Abstract
Silencing of transposable elements (TEs) is an essential process to maintain genomic integrity within the cell. In Arabidopsis, together with canonical epigenetic pathways such as DNA methylation and modifications of histone tails, the plant mobile domain (PMD) proteins MAINTENANCE OF MERISTEMS (MAIN) and MAIN-LIKE 1 (MAIL1) are involved in TE silencing. In addition, the MICRORCHIDIA (MORC) ATPases, including MORC1, are important cellular factors repressing TEs. Here, we describe the genetic interaction and connection between the PMD and MORC pathways by showing that MORC1 expression is impaired in main and mail1 mutants. Transcriptomic analyses of higher order mutant plants combining pmd and morc1 mutations, and pmd mutants in which MORC1 expression is restored, show that the silencing defects of a subset of TEs in pmd mutants are most likely the consequence of MORC1 down-regulation. Besides, a significant fraction of up-regulated TEs in pmd mutants are not targeted by the MORC1 pathway.
Introduction
Transposable elements (TEs) are highly repeated, self-replicating genetic elements that are capable of invading the host genome through the process of transposition (1). TEs are predominantly enriched in pericentromeric constitutive heterochromatin, although they can also occupy chromosome arms (2). When occurring within a gene, TE transposition can disrupt gene sequence and function with dramatic consequences for the host cell. Thus, to maintain its genome integrity, the cell has elaborated several epigenetic pathways, such as DNA methylation and histone modifications that repress TEs (3, 4). In plants such as Arabidopsis thaliana, DNA methylation occurs in three different cytosine contexts that are mCG, mCHG, and mCHH (where H is A, T, or C), involving specialized DNA methyltransferases (5). DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) is required for de novo DNA methylation in all sequence contexts through the RNA-directed DNA methylation (RdDM) pathway and in the maintenance of mCHH. METHYLTRANSFERASE 1 (MET1) is essential for the maintenance of virtually all mCG, whereas CHROMOMETHYLASE 2 (CMT2) and CMT3 are involved in mCHG maintenance. CMT2 can also mediate mCHH maintenance at specific genomic locations (6, 7). Besides DNA methylation and histone modifications, several epigenetic factors cooperate to repress TEs. These sophisticated epigenetic pathways converge toward TEs to maintain them silenced, acting either synergistically or redundantly (1). MICRORCHIDIA (MORC) proteins are ATPases conserved in most eukaryotes, playing a major role in TE and gene silencing in plants, nematodes, and mammals (8, 9). In A. thaliana, MORC1 physically interacts with MORC6 and with MORC4, MORC7, and RdDM factors to maintain heterochromatic TEs condensed (10, 11). It has been proposed that MORC proteins would repress TEs using a DNA loop-trapping mechanism to compact chromatin (12). Another pathway involves MAINTENANCE OF MERISTEMS (MAIN) and MAIN-LIKE 1 (MAIL1) that are two plant mobile domain (PMD) proteins, originally identified as essential factors for plant development and genome integrity (13, 14). MAIN and MAIL1 physically interact together, forming a molecular complex with the presumably inactive serine/threonine phosphoprotein phosphatase (PPP) called PP7-LIKE (PP7L). The three proteins are required for TE silencing and the proper expression of a common subset of genes (15, 16, 17). Synergistic effects were described between MAIN, DRM2, and CMT3 pathways (16). Nevertheless, the mode of action of PMD proteins remains largely unclear, and their involvement in TE silencing is elusive.
In this study, we report the complex interplay between the PMD and MORC1 pathways. We show that MORC1 belongs to the genes that are commonly down-regulated in several single- and higher order pmd mutants. Based on these observations and considering the major role of MORC1 in TE silencing, we hypothesized that MORC1 down-regulation could at least partially explain the TE silencing defects observed in the pmd mutants. To address this question, we decided to undertake two approaches: first, to decipher the genetic interaction between the PMD and MORC1 pathways by analyzing misregulation of TE and gene expression in main morc1 and mail1 morc1 double mutants; and second, to use a transgene-based approach to rescue MORC1 expression in pmd mutants, which demonstrates that silencing of a fraction of up-regulated TEs in main and mail1 mutants can be complemented by supplying MORC1 in trans.
Results
MORC1 is down-regulated in pmd mutants
By surveying the genes that were misregulated in the main-3 hypomorphic mutant, in the main-2 mail1-1 and pp7l-2 null mutants (hereafter called main, mail1, and pp7l in the text), and in higher order mutants thereof, we identified 26 genes that were commonly down-regulated in all the genetic backgrounds (Fig 1A and Table S1) (16). 25 of them carry a DNA motif in their promoter that was previously named the “DOWN” motif (16). Although we could not define any enrichment of gene ontology (GO) term among these genes, we found out that MORC1, which carries the “DOWN” motif in its promoter, belonged to the list of down-regulated loci (Table S1). This was further confirmed by reverse transcription coupled to quantitative PCR (RT–qPCR) experiments showing a fivefold decrease in all tested mutants in comparison with WT Columbia (Col) control (Fig 1B). Furthermore, MORC1 expression could be rescued in main, mail1, and pp7l null mutants that were complemented with the respective epitope-tagged genomic PMD or PP7L constructs (Fig 1B). Thus, the two PMD MAIN and MAIL1 proteins, and their interactor PP7L, are required for the proper expression of MORC1, and to some extent, pmd and pp7l mutants can be seen as morc1 knocked-down mutants.
(A) UpSet plot analyses allowing to visualize in a matrix layout the intersections of down-regulated gene datasets in the main-3 hypomorphic mutant, main-2, mail1-1, pp7l-2 single-null, and mail1-1 pp7l-2 double-null mutants as described in reference 16. (B) Relative expression of MORC1 mRNA levels assayed by reverse transcription coupled to quantitative PCR (RT–qPCR) in corresponding pmd and pp7l mutants and complementing lines. RT–qPCR analyses were normalized using the housekeeping RHIP1 gene, and transcript levels are represented relative to WT Columbia (Col) or ATCOPIA28::GFP in WT controls (16). Error bars indicate SD based on three independent biological replicates.
Source data are available for this figure.
Source Data for Figure 1[LSA-2022-01539_SdataF1.xlsx]
The pmd morc1 double-null mutants do not exacerbate TE silencing defects
To evaluate the effect of MORC1 down-regulation on TE activation observed in the pmd mutants, we decided to analyze the genetic interaction between PMD and MORC1 pathways by creating main morc1 and mail1 morc1 double-null mutants using the morc1-2 null allele (hereafter called morc1). Although morc1 mutant and WT Col plants are undistinguishable, main-2 and mail1 single-null mutants display a strong developmental phenotype that is not exacerbated by introducing the morc1 null mutant allele (Figs 2A and S1A). We then performed RNA-sequencing (RNA-seq) analyses using main, morc1 single-null, and main morc1 double-null mutants, and evaluated TE and gene misregulation in comparison with WT control plants (Fig 2B–D and Table S2). Principal component analyses showed that biological replicates of each genetic background clustered together, and remarkably, replicates of main and main morc1 mutants tend to group together (Fig S1B). Although up-regulated TEs were mostly pericentromeric, misregulated genes spanned the whole five chromosomes (Fig S1C). Furthermore, for up-regulated TEs and genes, comparative analyses identified significant numbers of loci that were commonly misregulated in the three mutant backgrounds (Fig 2E). We noticed that several TEs were apparently up-regulated only in the main single but not in the main morc1 double mutant or vice versa (Fig 2B and E). However, by analyzing more precisely the expression level of these TEs in each mutant background, we observed that overall, they seemed to be similarly up-regulated in main and main morc1 mutants, but, most likely, did not pass our stringent RNA-seq threshold (log2 ≥ 2 or log2 ≤ −2 fold change, adjusted P < 0.01; Fig 2D). To statistically validate this hypothesis, we performed boxplot analyses using TEs that were up-regulated in main or main morc1 mutants, which confirmed that there was no significant difference between these two genetic backgrounds regarding the extent of TE up-regulation (Fig 2F and I). In contrast, the morc1 null mutant showed a milder up-regulation of TEs than the main mutant, in which MORC1 is knocked down, suggesting that MAIN plays a broader role in TE silencing than MORC1 (Fig 2B, F, and I). Similar analyses with genes that were down-regulated in main or main morc1 mutants showed comparable results, with no significant difference between the two genetic backgrounds (Fig 2G and J). However, for up-regulated genes in main or main morc1 mutants, we observed significant differences between the main single and main morc1 double mutants, indicating a possible synergistic effect of the two mutations at these genomic locations (Fig 2H and K). This could also be explained as a consequence of another down-regulated gene deriving from the main background. Search for GO term enrichment revealed that up-regulated genes were significantly associated with the term “response to stress,” whereas down-regulated genes in main and main morc1 mutants were related to “response to red light” and “oxidoreductase activity” terms (Fig 2E and Table S3).
(A) Representative pictures of 3-wk-old main-2, morc1-2 single, and main-2 morc1-2 double mutants in comparison with WT Col plant. (B) Number of up-regulated TEs in main-2, morc1-2, and main-2 morc1-2, classified by the TE superfamily. (C) Number of misregulated genes in main-2, morc1-2, and main-2 morc1-2. (D) Heatmap showing misregulated loci in several biological replicates of main-2, morc1-2, and main-2 morc1-2 in comparison with WT Col. * represents loci that are commonly misregulated in the three mutant backgrounds. ** represents loci that are misregulated in main-2 morc1-2. (E) Venn diagram analyses representing the overlaps between misregulated loci in main-2, morc1-2, and main-2 morc1-2. Fisher’s exact test statistically confirmed the significance of overlaps (P < 10−3). (F, G, H) Boxplot analyses between main-2, morc1-2, and main-2 morc1-2 mutants in comparison with WT Col showing average RPKM values of up-regulated TEs (F), up-regulated genes (G), and down-regulated genes (H) in main-2. (I, J, K) Same as (F, G, H) using misregulated loci in main-2 morc1-2 as defined by ** in panel (D). P-values were calculated using a Wilcoxon test; n.s, not significant; *P < 0.05; **P < 10−6; and ***P < 10−12. (L, M, N) Relative expression analyses of up-regulated TEs, down-regulated genes, and up-regulated genes in the different genotypes assayed by RT–qPCR. RT–qPCR analyses were normalized using the housekeeping RHIP1 gene, and transcript levels in the different mutants are represented relative to WT Col. Error bars indicate SD based on three independent biological replicates.
Source data are available for this figure.
Source Data for Figure 2[LSA-2022-01539_SdataF2.xlsx]
Fig S1 supporting Fig 2. (A) Pictures of 3-wk-old mail1-1 single and mail1-1 morc1-2 double mutants. (B) Principal component analyses of RNA-seq data showing clustering of biological replicates of main-2, morc1-2, main-2 morc1-2, and WT Col plants. The batch effect because of data separated in two independent experiments was eliminated in the principal component analyses. (C) Chromosomal distributions of misregulated loci in main-2, morc1-2, and main-2 morc1-2 over WT Col. Chromosome arms are depicted in light gray, and pericentromeric regions, in dark gray as defined in reference 31. Up-regulated genes and TEs are represented in blue and red, respectively; down-regulated genes are represented in green. Log2 fold change ≥2 or ≤−2, adjusted P < 0.01.
These observations were further confirmed at several TEs and misregulated genes by performing RT–qPCR experiments, which also included mail1 and mail1 morc1 mutant backgrounds (Fig 2L–N). Altogether, these analyses revealed that pmd mutants showed a wilder up-regulation of TEs and misregulation of genes in comparison with the morc1 mutant. However, cumulating the pmd and morc1 mutations did not significantly aggravate the TE silencing defects observed in the main single mutant.
The pUBQ-MORC1 construct complements the TE silencing defects of morc1 null mutant
We showed that MORC1 is down-regulated in the pmd mutants, the pmd and morc1 null mutants share a common subset of up-regulated TEs and genes, and there is no significant difference in TE silencing defects between main single and main morc1 double mutants (Figs 1 and 2). This suggests that MAIN and MAIL1 are epistatic to MORC1, and MORC1 down-regulation might contribute, at least partially, to the TE silencing defects observed in the pmd mutant. To test this hypothesis, we engineered the pUBQ-MORC1 construct, in which the MORC1 coding sequence fused to a 3xFLAG epitope was cloned under the control of the housekeeping gene UBIQUITIN10 promoter (pUBQ) (Fig 3A) (18). The rationale was that placing MORC1 under pUBQ control would efficiently restore MORC1 expression because UBQ10 transcription is not impaired in main and mail1 mutants as seen in RNA-seq data (Fig S2A). pUBQ-MORC1 was thus introduced in main and mail1 mutants by plant transformation to generate pUBQ-MORC1/main line 1 and line 2, and pUBQ-MORC1/mail1 line 1 and line 2. To assay pUBQ-MORC1 functionality, the transgenes deriving from pUBQ-MORC1/main line 2 and pUBQ-MORC1/mail1 line 1 were introduced into the morc1 null mutant by crosses to generate pUBQ-MORC1/morc1 line 1 and pUBQ-MORC1/morc1 line 2, respectively. The accumulation of the MORC1-FLAG protein in each line was confirmed by Western blots, and RT–qPCR experiments demonstrated that the accumulation of MORC1-FLAG in both lines was sufficient to restore the silencing of several misregulated TEs and DNA-methylated genes in morc1-2 (Fig 3B and C). Thus, pUBQ-MORC1–derived MORC1-FLAG is a functional protein.
(A) Schematic representation of the pUBQ-MORC1 transgene. MORC1 CDS-3xFLAG is under the control of UBQ10 promoter and octopine synthase terminator. (B) Western blots using anti-FLAG antibody showing the accumulation of FLAG-tagged MORC1 protein in two pUBQ- independent MORC1/morc1-2 lines. WT Col and morc1-2 plants are used as negative controls. Coomassie staining of the membrane is used as a loading control; kD, kilodalton. (C) Relative expression levels of up-regulated TEs and DNA-methylated genes in the two pUBQ-MORC1/morc1-2 lines and morc1-2 control plants assayed by RT–qPCR. RT–qPCR analyses were normalized using the housekeeping RHIP1 gene, and transcript levels in the two genetic backgrounds are represented relative to WT Col. Error bars indicate SD based on three independent biological replicates. (D) Pictures of 3-wk-old pUBQ-MORC1/main-2, pUBQ-MORC1/mail1-1, and corresponding untransformed pmd mutants. (E) Same as (B) using two independent lines of pUBQ-MORC1/main-2 and pUBQ-MORC1/mail1-1 and WT Col, main-2, and mail1-1 as controls. (F, G) Same as (C) using pUBQ-MORC1/main-2 and pUBQ-MORC1/mail1-1 lines in comparison with main-2 and mail1-1 mutants and relative to WT Col.
Source data are available for this figure.
Source Data for Figure 3[LSA-2022-01539_SdataF3.1.pdf]
Source Data for Figure 3[LSA-2022-01539_SdataF3.2.xlsx]
Fig S2 supporting Fig 3. (A) Reads Per Kilobase of transcript per Million mapped reads (RPKM) of MAIN, MAIL1, MORC1, and UBQ10 in WT Col, main-2, mail1-1, and four independent pUBQ-MORC1/pmd lines. (B) Relative expression analyses of MORC1 in the four independent pUBQ-MORC1/pmd lines using main-2 and mail1-1 as respective controls, and relative to WT Col. RT–qPCR analyses were normalized using the housekeeping RHIP1 gene. Error bars indicate SD based on three independent biological replicates. (C) Western blot using anti-FLAG antibody to detect FLAG-tagged MORC1 protein in different pUBQ-MORC1/main-2 and pUBQ-MORC1/mail1-1 lines. WT Col, main-2, and mail1-1 are used as negative controls. (D) Relative expression levels of up-regulated TEs and up-regulated DNA-methylated genes in different pUBQ-MORC1/pmd lines assayed by RT–qPCR. RT–qPCR analyses were normalized using the housekeeping RHIP1 gene, and transcript levels in the different mutants are represented relative to WT Col. Error bars indicate SD based on three independent biological replicates.
pUBQ-MORC1 expression is sufficient to rescue the silencing at a subset of TEs in pmd mutants
To assess the effect of the functional MORC1-FLAG protein in the pmd mutants, we analyzed the four pUBQ-MORC1/main and pUBQ-MORC1/mail1 lines. As expected, the developmental phenotype of main and mail1 mutants was not complemented in pUBQ-MORC1/main and pUBQ-MORC1/mail1 lines (Fig 3D). pUBQ-MORC1 expression in the four lines was checked at the RNA and protein levels, confirming the accumulation of the MORC1-FLAG protein (Figs 3E and S2B). We then investigated the capacity of MORC1-FLAG to rescue the silencing defects of several TEs and DNA-methylated genes by RT–qPCR experiments. Remarkably, the four main and mail1 mutant lines expressing the pUBQ-MORC1 transgene showed a significant reduction in the expression of TEs and DNA-methylated genes in comparison with respective control mutant backgrounds (Fig 3F and G). Furthermore, at loci such as ATREP18 or UNK1, the strength of silencing was back to the WT level (Fig 3F and G). Conversely, in two additional independent lines called pUBQ-MORC1/main negative (neg.) and pUBQ-MORC1/mail1 neg. that did not accumulate the MORC1-FLAG protein, TE and DNA-methylated gene silencing was not rescued, with expression levels similar to the main and mail1 mutant controls (Fig S2C and D).
To fully evaluate the effect of rescuing MORC1 expression on TE silencing in the pmd mutants, we decided to extend our analyses by performing RNA-seq using the four pUBQ-MORC1/pmd lines accumulating the MORC1 protein. In each line, we could identify several complemented TEs, that is, TEs that were repressed in pUBQ-MORC1/pmd lines while identified as up-regulated in the respective pmd mutants (Fig 4A and B and Tables S2, S4, and S5). Although we could observe variation between independent lines, they shared significant fractions of complemented TEs, all of them being pericentromeric (Fig 4B and Table S5). As expected, boxplot analyses did not show any differences in TE up-regulation between main and mail1 (Fig 4C). Although rescuing MORC1 expression in the two pmd mutants did not fully restore TE silencing to the WT level, we could, however, observe that TE complementation was statistically significant for three of the four lines in comparison with their respective mutants (Fig 4D and E).
(A) Heatmap representing up-regulated TEs in main-2 and mail1-1 mutants and their expression levels in four independent pUBQ-MORC1/pmd lines. (B) Venn diagram analyses showing the overlaps between down-regulated TEs in pUBQ-MORC1/main-2 or pUBQ-MORC1/mail1-1 lines over their respective mutant backgrounds and up-regulated TEs in main-2 or mail1-1 over WT Col. Fisher’s exact test statistically confirmed the significance of overlaps (P < 10−3). (C) Boxplot analyses between main-2 and mail1-1 showing average RPKM values of up-regulated TEs in main-2 and mail1-1 union in comparison with WT Col. (D, E) Boxplot analyses between two independent pUBQ-MORC1/main-2 (D) or pUBQ-MORC1/mail1-1 (E) lines and their respective pmd mutants showing average RPKM values of up-regulated TEs in main-2 and mail1-1 union in comparison with WT Col. (F) Same as (D, E) for up-regulated TEs in morc1-2 as defined in Fig 2. (C, D, E, F) P-values of panels (C, D, E, F) were calculated using a Wilcoxon test; n.s, not significant; *P < 0.05; **P < 10−6; and ***P < 10−12. (G) In this model explaining the connection between the PMD and MORC1 pathways to repress TEs, MORC1 transcription requires the MAIN/MAIL1/PP7L complex. This latter could either directly recognize the “DOWN” motif located within the MORC1 promoter (CATGCAGTTT) or be recruited by an elusive transcription factor at this genomic location. Alternatively, MORC1 expression would indirectly depend on the MAIN/MAIL1/PP7L complex through the action of a downstream transcription factor. Upon translation, the MORC1 protein associates with other MORC proteins to ensure efficient silencing of a subset of TEs (MORC1-dependent TE silencing). Importantly, the silencing of a significant fraction of TEs requires another pathway independent of MORC1 yet to be deciphered. This MORC1-independent TE silencing pathway could directly involve the PMD proteins or another factor regulated by the MAIN/MAIL1/PP7L complex.
Source data are available for this figure.
Source Data for Figure 4[LSA-2022-01539_SdataF4.xlsx]
Table S4 List of misregulated loci in pUBQ-MORC1/pmd lines compared with their respective pmd mutants.
Table S5 List of complemented loci in pUBQ-MORC1/pmd lines compared with their respective pmd mutants.
We next determined the fractions of misregulated genes in main and mail1 mutants that were complemented by the pUBQ-MORC1 transgene (Fig S3A and Tables S2, S4, and S5). Unlike TEs, we could only identify a handful of commonly complemented genes between independent pUBQ-MORC1/main and pUBQ-MORC1/mail1 lines, with bigger variations between the lines (Fig S3B and C). Nevertheless, some of these lines showed complementation of misregulated genes that were statistically significant (Fig S3D–G). To explain the discrepancies between independent lines, we hypothesize that these variations are consequences of MORC1-unspecific effects occurring in each pUBQ-MORC1 line. Furthermore, three stress response–related genes LURP1, BG3, and WRKY38 identified by RNA-seq as complemented in pUBQ-MORC1/main line 2 were not validated by RT–qPCR analyses, neither were HAC4 and FAP4 that are two genes previously identified as down-regulated in main and mail1 (Fig S3H and I) (16). Importantly, up-regulated genes that were commonly complemented in the independent pUBQ-MORC1/main and pUBQ-MORC1/mail1 lines are mostly DNA-methylated genes that are enriched in the pericentromeric regions with no GO term enrichment (Fig S3B and Tables S5 and S6). Among these genes, we found the DNA-methylated gene UNK, and the two transposable element genes AT2G04460 and AT2G10980, validated by RT–qPCR (Fig 3G). Finally, we performed boxplot analyses using up-regulated TEs in morc1, which showed that for most of these TEs, the silencing was back to the WT level in the four pUBQ-MORC1/pmd lines (Figs 4F and S4A). Moreover, comparative analyses between commonly complemented TEs in pUBQ-MORC1/main or pUBQ-MORC1/mail1 lines and up-regulated TEs in pp7l or main-3 mutants showed significant overlaps (Fig S4B and C). Altogether, these results demonstrate that supplying MORC1 in trans in pmd mutants (i) efficiently restores the silencing of a fraction of TEs that are pericentromeric and up-regulated in morc1 mutant; (ii) to a lesser extent rescues the expression of several up-regulated genes that are mostly repressed genes targeted by DNA methylation in WT; and (iii) finally has a minor effect on genes that are down-regulated in the pmd mutants.
Fig S3 supporting Fig 4. (A) Heatmap representing up-regulated and down-regulated genes in main-2 and mail1-1 mutants and their expression levels in four independent pUBQ-MORC1/pmd lines. (B) Venn diagram analyses showing the intervals between down-regulated genes in pUBQ-MORC1/main-2 or pUBQ-MORC1/mail1-1 lines over their respective mutant backgrounds and up-regulated genes in main-2 or mail1-1 over WT Col. (C) Same as (B) for up-regulated genes in pUBQ-MORC1/main-2 or pUBQ-MORC1/mail1-1 lines and down-regulated genes in main-2 or mail1-1. Fisher’s exact test statistically confirmed the significance of overlaps (P < 10−3), except for the interval between down-regulated genes in main-2 and up-regulated genes in pUBQ-MORC1/main-2 line 1. (D, E) Boxplot analyses between two independent pUBQ-MORC1/main-2 (D) or pUBQ-MORC1/mail1-1 (E) lines and their respective pmd mutants showing average RPKM values of up-regulated genes in main-2 or mail1-1 in comparison with WT Col as depicted in panel (A). (F, G) Same as (D, E) for down-regulated genes in main-2 or mail1-1. P-values were calculated using a Wilcoxon test; n.s, not significant; *P < 0.05; and ***P < 10−12. (H, I) Relative expression levels of several up-regulated (H) and down-regulated (I) genes in pmd mutants in the four pUBQ-MORC1/pmd lines. RT–qPCR analyses were normalized using the housekeeping RHIP1 gene, and transcript levels in the different mutants are represented relative to WT Col. Error bars indicate SD based on three independent biological replicates.
Fig S4 supporting Fig 4. (A) Venn diagram analyses showing the intervals between down-regulated TEs in pUBQ-MORC1/main-2 or pUBQ-MORC1/mail1-1 lines over their respective mutant backgrounds and up-regulated TEs in morc1-2 over WT Col. (B, C) Same as (A) for up-regulated TEs in pp7l-2 (B) or in the hypomorphic main-3 mutant (C) as defined in reference 16. Fisher’s exact test statistically confirmed the significance of overlaps (P < 10−3).
Discussion
MORC1 down-regulation in pmd mutants cannot explain their abnormal developmental phenotype
The PMD proteins MAIN and MAIL1 are involved in several aspects of plant development, and the massive misregulation of gene expression in main and mail1 mutants is most likely accountable for their strong developmental phenotype (13, 14, 16). We showed here that MORC1 is down-regulated in the pmd mutants and in pp7l, which is mutated for the MAIN and MAIL1 interactor, PP7L (Fig 1) (16, 17). By analyzing higher order combinations of pmd and morc1 null mutations together with pmd plants in which MORC1 expression is restored (pUBQ-MORC1/pmd lines), we conclude that MORC1 down-regulation cannot account for the abnormal developmental phenotype of pmd mutants because pmd single and pmd morc1 double mutants and pUBQ-MORC1/pmd lines are undistinguishable (Figs 2A and 3D). Moreover, the subset of genes whose expression is rescued in the pUBQ-MORC1/pmd lines are not the causal factors of the pmd developmental phenotype, and further work will be needed to address this question.
Combining pmd and morc1 null mutations does not exacerbate TE silencing defects of pmd mutants
Distinct epigenetic pathways cooperate to efficiently silence TEs, forming the so-called epigenetic “mille-feuille” (1, 19). Generally, cumulating mutations in different epigenetic pathways acting redundantly or synergistically leads to a dramatic aggravation of TE silencing defects. For instance, a synergistic effect was observed in plants combining the morc6 and morpheus’ molecule 1 (mom1) mutations (8). Similarly, introducing drm2 and cmt3 mutations into the hypomorphic main-3 mutant allele led to a dramatic derepression of TEs (16). It was also shown for a handful of TEs that combining mail1 and morc6 mutations had a mild synergistic effect (15). Conversely, we observed that main morc1 double mutant did not show genome-wide massive up-regulation of TEs in comparison with main (Fig 2B–E). Indeed, focusing on the subsets of up-regulated TEs in main or main morc1 showed that there was no significant difference in misregulation between main morc1 and main mutants, which was further confirmed by RT–qPCR analyses including mail1 and mail1 morc1 mutants (Fig 2F, I, and L). However, TE silencing defects appeared stronger in main in comparison with the morc1 mutant (Fig 2B, F, and I). We propose that the stronger effect of morc6 mutation in comparison with morc1 on TE derepression could explain the discrepancy observed between mail1 morc1 and mail1 morc6 (8, 10). Thus, combining pmd and morc1 mutations does not exacerbate TE silencing defects, suggesting that the two pathways are connected, which is consistent with the fact that MORC1 is down-regulated in pmd mutants.
Rescuing MORC1 expression in pmd mutants is sufficient to restore the silencing of a subset of TEs
To re-establish MORC1 expression in pmd mutants, we introduced a FLAG-tagged MORC1 construct (pUBQ-MORC1) under the control of the UBQ10 promoter (Fig 3A). We first showed that pUBQ-MORC1 expression efficiently complemented the up-regulation of several TEs in the morc1 null mutant, confirming that the protein is functional (Fig 3C). We then analyzed the effect of pUBQ-MORC1 in main and mail1 in four independent lines and found that the silencing of a subset of TEs was restored in this genetic material (Figs 3F and 4A–E). Remarkably, these TEs corresponded to a significant fraction of TEs that were also up-regulated in morc1, which is consistent with the fact that (i) pmd mutants can be seen as morc1 knocked-down mutants and (ii) main and main morc1 mutants display similar TE up-regulation phenotypes (Figs 4F and S4A). Based on these results, we propose a model in which the MAIN/MAIL1/PP7L complex is required for the proper expression of the MORC1 protein, which in turn ensures the silencing of a subset of TEs together with other MORC proteins, including MORC6 (Fig 4G). It is not known at the moment whether the MAIN/MAIL1/PP7L complex interacts with chromatin. This interaction could be direct or indirect through the recruitment of an unknown transcription factor that would recognize, for instance, the cis-regulatory DNA elements called “DOWN” motif that is enriched in the promoter of down-regulated genes—including MORC1—in pmd and pp7l mutants (Fig 4G and Table S1) (16). Another hypothesis would be that MORC1 expression is regulated by a transcription factor acting downstream of the MAIN/MAIL1/PP7L complex.
Finally, this study revealed that a significant fraction of up-regulated TEs in the pmd mutants are not targeted by MORC1 (Fig 4G). One possibility is that the PMD proteins directly repress these TEs. A non-exclusive alternative would be that these TEs could also be targeted by an unknown factor that is impaired in the pmd mutants. Further studies will be essential to address these questions and to clarify the essential role of PMD proteins in TE silencing.
Materials and Methods
Plant material and growing conditions
WT and mutant lines are in the Columbia (Col) ecotype and were grown on soil under a 16 h- light/8-h dark cycle. The main-2 (GK-728H05), main-3 (hypomorphic allele), mail1-1 (GK-840E05), pp7l-2 (SALK_003071), morc1-2 (SAIL_893_B06), and mail1-1 pp7l-2 null mutant lines were previously described (10, 13, 14, 15, 16, 20, 21). The main-2 morc1-2 and mail1-1 morc1-2 double mutants were obtained by crosses and confirmed by PCR-based genotyping and RT–qPCR analyses. The pUBQ-MORC1/main-2 and pUBQ-MORC1/mail1-1 lines were obtained by plant transformation using the Agrobacterium-mediated floral dip method (22). The two pUBQ-MORC1/morc1-2 lines #1 and #2 were obtained by crossing morc1-2 with pUBQ-MORC1/main line #2 and pUBQ-MORC1/mail1 line #1, respectively, followed by subsequent PCR-based genotyping. The complementing lines expressing an epitope-tagged genomic version of PMD or PP7L in corresponding mutant backgrounds were previously described (16).
Cloning of pUBQ-MORC1
The pENTR Gateway (GW) vector carrying MORC1 CDS without STOP codon was obtained from the Jacobsen laboratory. The 3xFLAG tag was subcloned using an AscI site downstream of the cDNA, and DNA integrity was verified by Sanger sequencing (Eurofins). To generate pUBQ-MORC1, the MORC1-3xFLAG construct was then mobilized into the GW-compatible pUBQ10:GW vector by LR Clonase (Thermo Fisher Scientific) according to the manufacturer’s instruction (18). pUBQ-MORC1/main-2 and pUBQ-MORC1/mail1-1 primary transformants were selected by spraying glufosinate as a selection marker, and resistant plants were saved for further characterization. Primer sequences are described in Table S7.
Table S7 List of primers used in this study.
Immunoblotting
Total proteins were extracted from leaves of 3-wk-old seedlings using 8 M urea and denatured in Laemmli buffer for 5 min at 95°C. 10–15 μl of protein extracts were run on 10% SDS–PAGE, and proteins were detected by Western blotting using Anti-FLAG M2 monoclonal antibody–peroxidase conjugate (A8592; Sigma-Aldrich) at a dilution of 1:10,000. Western blots were developed using Substrat HRP Immobilon Western (WBKLS0500; Merck Millipore).
RNA extraction
Total RNA was extracted from leaves of 3-wk-old seedlings grown on soil using Monarch Total RNA Miniprep Kit (T2010; New England Biolabs) according to the manufacturer’s protocol.
RNA sequencing
RNA-seq libraries were generated from 1 µg of input RNA using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7490; New England Biolabs) according to the manufacturer’s protocols. Libraries were sequenced on an Illumina NextSeq 550 machine (Bio-environment platform, UPVD). Reads were trimmed using Trimmomatic (23) and mapped to the A. thaliana genome (Arabidopsis TAIR10 genome) using HISAT2 (24). The sequence alignment files were sorted by name and indexed using SAMtools (25). Files were converted to BAM files and a number of reads mapped onto a gene calculated using HTSeq-count (26). Differentially expressed genes were obtained with DESeq2 (27), using a log2 fold change ≥ 2 (up-regulated genes) or ≤ −2 (down-regulated genes) with an adjusted P < 0.01. Principal component analyses were produced using DESeq2 and ggplot2 R packages. Heatmap visualizations were realized using the heatmap2 function from the R gplots package. Boxplots were realized using the boxplot function from R. UpSet plot analyses were performed using the Intervene’s UpSet module interface described at https://asntech.shinyapps.io/intervene/ (28, 29). RNA sequencing mapping and coverage statistics are described in Table S8.
RT–qPCR
1 μg of input RNA was converted to cDNA using GoScript Reverse Transcriptase (A501C; Promega) according to the manufacturer’s protocol. The final reaction was diluted six times with RNase-free water. RT–qPCR experiments were performed with 4 μl of cDNA combined with the Power Track SYBR Green Master Mix (Thermo Fisher Scientific) using a LightCycler 480 instrument (Roche). Amplification conditions were as follows: 95°C for 5 min; 40 cycles of 95°C for 15 s and 60°C for 1 min; and melting curves. RT–qPCR analyses used the 2−∆∆Ct method. For each analysis, ∆Ct was first calculated based on the housekeeping RHIP1 gene Ct value (30). ∆∆Ct values were then obtained by subtracting the WT ∆Ct from the ∆Ct of each sample. Values were represented on bar charts relative to WT. Three technical replicates were performed per biological replicate, and three biological replicates were used in all experiments. Primer sequences are described in Table S7.
Data Availability
Nucleotide sequencing data generated in this study have been deposited in European Nucleotide Archive under the accession number PRJEB52795.
Acknowledgements
The authors thank the Steve Jacobsen laboratory who provided the MORC1 CDS construct as a gift. They also want to acknowledge the LGDP platform members and UPVD Bio-environment facility for their outstanding technical assistance and plant care. This study was supported by “Laboratoires d’Excellence” (LABEX) AGRO (10-LABX-0001)—Agropolis Foundation (project ID 2101-009) and by a UPVD BQR grant. It was set within the framework of the LABEX TULIP (ANR-10-LABX-41) and “Ecole Universitaire de Recherche (EUR)” TULIP-GS (ANR-18-EURE-0019). L Jarry is a recipient of the PhD fellowship “Bourse région Occitanie 2021.” The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
Author Contributions
L Jarry: conceptualization, resources, data curation, formal analysis, validation, investigation, visualization, methodology, and writing—original draft, review, and editing.
J Descombin: conceptualization, validation, investigation, visualization, methodology, and writing—review and editing.
M Nicolau: conceptualization, validation, investigation, and writing—review and editing.
A Dussutour: validation and investigation.
N Picault: conceptualization, resources, data curation, investigation, visualization, and writing—review and editing.
G Moissiard: conceptualization, supervision, funding acquisition, investigation, visualization, methodology, project administration, and writing—original draft, review, and editing.
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
- Received May 30, 2022.
- Revision received January 10, 2023.
- Accepted January 11, 2023.
- © 2023 Jarry et al.
https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).
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