Search

With the widespread use of CRISPR/Cas technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called WDV-Gate (Wheat Dwarf Virus-surrogate), to facilitate high-throughput genome editing. WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene (HygM) for surrogate selection; the other is a WDV-based construct expressing two sgRNAs targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T

Maintaining proper DNA methylation levels in the genome requires active demethylation of DNA. However, removing the methyl group from a modified cytosine is chemically difficult and therefore, the underlying mechanism of demethylation had remained unclear for many years. The discovery of the first eukaryotic DNA demethylase, Arabidopsis thaliana REPRESSOR OF SILENCING 1 (ROS1), led to elucidation of the 5-methylcytosine base excision repair mechanism of active DNA demethylation. In the 20 years since ROS1 was discovered, our understanding of this active DNA demethylation pathway, as well as its regulation and biological functions in plants, has greatly expanded. These exciting developments have laid the groundwork for further dissecting the regulatory mechanisms of active DNA demethylation, with potential applications in epigenome editing to facilitate crop breeding and gene therapy.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

Improved cytosine and adenine base editors and an efficient dual editor were applied in targeted evolution of ACETYL COA CARBOXYLASE in rice, resulting in the generation of dozens of herbicide-resistant mutations, at least three of which, W2125L, W2125Q and C2186H, have not been reported previously.

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 detail 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 binds 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 deposit new and parental histones during nucleosome assembly, maintaining proper epigenetic modifications and genome stability during DNA replication. This article is protected by copyright. All rights reserved.

DNA cytosine methylation confers stable epigenetic silencing in plants and many animals. However, the mechanisms underlying DNA methylation-mediated genomic silencing are not fully understood. We conducted a forward genetic screen for cellular factors required for the silencing of a heavily methylated p35S:NPTII transgene in the Arabidopsis thaliana rdm1-1 mutant background, which led to the identification of a Hsp20 family protein, RDS1 (rdm1-1 suppressor 1). Loss-of-function mutations in RDS1 released the silencing of the p35S::NPTII transgene in rdm1-1 mutant plants, without changing the DNA methylation state of the transgene. Protein interaction analyses suggest that RDS1 exists in a protein complex consisting of the methyl-DNA binding domain proteins MBD5 and MBD6, two other Hsp20 family proteins, RDS2 and IDM3, a Hsp40/DNAJ family protein, and a Hsp70 family protein. Like rds1 mutations, mutations in RDS2, MBD5, or MBD6 release the silencing of the transgene in the rdm1 mutant background. Our results suggest that Hsp20, Hsp40, and Hsp70 proteins may form a complex that is recruited to some genomic regions with DNA methylation by methyl-DNA binding proteins to regulate the state of silencing of these regions.

A new deaminase, TadA8e, was recently evolved in the laboratory. TadA8e catalyzes DNA deamination over 1,000 times faster than the widely used ABE7.10. Here, we took advantage of this speed to develop a high-efficiency adenine base editor, rABE8e (rice ABE8e), which combines monomeric TadA8e, bis-bpNLS and codon optimization. rABE8e had substantially increased editing efficiencies at both NG-protospacer adjacent motif (PAM) and NGG-PAM target sequences compared with ABEmax. For most targets, rABE8e exhibited close to 100% editing efficiency and high homozygous substitution rates in the specific editing window, especially at positions A5 and A6. The ability to rapidly generate plant materials with homozygous base substitutions will benefit gene function research and precision molecular breeding. This article is protected by copyright. All rights reserved.

DNA methylation is an epigenetic mark important for genome stability and gene expression. In Arabidopsis thaliana, the 5‐methylcytosine DNA glycosylase/demethylase DEMETER (DME) controls active DNA demethylation during the reproductive stage; however, the lethality of loss‐of‐function dme mutations has made it difficult to assess DME function in vegetative tissues. Here, we edited DME using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR‐associated protein 9 and created three weak dme mutants that produced a few viable seeds. We also performed central cell‐specific complementation in a strong dme mutant and combined this line with mutations in the other three Arabidopsis demethylase genes to generate the dme ros1 dml2 dml3 (drdd) quadruple mutant. A DNA methylome analysis showed that DME is required for DNA demethylation at hundreds of genomic regions in vegetative tissues. A transcriptome analysis of the drdd mutant revealed that DME and the other three demethylases are important for plant responses to biotic and abiotic stresses in vegetative tissues. Despite the limited role of DME in regulating DNA methylation in vegetative tissues, the dme mutants showed increased susceptibility to bacterial and fungal pathogens. Our study highlights the important functions of DME in vegetative tissues and provides valuable genetic tools for future investigations of DNA demethylation in plants., The Arabidopsis demethylase DEMETER acts on some target loci in the vegetative stage and plays a vital role in positive regulation of the response to pathogen infection, acting synergistically with three other demethylases.

In Arabidopsis, RNA-directed DNA methylation (RdDM) is required for the maintenance of CHH methylation, and for de novo methylation in all (CG, CHG, and CHH) contexts, but no obvious effect of RdDM deficiency on plant development has been found to date. We show that the combination of mutations in the chromatin remodeler PKL and RdDM components results in developmental alterations, which appear in a SUPPRESSOR OF DRM1 DRM2 CMT3 (SDC)-dependent manner.

The extensive phenotypic diversity within natural populations of Arabidopsis is associated with differences in gene expression. Transcript levels can be considered as inheritable quantitative traits, and used to map expression quantitative trait loci (eQTL) in genome-wide association studies (GWASs). In order to identify putative genetic determinants for variations in gene expression, we used publicly available genomic and transcript variation data from 665 Arabidopsis accessions and applied the single nucleotide polymorphism-set (Sequence) Kernel Association Test (SKAT) method for the identification of eQTL. Moreover, we used the penalized orthogonal-components regression (POCRE) method to increase the power of statistical tests. Then, gene annotations were used as test units to identify genes that are associated with natural variations in transcript accumulation, which correspond to candidate regulators, some of which may have a broad impact on gene expression. Besides increasing the chances to identify real associations, the analysis using POCRE and SKAT significantly reduced the computational cost required to analyze large datasets. As a proof of concept, we used this approach to identify eQTL that represent novel candidate regulators of immune responses. The versatility of this approach allows its application to any process that is subjected to natural variation among Arabidopsis accessions.

DNA methylation is typically regarded as a repressive epigenetic marker for gene expression. Genome-wide DNA methylation patterns in plants are dynamically regulated by the opposing activities of DNA methylation and demethylation reactions. In Arabidopsis, a DNA methylation monitoring sequence (MEMS) in the promoter of the DNA demethylase gene ROS1 functions as a methylstat that senses these opposing activities and regulates genome DNA methylation levels by adjusting ROS1 expression. How DNA methylation in the MEMS region promotes ROS1 expression is not known. Here, we show that several Su(var)3-9 homologs (SUVHs) can sense DNA methylation levels at the MEMS region and function redundantly to promote ROS1 expression. The SUVHs bind to the MEMS region, and the extent of binding is correlated with the methylation level of the MEMS. Mutations in the SUVHs lead to decreased ROS1 expression, causing DNA hypermethylation at more than 1,000 genomic regions. Thus, the SUVHs function to mediate the activation of gene transcription by DNA methylation.

We developed simplified single transcriptional unit (SSTU) CRISPR systems for multiplex gene editing in rice using FnCpf1, LbCpf1 or Cas9, in which the nuclease and its crRNA array are co-expressed from a single Pol II promoter, without any additional processing machinery. Our SSTU systems are easy to construct and effective in mediating multiplex genome editing.

Stomata, the pores formed by a pair of guard cells, are the main gateways for water transpiration and photosynthetic CO2 exchange, as well as pathogen invasion in land plants. Guard cell movement is regulated by a combination of environmental factors, including water status, light, CO2 levels and pathogen attack, as well as endogenous signals, such as abscisic acid and apoplastic reactive oxygen species (ROS). Under abiotic and biotic stress conditions, extracellular ROS are mainly produced by plasma membrane-localized NADPH oxidases, whereas intracellular ROS are produced in multiple organelles. These ROS form a sophisticated cellular signaling network, with the accumulation of apoplastic ROS an early hallmark of stomatal movement. Here, we review recent progress in understanding the molecular mechanisms of the ROS signaling network, primarily during drought stress and pathogen attack. We summarize the roles of apoplastic ROS in regulating stomatal movement, ABA and CO2 signaling, and immunity responses. Finally, we discuss ROS accumulation and communication between organelles and cells. This information provides a conceptual framework for understanding how ROS signaling is integrated with various signaling pathways during plant responses to abiotic and biotic stress stimuli.

In rice, amylose content (AC) is controlled by a single dominant Waxy gene. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) to introduce a loss-of-function mutation into the Waxy gene in two widely cultivated elite japonica varieties. Our results show that mutations in the Waxy gene reduce AC and convert the rice into glutinous ones without affecting other desirable agronomic traits, offering an effective and easy strategy to improve glutinosity in elite varieties. Importantly, we successfully removed the transgenes from the progeny. Our study provides an example of generating improved crops with potential for commercialization, by editing a gene of interest directly in elite crop varieties.

DNA methylation is typically regarded as a repressive epigenetic marker for gene expression. Genome-wide DNA methylation patterns in plants are dynamically regulated by the opposing activities of DNA methylation and demethylation reactions. In Arabidopsis, a DNA methylation monitoring sequence (MEMS) in the promoter of the DNA demethylase gene ROS1 functions as a methylstat that senses these opposing activities and regulates genome DNA methylation levels by adjusting ROS1 expression. How DNA methylation in the MEMS region promotes ROS1 expression is not known. Here, we show that several Su(var)3-9 homologs (SUVHs) can sense DNA methylation levels at the MEMS region and function redundantly to promote ROS1 expression. The SUVHs bind to the MEMS region, and the extent of binding is correlated with the methylation level of the MEMS. Mutations in the SUVHs lead to decreased ROS1 expression, causing DNA hypermethylation at more than 1,000 genomic regions. Thus, the SUVHs function to mediate the activation of gene transcription by DNA methylation.

Stomata, the pores formed by a pair of guard cells, are the main gateways for water transpiration and photosynthetic CO