Unusual Case of Apparent Hypermutation in Arabidopsis thaliana

Abstract

The dms4 (defective in meristem silencing 4) mutant of Arabidopsis thaliana is unique in having defects in both RNA-directed DNA methylation (RdDM) and plant development. DMS4 is an evolutionarily conserved, putative transcription factor of the Iwr1 (interacts with RNA polymerase II) type. DMS4 interacts with Pol II and also with RNA polymerases IV and V, which function in RdDM. Interactions with multiple polymerases may account for the diverse phenotypic effects of dms4 mutations. To dissect further the roles of DMS4 in RdDM and development, we performed a genetic suppressor screen using the dms4-1 allele, which contains in the sixth intron a splice site acceptor mutation that alters splicing and destroys the open reading frame. Following mutagenesis of dms4-1 seeds using ethyl methanesulfonate (EMS), we retrieved four dominant intragenic suppressor mutations that restored DMS4 function and wild-type phenotypes. Three of the four intragenic suppressor mutations created new splice site acceptors, which resulted in reestablishment of the wild-type open reading frame. Remarkably, the intragenic suppressor mutations were recovered at frequencies ranging from 35 to 150 times higher than expected for standard EMS mutagenesis in Arabidopsis. Whole-genome sequencing did not reveal an elevated mutation frequency genome-wide, indicating that the apparent hypermutation was confined to four specific sites in the dms4 gene. The localized high mutation frequency correlated with restoration of DMS4 function implies an efficient mechanism for targeted mutagenesis or selection of more fit revertant cells in the shoot apical meristem, thereby rapidly restoring a wild-type phenotype that is transmitted to future generations.

Distinct and concurrent pathways of Pol II‐ and Pol IV‐dependent siRNA biogenesis at a repetitive trans‐silencer locus in Arabidopsis thaliana

“…The protein extraction, SDS‐PAGE and western blotting to detect GFP protein were carried out as previously described (Sasaki et al ., ).…”

“…The sequence of the silencer locus in the T+S D35S line ( Figure S2) was deduced from whole-genome sequence data. Paired-end sequencing of a genomic DNA library was performed with 72-bp read length using an Illumina Genome Analyzer II, as described previously (Eun et al, 2011;Sasaki et al, 2012). For assembling the sequence of the silencer (S D35S ) locus in the T+S D35S line, the sequence of the S WT locus (accession number HE584556) was used as a reference sequence (Eun et al, 2011).…”

GFP Loss-of-Function Mutations inArabidopsis thaliana

“… Amino acid residues (column 1) are numbered according to wild-type GFP. In the present study and two previous studies ( Sun et al 2012 and Sasaki et al 2012 ), a modified GFP containing an additional GTG (Val) codon in position 2 after the ATG (Met) start codon was used to optimize translation initiation in eukaryotes. However, for consistency of numbering with wild-type GFP, we have treated in this article the Val in position 2 as 1a ( Zacharias and Tsien 2006 ).Therefore, amino acid numbers in the previous studies ( Sun et al 2012 , Sasaki et al 2012 ) are one higher compared to wild-type GFP.…”

“…In this study we used an Arabidopsis thaliana transgenic line in a Columbia (Col) ecotype background that is homozygous for a previously described target ( T ) locus encoding enhanced GFP protein (EGFP). This reporter gene is expressed primarily in shoot and root meristem regions and also in the hypocotyl (part of stem between seed leaves and root) of young seedlings ( Kanno et al 2008 , 2010 ; Sasaki et al 2012 , 2015 ). EGFP was purchased from Clontech as plasmid pIRES2-EGFP.…”

“…For seedlings showing very weak or no visible fluorescence, the GFP gene was sequenced to identify possible gfp loss-of-function mutations. From a screen of approximately 280,000 M2 seedlings (approximately seven M2 progeny were tested from each M1 plant) ( Haughn and Somerville 1990 ; Jander et al 2003 ), we retrieved 20 unique gfp mutants, which were combined for further analysis with eight additional unique, uncharacterized gfp mutants identified in a separate screen based on the same GFP reporter gene ( Sasaki et al 2012 ). Images of selected gfp mutants shown in Figure S2 were made using a Leica TCS LSI-III Confocal Microscope System.…”