Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas

Abstract

SummaryRNA interference (RNAi), the double-stranded RNA (dsRNA) triggered post-transcriptional gene silencing, is becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of inverted repeat (IR) transgenes, has been achieved in protozoa, algae, fungi, plants, and metazoans. However, the level of gene silencing is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. This is a hindrance in functional genomics studies, where it is desirable to suppress target genes reliably to analyze unknown phenotypes. Consequently, we explored strategies for direct selection of effective transgenic RNAi lines in Chlamydomonas reinhardtii. We initially attempted to suppress expression of the Rubisco small subunit multigene family by placing an IR, homologous to the conserved coding sequence, in the 3¢UTR of a transgene conferring resistance to bleomycin. However, this approach was fairly inefficient at inducing RNAi as many strains displayed defective transgene integration, resulting in partial or complete deletion of the IR, or low levels of dsRNA expression, presumably due to transcriptional silencing of the integrated IR transgenes. To overcome these problems we designed a system consisting of tandem IR transgenes that consistently triggered co-silencing of a gene with a selectable RNAiinduced phenotype (encoding tryptophan synthase b subunit) and another gene of interest (encoding either Ku80, an RNA-binding protein, or a thioredoxin isoform). We anticipate that this approach will be useful for generating stable hypomorphic epi-mutants in high-throughput phenotypic screens.

On the origin and functions of RNA-mediated silencing: from protists to man

“…Archaeplastida Chlamydomonas reinhardtii Rohr et al (2004) and Schroda (2006) Arabidopsis thaliana Llave et al (2002) and and Watson et al (2005) Direct b Matzke and Birchler (2005) and Zilberman et al (2003) Chan et al (2004) and Matzke and Birchler (2005) Chen ( Aspergillus nidulans Hammond and Keller (2005) among the RNAi-positive organisms listed in Table 1 is Giardia intestinalis that encodes a protein with a well-defined PIWI domain fused to a highly divergent PAZ domain. However, Giardia protein-encoding genes are notoriously fast-evolving compared with those of most other eukaryotes (Richards and Cavalier-Smith 2005), which might explain the poor conservation of the PAZ domain.…”

“…RdRPs are not as widely distributed among eukaryotes as Ago-Piwi and Dicer-like proteins (Table 1). Even though RNAi occurs in animals (Schwarz et al 2002;Stein et al 2003), Chlamydomonas reinhardtii (a green alga) (Rohr et al 2004;Schroda 2006), and T. brucei ), RdRPs were not detected in their genomes (Table 1), with the exception of C. elegans and Branchiostoma floridae (AAQ10792). In Aspergillus nidulans it has been experimentally demonstrated that RNAi induced by an inverted repeat transgene does not require any of the two RdRPs encoded in the genome (Hammond and Keller 2005).…”

Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo‐oxidative stress in response to iron availability in Chlamydomonas reinhardtii

“…Both constructs were transformed into wild-type cells. In the following section, we present data that were obtained with the RNAi construct designed according to the method described by Rohr et al (2004) due to its higher stability compared to the classical RNAi approach. Nevertheless, both RNAi approaches showed similar results.…”

“…A classical RNAi construct, using the expression vector pCB740 (Schroda et al, 1999) with 440 bp of the 5¢ coding sequence of ferritin in the antisense orientation, was generated. The second RNAi construct contained a cassette consisting of 410 bp of the coding region of ferritin in antisense orientation separated by a 310-bp long spacer and was inserted in the RNAi vector designed by Rohr et al (2004). Both constructs were transformed into wild-type cells.…”

“…Its ability to grow photo-heterotrophically or photo-autotrophically, the possibility of transforming all three genomes (Rochaix, 1995), all of which are sequenced (Maul et al, 2002;Merchant et al, 2007;Michaelis et al, 1990), and numerous molecular tools such as RNAi (Rohr et al, 2004) as well as proteomic approaches (Hippler et al, 2001) make Chlamydomonas a versatile model organism. Numerous studies have been published with respect to its iron homeostasis and consequences of iron deficiency (Allen et al, 2007;La Fontaine et al, 2002;Moseley et al, 2002;Naumann et al, 2005Naumann et al, , 2007, showing that Chlamydomonas reacts in a fine-tuned manner to changing iron availability.…”