Cucumber mosaic virus-induced gene silencing in banana
Banana (Musa spp.) is one of the world's most important staple and cash crops, providing food and economic security for more than 400 million people globally. Despite accumulating genetic and transcriptomic data, low transformation efficiency and polyploidy genome in agronomically important Musa spp. render translational researches in banana difficult by using conventional reverse genetic knockout approaches. To develop tools for translational research in bananas, we selected a naturally banana-infecting Cucumber mosaic virus (CMV) strain, CMV 20, for developing virus induced-gene silencing (VIGS) vector. The CMV 20 genomic RNAs, RNA 1, 2, and 3, were separately constructed on a binary vector, and an AfeI restriction enzyme site was introduced on RNA2 immediately after 2a open reading frame for cloning of foreign gene. Genes inserted in RNA2 will truncate the expression of RNA silencing suppressor, 2b. CMV 20 VIGS vector with truncated 2b still systematically infects Nicotiana benthamiana and banana, and is symptomless or only induces mild symptoms. An efficient Agrobacterium inoculation method was developed for bananas based on optimization of the inoculation buffer and needle-injection of Agrobacterium at the pseudostem-rhizome junction, which enabled CMV 20 VIGS vector infection rate to reach 95% in our repeated experiments. When CMV 20 VIGS vector with M. acuminate cv. Cavendish (AAA group)a fragment of glutamate 1-semialdehyde aminotransferase (GSA) or phytoene desaturase (PDS) gene is inserted in pJLCMV20-R2E for inoculation, extended GSA- and PDS-silenced phenotype can be observed starting at around 30 days post-inoculation (dpi), and this silencing phenotype is observed on emerging leaves of up to 63 dpi. We show this approach efficiently reduced GSA and PDS transcripts to 10% and 18% of the control, respectively. The high infection rate and extended silencing of this VIGS system will provide an invaluable tool to accelerate functional genomic studies in banana.
