Virus-induced gene-silencing in wheat spikes and grains and its application in functional analysis of HMW-GS-encoding genes

Meng Ma,Mingshun Chen,Li Huang,Yan Yan,Huixian Zhao

doi:10.1186/1471-2229-12-141

Abstract

BackgroundThe Barley stripe mosaic virus (BSMV)-based vector has been developed and used for gene silencing in barley and wheat seedlings to assess gene functions in pathogen- or insect-resistance, but conditions for gene silencing in spikes and grains have not been evaluated. In this study, we explored the feasibility of using BSMV for gene silencing in wheat spikes or grains.ResultsApparent photobleaching on the spikes infected with BSMV:PDS at heading stage was observed after13 days post inoculation (dpi), and persisted until 30dpi, while the spikes inoculated with BSMV:00 remained green during the same period. Grains of BSMV:PDS infected spikes also exhibited photobleaching. Molecular analysis indicated that photobleached spikes or grains resulted from the reduction of endogenous PDS transcript abundances, suggesting that BSMV:PDS was able to induce PDS silencing in wheat spikes and grains. Inoculation onto wheat spikes from heading to flowering stage was optimal for efficient silencing of PDS in wheat spikes. Furthermore, we used the BSMV-based system to reduce the transcript level of 1Bx14, a gene encoding for High-molecular-weight glutenin subunit 1Bx14 (HMW-GS 1Bx14), by 97 % in the grains of the BSMV:1Bx14 infected spikes at 15dpi, compared with that in BSMV:00 infected spikes, and the reduction persisted until at least 25 dpi. The amount of the HMW-GS 1Bx14 was also detectably decreased. The percentage of glutenin macropolymeric proteins in total proteins was significantly reduced in the grains of 1Bx14-silenced plants as compared with that in the grains of BSMV:00 infected control plants, indicating that HMW-GS 1Bx14 is one of major components participating in the formation of glutenin macropolymers in wheat grains.ConclusionThis is one of the first reports of successful application of BSMV-based virus-induced-gene-silencing (VIGS) for gene knockdown in wheat spikes and grains and its application in functional analysis of the 1Bx14 gene. The established BSMV-VIGS system will be very useful in future research on functional analysis of genes contributing to grain quality and the metabolic networks in developing seeds of wheat.

Highlights

The Barley stripe mosaic virus (BSMV)-based vector has been developed and used for gene silencing in barley and wheat seedlings to assess gene functions in pathogen- or insect-resistance, but conditions for gene silencing in spikes and grains have not been evaluated
The results in this study indicate that BSMV-VIGS system is a useful tool for functional analysis of genes expressed in wheat spikes and grains
Unlike Cucumber mosaic virus (CMV)- or Apple latent spherical virus (ALSV)-vector for VIGS in soybean (Glycine max), with which inoculation onto the first leaf at the seedling stage or two cotyledons at emergence stage of soybean plants could effectively lead to gene silence in seeds [28,29], inoculation with BSMV:phytoene desaturase gene (PDS) recombinant virus onto the first leaf of wheat or barley plants at the seedling stage led to the most efficient PDS silencing in the third leaf, but the silence effect largely disappeared before the onset of flowering in most plants

Summary

Introduction

The Barley stripe mosaic virus (BSMV)-based vector has been developed and used for gene silencing in barley and wheat seedlings to assess gene functions in pathogen- or insect-resistance, but conditions for gene silencing in spikes and grains have not been evaluated. As a result of rapid advance in structural genomics, the availability of powerful tools for gene function analysis has become a bottleneck, especially for important crops beyond the few model plant species. In model plants such as Arabidopsis and rice, two methods, i.e. T-DNA knockout libraries [4] and T-DNA activation libraries [5], have significantly accelerated the speed of gene functional identification. An additional complication for determining gene function through the analysis of loss-of-function mutations in wheat is the fact that all cultivated varieties are polyploid; in most cases, expression of homeologous genes could mask loss-of-function phenotypes resulting from the disruption of the other homeologous alleles [1]

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Discussion

Conclusion