Efficiency of gene silencing in Arabidopsis: direct inverted repeats vs. transitive RNAi vectors.
We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1, ETTIN and TTG1). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required.
Filichkin
Sergei A
SA
Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.
DiFazio
Stephen P
SP
Brunner
Amy M
AM
Davis
John M
JM
Yang
Zamin K
ZK
Kalluri
Udaya C
UC
Arias
Renee S
RS
Etherington
Elizabeth
E
Tuskan
Gerald A
GA
Strauss
Steven H
SH
D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics*; D016000:Cluster Analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D035264:Flowers / Q000235:genetics / Q000254:growth & development; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics; D026161:MADS Domain Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D034622:RNA Interference; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C091535:AP1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; C109765:ETT protein, Arabidopsis; D026161:MADS Domain Proteins; D009687:Nuclear Proteins; C121067:TTG1 protein, Arabidopsis