Cucumber mosaic virus-induced gene silencing in banana

Yuh Tzean,Yi-Shu Chiu,Ming-Chi Lee,Chun-Nan Chou,Ho-Ming Chen,Hsin-Hung Yeh,Hsiao-Hsuan Jan,Tsui-Chin Tu,Bo-Han Hou

doi:10.1038/s41598-019-47962-3

Abstract

Banana (Musa spp.) is one of the world’s most important staple and cash crops. Despite accumulating genetic and transcriptomic data, low transformation efficiency in agronomically important Musa spp. render translational researches in banana difficult by using conventional knockout approaches. To develop tools for translational research in bananas, we developed a virus induced-gene silencing (VIGS) system based on a banana-infecting cucumber mosaic virus (CMV) isolate, CMV 20. CMV 20 genomic RNA 1, 2, and 3, were separately cloned in Agrobacterium pJL89 binary vectors, and a cloning site was introduced on RNA 2 immediately after the 2a open reading frame to insert the gene targeted for silencing. An efficient Agrobacterium inoculation method was developed for banana, which enabled the CMV 20 VIGS vector infection rate to reach 95% in our experiments. CMV 20-based silencing of Musa acuminata cv. Cavendish (AAA group) glutamate 1-semialdehyde aminotransferase (MaGSA) produced a typical chlorotic phenotype and silencing of M. acuminata phytoene desaturase (MaPDS) produced a photobleachnig phenotype. 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.

Highlights

Banana (Musa spp.) is one of the world’s most important staple and cash crops
No modification to full-length cDNA of cucumber mosaic virus (CMV) 20 RNAs 1 and 3 were made, whereas an AfeI restriction enzyme site was introduced in a position similar to that previously described[55], which is immediately downstream of 2a and in the 2b ORF of CMV to generate pCMV20-R2E
We report the development of a robust and efficient CMV-virus induced-gene silencing (VIGS) system for bananas and demonstrated that this approach enabled efficient silencing of genes using PDS and GSA

Summary

Introduction

Despite accumulating genetic and transcriptomic data, low transformation efficiency in agronomically important Musa spp. render translational researches in banana difficult by using conventional knockout approaches. Cavendish (AAA group) glutamate 1-semialdehyde aminotransferase (MaGSA) produced a typical chlorotic phenotype and silencing of M. acuminata phytoene desaturase (MaPDS) produced a photobleachnig phenotype We show this approach efficiently reduced GSA and PDS transcripts to 10% and 18% of the control, respectively. Despite the success of banana genomic and transcriptomic sequencing projects, reverse genetic approaches for identifying and characterizing gene functions in banana remain a challenge. Virus-induced gene silencing (VIGS) has been used extensively as a tool for reverse genetic study in a wide range of plant species to characterize gene functions or for high throughput phenotypic screening of genes[23–34]. The ease of the VIGS system has led to the development of www.nature.com/scientificreports/

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