A genetic screen for suppressors of a hypomorphic allele of Arabidopsis ARGONAUTE1

Abstract

ARGONAUTE1 (AGO1) encodes a key component of the complexes mediating microRNA (miRNA) function in Arabidopsis. To study the regulation, action and interactions of AGO1, we conducted a genetic screen to identify second-site mutations modifying the morphological phenotype of ago1-52, a partial loss-of-function allele of AGO1. Unlike null ago1 mutations, the hypomorphic ago1-52 allele does not cause lethality or sterility; however, ago1-52 does produce a morphological phenotype clearly distinct from wild type. In our screen for modifiers of ago1-52, we identified suppressor mutations that partially restore wild-type morphology in the ago1-52 background and we termed these mas (morphology of argonaute1-52 suppressed). We focused on 23 of these putative suppressors. Linkage analysis of the mas mutations together with sequencing of the AGO1 gene in genomic DNA and cDNA from ago1-52 mas plants indicated that 22 of the mas lines contain extragenic suppressors, and one contains an intragenic suppressor that affects splicing of ago1-52. In the presence of the wild-type allele of AGO1, most of the mas mutations cause a mild or no mutant phenotype on their own, indicating that the ago1-52 mutant may provide a sensitized background for examining the interactions of AGO1.

Figure 1. Some phenotypes of the ago1-52 mutant.

(a–c) Rosettes from (a) the wild type Ler and (b, c) ago1-52. An arrow in c highlights a trumpet-shaped, radialised leaf. (d) Detail of a radialised ago1-52 leaf. (e, f) Terminal region of the inflorescences of (e) Ler and (f) ago1-52. Scale bars: 1 mm. Pictures were taken (a–d) 21 das and (e, f) 42 das.

Figure 2. Flowchart describing the screening strategy for the isolation and genetic analysis of suppressors of ago1-52.

The ↓ and symbols indicate growth and selfing, respectively. The symbol indicates outcrossing or backcrossing.

Figure 3. Vegetative phenotype of the suppressor lines identified in this work.

Rosettes of M₃ plants are shown from 23 of the double mutants isolated, in which the morphological phenotype of ago1-52 is partially suppressed by a second-site mutation. Pictures were taken 21 das. Scale bar: 1 mm.

Figure 4. Examples of other phenotypes of the suppressor lines identified in this work.

Plant height of the (a) ago1-52 mas1-1 (P1 5.33), (b) ago1-52 mas2-1 (P2 11.1) and (c) ago1-52 mas3-1 (P8 14.1) double mutants was intermediate between those of the wild type Ler and the ago1-52 single mutant. As shown in the insets, the structure of the terminal region of the inflorescence in the double mutants was also intermediate between those of Ler (Figure 1e) and ago1-52 (Figure 1f). Pictures were taken (a) 54 das, (b) 43 das and (c) 48 das. Scale bars: (a–c) 1 cm and (insets) 1 mm.

Figure 5. The P9 3.3 line carries an intragenic suppressor mutation of ago1-52.

(a) Structure of the AGO1 gene with indication of the nature of the ago1-52 and ago1-52S mutations (green arrows and red letters). Positions of the start (ATG) and stop (TGA) codons are also indicated. Exons are shown as boxes, and introns as lines between boxes. Open boxes represent untranslated exon sequences. (b) Effects of the ago1-52 and ago1-52S mutations on splicing and mRNA translation. Intron sequences are shown in small case, and exon sequences in capital letters. The preferred splicing acceptor sites are boxed in green. Amino acids different from those of the wild type are shown in red. The asterisk indicates a premature stop codon. (c) Sequencing electropherograms obtained from Ler (AGO1), ago1-52 and P9 3.3 genomic DNA (gDNA; top) and cDNA (bottom). Nucleotide sequences that are intronic in the AGO1 wild type allele but exonic in the ago1-52 mutant and the ago1-52 ago1-52S double mutant are boxed in orange.