Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Abstract

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69(159) and amiR-HC-Pro(159) are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP(159) targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69(159) and amiR-HC-Pro(159) from a dimeric pre-amiR-P69(159)/amiR-HC-Pro(159) transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 degrees C, a temperature that compromises small interfering RNA-mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.

Rapid Assessment of Gene Function in the Circadian Clock Using Artificial MicroRNA in Arabidopsis Mesophyll Protoplasts

“…The advantages and effectiveness of amiRNA technology in plant systems have been verified from several studies using transgenic approaches (Alvarez et al, 2006;Niu et al, 2006;Schwab et al, 2006;Choi et al, 2007;Ossowski et al, 2008) since the first finding of amiRNA activity in Arabidopsis (Parizotto et al, 2004). Our study extends amiRNA versatility into Arabidopsis protoplasts.…”

Gene silencing: concepts, applications, and perspectives in woody plants

“…Recently, it has been demonstrated that the modification of plant endogenous miRNA precursors to interfere with viral mRNA sequences can confer virus resistance in Arabidopsis thaliana (Niu et al, 2006). These modified miRNA are termed artificial miRNA and this technique will open new perspectives for engineering viral resistant plants.…”

“…VIGS can be defined as the silencing of endogenous plant genes initi- repeats (Meister & Tuschl, 2004); (B) Dicer-like protein (DCL2, DCL3) processing of intermediates formed during RNA virus replication (Hannon, 2002); (C) Long dsRNA; (D) Dicer-like protein (DCL1) processing of miRNAs precursors ; (E) RNA-induced silencing complex (RISC) (Hammond et al, 2000;Nykänen et al, 2001); (F) Targeting and cleavage of sequence specific mRNA by RISC (Martinez & Tuschl, 2004); (G) mRNA destruction after RISC processing (Tolia & Joshua-Tor, 2006); (H) Possible systemic signal composed by siRNA + movement proteins; (I) Possible systemic signal composed by long dsRNA + movement proteins (Waterhouse et al, 2001); (J) Primer dependent RdRP amplification (Matzke et al, 2001;Ceruti, 2003;Baulcombe, 2004); (K) Primer-independent (aberrant RNA features) RdRP amplification (Baulcombe, 2004); (L) Primary siRNA (Pak & Fire, 2007); (M) Secondary siRNA processing by Dicer-like enzymes (Pak & Fire, 2007); (N) RNA-directed DNA methylation (RdDM) signal transmitted from the cytoplasm to the nucleus is most likely siRNA ; (O) RNAi effector complex termed RITS (RNA-induced Initiation of Transcriptional gene Silencing) required for heterochromatin assembly in fission yeast (Schizosaccharomyces pombe) (Verdel et al, 2004). RITS is composed by repeat-associated short interfering RNA (rasiRNA) (Meister & Tuschl, 2004); (P) Translational repression of mRNA by miRNP (Meister & Tuschl, 2004 In early approaches on VIGS studies, gene sequences were individually subcloned into viral genomes, and plants were physically inoculated using viral RNA produced by in-vitro transcriptional reactions (Kumagai et al, 1995).…”

“…After Dicer processes the dsRNA, the siRNAs are subsequently rearranged into the RNA-induced silencing complex (RISC) ( Figure 1E) (Hammond et al, 2000;Nykänen et al, 2001). The RISC complex was originally identified through fractionation of sequence specific nuclease activity from D. melanogaster extracts Tolia & Joshua-Tor, 2006).…”

Alternate approaches to repress endogenous microRNA activity inArabidopsis thaliana

“…17,[45][46][47][48][49] The structural features of the endogenous miRNA precursor transcript on which an amiRNA plant expression vector is based are maintained while the miRNA/miRNA* duplex is replaced with amiRNA and amiRNA* sequences. Such a design ensures that the modified precursor transcripts are still recognized and processed by the protein machinery of the miRNA biogenesis pathway to produce a single specific 21-nt silencing signal.…”