Development of Agrobacterium-Mediated Virus-Induced Gene Silencing and Performance Evaluation of Four Marker Genes in Gossypium barbadense

Yingchuan Tian,Jinzhi Liu,Yule Liu,Jinhuan Pang,Yue Zhu,Qing Li,Jiahe Wu,Y Adam Yuan

doi:10.1371/journal.pone.0073211

Abstract

Gossypium barbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G . barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G . barbadense . In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G . barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G . barbadense . The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G . barbadense , and help to contribute desirable traits for breeding of G . barbadense and G. hirsutum.

Highlights

Virus-induced gene silencing (VIGS) vector technology exploits the plant defense system against virus RNA
Gao et al [16,17] used the Tobacco rattle virus (TRV) as a vector to silence another marker gene, cloroplastos alterados 1 (CLA1). Both VIGS systems were shown to work in G. hirsutum, it was not known whether these methods could be used to assess gene function in G. barbadense
In our initial experiment to develop an Agrobacteriummediated VIGS system in G. barbadense, we investigated the optimal conditions under which tobacco rattle virus (TRV)-based VIGS in G. barbadense cv 3-79 was effective by attempting to silence the G. barbadense Phytoene desaturase (GaPDS) gene as a marker

Summary

Introduction

Virus-induced gene silencing (VIGS) vector technology exploits the plant defense system against virus RNA. When a partial fragment of a candidate gene is inserted into a virus vector, plants inoculated with the recombinant virus generate virus-related siRNAs [2]. Gao et al [16,17] used the Tobacco rattle virus (TRV) as a vector to silence another marker gene, cloroplastos alterados 1 (CLA1). Both VIGS systems were shown to work in G. hirsutum, it was not known whether these methods could be used to assess gene function in G. barbadense

Methods

Results

Conclusion