Abstract
In Arabidopsis, the RING finger-containing E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) functions as a main regulator of the cold signaling. In this study, CRISPR/Cas9-mediated targeted mutagenesis of the HOS1 gene in the first exon was performed. DNA sequencing showed that frameshift indels introduced by genome editing of HOS1 resulted in the appearance of premature stop codons, disrupting the open reading frame. Obtained hos1Cas9 mutant plants were compared with the SALK T-DNA insertion mutant, line hos1-3, in terms of their tolerance to abiotic stresses, accumulation of secondary metabolites and expression levels of genes participating in these processes. Upon exposure to cold stress, enhanced tolerance and expression of cold-responsive genes were observed in both hos1-3 and hos1Cas9 plants. The hos1 mutation caused changes in the synthesis of phytoalexins in transformed cells. The content of glucosinolates (GSLs) was down-regulated by 1.5-times, while flavonol glycosides were up-regulated by 1.2 to 4.2 times in transgenic plants. The transcript abundance of the corresponding MYB and bHLH transcription factors, which are responsible for the regulation of secondary metabolism in Arabidopsis, were also altered. Our data suggest a relationship between HOS1-regulated downstream signaling and phytoalexin biosynthesis.
Highlights
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has become a versatile approach of targeted genetic manipulations in plants [1,2]
We constructed plant CRISPR/Cas9 all-in-one binary vectors harboring components for HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) mutagenesis, including the plant codon-optimized Cas9 and enhanced green fluorescent protein (EGFP), both controlled by the 35S CaMV promoter, a sgRNA
Two gRNAs targeting exon 1 and 2 of the HOS1 gene were designed (Figure 1C,D), CRISPR-OFFinder was used for off-target scoring [52]; no potential off-targets were detected with 0 to 2 mismatches in the core sequence
Summary
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has become a versatile approach of targeted genetic manipulations in plants [1,2]. In contrast to the classical loss-of-function approach introducing random mutations, Cas protein driven by an engineered 20-nt RNA sequence (short guide RNA; sgRNA) performs strand scission of a specific target sequence within the genome, enabling precise corrections [3]. Cas nickase and dCas, coupled with other functional enzymes, may be used as carriers to a certain region in the genome targeted by sgRNA. Using this technique, gene activation or repression, single nucleotide substitutions and epigenetic editing of chromatin status have been reported [8]
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