virus-induced gene silencing is a promising technique for functional genomics studies. Citrus tristeza virus was employed successfully to create an infectious clone that was used to silence endogenous citrus genes. Phytoene desaturase (PDS) and delta (δ)-aminolevulinic acid dehydratase (ALAD) were targeted successfully in citrus. Silencing PDS usually results in a photo-bleached leaf phenotype while silencing ALAD causes discrete yellow spots in leaves. Silencing two or more genes simultaneously using the same infectious clone could be difficult due to the capacity of the plasmid and subsequent cloning. On the other hand, inoculating a new construct into a citrus plant pre-infected with another construct fails due to the superinfection exclusion phenomenon. Herein, I report our successful trials whereby we simultaneously graft-inoculate constructs targeting PDS and ALAD. The budwoods were graft-inoculated into the same tree but on two different branches. Interestingly, a new phenotype was produced because of the silencing of the two genes, which we called "color-breaking". The phenotype was observed in both branches. Gene expression analysis showed a significant reduction of PDS and ALAD transcripts. This finding suggests the possibility of targeting more than one gene using different constructs, however, the graft-inoculation must be at the same time.