CRISPR/FnCas12a-mediated efficient multiplex and iterative genome editing in bacterial plant pathogens without donor DNA templates.

CRISPR-based genome editing technology is revolutionizing prokaryotic research, but it has been rarely studied in bacterial plant pathogens. Here, we have developed a targeted genome editing method with no requirement of donor templates for convenient and efficient gene knockout in Xanthomonas oryzae pv. oryzae (Xoo), one of the most important bacterial pathogens on rice, by employing the heterologous CRISPR/Cas12a from Francisella novicida and NHEJ proteins from Mycobacterium tuberculosis. FnCas12a nuclease generated both small and large DNA deletions at the target sites as well as it enabled multiplex genome editing, gene cluster deletion, and plasmid curing in the Xoo PXO99A strain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25 xop genes involved in Xoo pathogenesis, has been engineered through iterative genome editing. Whole-genome sequencing analysis indicated that FnCas12a did not have a noticeable off-target effect. In addition, we revealed that these strategies are also suitable for targeted genome editing in another bacterial plant pathogen Pseudomonas syringae pv. tomato (Pst). We believe that our bacterial genome editing method will greatly expand the CRISPR study on microorganisms and advance our understanding of the physiology and pathogenesis of Xoo.

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis.

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

T-LOC: A comprehensive tool to localize and characterize T-DNA integration sites.

Scientists have developed many approaches based on PCR or next-generation sequencing to localize and characterize integrated T-DNAs in transgenic plants generated by Agrobacterium tumefaciens-mediated T-DNA transfer. However, none of these methods has the robust ability to handle all transgenic plants with diversified T-DNA patterns. Utilizing the valuable information in the whole-genome sequencing data of transgenic plants, we have developed a comprehensive approach (T-LOC) to localize and characterize T-DNA integration sites (TISs). We evaluated the performance of T-LOC on genome sequencing data from 48 transgenic rice (Oryza sativa) plants that provide real and unbiased resources of T-DNA integration patterns. T-LOC discovered 75 full TISs and reported a diversified pattern of T-DNA integration: the ideal single-copy T-DNA between two borders, multiple-copy of T-DNAs in tandem or inverted repeats, truncated partial T-DNAs with or without the selection hygromycin gene, the inclusion of T-DNA backbone, the integration at the genome repeat region, and the concatenation of multiple ideal or partial T-DNAs. In addition, we reported that DNA fragments from the two A. tumefaciens plasmids can be fused with T-DNA and integrated into the plant genome. Besides, T-LOC characterizes the genomic changes at TISs, including deletion, duplication, accurate repair, and chromosomal rearrangement. Moreover, we validated the robustness of T-LOC using PCR, Sanger sequencing, and Nanopore sequencing. In summary, T-LOC is a robust approach to studying the TISs independent of the integration pattern and can recover all types of TISs in transgenic plants.

The Arabidopsis Nodulin Homeobox Factor AtNDX Interacts with AtRING1A/B and Negatively Regulates Abscisic Acid Signaling.

The phytohormone abscisic acid (ABA) and the Polycomb group proteins have key roles in regulating plant growth and development; however, their interplay and underlying mechanisms are not fully understood. Here, we identified an Arabidopsis (Arabidopsis thaliana) nodulin homeobox (AtNDX) protein as a negative regulator in the ABA signaling pathway. AtNDX mutants are hypersensitive to ABA, as measured by inhibition of seed germination and root growth, and the expression of AtNDX is downregulated by ABA. AtNDX interacts with the Polycomb Repressive Complex1 (PRC1) core components AtRING1A and AtRING1B in vitro and in vivo, and together, they negatively regulate the expression levels of some ABA-responsive genes. We identified ABA-INSENSITIVE (ABI4) as a direct target of AtNDX. AtNDX directly binds the downstream region of ABI4 and deleting this region increases the ABA sensitivity of primary root growth. Furthermore, ABI4 mutations rescue the ABA-hypersensitive phenotypes of ndx mutants and ABI4-overexpressing plants are hypersensitive to ABA in primary root growth. Thus, our work reveals the critical functions of AtNDX and PRC1 in some ABA-mediated processes and their regulation of ABI4.

The PROTEIN PHOSPHATASE4 Complex Promotes Transcription and Processing of Primary microRNAs in Arabidopsis.

PROTEIN PHOSPHATASE4 (PP4) is a highly conserved Ser/Thr protein phosphatase found in yeast, plants, and animals. The composition and functions of PP4 in plants are poorly understood. Here, we uncovered the complexity of PP4 composition and function in Arabidopsis (Arabidopsis thaliana) and identified the composition of one form of PP4 containing the regulatory subunit PP4R3A. We show that PP4R3A, together with one of two redundant catalytic subunit genes, PROTEIN PHOSPHATASE X (PPX)1 and PPX2, promotes the biogenesis of microRNAs (miRNAs). PP4R3A is a chromatin-associated protein that interacts with RNA polymerase II and recruits it to the promoters of miRNA-encoding (MIR) genes to promote their transcription. PP4R3A likely also promotes the cotranscriptional processing of miRNA precursors, because it recruits the microprocessor component HYPONASTIC LEAVES1 to MIR genes and to nuclear dicing bodies. Finally, we show that hundreds of introns exhibit splicing defects in pp4r3a mutants. Together, this study reveals roles for Arabidopsis PP4 in transcription and nuclear RNA metabolism.

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.

NAD+-capped RNAs are widespread in the Arabidopsis transcriptome and can probably be translated.

As the most common RNA cap in eukaryotes, the 7-methylguanosine (m7G) cap impacts nearly all processes that a messenger RNA undergoes, such as splicing, polyadenylation, nuclear export, translation, and degradation. The metabolite and redox agent, nicotinamide adenine diphosphate (NAD+), can be used as an initiating nucleotide in RNA synthesis to result in NAD+-capped RNAs. Such RNAs have been identified in bacteria, yeast, and human cells, but it is not known whether they exist in plant transcriptomes. The functions of the NAD+ cap in RNA metabolism or translation are still poorly understood. Here, through NAD captureSeq, we show that NAD+-capped RNAs are widespread in Arabidopsis thaliana NAD+-capped RNAs are predominantly messenger RNAs encoded by the nuclear and mitochondrial genomes, but not the chloroplast genome. NAD+-capped transcripts from the nuclear genome appear to be spliced and polyadenylated. Furthermore, although NAD+-capped transcripts constitute a small proportion of the total transcript pool from any gene, they are enriched in the polysomal fraction and associate with translating ribosomes. Our findings implicate the existence of as yet unknown mechanisms whereby the RNA NAD+ cap interfaces with RNA metabolic processes as well as translation initiation. More importantly, our findings suggest that cellular metabolic and/or redox states may influence, or be regulated by, mRNA NAD+ capping.

Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis.

Twenty-four-nucleotide small interfering (si)RNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes and maintains DNA methylation at transposable elements to ensure genome stability in plants. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is believed to transcribe RdDM loci to produce primary transcripts that are converted to double-stranded RNAs (dsRNAs) by RDR2 to serve as siRNA precursors. Yet, no such siRNA precursor transcripts have ever been reported. Here, through genome-wide profiling of RNAs in genotypes that compromise the processing of siRNA precursors, we were able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci. We show that Pol IV/RDR2-dependent transcripts correspond to both DNA strands, whereas the RNA polymerase II (Pol II)-dependent transcripts produced upon derepression of the loci are derived primarily from one strand. We also show that Pol IV/RDR2-dependent transcripts have a 5' monophosphate, lack a poly(A) tail at the 3' end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Like Pol II-transcribed genic regions, Pol IV-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes, which highlights similarities in Pol II- and Pol IV-mediated transcription. Computational analysis of siRNA abundance from various mutants reveals differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation via two different DNA methyltransferases. These findings begin to reveal features of Pol IV/RDR2-mediated transcription at the heart of genome stability in plants.

SUVH1, a Su(var)3-9 family member, promotes the expression of genes targeted by DNA methylation.

Transposable elements are found throughout the genomes of all organisms. Repressive marks such as DNA methylation and histone H3 lysine 9 (H3K9) methylation silence these elements and maintain genome integrity. However, how silencing mechanisms are themselves regulated to avoid the silencing of genes remains unclear. Here, an anti-silencing factor was identified using a forward genetic screen on a reporter line that harbors a LUCIFERASE (LUC) gene driven by a promoter that undergoes DNA methylation. SUVH1, a Su(var)3-9 homolog, was identified as a factor promoting the expression of the LUC gene. Treatment with a cytosine methylation inhibitor completely suppressed the LUC expression defects of suvh1, indicating that SUVH1 is dispensable for LUC expression in the absence of DNA methylation. SUVH1 also promotes the expression of several endogenous genes with promoter DNA methylation. However, the suvh1 mutation did not alter DNA methylation levels at the LUC transgene or on a genome-wide scale; thus, SUVH1 functions downstream of DNA methylation. Histone H3 lysine 4 (H3K4) trimethylation was reduced in suvh1; in contrast, H3K9 methylation levels remained unchanged. This work has uncovered a novel, anti-silencing function for a member of the Su(var)3-9 family that has previously been associated with silencing through H3K9 methylation.

DNA topoisomerase 1α promotes transcriptional silencing of transposable elements through DNA methylation and histone lysine 9 dimethylation in Arabidopsis.

RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.

Ancient Origin and Recent Innovations of RNA Polymerase IV and V.

Small RNA-mediated chromatin modification is a conserved feature of eukaryotes. In flowering plants, the short interfering (si)RNAs that direct transcriptional silencing are abundant and subfunctionalization has led to specialized machinery responsible for synthesis and action of these small RNAs. In particular, plants possess polymerase (Pol) IV and Pol V, multi-subunit homologs of the canonical DNA-dependent RNA Pol II, as well as specialized members of the RNA-dependent RNA Polymerase (RDR), Dicer-like (DCL), and Argonaute (AGO) families. Together these enzymes are required for production and activity of Pol IV-dependent (p4-)siRNAs, which trigger RNA-directed DNA methylation (RdDM) at homologous sequences. p4-siRNAs accumulate highly in developing endosperm, a specialized tissue found only in flowering plants, and are rare in nonflowering plants, suggesting that the evolution of flowers might coincide with the emergence of specialized RdDM machinery. Through comprehensive identification of RdDM genes from species representing the breadth of the land plant phylogeny, we describe the ancient origin of Pol IV and Pol V, suggesting that a nearly complete and functional RdDM pathway could have existed in the earliest land plants. We also uncover innovations in these enzymes that are coincident with the emergence of seed plants and flowering plants, and recent duplications that might indicate additional subfunctionalization. Phylogenetic analysis reveals rapid evolution of Pol IV and Pol V subunits relative to their Pol II counterparts and suggests that duplicates were retained and subfunctionalized through Escape from Adaptive Conflict. Evolution within the carboxy-terminal domain of the Pol V largest subunit is particularly striking, where illegitimate recombination facilitated extreme sequence divergence.

Activated carbon fibers functionalized with superhydrophilic coated pDA/TiO2/SiO2 with photoluminescent self-cleaning properties for efficient oil-water separation.

The adhesion of high-viscosity oil contamination poses limitations on three-dimensional (3D) materials' practical use in treating oilfield-produced water (OPW). In this study, we developed a hybrid pDA/TiO2/SiO2 coating (PTS) on the surface of hydrophilic activated carbon (ACF1) through a combination of dopamine (DA) polymerization, ethyl orthosilicate (TEOS) hydrolysis, and the condensation of TiO2 nanoparticles (NPs) with SiO2 NPs. This coating was designed for gravity-based oil-water separation. The inherent porosity and generous pore size of ACF1-PTS conferred it an ultra-high permeation flux (pure water flux of 3.72 × 105 L∙m-2∙h-1), allowing it to effectively separate simulated oil-water mixtures and oil-water emulsions while maintaining exceptional permeation flux and oil rejection efficiency. When compared to cleaning methods involving ethanol aqueous solutions and NaClO, ultraviolet (UV) illumination cleaning proved superior, enabling oil-contaminated ACF1-PTS to exhibit remarkable flux recovery efficiency and oil-removal capabilities during cyclic separation of actual OPW. Furthermore, the ACF1-PTS material demonstrated impressive stability and durability when exposed to acidic environments (acid, alkali, and salt), robust hydraulic washout conditions, and 25-cycle tests. This study offers valuable insights and research avenues for the development of highly efficient and environmentally friendly 3D oil-water separation materials for the actual treatment of OPW.

Diverse nucleotide substitutions in rice base editing mediated by novel TadA variants.

CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops. The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes. Compared with the well-developed adenine base editors (ABEs), cytosine base editors (CBEs) and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice, significantly limiting their application. Here, we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice. We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID∗Δ, and TadA-CDd outperformed TadA-E27R/N46L. A C-to-G base editor (CGBE) engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P. In addition, a dual base editor constructed with a single protein, TadDE, enabled simultaneous, highly efficient C-to-T and A-to-G editing in rice. Collectively, our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice, providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.

Molecular recognition and quantitative analysis of xylene isomers utilizing cataluminescence sensor array.

A facile and effective sensor array consisting of three cataluminescence (CTL) sensors based on nanomaterial Y(2)O(3), γ-Al(2)O(3) and ZrO(2) as probes was firstly proposed for the molecular recognition and quantitative analysis of xylene isomers. Under the optimized conditions, the linear range of CTL intensity versus concentration of xylene isomers was 86.70-8670.00 mg m(-3). The use of a sensor array instead of a single sensor has provided a novel strategy for the process of identifying similar chemical compounds, which should have a bright future in environmental and industrial monitoring.

Genetic Screens for Floral Mutants in Arabidopsis thaliana: Enhancers and Suppressors.

The flower is a hallmark feature that has contributed to the evolutionary success of land plants. Diverse mutagenic agents have been employed as a tool to genetically perturb flower development and identify genes involved in floral patterning and morphogenesis. Since the initial studies to identify genes governing processes such as floral organ specification, mutagenesis in sensitized backgrounds has been used to isolate enhancers and suppressors to further probe the molecular basis of floral development. Here, we first describe two commonly employed methods for mutagenesis (using ethyl methanesulfonate (EMS) or T-DNAs as mutagens), and then describe three methods for identifying a mutation that leads to phenotypic alterations: traditional map-based cloning, modified high-efficiency thermal asymmetric interlaced PCR (mhiTAIL-PCR), and deep sequencing in the plant model Arabidopsis thaliana.

SUMOylation-modified Pelota-Hbs1 RNA surveillance complex restricts the infection of potyvirids in plants.

RNA quality control nonsense-mediated decay is involved in viral restriction in both plants and animals. However, it is not known whether two other RNA quality control pathways, nonstop decay and no-go decay, are capable of restricting viruses in plants. Here, we show that the evolutionarily conserved Pelota-Hbs1 complex negatively regulates infection of plant viruses in the family Potyviridae (termed potyvirids), the largest group of plant RNA viruses that accounts for more than half of the viral crop damage worldwide. Pelota enables the recognition of the functional G1-2A6-7 motif in the P3 cistron, which is conserved in almost all potyvirids. This allows Pelota to target the virus and act as a viral restriction factor. Furthermore, Pelota interacts with the SUMO E2-conjugating enzyme SCE1 and is SUMOylated in planta. Blocking Pelota SUMOylation disrupts the ability to recruit Hbs1 and inhibits viral RNA degradation. These findings reveal the functional importance of Pelota SUMOylation during the infection of potyvirids in plants.