The Arabidopsis DNA glycosylase MBD4L repairs the nuclear genome in vivo.

DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl-CpG-binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4-like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5-fluorouracil (5-FU) and 5-bromouracil (5-BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5-FU and 5-BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER-associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5-BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5-FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG-GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L-deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions.

Convergent Epigenetic Mechanisms Avoid Constitutive Expression of Immune Receptor Gene Subsets.

The gene pool encoding PRR and NLR immune receptors determines the ability of a plant to resist microbial infections. Basal expression of these genes is prevented by diverse mechanisms since their hyperactivity can be harmful. To approach the study of epigenetic control of PRR/NLR genes we here analyzed their expression in mutants carrying abnormal repressive 5-methyl cytosine (5-mC) and histone 3 lysine 9 dimethylation (H3K9me2) marks, due to lack of MET1, CMT3, MOM1, SUVH4/5/6, or DDM1. At optimal growth conditions, none of the mutants showed basal expression of the defense gene marker PR1, but all of them had greater resistance to Pseudomonas syringae pv. tomato than wild type plants, suggesting they are primed to stimulate immune cascades. Consistently, analysis of available transcriptomes indicated that all mutants showed activation of particular PRR/NLR genes under some growth conditions. Under low defense activation, 37 PRR/NLR genes were expressed in these plants, but 29 of them were exclusively activated in specific mutants, indicating that MET1, CMT3, MOM1, SUVH4/5/6, and DDM1 mediate basal repression of different subsets of genes. Some epigenetic marks present at promoters, but not gene bodies, could explain the activation of these genes in the mutants. As expected, suvh4/5/6 and ddm1 activated genes carrying 5-mC and H3K9me2 marks in wild type plants. Surprisingly, all mutants expressed genes harboring promoter H2A.Z/H3K27me3 marks likely affected by the chromatin remodeler PIE1 and the histone demethylase REF6, respectively. Therefore, MET1, CMT3, MOM1, SUVH4/5/6, and DDM1, together with REF6, seemingly contribute to the establishment of chromatin states that prevent constitutive PRR/NLR gene activation, but facilitate their priming by modulating epigenetic marks at their promoters.