Assessing the Efficiency of RNA Interference for Maize Functional Genomics

Abstract

A large-scale functional genomics project was initiated to study the function of chromatin-related genes in maize (Zea mays). Transgenic lines containing short gene segments in inverted repeat orientation designed to reduce expression of target genes by RNA interference (RNAi) were isolated, propagated, and analyzed in a variety of assays. Analysis of the selectable marker expression over multiple generations revealed that most transgenes were transmitted faithfully, whereas some displayed reduced transmission or transgene silencing. A range of target-gene silencing efficiencies, from nondetectable silencing to nearly complete silencing, was revealed by semiquantitative reverse transcription-PCR analysis of transcript abundance for the target gene. In some cases, the RNAi construct was able to cause a reduction in the steady-state RNA levels of not only the target gene, but also another closely related gene. Correlation of silencing efficiency with expression level of the target gene and sequence features of the inverted repeat did not reveal any factors capable of predicting the silencing success of a particular RNAi-inducing construct. The frequencies of success of this large-scale project in maize, together with parameters for optimization at various steps, should serve as a useful framework for designing future RNAi-based functional genomics projects in crop plants.

RNA interference for wheat functional gene analysis

“…1a, Yan et al 2004). Results from the previous two studies indicate that in order to trigger RNAi the two sequences need to share a continuous stretch of identical nucleotides longer than 18-nt, which is consistent with the ''21-nt rule'' that indicates that at least 21-nt of continuous perfect identity are required to trigger RNAi (Miki et al 2005;McGinnis et al 2007).…”

“…Other factors, such as GC content and predicted melting temperature of the RNA hairpin, have been suggested to play a role in determining the efficacies of RNAi triggers (Reynolds et al 2004). However, these characteristics were not significantly correlated to non-target silencing efficiency in a comprehensive study in maize (McGinnis et al 2007), suggesting that additional experiments will be required to identify the factors that affect RNAi silencing efficiency beyond the ''21-nt rule.'' For example, it would be interesting to create artificial RNAi triggers with high sequence identity to the different homoeologues, but with mutations spaced out evenly at intervals of 19-23-nt.…”

“…During the RNAi response in plants, cleavage of dsRNA produces siRNAs of 21-26 nt (Hamilton et al 2002;Llave et al 2002;Tang et al 2003;Qi et al 2005). Therefore, the presence of a continuous stretch of at least 21 identical nucleotides between the trigger and the target gene is required, although it is not always sufficient to produce efficient silencing (Miki et al 2005;Yue et al 2007;McGinnis et al 2007).…”

“…The SBEII-a transgenic plants showed reduced levels of both proteins, whereas SBEII-b transgenic plants showed reduced protein levels only for SBEII-b. In addition, two recent studies in wheat (Yue et al 2007) and maize (McGinnis et al 2007) presented examples of genes whose transcript levels were not significantly affected by RNAi in spite of including 21-nt of perfect identity with the RNAi trigger sequence. Other factors, such as GC content and predicted melting temperature of the RNA hairpin, have been suggested to play a role in determining the efficacies of RNAi triggers (Reynolds et al 2004).…”

Interspecific RNA Interference of SHOOT MERISTEMLESS-Like Disrupts Cuscuta pentagona Plant Parasitism

“…Dodder growing on plants transformed with this construct showed no downregulation of the KNAT1-3 homolog. This could be due to possible secondary structure that makes the target message inaccessible to the siRNAs, inefficient selection of the targeted region, or other as yet unknown reasons (McGinnis et al, 2007). The dodder strands at the attachment points with mature haustoria had low levels of STM expression and did not show differences between dodder grown on the wild type compared with transgenic hosts.…”