Elucidating the role of highly homologous Nicotiana benthamiana ubiquitin E2 gene family members in plant immunity through an improved virus-induced gene silencing approach

Bangjun Zhou,Lirong Zeng

doi:10.1186/s13007-017-0210-6

Abstract

BackgroundVirus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a high-throughput manner. However, the specificity and efficiency for knocking down members of a highly homologous gene family have remained to date a significant challenge in VIGS due to silencing of off-targets.ResultsHere we present an improved method for the selection and evaluation of gene fragments used for VIGS to specifically and efficiently knock down members of a highly homologous gene family. Using this method, we knocked down twelve and four members, respectively of group III of the gene family encoding ubiquitin-conjugating enzymes (E2) in Nicotiana benthamiana. Assays using these VIGS-treated plants revealed that the group III E2s are essential for plant development, plant immunity-associated reactive oxygen species (ROS) production, expression of the gene NbRbohB that is required for ROS production, and suppression of immunity-associated programmed cell death (PCD) by AvrPtoB, an effector protein of the bacterial pathogen Pseudomons syringae. Moreover, functional redundancy for plant development and ROS production was found to exist among members of group III E2s.ConclusionsWe have found that employment of a gene fragment as short as approximately 70 base pairs (bp) that contains at least three mismatched nucleotides to other genes within any 21-bp sequences prevents silencing of off-target(s) in VIGS. This improved approach in the selection and evaluation of gene fragments allows for specific and efficient knocking down of highly homologous members of a gene family. Using this approach, we implicated N. benthamiana group III E2s in plant development, immunity-associated ROS production, and suppression of multiple immunity-associated PCD by AvrPtoB. We also unraveled functional redundancy among group III members in their requirement for plant development and plant immunity-associated ROS production.

Highlights

Virus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a highthroughput manner
Various tools for knocking out or knocking down genes of interest have been developed. Examples of such tools include the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated9 (Cas9) system [1,2,3], silencing of genes by RNA interference (RNAi) [4], and mutation of genes by transposable element [5], Agrobacterium-mediated T-DNA insertion [6, 7], or chemicals, such as ethyl methanesulfonate [8] and targeting induced local lesions in genomes (TILLING) [9]. Among these tools, silencing of genes via RNAi has largely been used in reverse genetics and involves direct alteration in the expression of a gene and subsequent identification of gene function based on the phenotypes that result from such change in gene expression
Due to high percentage of identity in their DNA sequences, a DNA fragment derived from NbUBC9 can theoretically be used for silencing the NbUBC8, NbUBC9 and NbUBC38 genes that are from the same clade of the phylogenetic tree (Fig. 1B) and was designed for building the Tobacco rattle virus (TRV)-III construct

Summary

Introduction

Virus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a highthroughput manner. Various tools for knocking out or knocking down genes of interest have been developed Examples of such tools include the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system [1,2,3], silencing of genes by RNA interference (RNAi) [4], and mutation of genes by transposable element [5], Agrobacterium-mediated T-DNA insertion [6, 7], or chemicals, such as ethyl methanesulfonate [8] and targeting induced local lesions in genomes (TILLING) [9]. During VIGS, as the recombinant virus spreads out systemically in the plant, dsRNAs corresponding to the host gene are produced and cleaved into small interference RNAs (siRNAs) in the length of 21–24 nucleotides (nt) These siRNAs are incorporated into the RNAinduced silencing complex (RISC) to degrade the target mRNAs [16]. It is well accepted that VIGS is a simple, quick and cost-effective method for high throughput study of gene function

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Discussion

Conclusion