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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 inXanthomonas oryzaepv.oryzae(Xoo), one of the most important bacterial pathogens on rice, by employing the heterogenous CRISPR/Cas12a fromFrancisella novicidaand NHEJ proteins fromMycobacterium 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 cure in theXooPXO99Astrain. Accordingly, a non-TAL effector-free polymutant strain PXO99AD25E, which lacks all 25Xopgenes involved inXoopathogenesis, 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 pathogenPseudomonas syringaepv.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 ofXoo.

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.

Objective This study aimed to explore the prevalence of hyperuricemia among the general adult population in Shenzhen and to determine the risk factors of hyperuricemia. Methods A total of 9337 cases who underwent physical examination in The Seventh Affiliated Hospital of Sun Yat-Sen University in 2020 were enrolled in this study. The binary logistic regression analysis was performed to determine potential risk factors of hyperuricemia. Result The overall prevalence rate of hyperuricemia in the studied population was 40.5% (3783/9337). Subgroup analysis showed that the prevalence of males was 52.8% (3046/5769) and the prevalence of females was 20.7% (737/3568). Moreover, among different age groups (≤29 years old, 30-39 years old, 40-49 years old, 50-59 years old, and ≥60 years old), the prevalence rate of males was significantly higher than that of females (P

RNA quality control (RQC) nonsense-mediated decay (NMD) is involved in viral restriction in both plants and animals. However, it is not known whether two other RQC pathways, nonstop decay (NSD) and no-go decay (NGD), 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 G

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.

Using cross-country data for 1999–2019, we investigate how bank capital structure and institutional environment affect bank competition. Banks with a higher Tier 1 ratio have greater market power, ...

DNA methylation regulates gene expression under abiotic and biotic stresses. Recently, Gui et al. discovered that geminiviruses subverted DNA methylation-mediated defense through boosting the active DNA demethylation mediated by host DNA glycosylases to promote viral virulence. Their findings reveal a distinctive counter-defense strategy exploited by invading pathogens to achieve successful infection.

This study investigates the impact of bank competition and regulation on bank efficiency in the Asia-Pacific region during 2001–2016. The result reveals that market power is positively related to b...

RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription. However, co-transcriptional splicing has not yet been explored in plants. Here, we built transcriptomes of nascent chromatin RNAs in Arabidopsis thaliana and showed that nearly all introns undergo co-transcriptional splicing, which occurs with higher efficiency for introns in protein-coding genes than for those in noncoding RNAs. Total intron number and intron position are two predominant features that correlate with co-transcriptional splicing efficiency, and introns with alternative 5′ or 3′ splice sites are less efficiently spliced. Furthermore, we found that mutations in genes encoding trans-acting proteins lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs, and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level. Collectively, our results not only uncovered widespread co-transcriptional splicing in Arabidopsis but also identified features that may affect or be affected by co-transcriptional splicing efficiency.

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.

This study empirically investigates how bank capital and competitive conditions affect bank risk-taking. Using financial data of 7620 banks on 118 countries from 2001 to 2016, we show that banks wi...

Background The high-activity adenine base editors (ABEs), engineered with the recently-developed tRNA adenosine deaminases (TadA8e and TadA9), show robust base editing activity but raise concerns about off-target effects. Results In this study, we perform a comprehensive evaluation of ABE8e- and ABE9-induced DNA and RNA mutations in Oryza sativa. Whole-genome sequencing analysis of plants transformed with four ABEs, including SpCas9n-TadA8e, SpCas9n-TadA9, SpCas9n-NG-TadA8e, and SpCas9n-NG-TadA9, reveal that ABEs harboring TadA9 lead to a higher number of off-target A-to-G (A>G) single-nucleotide variants (SNVs), and that those harboring CRISPR/SpCas9n-NG lead to a higher total number of off-target SNVs in the rice genome. An analysis of the T-DNAs carrying the ABEs indicates that the on-target mutations could be introduced before and/or after T-DNA integration into plant genomes, with more off-target A>G SNVs forming after the ABEs had integrated into the genome. Furthermore, we detect off-target A>G RNA mutations in plants with high expression of ABEs but not in plants with low expression of ABEs. The off-target A>G RNA mutations tend to cluster, while off-target A>G DNA mutations rarely clustered. Conclusion Our findings that Cas proteins, TadA variants, temporal expression of ABEs, and expression levels of ABEs contribute to ABE specificity in rice provide insight into the specificity of ABEs and suggest alternative ways to increase ABE specificity besides engineering TadA variants.

MicroRNAs (miRNAs) play crucial roles in gene expression regulation through RNA cleavage or translation repression. Although miRNA biogenesis has been extensively studied, new factors involved in miRNA biogenesis are still being found from various genetic screens, suggesting that knowledge of miRNA biogenesis or its regulation is still incomplete. Here, we report the identification of a previously uncharacterized and evolutionarily conserved WD40 domain protein as a player in miRNA biogenesis in Arabidopsis thaliana. A mutation in the REDUCTION IN BLEACHED VEIN AREA (RBV) gene encoding a WD40 domain protein led to the suppression of leaf bleaching caused by an artificial miRNA; the mutation also led to a global reduction in the accumulation of endogenous miRNAs. RBV may act at multiple steps in miRNA biogenesis. RBV promotes the transcription of MIR genes into pri-miRNAs by enhancing the occupancy of RNA polymerase II (Pol II) at MIR gene promoters. The nuclear protein RBV may also participate in pri-miRNA processing, as the mutation of RBV leads to the reduction of dicing body number. Moreover, mutation of RBV leads to a defect of miRNA loading into AGO1.RBV function is required in messenger RNA splicing, as RNA-seq uncovered a global intron retention defect in the mutant. Thus, this previously uncharacterized, evolutionarily conserved, nuclear WD40 domain protein acts in miRNA biogenesis and RNA splicing.

As the most common RNA cap in eukaryotes, the 7-methylguanosine (m 7 G) 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.

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 β-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 β-oxidation pathway, lead to similar patterns of DNA hypermethylation as in acx4 . Thus, metabolites from fatty acid β-oxidation in peroxisomes are closely linked to nuclear epigenetic modifications, which may affect diverse cellular processes in plants.

This article empirically investigates the influence of bank regulation and supervision on the competitive landscape in banking systems. Using the information on 23 emerging economies from 1996 to 2...

This study tests the impact of banking sector reform and competition on bank stability based on unbalanced data from 22 transition countries from 1998 to 2016. The initial results not only ...

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.

Three-dimensional visualization technology converts engineering design drawings and data into graphics or images, realizes virtual reality perception of simulated users in future construction scene, enhances the interaction between project management and technical personnel and engineering construction achievement, and provides intuitive, flexible and strong realistic experience for project management. It can effectively improve the level of project communication, and assist the needs of project construction planning management, training, exhibition, etc. As a tool to help improve project management skills, it has good application effect and prospects.

This chapter empirically investigates how bank capital and competitive conditions affect bank risk-taking. Using financial data of 7620 banks on 118 countries from 2001 to 2016, we show that banks with a higher Tier 1 ratio and a lower Tier 2 ratio are lower risk-takers. A bank with greater market power in a banking system tends to reduce its risk-taking activities. Our findings also highlight that the negative relationship between Tier 1 ratio and bank risk are more pronounced in more competitive conditions. During the financial crisis, Tier 1 capital acted as a stable funding source and reduced bank risk, but the evidence on Tier 2 capital shows that a higher Tier 2 ratio results in a higher level of risk and increases bank instability.

This study investigates the impact of bank competition and regulation on bank efficiency in the Asia–Pacific region during 2001–2016. The result reveals that market power is positively related to bank efficiency. We also find that stringent activity restrictions, strong official supervisory power, and low capital requirements are associated with high bank efficiency.

This article empirically investigates the influence of bank regulation and supervision on the competitive landscape in banking systems. Using the information on 23 emerging economies from 1996 to 2016, we confirm that banking systems with fewer activity restrictions and (foreign) bank entry barriers are more competitive. Greater capital strictness and official supervision enhance competition in the banking industry.

This study tests the impact of banking sector reform and competition on bank stability based on unbalanced data from 22 transition countries from 1998 to 2016. The initial results not only highlight the positive relationship between market power and bank fragility, but also confirm the positive relationship between bank reform and stability. Our findings also show that both higher activity restrictions and more explicit guidelines for asset diversification increase bank stability, but this positive effect significantly weakens for banks with higher market power. More stringent capital requirements in combination with higher market power increase the risk of bank insolvency.

This study finds a negative association between firm-level political risk (PRISK) and firm-level patents and citations in general. The PRISK extracted from different sectors also shows similar effects. This impact is more pronounced when a firm belongs to economically uncertain states, politically corrupted regions, and competitive industries. Besides, we identify that increasing cost of capital is a transmission channel that surges the negative impact of PRISK on corporate innovations. We also find the robust negative impact of PRISK on innovation when firms mainly rely on external finances. Our findings have policy implications for the firm’s manager, market participants, and practitioners.

Recently reported adenine base editors (ABEs) exhibit powerful potential for targeted gene correction as well as developing gain-of-function mutants and novel germplasms for both gene function studies and crop breeding. However, editing efficiency varies significantly among different target sites. Here, we investigated the activities of three evolved E. coli adenosine deaminase TadA variants (TadA8e, TadA8.17, and TadA8.20) side-by-side in transgenic rice. We found that TadA8e outperforms TadA8.17 and TadA8.20, and induces efficient A-to-G conversion at all tested sites in the rice genome, including those that were uneditable by ABE7.10 in our previous experiments. Furthermore, V82S/Q154R mutations were incorporated into TadA8e, resulting in a new variant that we named TadA9. Our data show that TadA9 is broadly compatible with CRISPR/SpCas9, CRISPR/SpCas9-NG, and CRISPR/SpRY, as well as CRISPR/ScCas9 nickase systems, achieving comparable or enhanced editing in a larger editing window at diverse PAM sites as compared with TadA8e. Finally, TadA9 was used to simultaneously install novel SNPs in four endogenous herbicide target genes in the commercial rice cultivar Nangeng 46 for potential field application in weed control. Collectively, we successfully generated a series of novel ABEs that can efficiently edit adenosines in the rice genome. Our findings suggest that TadA9 and TadA8e have great potentials in the development of plant base editors and crop molecular breeding.

Eurasian avian-like (EA) H1N1 swine influenza viruses (SIVs) are currently the most prevalent SIVs in Chinese swine populations, but recent human-like H3N2 SIV subtypes have also been frequently isolated. Hence, there is an urgent need to develop an effective vaccine against both EA H1N1 and recent human-like H3N2 infections. In this study, we utilized the baculovirus expression system to produce virus-like particles (VLPs) containing hemagglutinin protein (HA) and matrix protein (M1) based on A/Swine/Guangdong/YJ4/2014 (H1N1) and A/swine/Guangdong/L22/2010 (H3N2). An immunological experiment showed that in a mouse model, bivalent VLP vaccines against H1N1 and H3N2 can induce stronger humoral and cellular immune responses than whole influenza virus vaccines. Compared with monovalent inactivated vaccines that cannot offer protection against different SIV subtypes, monovalent H1N1 or H3N2 VLP vaccines can provide partial protection against lethal challenge by viruses of different subtypes. Meanwhile, bivalent VLP vaccines against H1N1 and H3N2 can provide full protection against lethal doses of homologous and heterologous viruses belonging to the EA H1N1 or recent human-like H3N2 lineage. These results suggest a promising approach to the development of vaccines against SIVs.