The role of AtMUS81 in interference-insensitive crossovers in A. thaliana

Abstract

MUS81 is conserved among plants, animals, and fungi and is known to be involved in mitotic DNA damage repair and meiotic recombination. Here we present a functional characterization of the Arabidopsis thaliana homolog AtMUS81, which has a role in both mitotic and meiotic cells. The AtMUS81 transcript is produced in all tissues, but is elevated greater than 9-fold in the anthers and its levels are increased in response to gamma radiation and methyl methanesulfonate treatment. An Atmus81 transfer-DNA insertion mutant shows increased sensitivity to a wide range of DNA-damaging agents, confirming its role in mitotically proliferating cells. To examine its role in meiosis, we employed a pollen tetrad-based visual assay. Data from genetic intervals on Chromosomes 1 and 3 show that Atmus81 mutants have a moderate decrease in meiotic recombination. Importantly, measurements of recombination in a pair of adjacent intervals on Chromosome 5 demonstrate that the remaining crossovers in Atmus81 are interference sensitive, and that interference levels in the Atmus81 mutant are significantly greater than those in wild type. These data are consistent with the hypothesis that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive.

Figure 1. The AtMUS81 Gene Structure, T-DNA Insertion Mutant, and Expression

(A) An illustration of the AtMUS81 (At4g30870) locus showing the exon/intron organization of AtMUS81. Solid boxes represent transcribed regions including protein coding (black) and untranslated regions (gray). The T-DNA insertion site for the mutant used in this study is shown. Conserved domains are shown above. Below are products for the following primers that were used for genotyping (M81_F/M81_R), RT-PCR (M81S1_F/M81S1R and M81S2_F/M81S2_R), and real-time qPCR (M81RTRT3_F/M81RTRT_R). (B) Whole seedling (10-d) RT-PCR of wild type and the Atmus81 mutant. Primers (S1 and S2) downstream of the T-DNA insertion site were used in the RT-PCR reaction with and without reverse transcriptase (RT) using RNA from wild-type (WT) and mutant (mus) plants. The APT1 transcript was used as a control.

Figure 2. Hypersensitivity of Atmus81 Mutants to MMS, Cisplatin, and Gamma Radiation

(A) Wild-type Col-0 (rows one and three) and mutant Atmus81/Atmus81 (rows two and four) were subjected to a gradient (0–75 ppm) of MMS. The photograph was taken after 20 d. The wild-type plants can grow at each concentration tested while the mutants cannot grow at >30 ppm MMS. (B) Wild-type Col-0 (right side of plate) and mutant Atmus81/Atmus81 (left side of plate) were subjected to various concentrations of cisplatin (0–15 ppm). The photograph was taken after 12 d. Wild-type plants consistently outperformed the mutants at all concentrations tested. (C) Wild-type Col-0 (upper left third), mutant Atmus81/Atmus81 (upper right third), and gamma-hypersensitive mutant atm-2/atm-2 (bottom third) were exposed to various levels of gamma radiation (0–150 Gy). At 75–100 Gy, the Atmus81 mutants resembled the atm-2 mutants rather than the wild-type plants.

Figure 3. Real-Time qPCR Analysis of AtMUS81 Transcription

(A) RNA from untreated (NT) plants, plants harvested immediately after gamma radiation treatment (R0), plants harvested 6 h after gamma radiation treatment (R6), and plants treated with 50 ppm MMS (MS) was used to measure the induction of the AtMUS81 transcript. (B) Real-time qPCR analysis of RNA from wild-type root (RT), stem (ST), leaf (LF), inflorescence (IN), anthers (AN), and silique (SL) tissue as a measure of tissue-specific AtMUS81 expression. The EF1 gene was used as a control. Error bars are +/− the standard error of the mean calculated from four replicates.

Figure 4. Pollen Viability in Wild Type and Atmus81 Mutants

(A) Pollen tetrads from plants homozygous for three different fluorescent markers were examined. Pollen was classified as nonviable if grains were aborted (bottom row) or if all of the fluorescent proteins were not expressed (top row). (B) The Atmus81 mutant has lower levels of pollen viability. Viability of wild type (open bars) is compared to Atmus81/Atmus81 plants (gray bars) and is also broken into tetrad categories (4:0, 3:1, 2:2, 1:3, 0:4; viable:nonviable). Error bars are +/− the standard error of the mean.

Figure 5. Meiotic Recombination in Atmus81 and Atmsh4 Mutants

(A) Characterization of the meiotic recombination phenotype using intervals on Chromosomes 1 (left) and 3 (right). Green bars represent pooled data from wild type and heterozygotes, orange bars represent Atmus81 mutants, blue bars represent Atmsh4 mutants, and purple bars represent Atmus81/Atmsh4 double mutants. Error bars are +/− the standard error of the mean. (B) Interference analysis of the Atmus81 mutant using three linked markers on Chromosome 5. Each pair of graphs (wild type and heterozygotes top; Atmus81 bottom) shows the genetic distances of an interval without and with an adjacent CO. The ratios of these genetic distances with adjacent CO: without a CO were significantly different between the pooled wt/heterozygotes and Atmus81 mutants with a one-tailed p-value of 0.032 (see Materials and Methods for calculation of the p-value).