CRISPR/Cas9 generates viable mutant alleles of DRE2

Because complete loss of DRE2 functions leads to embryonic lethality [32], which precludes investigation of DRE2’s functions, we set out to create dre2 hypomorphic mutants. For this purpose, we designed sgRNA1 to sgRNA3 targeting the first exon (corresponding to the N-terminal Methyltransferase-like domain [33]), the sixth exon (corresponding to the C-terminal CIAPIN1 domain [33]) and the junction of the fourth intron and exon (corresponding to the start of linker region), respectively (S1A and S1B Fig). Mutant lines carrying sgRNA1 or sgRNA2 developed chlorotic leaf spots or stripes reminiscent of cell death (S1C Fig). However, we were unable to obtain homozygous lines, indicating that these homozygous lines have lethal defects. Fortunately, we obtained two homozygous dre2 mutant lines from the transformants expressing sgRNA3. The first line, named as dre2-3, harbors a 27-nucleotides deletion in the fourth exon (Fig 1A), which causes a deletion of the amino acids ‘IKAKKPSWK’ (Fig 1B). The second line, named as dre2-4, harbors a deletion of the 3′ splicing site ‘AG’ in the fourth intron (Fig 1A), which disrupts splicing of the DRE2 mRNA (Fig 1C). We cloned DRE2 cDNA from dre2-4. Seven splicing variants were identified from 50 clones we sent for sequencing. Two of them are deletion variants which deletes amino acids (DRE2^Δ6 and DRE2^Δ75). The rest of them all contain premature stop codons, which are resulted from either deletion (DRE2^Δ85, DRE2^Δ112 and DRE2^Δ127), insertion (DRE2⁺²⁰) or one intron-retention (DRE2⁺²²¹) (Fig 1D; S1F and S1G Fig). No normal DRE2 transcripts was identified from those clones, suggesting that the primary 3′ splicing site ‘AG’ is important for correct splicing of DRE2 mRNA (S1F Fig). DRE2 transcripts are slightly decreased in dre2-3, while reduced to about 40% in dre2-4 (Fig 1E; S1D Fig). Like mutant lines carrying sgRNA1 or sgRNA2 (S1C Fig), dre2-4 has chlorotic mosaics and wrinkled leaves (S1E Fig). To verify that the CIA-dependent function of DRE2 is at least partially compromised, we analyzed the activities of aldehyde oxidases (AO) [17], a representative Fe-S containing enzyme, in dre2-3 and dre2-4. In-gel AO activity staining revealed that the AO activities are indeed lower in dre2-3 and dre2-4 than that in Col-0 (Fig 1F). However, we did not find seed abortion in dre2-3 and dre2-4. We further introduced the pFWA::ΔFWA-GFP reporter into dre2-4. The expression of ΔFWA-GFP in the central cell and endosperm was normal in dre2-4 (S2 Fig), suggesting that dre2-4 does not affect epigenetic activation of FWA. Thus, dre2-3 and dre2-4 may only lose some of DRE2’s functions.

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Fig 1. Generation of dre2 hypomorphic mutants using the CRISPR/Cas9 system.

(A) CRISPR/Cas9-induced deletions in dre2-3 and dre2-4. The sgRNA-3 targeting sequence is highlighted in red and the PAM site is underlined. Exons are in upper case letters and introns in lower case. (B) The dre2-3 mutation causes a deletion of nine amino acids. (C) The dre2-4 mutation causes a splicing defect of DRE2 pre-mRNA. RT-PCR shows that the splicing variants of DRE2 mRNA in dre2-4. The positions of primers for RT-PCR are indicated by blue arrows in S1F Fig. (D) Summary of the splicing variants of DRE2 mRNA in dre2-4. (E) Relative expression levels of DRE2 in the indicated genotypes as determined by RT-qPCR. Data are presented as mean ± SD of four technical replicates. Asterisks indicate two-tailed Student’s t-test, *P < 0.05, **P < 0.01. Results from the second biological replicate is shown in S1D Fig. (F) In-gel activities of AO in Col-0 and dre2 mutants. Equal amounts of protein (400 μg) extracted from seedlings were separated on nondenaturing PA gels and stained using synthetic aldehydes (1-naphthaldehyde) as substrates. Coomassie brilliant blue staining shows equal loading. The positions of the three bands, AO1, AO2, and AO3, are indicated with arrows.

https://doi.org/10.1371/journal.pgen.1008094.g001

The viable mutants of DRE2 have accumulation of DNA damage and constitutively activation of DNA damage response

The CIA pathway mutants, including the ae7, met18 and grxs17 mutants, are characteristic of a compromise in DNA repair and accumulation of DNA damage [19,20,22,30]. To test whether the dre2 mutants have the same defects, we conducted comet assays using the alkaline unwinding/neutral electrophoresis (A/N) protocol, which measures DNA SSBs as well as double strand breaks (DSBs) (Fig 2A). Based on relative signal intensities of the comet tail, we classified a broad spectrum of DNA damage into four levels: 1%, 10%, 30% and 50%. The majority of cells had 1% DNA damage and less than 2% cells had 30% and higher level of DNA damage in Col-0. However, the percentages of cells with 1% DNA damage dramatically decreased and the percentages of cells with 30% and 50% DNA damage increased to 65% or higher in dre2-3, dre2-4 and met18-2 (Fig 2A and 2B), suggesting accumulation of DNA damage in these mutants. In line with the comet assay results, dre2-4 showed higher sensitivity to MMS, a DNA double strand break (DSB)-inducing chemical, than Col-0 (S3A Fig).

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Fig 2. The dre2 mutants have accumulation of DNA damage and constitutive activation of DNA damage response.

(A) Typical comet images of nuclei from 21-day-old leaves of Col-0, dre2-3, dre2-4 and met18-2. DNA damage was expressed as the ratio of the signal intensity of a comet tail versus that of the entire nucleus. (B) Frequency distribution of different grades of DNA damage in Col-0, dre2-3, dre2-4 and met18-2. At least 200 comets were counted for each genotype. (C) Relative expression levels of the indicated genes in the indicated genotypes as determined by RT-qPCR. Data are presented as mean ± SD of four technical replicates. Asterisks indicate two-tailed Student’s t-test, *P < 0.05, **P < 0.01. Statistic tests are performed between Col-0 and the dre2 mutants either with or without MMS treatment. Results from the second biological replicate are shown in S4 Fig. (D) Short root phenotype of the dre2 mutants. Images of 7-day-old Col-0, dre2-3, dre2-4 and 35S::DRE2/dre2-4 grown on vertical 1/2 MS plates were taken. (E) Statistic analysis of primary root lengths of 7-day-old Col-0, dre2-3, dre2-4 and 35S::DRE2/dre2-4 seedlings. Mean ± SD, n = 10; Asterisks indicate two-tailed Student’s t-test, *P < 0.05, **P < 0.01.

https://doi.org/10.1371/journal.pgen.1008094.g002

We further detected the expression levels of multiple genes involved in DNA Damage Response (DDR), including PARP1, PARP2 [34], PCNA [35], BRCA1, GR1, DMC1, RAD51, RAD54, TSO2 and BARD1 [36] in Col-0, dre2-3 and dre2-4 without or with the application of MMS. We found that PARP1, PARP2, PCNA, BRCA1, DMC1 and RAD51 were activated in dre2-3 and dre2-4 even without MMS treatment, whereas the rest were activated only after MMS treatment (Fig 2C). Multiple cyclin genes, including Cyclin-B1-1, Cyclin-B1-2, Cyclin-A1-1, Cyclin-A2-4 and Cyclin-B2-4 were also upregulated in dre2-3 and dre2-4 (S3B Fig), suggesting that the accumulation of DNA damage and activation of DDR in these mutants arrested cell cycle progression. The short root phenotype is often associated with activation of DNA damage and cell cycle arrest. We also observed that dre2-3 and dre2-4 developed shorter roots than Col-0 (Fig 2D and 2E). It appeared that dre2-4 is a stronger allele compared to dre2-3, as dre2-4 had more DNA damage, more significant upregulation of DDR and cyclin genes and more severe short root phenotype (Fig 2). To exclude the possibility that these phenotypes are off-target effects arose from the CRISPR/Cas9 strategy, we introduced the 35S::DRE2 transgene into dre2-4. The upregulation of DDR genes and cyclin genes and the short root phenotype in dre2-4 were all rescued by overexpression of wild type DRE2 in dre2-4 (Fig 2D and 2E; S4 Fig), suggesting that these phenotypes result from DRE2 dysfunction.

The viable mutants of DRE2 have DNA hypermethylation at hundreds of loci

The CIA pathway mutants, including the ae7, met18 and nbp35 mutants, have DNA hypermethylation at specific loci due to impaired activity of ROS1 [19,20,22,31], a DNA glycosylase which catalyzes the first step of active DNA demethylation [37]. To assess the effects of mutation of DRE2, an early-acting CIA factor, on DNA methylation patterns, we performed whole genome bisulfite sequencing on dre2-4. In total, we identified 1459 differentially methylated regions (DMRs) in dre2-4, including 951 hyper-DMRs and 508 hypo-DMRs (S5A Fig; S1 Dataset). Of the 951 hyper-DMRs, 633 (66.6%) are overlapping with those in ros1-4 (Fig 3A; S5A Fig). Hyper-DMRs unique to dre2-4 are mainly hyper-methylated in CG context (Fig 3A). The CG methylation levels of these dre2-4 specific hyper-DMRs in ros1-4 are also higher than those in Col-0, suggesting that hyper-methylated regions in dre2-4 and ros1-4 actually overlap to a larger extent (Fig 3A). Heat maps show that the methylation levels of dre2-4 hyper-DMRs in met18-2 and ros1-4 are also higher than that in Col-0 in CG, CHG, and CHH contexts (Fig 3B). Analysis of the distribution of the dre2-4 hyper-DMRs in different genomic regions (gene body region, intergenic region, TEs out of gene region and TEs overlapping with gene region) revealed that more than 60% of the hyper-DMRs are distributed in gene body regions in dre2-4 (S5B Fig) much more than that of ros1-4. Collectively, these results suggest that mutation of the early-acting CIA factor DRE2 has a similar effect on overall DNA methylation patterns as mutation of the late-acting MET18. We further examined whether ROS1 promoter, hypermethylation of which induces ROS1 expression [38,39], is hypermethylated in dre2-4, as in met18-2. We found similar hypermethylation of ROS1 promoter in dre2-4 and met18-2 (S5C Fig). Results of Locus-specific bisulfite sequencing confirmed this (Fig 3C). As a result of ROS1 promoter hypermethylation, ROS1 transcripts are upregulated in dre2-3, dre2-4, met18-2 and ros1-4 (Fig 3D). The upregulation of ROS1 expression was also rescued by overexpression of wild type DRE2 in dre2-4 (S5D Fig). Despite upregulation of ROS1 transcripts, the DNA hypermethylation phenotype of dre2-4 exists and persists, suggesting impaired ROS1 activity.

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Fig 3. Dysfunction of DRE2 causes genome-wide DNA hypermethylation.

(A) Venn diagram showing the numbers of hyper-DMRs that either overlap between or are unique in dre2-4 and ros1-4. Boxplots showing the distribution and the average CG, CHG, or CHH methylation levels calculated from hyper-DMRs in the respective subgroups. Dark horizontal line, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; whiskers, minimum and maximum percentage of DNA methylation. (B) Heat-map showing the methylation levels of dre2-4-associated hyper-DMRs in Col-0, dre2-4, met18-2 and ros1-4. The color key is presented at right. Light yellow indicates low methylation and black indicates high methylation. (C) Locus-specific bisulfite sequencing results showing the DNA methylation levels of the ROS1 promoter in different genotypes. (D) Relative expression levels of ROS1 in the indicated genotypes as determined by RT-qPCR. Asterisks indicate two-tailed Student’s t-test, *P < 0.05, **P < 0.01. Results from the second biological replicate are shown in S5D Fig.

https://doi.org/10.1371/journal.pgen.1008094.g003

DRE2 mutations affect protein-protein interactions in the CIA pathway

To investigate the underlying cause for defects in the dre2-3 and dre2-4 mutants, we carried out yeast two-hybrid (Y2H) assays to assess whether crucial protein-protein interactions are affected in dre2-3 and dre2-4. As characterized above, a deletion mutant of DRE2, referred to as DRE2-3, is produced in dre2-3, whereas, two deletion mutants of DRE2, referred to as DRE2^Δ6 and DRE2^Δ75, are produced in dre2-4 (Fig 1). Alignment of the deletion mutants with wild type DRE2 revealed that all of the deletions are located within the unstructured linker region that separate the N-terminal methyltransferase domain and the C-terminal CIAPIN1 domain [40] (S1B Fig). They start from Isoleucine139 (Ile139), but they end at different lysines (Ks) (S1G Fig). We first detected the interactions of these deletion mutants with TAH18 and NBP35. All of the deletion mutants interacted with TAH18, like wild type DRE2 (Fig 4A). However, they showed much stronger interaction with NBP35 compared with wild type DRE2 (Fig 4A). We next examined the interactions of these deletion mutants with GRXS17. While DRE2^Δ6 interacted normally with GRXS17, DRE2-3 and DRE2^Δ75 lost interaction with GRXS17 (Fig 4B). This is consistent with previous findings that the unstructured linker region of anamorsin in human cells reinforces its interaction with GRX3 [27]. Together, our results suggest that the linker region in DRE2 inhibits DRE2-NBP35 interaction but promotes DRE2-GRXS17 interaction.

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Fig 4. The linker region of DRE2 coordinates protein-protein interactions in the CIA pathway.

(A-B) Interactions of DRE2, DRE2^Δ6, DRE2-3 and DRE2^Δ75 with TAH18, NBP35 and GRXS17 as determined by Y2H assays. Yeast cells harboring different fusion protein combinations (listed at the left) in pGBK-T7 (BD) and pGAD-T7 (AD) vectors were plated on medium lacking Leu, Trp (SD-TL), medium lacking Leu, Trp and His (SD-TLH) and medium lacking Leu, Trp, His and Ade (SD-TLHA) (A) or SD-TLH medium with 1 mM 3AT (B).

https://doi.org/10.1371/journal.pgen.1008094.g004

DRE2 is involved in auxin response

To uncover more biological functions of DRE2, we performed RNA-seq to identify the differentially expressed genes between 12-day-old Col-0 and dre2-4 seedlings. Three biological replicates were performed and over 50 M high quality reads were obtained from the RNA library constructed from each sample. More than 90% of the reads could be uniquely mapped to the Tair10 Arabidopsis genome. In total, we identified 1593 genes that were significantly upregulated and 1111 genes that were significantly downregulated (fold-change>2, q<0.01) in dre2-4 as compared to Col-0 (S2 Dataset). Gene Ontology (GO) analysis of the upregulated genes revealed 77 enriched biological processes (FDR<0.05), including ‘response to ionizing radiation’, ‘response to gamma radiation’, ‘cell cycle’ and ‘DNA replication’ (S6A and S6C Fig). This is in accordance with our results that mutations in DRE2 cause accumulation of DNA damage and constitutive activation of DDR (Fig 2). GO analysis of the downregulated genes revealed 22 enriched biological processes (FDR<0.05), among which ‘response to auxin stimulus’ is enriched (Fig 5A; S6B and S6D Fig).

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Fig 5. DRE2 is involved in auxin response.

(A) Biological processes associated with downregulated genes in dre2-4. (B) Relative expression levels of CIA pathway genes after 10 μM IAA treatment. The expression level of MET18 at 0 h was set as 1. Data are presented as mean ± SD of four technical replicates. (C) Primary and lateral root phenotypes of 9-day-old Col-0, dre2-3, dre2-4 and met18-2 without or with the application of 10 μM IAA. (D) Statistical analysis of primary root length (upper) and lateral root numbers (lower) of Col-0, dre2-3, dre2-4 and met18-2 seedlings. Mean ± SD, n ≥ 20; Asterisks indicate two-tailed Student’s t-test, *P < 0.05, **P < 0.01.

https://doi.org/10.1371/journal.pgen.1008094.g005

To confirm that DRE2 is involved in auxin responses, we first performed RT-qPCR to detect the expression levels of DRE2 and other CIA pathway components after treatment with IAA, the natural auxin. While the expression of other CIA pathway proteins were mildly induced, the expression of DRE2 was greatly induced by IAA, especially at early stage after IAA treatment (Fig 5B). Secondly, we tested whether the growth of the primary root, which is inhibited by auxin, is affected in the dre2 mutants. The effect of IAA treatment on primary root length was much weaker in the dre2 mutant compared to that in wild type and met18-2 (Fig 5C and 5D; S7 Fig), indicating that the dre2 mutations confer auxin insensitivity. Third, we tested whether the increase of lateral root formation, that is promoted by auxin [41,42], is affected in dre2-3, dre2-4 and met18-2. There is no significant difference of the lateral root numbers between Col-0 and the three mutants without the application of IAA (Fig 5C and 5D). IAA treatment greatly increased the lateral root numbers in Col-0 and the three mutants. The increase of lateral root number in met18-2 is comparable to that in Col-0. However, the increase of lateral root numbers in dre2-3 and dre2-4 mutants was significantly less than that in Col-0 (Fig 5C and 5D). Together, our results suggest that DRE2 participates in auxin response. This could be a CIA-independent function of DRE2.

DRE2 localizes in both the cytoplasm and the nucleus and nuclear DRE2 directly binds several auxin responsive genes

Previous studies showed that DRE2 mainly localizes in the cytoplasm, like other CIA pathway proteins, but weak DRE2-GFP signal can be detected in the nucleus [32]. To ascertain the subcellular localization of DRE2, we generated pDRE2::DRE2-GFP transgenic plants in Col-0 background. Consistently, we found that DRE2-GFP mainly localized in the cytoplasm in the differentiation zone of root (S8A Fig). Strong nuclear DRE2-GFP signal could be detected after Leptomycin B (an exportin inhibitor) treatment (S8A Fig), suggesting that DRE2 shuttles between the nucleus and the cytoplasm. In the meristematic zone of root, DRE2-GFP appeared to be more abundant in the nucleoplasm than in the cytoplasm (S8B Fig). Unexpectedly, strong DRE2-GFP signal could be detected in the nucleolar cavity (S8B Fig). We further performed subcellular fractionation experiments with HSP90 and Histone H3 as cytoplasmic and nuclear protein markers, respectively. We found that DRE2 was present in both the cytoplasm and the nucleus (Fig 6A; S9 Fig). Importantly, when we separated the nuclear fraction into nucleoplasm and chromatin-bound fractions, we found that DRE2 was associated with chromatin (Fig 6B; S9 Fig). To further visualize the nuclear localization of DRE2, we immunostained nuclei isolated from Col-0 and the DRE2-HA transgenic plants. The DRE2-HA signal did not overlap with compacted heterochromatin regions that are intensely stained by DAPI, but overlapped with open euchromatin regions that are weakly stained by DAPI (Fig 6C), suggesting that DRE2 is specifically associated with euchromatin. To test whether DRE2 can directly bind auxin responsive genes, we performed chromatin immunoprecipitation (ChIP)-qPCR using pDRE2::DRE2-GFP transgenic plants. Our results revealed that DRE2 were enriched at some auxin responsive genes, including SAUR16, IAA14, PIN4 and PIN7 (Fig 6D). In dre2-4 mutants, the transcript levels of SAUR16, IAA14 and PIN4 were reduced, while PIN7 remained unaltered (Fig 6E). These results suggest that DRE2 binding is important for the expression of auxin responsive genes.

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Fig 6. DRE2 associates with chromatin and some auxin responsive genes.

(A-B) Detection of DRE2 in cytoplasmic, nucleoplasmic and chromatin-associated fractions by Western blot. DRE2-HA fusion protein was detected using an anti-HA antibody. DRE2-GFP fusion protein was detected using an anti-GFP antibody. HSP90 and histone H3 were detected as markers for cytoplasmic proteins and chromatin proteins, respectively. AGO1 was detected as markers for proteins present in all the fractions. (C) Immunostaining results showing the nuclear distribution of DRE2. DRE2-HA in the pDRE2::DRE2-HA transgenic plants was stained with an anti-HA antibody and an Alexa Fluor 488-conjugated secondary antibody (green). DNA was stained with DAPI (blue). (D) ChIP-qPCR results showing the enrichment of DRE2-GFP at four auxin responsive genes. (E) Relative expression levels of the indicated genes in the indicated genotypes as determined by RT-qPCR. Data are presented as mean ± SD of four technical replicates. Asterisks indicate two-tailed Student’s t-test, *P < 0.05, **P < 0.01.

https://doi.org/10.1371/journal.pgen.1008094.g006

CRISPR/Cas9 technology creates hypomorphic dre2 mutants

For most of the essential genes, no homozygous cell lines or mutants can be obtained. This impedes the functional studies of these genes. RNAi and CRISPR/Cas9 are two technologies for gene silencing, with the former knock down genes and the latter knock out genes or introduce deletions/mutations. Because RNAi only reduces the expression of genes, it is a better choice for investigating the functions of essential genes. However, RNAi-mediated reduction of gene expression sometimes is not sufficient to result in desired molecular and/or phenotypic changes. For instance, the RNAi line for NBP35 have unaltered activities of Fe-S enzymes in leaves [31]. Another drawback of RNAi is that it often has off-target effects and may produce false positive results [43]. CRISPR/Cas9 is effective for editing of specific genomic regions and has less off-target effects. Thus, it could be ideal for creation of hypomorphic mutants for essential genes. Usually, sgRNAs targeting exons are designed to introduce insertion or deletions mutations in protein-coding regions of genes. Different from the popular strategy, we designed a sgRNA targeting an intron-exon junction region of DRE2 to create mis-splicing mutants for DRE2. We successfully created mis-splicing mutants for DRE2 and obtained viable homozygous lines. The mutant lines were found to exhibit hallmark features of the CIA pathway mutants. Moreover, we found that DRE2 participates in auxin response independently of its CIA role using these mutant lines. Our results suggest that generating viable mutant lines using CRISPR/Cas9 technology is a good strategy for studying essential genes.

The dre2 mutants exhibit hallmark features of the CIA pathway mutants

To clearly identify DRE2 as a CIA factor, we measured the activities of AO in dre2-3 and dre2-4. Like the atm3 mutant [17], dre2-3 and dre2-4 have low AO activities, suggesting that the CIA function of DRE2 is conserved in plants. Like the ae7, met18, atm3 and grxs17 mutants, the dre2 mutants have accumulation of DNA damage and activation of DDR. In eukaryotes, Fe-S clusters have been found to be assembled on DNA replication/repair proteins, including Pol α, δ, ε, and ζ, which are DNA polymerases for DNA replication and repair [44], PriL, which is a subunit of eukaryotic primase for DNA replication and telomere maintenance [45,46], XPD and FancJ, which are DNA helicases involved in nucleotide excision repair [47], RTEL1, which is a DNA helicase involved in homologous recombination [48]. The accumulation of DNA damage and activation of DDR in the ae7 [19], met18 [15], atm3 [19] and grxs17 mutants have been proposed to be caused by defective assembly of Fe-S clusters on plant counterparts of these proteins. It is possible that the DNA damage phenotype of the dre2 mutants is also a result of defective assembly of Fe-S clusters on DNA replication/repair proteins. Previous findings indicate that different Fe-S proteins are differentially affected by certain CIA mutation, future investigations are needed to determine which DNA replication/repair proteins are preferentially affected by DRE2.

The ae7, nbp35 and met18 mutants have low activity of ROS1 and whole genome bisulfite sequencing analysis revealed DNA hypermethylation at hundreds of loci in met18 [19,20,22,31]. The dre2 mutants also exhibit DNA hypermethylation and the pattern of DNA hypermethylation in dre2-4 resembles that in met18-2 (Fig 3B). Our results suggest that the action of DRE2, an early acting CIA factor, is also required for full enzymatic activity of ROS1. ROS1 transcript levels did not decrease in the dre2 mutants, excluding the possibility that decreased expression of ROS1 caused DNA hypermethylation. AE7 and MET18 interact with ROS1 and the physical interactions may mediate the transfer of Fe-S cluster to ROS1 and contribute to ROS1 activity [19,20,22,31]. As DRE2 is not a component of the CIA targeting complex, it is less likely that DRE2 directly interacts with ROS1. Impairment of Fe-S assembly on ROS1 is less likely a result of disrupted DRE2-ROS1 interaction. As electron transfer from DRE2 provides reducing power to Fe-S cluster assembly, impairment of Fe-S assembly on ROS1 could be due to inadequate reducing power.

The unstructured linker of DRE2 coordinates protein-protein interaction for proper CIA function of DRE2

The DRE2 protein contains a methyltransferase-like domain at the N-terminal, a CIAPIN1 domain at the C-terminal, and an unstructured linker in the middle (S1B Fig). In human cells, the unstructured linker is the only region of anamorsin that tightly interacts with Ndor1 (human homolog of Tah18) [49] and can stabilize the interaction with GRX3 [27,28]. The dre2-3 and dre2-4 mutations, we created by CRISPR/Cas9, lead to amino acid deletions in the unstructured liker region. Consistent with studies in human cells, our Y2H results revealed that the linker is required for DRE2’s interaction with GRXS17. However, in contrast to promoting DRE2-GRXS17 interaction, the linker region was found to inhibit DRE2’s interaction with NBP35. The opposing functions of the linker region on DRE2-GRXS17 interaction and DRE2-NBP35 interaction lead us to propose that before mature, DRE2 exposes its linker region to facilitate DRE2-GRXS17 interaction and Fe-S cluster transfer from GRXS17 to DRE2. Binding of Fe-S cluster and maturation of DRE2 may change the conformation of DRE2 and hide the linker region, thus disrupting DRE2-GRXS17 interaction to facilitate DRE2-NBP35 interaction and Fe-S cluster assembly on cytoplasmic or nuclear Fe-S proteins. Because the GRX3-anamorsin interaction promotes [2Fe-2S] cluster transfer [27], loss of DRE2-GRXS17 interaction before Fe-S transfer to DRE2 in dre2-4 may lead to reduced Fe-S binding on DRE2, eventually leading to the CIA-related defects in dre2-3 and dre2-4. The CIA-related defects in dre2-3 and dre2-4 may also arise from stronger interaction between DRE2 and NBP35.

Novel functions of DRE2

The CIA function of DRE2 is considered to be executed by cytoplasmic DRE2. Previous studies found that DRE2 also localizes in the nucleus [32]. In this study, we confirmed the nuclear localization of DRE2 and found that it can associate with chromatin (Fig 6A and 6B). Further immunostaining results indicate that DRE2 is associated with euchromatin (Fig 6C). The nuclear localization, especially the euchromatin-association, of DRE2 suggests that DRE2 plays non-CIA roles. Previous studies revealed one of the non-CIA roles that DRE2 plays is activation of the imprinting gene FWA in the central cell and endosperm [32]. We found that dre2 mutant lines created by sgRNA1 or sgRNA2 and dre2-4 show chlorotic leaf spots or stripes reminiscent of cell death. This phenotype was detected only in the dre2 mutants, but not in other CIA pathway mutants, suggesting that DRE2 may regulate signaling pathways related to cell death independently of its CIA role. Through RNA-seq analysis, we found that auxin response was disrupted in dre2 mutants. Because only DRE2 was greatly induced by auxin treatment and only dre2 mutants, but not Col-0 and met18-2, have alleviated inhibition of primary root growth and less increase of lateral root number, we propose that the involvement of DRE2 in auxin response might be independent of its CIA role. Our ChIP-qPCR results revealed that DRE2 directly binds multiple auxin responsive genes and DRE2 binding is required for their optimal expression. Thus, it is very likely that DRE2 participates in auxin response by controlling the expression of auxin responsive genes, although we could not exclude the possibility that the CIA-dependent function of DRE2 indirectly (via a FeS protein) contributes to the auxin response. Interestingly, the knockout line and RNAi line for GRXS17 also display reduced auxin sensitivity, albeit only at high temperature. Specifically, the inhibition of primary root length by auxin is alleviated in the knockout line and RNAi line for GRXS17. This was attributed to elevated levels of reactive oxygen species in these lines [50]. Our finding that DRE2, a downstream Fe-S transfer target of GRXS17, participates in auxin response suggesting that malfunction of DRE2 could be a cause of altered auxin response in these lines. Together, our findings define the nuclear localization of DRE2, its association with chromatin and its modulation of auxin responsive gene expression. It will be interesting to search for nuclear DRE2-interacting proteins and other nuclear DRE2 functions in the future.

Plant materials and growth conditions

All Arabidopsis materials used in this study are in Columbia background. The met18-2 (SALK_147068) mutant has been described previously [19,22]. The dre2-3 and dre2-4 mutants were generated by an egg cell-specific promoter-controlled CRISPR/Cas9 system [51] using sgRNA-3 (S1 Table). For horizontally-grown seedlings, surface-sterilized seeds were germinated on 1/2 Murashige-Skoog (MS) medium (7‰ Agar and 1% Suc) at 22°C with 16 h of light and 8 h of darkness. For vertically-grown seedlings, surface-sterilized seeds were germinated on 1/2 MS medium (12‰ Agar and 1% Suc) also at 22°C with 16 h of light and 8 h of darkness. The seedlings were harvested for experiments or transplanted into soil and grown at 22°C with the same photoperiod.

To generate the pDRE2::DRE2-GFP and pDRE2::DRE2-HA transgenic plants, DRE2 genomic DNA fragment with its native promoter was amplified from Col-0 genomic DNA by PCR and cloned into the pCAMBIA1300 or pCAMBIA1305 vectors for plant transformation. For complementation of dre2-4, DRE2 coding sequence (CDS) was amplified from Col-0 cDNA by PCR and cloned into the pCAM2300-35S-Ocs vector for plant transformation. Agrobacterium tumefaciens strain GV3101 carrying different constructs were used to transform the wild type or mutant plants via the standard floral dipping method [52]. Primary transgenic plants were selected on 1/2 MS plates containing 25 mg/L hygromycin (pCAMBIA1300 and pCAMBIA1305) or 50 mg/L kanamycin (pCAM2300-35S-Ocs). Homozygous transgenic lines were used for further experiments. The primers used for PCR are listed in S1 Table.

Enzyme extraction and AO activity staining

Enzyme extraction and AO activity staining were performed as previously described [53–55]. Twelve-day-old seedlings were ground into fine powder and 100 mg of powder were suspended in 400 μL of extraction buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM sodium molybdate, 10 μM flavin adenine dinucleotide, 2 mM DTT, protease inhibitor (one tablet/100 mL) and Polyclar AT (0.2 g/g fresh weight)). After centrifugation at 15,000 rpm for 20 min, the supernatant was concentrated by filtration using Amicon Ultra-0.5 Centrifugal Filters (3K) to achieve a final volume of 50 to 100 μL. The concentrated samples were used as crude enzyme preparations for native PAGE (6%). After electrophoresis, the gel was immersed in 0.1 M potassium phosphate (pH 7.4) for 5 min, and then the activity of AO was determined by developing in a reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.4), 1 mM 1-naphthaldehyde, 0.1 mM phenazine methosulfate, and 0.4 mM MTT at room temperature (about 25°C) for 1 h in the dark.

Real-time RT-PCR

Total RNA was extracted from 12-day-old seedlings grown on 1/2 MS medium using the TRIzol reagent (Invitrogen, 15596026). About 2 μg of total RNA was used for first-strand cDNA synthesis with the 5X All-In-One RT MasterMix (abm, G485) following the manufacturer’s instructions. The cDNA reaction mixture was then diluted 10 times, and 1 μL was used as a template in a 20 μL PCR reaction with PowerUp SYBR Green Master Mix (Applied Biosystems). The RNA transcript levels were determined by quantitative RT-PCR. TUB8 was used as an internal control. The primers used for PCR are listed in Table S1.

Comet assay

Comet assay was performed by using the Trevigen comet assay kit (Trevigen, 4250-050-K) following the manufacturer’s instruction. Briefly, nuclei extracted from 22-day-old rosette leaves at 1 x 10⁵/mL were combined with molten LMAgarose at a ratio of 1:10 (v/v) at 37°C. Samples (50 μL) were immediately pipetted onto CometSlide. The slides were placed flat at 4°C in the dark for 30 min and then immersed in prechilled Lysis Solution (Trevigen, 4250-050-01,) at 4°C for 60 min. After lysis, the slides were immersed in freshly prepared Alkaline Unwinding Solution, pH >13 (200 mM NaOH, 1 mM EDTA) for 20–60 min at room temperature in the dark. Electrophoresis was in Alkaline Electrophoresis Solution, pH >13 (200 mM NaOH, 1 mM EDTA) at 21 volts for 30 min at 4°C. After washing in dH₂O and 70% ethanol, samples were dried at ≤ 45°C for 10–15 min. DNA was stained by diluted SYBR Green I (Trevigen, 4250-050-05,) in refrigerator for 5 min. The slides were dried completely at room temperature in the dark and then viewed by Olympus BX53 fluorescence microscope. The Comet Score software (http://www.autocomet.com) was used to evaluate the levels of DNA damage.

Whole-genome bisulfite sequencing and data analysis

About 3 μg genomic DNA was extracted from 14-day-old seedlings using Hi-DNAsecure Plant Kit (TIANGEN, DP350-03) and then sent for bisulfite treatment, library preparation and sequencing (illumina Hiseq 4000, PE100) by the Beijing Genomics Institute (Shenzhen, China). Data analysis was performed according to Wang et al., 2016 [22].

Locus-specific bisulfite sequencing

About 100 ng of genomic DNA was modified using the BisulFlash DNA Modification Kit (Epigentek, P-1026-050) according to the manufacturer’s instructions. An aliquot (1 μL) of bisulfite-treated DNA was used for PCR using the TaKaRa EpiTaq HS (for bisulfite-treated DNA) (Takara, R110A) with gene-specific primers (S1 Table) in a reaction volume of 20 μL. The PCR products were cloned into the pClone007 Blunt vector kit vector (TSINGKE, TSV-007B) and at least 20 independent clones of each sample were sequenced.

Yeast two-hybrid assay

The CDSs of TAH18, NBP35 and GRXS17 were amplified by PCR and then subcloned into pGBK-T7 (Clontech, 630443). The CDSs of DRE2, DRE2-3, DRE2^Δ6 and DRE2^Δ75 were amplified by PCR and then subcloned into pGAD-T7 (Clontech, 630442). For protein interaction analysis, two combinatory constructs were transformed simultaneously into the yeast strain AH109 (Kept by our lab) and tested for Leu, Trp, Ade, and His auxotrophy according to the manufacturer’s protocols. The primers used for PCR are listed in S1 Table.

Leptomycin B treatment

Leptomycin B treatment was performed as previously described [56]. Briefly, pDRE2::DRE2-GFP transgenic plants were grown vertically on 1/2 MS-medium plates for 5 days and then transferred to 3 mL of liquid 1/2 MS-medium (pH 5.7). After 48 h, Leptomycin B was added to a final concentration of 2.5 mM/L and incubated for 8–24 h.

Fluorescence microscopy

FWA-GFP in the central cell and endosperm was detected using an Olympus BX53 fluorescence microscope equipped with an Olympus DP80 digital camera [57]. DRE2-GFP in the root was detected using the Nikon’s modular A1⁺/A1R⁺ confocal laser scanning microscope system (National Center for Protein Sciences at Peking University, Beijing).

Subcellular fractionation and Western blot analysis

Tissues of 14-day-old DRE2-HA transgenic plants (about 1 g) were ground into fine powder and then suspended in 2 mL of Honda buffer (400 mM sucrose, 2.5% Ficoll, 5% Dextran T40, 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 0.5% Triton X-100, 0.5 mM PMSF, 10 mM β-mercaptoethanol and protease inhibitor (1 tablet/100 mL)) The sample was centrifuged at 10,000 g for 20 min at 4°C. The homogenate was filtered through a double layer of Miracloth twice and then centrifuged at 1,500 g for 5 min at 4°C. The supernatant was further centrifuged at 10,000 g for 10 min at 4°C and the cytoplasmic fraction (supernatant) was collected. The pellet (nuclear fraction) was washed three times with Honda buffer and one time with PBS buffer (1 mM EDTA, 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄ (pH 7.4)) and then resuspended in 200 μL of glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 50% glycerol, 0.125 mM PMSF, 10 mM β-mercaptoethanol and protease inhibitor (1 tablet/100 mL)). Another 200 μL of nuclei lysis buffer (10 mM HEPES (pH 7.6), 1 mM DTT, 7.5 mM MgCl₂, 0.2 mM EDTA, 300 mM NaCl, 1 M urea, 1% NP-40, and 0.5 mM PMSF, 10 mM β-mercaptoethanol and protease inhibitor (1 tablet/100 mL)) was added and the sample was vortexed gently. The mixture was incubated on ice for 5 min and then centrifuged at 13,200 g for 2 min at 4°C. The supernatant was collected as the nucleoplasmic fraction. The pellet was washed one time with PBS buffer and collected as the chromatin-associated fraction. The cytoplasmic marker HSP90 was detected by a rabbit anti-HSP90 polyclonal antibody (Santa Cruz Biotechnology, at-115). The nuclear marker Histone H3 was detected by a mouse anti-H3 monoclonal antibody (EASYBIO, BE7004-100). The cytoplasmic, nucleoplasmic and chromatin-associated protein markers AGO1 was detected by a rabbit anti-AGO1 polyclonal antibody (provided by Dr. Xiaoming Zhang, Institute of Zoology, CAS) [58]. DRE2-HA was detected by a mouse anti-HA monoclonal antibody (Sigma, H3663).

Immunolocalization

Immunofluorescence staining was performed using leaves from 21-day-old DRE2-HA transgenic plant as previously described [59]. Nuclei preparations were incubated with a mouse anti-HA monoclonal antibody (Sigma, H3663) overnight at room temperature and then incubated with goat anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, A-11001) for 2 h at 37°C. DNA was counterstained using DAPI in Prolong Gold (Invitrogen, P36931). Pictures were taken using the Nikon’s modular A1⁺/A1R⁺ confocal laser scanning microscope system (National Center for Protein Sciences at Peking University, Beijing).

RNA sequencing and data analysis

Total RNA samples were used to generate RNA libraries for deep sequencing (HiSeq XTEN, Illumina). Quality control was performed with FastQC (v0.11.5). Adapters and low quality reads were removed by cutadapt (v1.11). The trimmed and quality filtered clean reads were mapped to the Arabidopsis reference genome (TAIR10, https://www.arabidopsis.org) under the guide of annotation from Araport11 (https://www.araport.org) using Tophat2 (v2.1.1). Reads were then sorted, indexed and compressed by samtools (v1.5) and bigwig files were generated by bam2wig.py with option–u (skip non-unique hit reads) from RseQC (v2.6.4). Cuffdiff (v2.2.1) was used to quantify the gene expression level and identify differentially expressed genes (DEGs) with option–u (use 'rescue method' for multi-reads). DEGs were filtered in CummeRbund (v2.16.0) under the criteria of FDR<0.05 and log2 fold change > 1. Gene ontology (GO) analysis was performed by AgriGO (http://bioinfo.cau.edu.cn/agriGO/analysis.php) with the default parameters. Heatmaps were generated by R package ComplexHeatmap (1.12.0). All statistical analysis analyses were performed in R (3.3.1).

ChIP assay

Chromatin immunoprecipitation (ChIP) assays were performed according to published protocols [60,61] with minor modifications. Briefly, seedling tissues (about 1.5 g) were ground in liquid N₂ and suspended in 20 mL of ChIP extraction buffer I (10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 400 mM sucrose, 0.1 mM PMSF, 1 mM DTT, protease inhibitor (1 tablet/100 mL)). The nuclei were fixed with 1% formaldehyde for 10 min at 4°C and then neutralized with 0.125 M glycine for 5 min. The nuclei were washed 4 times with ChIP extraction buffer II (10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 250 mM sucrose, 1% Triton X-100, 0.1 mM PMSF, 1 mM DTT, protease inhibitor (1 tablet/100 mL)) until they turned white, then layered on top of ChIP extraction buffer III (10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 1.7 M sucrose, 0.15% Triton-X100, 0.1 mM PMSF, 1 mM DTT, protease inhibitor (1 tablet/100 mL)) and centrifuged at 12000 rpm for 1 h. The purified nuclei were then suspended in nuclear lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS), and sonicated for 28 cycles (30s on, 30s off). After centrifugation, the chromatin supernatant was diluted 10 times with dilution buffer (16.7 mM Tris-HCl (pH 8.0), 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100). An anti-GFP antibody (Abcam, ab290) was used for ChIP assays. After washing, the reversal of crosslinking and phenol chloroform extraction, the purified DNA was suspended in 50 μL of ddH₂O and diluted 5 times. A 1 μL aliquot was used for quantitative PCR.

Accession numbers

The RNA-seq data for Col-0 and dre2-4 and the whole-genome bisulfite sequencing data for dre2-4 was deposited at NCBI (SRP153123). The Col-0 and ros1-4 whole-genome bisulfite sequencing data used were from NCBI (SRP119887).