The Plant Journal (2010) 61, 873–882
doi: 10.1111/j.1365-313X.2009.04104.x
TECHNICAL ADVANCE
Targeted forward mutagenesis by transitive RNAi Katherine A. Petsch1,†, Chonglie Ma2, Michael J. Scanlon1,* and Richard A. Jorgensen2,3 Department of Plant Biology, Cornell University, Ithaca, NY 14853, USA, 2 Department of Plant Sciences, University of Arizona, Tucson, AZ 85721-0036, USA, and 3 BIO5 Institute, University of Arizona, Tucson, AZ 85721-0240, USA
1
Received 23 August 2009; revised 17 November 2009; accepted 24 November 2009; published online 22 January 2010. * For correspondence (fax 607 255 5407; e-mail
[email protected]). † Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
SUMMARY A novel technique is described that targets specific populations of transcripts for homology-based gene silencing using transitive RNAi. This approach is designed to target a subset of the transcriptome in order to identify genes involved in a particular localized process, such as photosynthesis. As a proof-of-concept approach, mesophyll cells from Arabidopsis thaliana were laser-microdissected from whole leaves to generate a focused cDNA library that was bi-directionally cloned into a transitive RNAi vector that had been designed to induce silencing of homologous, endogenous genes. Approximately 15% of the transformant plants identified from both sense and antisense libraries exhibited visible phenotypes indicative of photosynthetic defects. Amplification from the genome and sequencing of cDNA inserts identified candidate genes underlying the phenotypes. For 10 of 11 such mutants, re-transformation with an RNAi construct corresponding to the candidate gene recapitulated the original mutant phenotype, and reduction of corresponding endogene transcripts was confirmed. In addition, one of the re-transformed transgenes also silenced transcripts of closely related family members, thereby demonstrating the utility of this approach for mutagenesis of redundant gene functions. Preliminary results using tissue-specific transitive RNAi forward mutagenesis of the Arabidopsis vegetative shoot apical meristem demonstrate the broad applicability of this forward mutagenesis technique for a variety of plant cell types. Keywords: RNAi, forward mutagenesis, Arabidopsis thaliana, laser capture microdissection, mesophyll cell.
INTRODUCTION RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that is triggered by double-stranded RNA (dsRNA) and results in the degradation of homologous transcripts. RNAi has been widely exploited as a technique for reverse mutagenesis in plants (Hilson et al., 2004; McGinnis et al., 2005, 2007; Fu et al., 2007b; Tang et al., 2007). The two modes that are most commonly used are inverted repeat RNAi (IR-RNAi) (Waterhouse and Helliwell, 2003) and virus-induced gene silencing (VIGS) (Bernacki et al., 2008). However, both these strategies have limitations for use in high-throughput mutagenesis on a global scale. IR-RNAi is restrictive in that sense and antisense copies of a genic segment must be individually cloned into the vector of interest, a strategy that is not suitable for high-throughput analyses in which it is desirable to produce RNAi constructs for a large population of target transcripts. Although VIGS does permit the generation of ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd
large, comprehensive genic libraries in viral vectors, RNA silencing is limited to those cells and tissues that the virus can reach and locations and conditions that allow viral replication, and therefore it cannot be precisely controlled. An alternative mode of RNAi, termed sense RNAi, is triggered by high levels of translatable ‘sense’ transcripts (Napoli et al., 1990; Que et al., 1997). In Arabidopsis, sense RNAi requires RDR6, an RNA-dependent RNA polymerase (RDRP) that is believed to recognize abundant or aberrant transcripts and produce an antisense copy of RNA, and thereby double-stranded RNA, which then triggers RNA interference. RDR6 acts in a ‘transitive’ manner, i.e. silencing spreads from the site of initiation of synthesis of copy RNA, generally in the 3¢ UTR, to sequences upstream in the sense transcript, but also from internal sites (Lipardi et al., 2001; Sijen et al., 2001; Vaistij et al., 2002; Petersen and 873
874 Katherine A. Petsch et al. Albrechtsen, 2005). A more efficient modification of sense RNAi has been termed transitive RNAi, in which an inverted repeat sequence is placed in the 3¢ UTR (Brummell et al., 2003). This 3¢ inverted repeat is thought to increase the efficiency by which RDR6 initiates copying of the sense transcript to produce dsRNA. The siRNA cleavage products of the inverted repeat region may act as primers for efficient RDRP initiation in the 3¢ UTR of other copies of the same transcript. Significantly, when full-length coding sequences are utilized upstream of a 3¢ UTR inverted repeat, the efficiency of this method as a gene silencing tool approaches that of IR-RNAi (Brummell et al., 2003). Here, we describe a novel forward genetic approach for functional genomics using transitive RNAi that allows targeted mutagenesis of a functionally related subset of the genome by choosing a particular population of RNAs expressed under particular conditions, such as in a specific cell-type or tissue, or as a physiological or environmental response. In the proof-of-concept experiments presented here, we used laser microdissection to select the population of transcripts for targeted mutagenesis by transitive RNAi, but any approach that selects a subset of genes could also be used. By targeting only a specific set of genes that are represented in a selected RNA population, transitive RNAi forward mutagenesis provides an efficient and complementary approach to other forward mutagenesis methods that target the whole genome, such as chemical or insertion mutagenesis. In addition, RNAi silencing methods act in a dominant fashion in trans on all homologous sequences with 80–90% identity. Thus, in addition to circumventing the limitations imposed by existing technologies, this targeted RNAi strategy holds particular promise for use in polyploid crop plants in which genetic redundancy hampers the efficiency of mutagenesis, as well as in diploid plants with frequent recent segmental duplications.
mesophyll cells of approximately 0.5 mm2 (8–10 leaf sections at a thickness of 10 lm) was microdissected (Figure 1a–c), after which the extracted RNA transcripts were converted into cDNA and amplified to generate both sense and antisense mesophyll libraries. Bi-directional (i.e. sense and antisense) libraries were constructed because our preliminary studies indicated that some genes are more efficiently silenced when cloned in one orientation rather than the other (Table S1 and Figure S1). Ten random clones from each of the bi-directional mesophyll tissue libraries were sequenced in order to assess the quality and genic diversity represented therein (Table S2). Transcripts ranged in size from
