Mutual upregulation of HY5 and TZP in mediating phytochrome A signaling.

Phytochrome A (phyA) is the far-red (FR) light photoreceptor in plants that is essential for seedling de-etiolation under FR-rich environments, such as canopy shade. TANDEM ZINC-FINGER/PLUS3 (TZP) was recently identified as a key component of phyA signal transduction in Arabidopsis thaliana; however, how TZP is integrated into the phyA signaling networks remains largely obscure. Here, we demonstrate that ELONGATED HYPOCOTYL5 (HY5), a well-characterized transcription factor promoting photomorphogenesis, mediates FR light induction of TZP expression by directly binding to a G-box motif in the TZP promoter. Furthermore, TZP physically interacts with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), an E3 ubiquitin ligase targeting HY5 for 26S proteasome-mediated degradation, and this interaction inhibits COP1 interaction with HY5. Consistent with those results, TZP post-translationally promotes HY5 protein stability in FR light, and in turn, TZP protein itself is destabilized by COP1 in both dark and FR light conditions. Moreover, tzp hy5 double mutants display an additive phenotype relative to their respective single mutants under high FR light intensities, indicating that TZP and HY5 also function in largely independent pathways. Together, our data demonstrate that HY5 and TZP mutually upregulate each other in transmitting the FR light signal, thus providing insights into the complicated but delicate control of phyA signaling networks.

The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures.

Light and temperature are two core environmental factors that coordinately regulate plant growth and survival throughout their entire life cycle. However, the mechanisms integrating light and temperature signaling pathways in plants remain poorly understood. Here, we report that CBF1, an AP2/ERF-family transcription factor essential for plant cold acclimation, promotes hypocotyl growth under ambient temperatures in Arabidopsis. We show that CBF1 increases the protein abundance of PIF4 and PIF5, two phytochrome-interacting bHLH-family transcription factors that play pivotal roles in modulating plant growth and development, by directly binding to their promoters to induce their gene expression, and by inhibiting their interaction with phyB in the light. Moreover, our data demonstrate that CBF1 promotes PIF4/PIF5 protein accumulation and hypocotyl growth at both 22°C and 17°C, but not at 4°C, with a more prominent role at 17°C than at 22°C. Together, our study reveals that CBF1 integrates light and temperature control of hypocotyl growth by promoting PIF4 and PIF5 protein abundance in the light, thus providing insights into the integration mechanisms of light and temperature signaling pathways in plants.

Verticillium dahliae effector VDAL protects MYB6 from degradation by interacting with PUB25 and PUB26 E3 ligases to enhance Verticillium wilt resistance.

Verticillium wilt is a severe plant disease that causes massive losses in multiple crops. Increasing the plant resistance to Verticillium wilt is a critical challenge worldwide. Here, we report that the hemibiotrophic Verticillium dahliae-secreted Asp f2-like protein VDAL causes leaf wilting when applied to cotton leaves in vitro but enhances the resistance to V. dahliae when overexpressed in Arabidopsis or cotton without affecting the plant growth and development. VDAL protein interacts with Arabidopsis E3 ligases plant U-box 25 (PUB25) and PUB26 and is ubiquitinated by PUBs in vitro. However, VDAL is not degraded by PUB25 or PUB26 in planta. Besides, the pub25 pub26 double mutant shows higher resistance to V. dahliae than the wild-type. PUBs interact with the transcription factor MYB6 in a yeast two-hybrid screen. MYB6 promotes plant resistance to Verticillium wilt while PUBs ubiquitinate MYB6 and mediate its degradation. VDAL competes with MYB6 for binding to PUBs, and the role of VDAL in increasing Verticillium wilt resistance depends on MYB6. Taken together, these results suggest that plants evolute a strategy to utilize the invaded effector protein VDAL to resist the V. dahliae infection without causing a hypersensitive response (HR); alternatively, hemibiotrophic pathogens may use some effectors to keep plant cells alive during its infection in order to take nutrients from host cells. This study provides the molecular mechanism for plants increasing disease resistance when overexpressing some effector proteins without inducing HR, and may promote searching for more genes from pathogenic fungi or bacteria to engineer plant disease resistance.

Phosphorylation of ZmAL14 by ZmSnRK2.2 regulates drought resistance through derepressing ZmROP8 expression.

Drought stress has negative effects on crop growth and production. Characterization of transcription factors that regulate the expression of drought-responsive genes is critical for understanding the transcriptional regulatory networks in response to drought, which facilitates the improvement of crop drought tolerance. Here, we identified an Alfin-like (AL) family gene ZmAL14 that negatively regulates drought resistance. Overexpression of ZmAL14 exhibits susceptibility to drought while mutation of ZmAL14 enhances drought resistance. An abscisic acid (ABA)-activated protein kinase ZmSnRK2.2 interacts and phosphorylates ZmAL14 at T38 residue. Knockout of ZmSnRK2.2 gene decreases drought resistance of maize. A dehydration-induced Rho-like small guanosine triphosphatase gene ZmROP8 is directly targeted and repressed by ZmAL14. Phosphorylation of ZmAL14 by ZmSnRK2.2 prevents its binding to the ZmROP8 promoter, thereby releasing the repression of ZmROP8 transcription. Overexpression of ZmROP8 stimulates peroxidase activity and reduces hydrogen peroxide accumulation after drought treatment. Collectively, our study indicates that ZmAL14 is a negative regulator of drought resistance, which can be phosphorylated by ZmSnRK2.2 through the ABA signaling pathway, thus preventing its suppression on ZmROP8 transcription during drought stress response.

Ca2+-independent ZmCPK2 is inhibited by Ca2+-dependent ZmCPK17 during drought response in maize.

Calcium oscillations are induced by different stresses. Calcium-dependent protein kinases (CDPKs/CPKs) are one major group of the plant calcium decoders that are involved in various processes including drought response. Some CPKs are calcium-independent. Here, we identified ZmCPK2 as a negative regulator of drought resistance by screening an overexpression transgenic maize pool. We found that ZmCPK2 does not bind calcium, and its activity is mainly inhibited during short term abscisic acid (ABA) treatment, and dynamically changed in prolonged treatment. Interestingly, ZmCPK2 interacts with and is inhibited by calcium-dependent ZmCPK17, a positive regulator of drought resistance, which is activated by ABA. ZmCPK17 could prevent the nuclear localization of ZmCPK2 through phosphorylation of ZmCPK2T60. ZmCPK2 interacts with and phosphorylates and activates ZmYAB15, a negative transcriptional factor for drought resistance. Our results suggest that drought stress-induced Ca2+ can be decoded directly by ZmCPK17 that inhibits ZmCPK2, thereby promoting plant adaptation to water deficit.

MYB30 Is a Key Negative Regulator of Arabidopsis Photomorphogenic Development That Promotes PIF4 and PIF5 Protein Accumulation in the Light.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants, and PHYTOCHROME-INTERACTING FACTORS (PIFs) are a group of basic helix-loop-helix family transcription factors that play central roles in repressing photomorphogenesis. Here, we report that MYB30, an R2R3-MYB family transcription factor, acts as a negative regulator of photomorphogenesis in Arabidopsis (Arabidopsis thaliana). We show that MYB30 preferentially interacts with the Pfr (active) forms of the phytochrome A (phyA) and phytochrome B (phyB) holoproteins and that MYB30 levels are induced by phyA and phyB in the light. It was previously shown that phytochromes induce rapid phosphorylation and degradation of PIFs upon R light exposure. Our current data indicate that MYB30 promotes PIF4 and PIF5 protein reaccumulation under prolonged R light irradiation by directly binding to their promoters to induce their expression and by inhibiting the interaction of PIF4 and PIF5 with the Pfr form of phyB. In addition, our data indicate that MYB30 interacts with PIFs and that they act additively to repress photomorphogenesis. In summary, our study demonstrates that MYB30 negatively regulates Arabidopsis photomorphogenic development by acting to promote PIF4 and PIF5 protein accumulation under prolonged R light irradiation, thus providing new insights into the complicated but delicate control of PIFs in the responses of plants to their dynamic light environment.

AtMCM10 promotes DNA replication-coupled nucleosome assembly in Arabidopsis.

Minichromosome Maintenance protein 10 (MCM10) is essential for DNA replication initiation and DNA elongation in yeasts and animals. Although the functions of MCM10 in DNA replication and repair have been well documented, the detailed mechanisms for MCM10 in these processes are not well known. Here, we identified AtMCM10 gene through a forward genetic screening for releasing a silenced marker gene. Although plant MCM10 possesses a similar crystal structure as animal MCM10, AtMCM10 is not essential for plant growth or development in Arabidopsis. AtMCM10 can directly bind to histone H3-H4 and promotes nucleosome assembly in vitro. The nucleosome density is decreased in Atmcm10, and most of the nucleosome density decreased regions in Atmcm10 are also regulated by newly synthesized histone chaperone Chromatin Assembly Factor-1 (CAF-1). Loss of both AtMCM10 and CAF-1 is embryo lethal, indicating that AtMCM10 and CAF-1 are indispensable for replication-coupled nucleosome assembly. AtMCM10 interacts with both new and parental histones. Atmcm10 mutants have lower H3.1 abundance and reduced H3K27me1/3 levels with releasing some silenced transposons. We propose that AtMCM10 deposits new and parental histones during nucleosome assembly, maintaining proper epigenetic modifications and genome stability during DNA replication.

Peroxisomal ß-oxidation regulates histone acetylation and DNA methylation in Arabidopsis.

Epigenetic markers, such as histone acetylation and DNA methylation, determine chromatin organization. In eukaryotic cells, metabolites from organelles or the cytosol affect epigenetic modifications. However, the relationships between metabolites and epigenetic modifications are not well understood in plants. We found that peroxisomal acyl-CoA oxidase 4 (ACX4), an enzyme in the fatty acid ß-oxidation pathway, is required for suppressing the silencing of some endogenous loci, as well as Pro35S:NPTII in the ProRD29A:LUC/C24 transgenic line. The acx4 mutation reduces nuclear histone acetylation and increases DNA methylation at the NOS terminator of Pro35S:NPTII and at some endogenous genomic loci, which are also targeted by the demethylation enzyme REPRESSOR OF SILENCING 1 (ROS1). Furthermore, mutations in multifunctional protein 2 (MFP2) and 3-ketoacyl-CoA thiolase-2 (KAT2/PED1/PKT3), two enzymes in the last two steps of the ß-oxidation pathway, lead to similar patterns of DNA hypermethylation as in acx4 Thus, metabolites from fatty acid ß-oxidation in peroxisomes are closely linked to nuclear epigenetic modifications, which may affect diverse cellular processes in plants.

The transcriptional co-repressor SEED DORMANCY 4-LIKE (AtSDR4L) promotes the embryonic-to-vegetative transition in Arabidopsis thaliana.

Repression of embryonic traits during the seed-to-seedling phase transition requires the inactivation of master transcription factors associated with embryogenesis. How the timing of such inactivation is controlled is unclear. Here, we report on a novel transcriptional co-repressor, Arabidopsis thaliana SDR4L, that forms a feedback inhibition loop with the master transcription factors LEC1 and ABI3 to repress embryonic traits post-imbibition. LEC1 and ABI3 regulate their own expression by inducing AtSDR4L during mid to late embryogenesis. AtSDR4L binds to sites upstream of LEC1 and ABI4, and these transcripts are upregulated in Atsdr4l seedlings. Atsdr4l seedlings phenocopy a LEC1 overexpressor. The embryonic traits of Atsdr4l can be partially rescued by impairing LEC1 or ABI3. The penetrance and expressivity of the Atsdr4l phenotypes depend on both developmental and external cues, demonstrating the importance of AtSDR4L in seedling establishment under suboptimal conditions.

Metabolomics-driven gene mining and genetic improvement of tolerance to salt-induced osmotic stress in maize.

The farmland of the world's main corn-producing area is increasingly affected by salt stress. Therefore, the breeding of salt-tolerant cultivars is necessary for the long-term sustainability of global corn production. Previous studies have shown that natural maize varieties display a large diversity of salt tolerance, yet the genetic variants underlying such diversity remain poorly discovered and applied, especially those mediating the tolerance to salt-induced osmotic stress (SIOS). Here we report a metabolomics-driven understanding and genetic improvement of maize SIOS tolerance. Using a LC-MS-based untargeted metabolomics approach, we profiled the metabolomes of 266 maize inbred lines under control and salt conditions, and then identified 37 metabolite biomarkers of SIOS tolerance (METO1-37). Follow-up metabolic GWAS (mGWAS) and genotype-to-phenotype modeling identified 10 candidate genes significantly associating with the SIOS tolerance and METO abundances. Furthermore, we validated that a citrate synthase, a glucosyltransferase and a cytochrome P450 underlie the genotype-METO-SIOS tolerance associations, and showed that their favorable alleles additively improve the SIOS tolerance of elite maize inbred lines. Our study provides a novel insight into the natural variation of maize SIOS tolerance, which boosts the genetic improvement of maize salt tolerance, and demonstrates a metabolomics-based approach for mining crop genes associated with this complex agronomic trait.

Hinge region of Arabidopsis phyA plays an important role in regulating phyA function.

Phytochrome A (phyA) is the only plant photoreceptor that perceives far-red light and then mediates various responses to this signal. Phosphorylation and dephosphorylation of oat phyA have been extensively studied, and it was shown that phosphorylation of a serine residue in the hinge region of oat phyA could regulate the interaction of phyA with its signal transducers. However, little is known about the role of the hinge region of Arabidopsis phyA. Here, we report that three sites in the hinge region of Arabidopsis phyA (i.e., S590, T593, and S602) are essential in regulating phyA function. Mutating all three of these sites to either alanines or aspartic acids impaired phyA function, changed the interactions of mutant phyA with FHY1 and FHL, and delayed the degradation of mutant phyA upon light exposure. Moreover, the in vivo formation of a phosphorylated phyA form was greatly affected by these mutations, while our data indicated that the abundance of this phosphorylated phyA form correlated well with the extent of phyA function, thus suggesting a pivotal role of the phosphorylated phyA in inducing the far-red light response. Taking these data together, our study reveals the important role of the hinge region of Arabidopsis phyA in regulating phyA phosphorylation and function, thus linking specific residues in the hinge region to the regulatory mechanisms of phyA phosphorylation.

High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis.

It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported endogenous reference gene hmg was analyzed using 633 maize inbred lines, and two SNPs were observed. These SNPs significantly increased the PCR efficiency, while the newly developed hmg gene assay (hmg-taq-F2/R2) excluding these SNPs reduced the efficiency into normal ranges. The TaqMan amplification efficiency of bar and hmg with newly developed primers was calculated as 0.993 and 1.000, respectively. The inter-assay coefficient of variation (CV) values for the bar and hmg genes varied from 1.18 to 2.94%. The copy numbers of the transgene bar using new TaqMan assays were identical to those using dPCR. Significantly, the precision of one repetition reached 96.7% of that of three repetitions of single-copy plants analyzed by simple random sampling, and the actual accuracy reached 95.8%, confirmed by T1 and T2 progeny. With the high-throughput DNA extraction and automated data analysis procedures developed in this study, nearly 2700 samples could be analyzed within eight hours by two persons. The combined results suggested that the new hmg gene assay developed here could be a universal maize reference gene system, and the new assay has high throughput and high accuracy for large-scale screening of maize varieties around the world.

METHIONINE SYNTHASE1 Is Involved in Chromatin Silencing by Maintaining DNA and Histone Methylation.

DNA methylation and histone modification are important epigenetic marks that coregulate gene expression and genome stability. To identify factors involved in chromatin silencing, we carried out a forward genetic screen for mutants that release the silenced Pro-35S:LUCIFERASE (35SP-LUC) in Arabidopsis (Arabidopsis thaliana). We identified an epigenetic regulator, METHIONINE SYNTHASE1 (ATMS1), which catalyzes the synthesis of methionine (Met) in the one-carbon metabolism pathway. The ATMS1 mutation releases the silenced 35SP-LUC and the majority of endogenous genes and transposons. The effect of ATMS1 on chromatin silencing is related to decreased levels of DNA methylation (CG, CHG, and CHH) and histone-3 lysine-9 dimethylation. The ATMS1 mutation caused a significant decrease in the ratio of S-adenosylmethionine to S-adenosylhomocysteine. Exogenous application of Met rescued the phenotype of atms1-1 ATMS1 plays a predominant role in DNA and histone methylations among the three Met synthetase homologs. These results suggest that ATMS1 is required for DNA and histone methylations through its function in the one-carbon metabolism pathway, indicating the complex interplay between metabolism and epigenetic regulation.

METHIONINE ADENOSYLTRANSFERASE4 Mediates DNA and Histone Methylation.

DNA and histone methylation coregulate heterochromatin formation and gene silencing in animals and plants. To identify factors involved in maintaining gene silencing, we conducted a forward genetic screen for mutants that release the silenced transgene Pro35S::NEOMYCIN PHOSPHOTRANSFERASE II in the transgenic Arabidopsis (Arabidopsis thaliana) line L119 We identified MAT4/SAMS3/MTO3/AT3G17390, which encodes methionine (Met) adenosyltransferase 4 (MAT4)/S-adenosyl-Met synthetase 3 that catalyzes the synthesis of S-adenosyl-Met (SAM) in the one-carbon metabolism cycle. mat4 mostly decreases CHG and CHH DNA methylation and histone H3K9me2 and reactivates certain silenced transposons. The exogenous addition of SAM partially rescues the epigenetic defects of mat4 SAM content and DNA methylation were reduced more in mat4 than in three other mat mutants. MAT4 knockout mutations generated by CRISPR/Cas9 were lethal, indicating that MAT4 is an essential gene in Arabidopsis. MAT1, 2, and 4 proteins exhibited nearly equal activity in an in vitro assay, whereas MAT3 exhibited higher activity. The native MAT4 promoter driving MAT1, 2, and 3 cDNA complemented the mat4 mutant. However, most mat4 transgenic lines carrying native MAT1, 2, and 3 promoters driving MAT4 cDNA did not complement the mat4 mutant because of their lower expression in seedlings. Genetic analyses indicated that the mat1mat4 double mutant is dwarfed and the mat2mat4 double mutant was nonviable, while mat1mat2 showed normal growth and fertility. These results indicate that MAT4 plays a predominant role in SAM production, plant growth, and development. Our findings provide direct evidence of the cooperative actions between metabolism and epigenetic regulation.

The involvement of the mGluR5-mediated JNK signaling pathway in rats with diabetic retinopathy.

OBJECTIVE: To understand the involvement of the mGluR5-mediated JNK signaling pathway in rats with diabetic retinopathy (DR). METHODS: This study established rat models of diabetes mellitus (DM), which were divided into Normal, DM, DM + CHPG (mGluR5 agonist CHPG), and DM + MTEP (mGluR5 antagonist MTEP) groups. The blood glucose and weight of rats were recorded. EB staining was used for observation of blood-retinal barrier (BRB) damage. Neural retina function was measured by pattern electroretinogram (ERG). PAS and NG2 immunohistochemistry were conducted to evaluate the retinal vascular morphology. The TUNEL assay and active caspase-3 immunohistochemistry were performed to detect retinal cell apoptosis. Additionally, the expression levels of superoxide dismutase (SOD) and methylenedioxyamphetamine (MDA) were measured. Moreover, expression levels of mGluR5 and JNK pathway-related proteins were detected by western blot. RESULTS: When compared with control rats, rats in the DM group showed decreased amplitude and latency of the peak times in the ERG test; further, DM group rats presented increases in blood glucose, BRB permeability, a retinal capillary area density, retinal cell apoptosis with an increased number of active caspase-3-positive cells, MDA level, mGluR5 levels, and the ratio of p-JNK/JNK, and they showed reductions in body weight and SOD activity, as well as in the number of pericytes and in the pericyte coverage (all P < 0.05). However, rats in DM + CHPG group had stronger negative effects than those in DM group (all P < 0.05). Rats from DM + MTEP group showed an opposite trend compared with the DM rats (all P < 0.05). CONCLUSION: The level of mGluR5 in DR rats was upregulated, whereas inhibition of mGluR5 alleviated retinal pathological damage and decreased cell apoptosis to improve DR via suppression of the JNK signaling pathway, which provided a scientific theoretical basis for the clinical treatment of DR.

The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation.

DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression.

AGP: a multimethods web server for alignment-free genome phylogeny.

Phylogenetic analysis based on alignment method meets huge challenges when dealing with whole-genome sequences, for example, recombination, shuffling, and rearrangement of sequences. Thus, various alignment-free methods for phylogeny construction have been proposed. However, most of these methods have not been implemented as tools or web servers. Researchers cannot use these methods easily with their data sets. To facilitate the usage of various alignment-free methods, we implemented most of the popular alignment-free methods and constructed a user-friendly web server for alignment-free genome phylogeny (AGP). AGP integrated the phylogenetic tree construction, visualization, and comparison functions together. Both AGP and all source code of the methods are available at http://www.herbbol.org:8000/agp (last accessed February 26, 2013). AGP will facilitate research in the field of whole-genome phylogeny and comparison.

CGAP: a new comprehensive platform for the comparative analysis of chloroplast genomes.

BACKGROUND: Chloroplast is an essential organelle in plants which contains independent genome. Chloroplast genomes have been widely used for plant phylogenetic inference recently. The number of complete chloroplast genomes increases rapidly with the development of various genome sequencing projects. However, no comprehensive platform or tool has been developed for the comparative and phylogenetic analysis of chloroplast genomes. Thus, we constructed a comprehensive platform for the comparative and phylogenetic analysis of complete chloroplast genomes which was named as chloroplast genome analysis platform (CGAP). RESULTS: CGAP is an interactive web-based platform which was designed for the comparative analysis of complete chloroplast genomes. CGAP integrated genome collection, visualization, content comparison, phylogeny analysis and annotation functions together. CGAP implemented four web servers including creating complete and regional genome maps of high quality, comparing genome features, constructing phylogenetic trees using complete genome sequences, and annotating draft chloroplast genomes submitted by users. CONCLUSIONS: Both CGAP and source code are available at http://www.herbbol.org:8000/chloroplast. CGAP will facilitate the collection, visualization, comparison and annotation of complete chloroplast genomes. Users can customize the comparative and phylogenetic analysis using their own unpublished chloroplast genomes.

An acoustic system for the individual recognition of insects.

Research into acoustic recognition systems for insects has focused on species identification rather than individual identification. In this paper, the feasibility of applying pattern recognition techniques to construct an acoustic system capable of automatic individual recognition for insects is investigated analytically and experimentally across two species of Orthoptera. Mel-frequency cepstral coefficients serve as the acoustic feature, and α-Gaussian mixture models were selected as the classification models. The performance of the proposed acoustic system is promising and displays high accuracy. The results suggest that the acoustic feature and classifier method developed here have potential for individual animal recognition and can be applied to other species of interest.

Chl1 coordinates with H3K9 methyltransferase Clr4 to reduce the accumulation of RNA-DNA hybrids and maintain genome stability.

A genome-wide analysis in Schizosaccharomyces pombe indicated that double-deletion mutants of Chl1 and histone H3K9 methyltransferase complex factors are synthetically sick. Here, we show that loss of Chl1 increases the accumulation of RNA-DNA hybrids at pericentromeric dg and dh repeats in the absence of the H3K9 methyltransferase Clr4, which leads to genome instability, including more severe defects in chromosome segregation and increased chromatin accessibility. Localization of Chl1 at pericentromeric regions depends on a subunit of replication protein A (RPA), Ssb1. In wild-type (WT) cells, transcriptionally repressed heterochromatin prevents the formation of RNA-DNA hybrids. When Clr4 is deleted, dg and dh repeats are highly transcribed. Then Ssb1 associates with the displaced single-stranded DNA (ssDNA) and recruits Chl1 to resolve the RNA-DNA hybrids. Together, our data suggest that Chl1 coordinates with Clr4 to eliminate RNA-DNA hybrids, which contributes to the maintenance of genome integrity.