Constructs and methods for high-throughput gene silencing in plants

Abstract

Gene silencing can be achieved by transformation of plants with constructs that express self-complementary (termed hairpin) RNA containing sequences homologous to the target genes. The DNA sequences encoding the self-complementary regions of hairpin (hp) RNA constructs form an inverted repeat. The inverted repeat can be stabilized in bacteria through separation of the self-complementary regions by a “spacer” region. When the spacer sequence encodes an intron, the efficiency of gene silencing is very high. There are at least three ways in which hpRNA constructs can be made. The construct may be generated from standard binary plant transformation vectors in which the hairpin-encoding region is generated de novo for each gene. Alternatively, generic gene-silencing vectors such as the pHANNIBAL and the pHELLSGATE series can be used. They simply require the insertion of PCR products, derived from the target gene, into the vectors by conventional cloning or by using the Gateway directed recombination system. In this article, we describe and evaluate the advantages of these vectors and then provide the protocols for their efficient use.

Introduction

Double-stranded RNA has been shown to be an effective trigger of gene silencing in vertebrate, invertebrate, and plant systems [1], [2]. This silencing operates by sequence-specific RNA degradation. In plants a particularly effective method of silencing an endogenous gene is to transform the plant with a gene construct encoding a hairpin RNA (hpRNA) consisting of an inverted repeat of a fragment of the gene sequence separated by a spacer to allow easier plasmid construction. Using an intron as this spacer fragment increases the frequency of obtaining silenced plants [3], [4]. Based on these findings we have adopted the intron-spliced hairpin RNA (ihpRNA) construct as our current system of choice for gene silencing and made a generic vector, pHANNIBAL, that facilitates the easy production of ihpRNA constructs. ihpRNA-mediated silencing is highly efficient in a number of plants that show gene silencing, with up to 100% of the transformants generated with a particular gene construct showing some degree of silencing. The silenced plants produced with a particular construct tend to have differing degrees of silencing ranging up to close to 100% silencing of the target gene.

The features of ihpRNA-mediated gene silencing make it particularly attractive for functional genomics applications in which large groups of genes are under study. In these cases a method to reduce or remove the function of these genes can reveal information about their function in the normal growth and development of plants. As hpRNA targets specific genes, each gene in the group under study can be targeted with an hpRNA construct. The hpRNA construct is genetically dominant and therefore phenotypes can be screened in T0 or T1 plants without the need to produce homozygous lines. Most plant transformation systems give rise to a number of transformation events that are propagated as separate transgenic lines. Thus if a phenotype is replicated among the population of plants generated using a particular hpRNA transgene it is highly likely that the phenotype is due to silencing of the target gene rather than a mutation introduced by the transformation procedure. The differing degrees of silencing obtained in the lines produced from a transformation event may allow survival of weakly silenced lines for genes for which a complete loss of function is lethal. The sequence specificity of gene silencing allows the use of unique sequences to target specific genes and the potential to use conserved sequences to target multigene families. We have also shown that constructs targeting two distinct sequences give silencing of both genes, providing a method for silencing two genes of redundant function that do not have a high degree of sequence conservation [5].

A major limitation to using hpRNA-mediated silencing for high-throughput applications is the number of cloning steps needed to produce hpRNA constructs. We therefore designed a vector system (pHELLSGATE) to address this problem by utilizing the Gateway recombination system. Using pHELLSGATE vectors the gene-specific arms of the hairpin construct are inserted in a single recombination step, removing the need for the use of restriction enzymes, greatly streamlining the construction procedure. We have shown that the presence of the recombination sites in the vectors does not adversely affect gene silencing performance compared to the conventional restriction enzyme cloning vectors on which they are based.

In this article we describe the pHANNIBAL and pHELLSGATE vectors, including a new vector that addresses the problem of inversions of the intron that separates the arms of the hairpin. We also discuss the methodology of producing hpRNA constructs using the pHELLSGATE vectors with a particular focus on application to large-scale projects.