Light controls mesophyll-specific post-transcriptional splicing of photoregulatory genes by AtPRMT5.

Light plays a central role in plant growth and development, providing an energy source and governing various aspects of plant morphology. Previous study showed that many polyadenylated full-length RNA molecules within the nucleus contain unspliced introns (post-transcriptionally spliced introns, PTS introns), which may play a role in rapidly responding to changes in environmental signals. However, the mechanism underlying post-transcriptional regulation during initial light exposure of young, etiolated seedlings remains elusive. In this study, we used FLEP-seq2, a Nanopore-based sequencing technique, to analyze nuclear RNAs in Arabidopsis (Arabidopsis thaliana) seedlings under different light conditions and found numerous light-responsive PTS introns. We also used single-nucleus RNA sequencing (snRNA-seq) to profile transcripts in single nucleus and investigate the distribution of light-responsive PTS introns across distinct cell types. We established that light-induced PTS introns are predominant in mesophyll cells during seedling de-etiolation following exposure of etiolated seedlings to light. We further demonstrated the involvement of the splicing-related factor A. thaliana PROTEIN ARGININE METHYLTRANSFERASE 5 (AtPRMT5), working in concert with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a critical repressor of light signaling pathways. We showed that these two proteins orchestrate light-induced PTS events in mesophyll cells and facilitate chloroplast development, photosynthesis, and morphogenesis in response to ever-changing light conditions. These findings provide crucial insights into the intricate mechanisms underlying plant acclimation to light at the cell-type level.

Integrated single-nucleus and spatial transcriptomics captures transitional states in soybean nodule maturation.

Legumes form symbiosis with rhizobium leading to the development of nitrogen-fixing nodules. By integrating single-nucleus and spatial transcriptomics, we established a cell atlas of soybean nodules and roots. In central infected zones of nodules, we found that uninfected cells specialize into functionally distinct subgroups during nodule development, and revealed a transitional subtype of infected cells with enriched nodulation-related genes. Overall, our results provide a single-cell perspective for understanding rhizobium-legume symbiosis.

Plant Intron-Splicing Efficiency Database (PISE): exploring splicing of ∼1,650,000 introns in Arabidopsis, maize, rice, and soybean from ∼57,000 public RNA-seq libraries.

Intron retention is the most common alternative splicing event in plants and plays a crucial role in the responses of plants to environmental signals. Despite a large number of RNA-seq libraries from different treatments and genetic mutants stored in public domains, a resource for querying the intron-splicing ratio of individual intron is still required. Here, we established the first-ever large-scale splicing efficiency database in any organism. Our database includes over 57,000 plant public RNA-seq libraries, comprising 25,283 from Arabidopsis, 17,789 from maize, 10,710 from rice, and 3,974 from soybean, and covers a total of 1.6 million introns in these four species. In addition, we manually curated and annotated all the mutant- and treatment-related libraries as well as their matched controls included in our library collection, and added graphics to display intron-splicing efficiency across various tissues, developmental stages, and stress-related conditions. The result is a large collection of 3,313 treatment conditions and 3,594 genetic mutants for discovering differentially regulated splicing efficiency. Our online database can be accessed at https://plantintron.com/ .

An atlas of plant full-length RNA reveals tissue-specific and monocots-dicots conserved regulation of poly(A) tail length.

Poly(A) tail is a hallmark of eukaryotic messenger RNA and its length plays an essential role in regulating mRNA metabolism. However, a comprehensive resource for plant poly(A) tail length has yet to be established. Here, we applied a poly(A)-enrichment-free, nanopore-based method to profile full-length RNA with poly(A) tail information in plants. Our atlas contains over 120 million polyadenylated mRNA molecules from seven different tissues of Arabidopsis, as well as the shoot tissue of maize, soybean and rice. In most tissues, the size of plant poly(A) tails shows peaks at approximately 20 and 45 nucleotides, while the poly(A) tails in pollen exhibit a distinct pattern with strong peaks centred at 55 and 80 nucleotides. Moreover, poly(A) tail length is regulated in a gene-specific manner-mRNAs with short half-lives in general have long poly(A) tails, while mRNAs with long half-lives are featured with relatively short poly(A) tails that peak at ~45 nucleotides. Across species, poly(A) tails in the nucleus are almost twice as long as in the cytoplasm. Our comprehensive dataset lays the groundwork for future functional and evolutionary studies on poly(A) tail length regulation in plants.

Mutations in PpAGO3 Lead to Enhanced Virulence of Phytophthora parasitica by Activation of 25-26 nt sRNA-Associated Effector Genes.

Oomycetes represent a unique group of plant pathogens that are destructive to a wide range of crops and natural ecosystems. Phytophthora species possess active small RNA (sRNA) silencing pathways, but little is known about the biological roles of sRNAs and associated factors in pathogenicity. Here we show that an AGO gene, PpAGO3, plays a major role in the regulation of effector genes hence the pathogenicity of Phytophthora parasitica. PpAGO3 was unique among five predicted AGO genes in P. parasitica, showing strong mycelium stage-specific expression. Using the CRISPR-Cas9 technology, we generated PpAGO3ΔRGG1-3 mutants that carried a deletion of 1, 2, or 3 copies of the N-terminal RGG motif (QRGGYD) but failed to obtain complete knockout mutants, which suggests its vital role in P. parasitica. These mutants showed increased pathogenicity on both Nicotiana benthamiana and Arabidopsis thaliana plants. Transcriptome and sRNA sequencing of PpAGO3ΔRGG1 and PpAGO3ΔRGG3 showed that these mutants were differentially accumulated with 25-26 nt sRNAs associated with 70 predicted cytoplasmic effector genes compared to the wild-type, of which 13 exhibited inverse correlation between gene expression and 25-26 nt sRNA accumulation. Transient overexpression of the upregulated RXLR effector genes, PPTG_01869 and PPTG_15425 identified in the mutants PpAGO3ΔRGG1 and PpAGO3ΔRGG3 , strongly enhanced N. benthamiana susceptibility to P. parasitica. Our results suggest that PpAGO3 functions together with 25-26 nt sRNAs to confer dynamic expression regulation of effector genes in P. parasitica, thereby contributing to infection and pathogenicity of the pathogen.

Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing.

BACKGROUND: The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods. RESULTS: Here, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5' Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant. CONCLUSIONS: Our method is applicable to establish a comprehensive termination landscape in a broad range of species.

FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing.

Elongation, splicing and polyadenylation are fundamental steps of transcription, and studying their coordination requires simultaneous monitoring of these dynamic processes on one transcript. We recently developed a full-length nascent RNA sequencing method in the model plant Arabidopsis that simultaneously detects RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale. This method allows calculation of the kinetics of cotranscriptional splicing and detects polyadenylated transcripts with unspliced introns retained at specific positions posttranscriptionally. Here we describe a detailed protocol for this method called FLEP-seq (full-length elongating and polyadenylated RNA sequencing) that is applicable to plants. Library production requires as little as one nanogram of nascent RNA (after rRNA/tRNA removal), and either Nanopore or PacBio platforms can be used for sequencing. We also provide a complete bioinformatic pipeline from raw data processing to downstream analysis. The minimum time required for FLEP-seq, including RNA extraction and library preparation, is 36 h. The subsequent long-read sequencing and initial data analysis ranges between 31 and 40 h, depending on the sequencing platform.

Multiplex CRISPR-Cas9 editing of DNA methyltransferases in rice uncovers a class of non-CG methylation specific for GC-rich regions.

DNA methylation in the non-CG context is widespread in the plant kingdom and abundant in mammalian tissues such as the brain and pluripotent cells. Non-CG methylation in Arabidopsis thaliana is coordinately regulated by DOMAINS REARRANGED METHYLTRANSFERASE (DRM) and CHROMOMETHYLASE (CMT) proteins but has yet to be systematically studied in major crops due to difficulties in obtaining genetic materials. Here, utilizing the highly efficient multiplex CRISPR-Cas9 genome-editing system, we created single- and multiple-knockout mutants for all the nine DNA methyltransferases in rice (Oryza sativa) and profiled their whole-genome methylation status at single-nucleotide resolution. Surprisingly, the simultaneous loss of DRM2, CHROMOMETHYLASE3 (CMT2), and CMT3 functions, which completely erases all non-CG methylation in Arabidopsis, only partially reduced it in rice. The regions that remained heavily methylated in non-CG contexts in the rice Os-dcc (Osdrm2/cmt2/cmt3a) triple mutant had high GC contents. Furthermore, the residual non-CG methylation in the Os-dcc mutant was eliminated in the Os-ddccc (Osdrm2/drm3/cmt2/cmt3a/cmt3b) quintuple mutant but retained in the Os-ddcc (Osdrm2/drm3/cmt2/cmt3a) quadruple mutant, demonstrating that OsCMT3b maintains non-CG methylation in the absence of other major methyltransferases. Our results showed that OsCMT3b is subfunctionalized to accommodate a distinct cluster of non-CG-methylated sites at highly GC-rich regions in the rice genome.

FlsnRNA-seq: protoplasting-free full-length single-nucleus RNA profiling in plants.

The broad application of single-cell RNA profiling in plants has been hindered by the prerequisite of protoplasting that requires digesting the cell walls from different types of plant tissues. Here, we present a protoplasting-free approach, flsnRNA-seq, for large-scale full-length RNA profiling at a single-nucleus level in plants using isolated nuclei. Combined with 10x Genomics and Nanopore long-read sequencing, we validate the robustness of this approach in Arabidopsis root cells and the developing endosperm. Sequencing results demonstrate that it allows for uncovering alternative splicing and polyadenylation-related RNA isoform information at the single-cell level, which facilitates characterizing cell identities.

Soybean DICER-LIKE2 Regulates Seed Coat Color via Production of Primary 22-Nucleotide Small Interfering RNAs from Long Inverted Repeats.

In plants, 22-nucleotide small RNAs trigger the production of secondary small interfering RNAs (siRNAs) and enhance silencing. DICER-LIKE2 (DCL2)-dependent 22-nucleotide siRNAs are rare in Arabidopsis (Arabidopsis thaliana) and are thought to function mainly during viral infection; by contrast, these siRNAs are abundant in many crops such as soybean (Glycine max) and maize (Zea mays). Here, we studied soybean 22-nucleotide siRNAs by applying CRISPR-Cas9 to simultaneously knock out the two copies of soybean DCL2, GmDCL2a and GmDCL2b, in the Tianlong1 cultivar. Small RNA sequencing revealed that most 22-nucleotide siRNAs are derived from long inverted repeats (LIRs) and disappeared in the Gmdcl2a/2b double mutant. De novo assembly of a Tianlong1 reference genome and transcriptome profiling identified an intronic LIR formed by the chalcone synthase (CHS) genes CHS1 and CHS3 This LIR is the source of primary 22-nucleotide siRNAs that target other CHS genes and trigger the production of secondary 21-nucleotide siRNAs. Disruption of this process in Gmdcl2a/2b mutants substantially increased CHS mRNA levels in the seed coat, thus changing the coat color from yellow to brown. Our results demonstrated that endogenous LIR-derived transcripts in soybean are predominantly processed by GmDCL2 into 22-nucleotide siRNAs and uncovered a role for DCL2 in regulating natural traits.

The Arabidopsis thaliana gene AtERF019 negatively regulates plant resistance to Phytophthora parasitica by suppressing PAMP-triggered immunity.

Phytophthora species are destructive plant pathogens that cause significant crop losses worldwide. To understand plant susceptibility to oomycete pathogens and to explore novel disease resistance strategies, we employed the Arabidopsis thaliana-Phytophthora parasitica model pathosystem and screened for A. thaliana T-DNA insertion mutant lines resistant to P. parasitica. This led to the identification of the resistant mutant 267-31, which carries two T-DNA insertion sites in the promoter region of the ethylene-responsive factor 19 gene (ERF019). Quantitative reverse transcription PCR (RT-qPCR) assays showed that the expression of ERF019 was induced during P. parasitica infection in the wild type, which was suppressed in the 267-31 mutant. Additional erf019 mutants were generated using CRISPR/Cas9 technology and were confirmed to have increased resistance to P. parasitica. In contrast, ERF019 overexpression lines were more susceptible. Transient overexpression assays in Nicotiana benthamiana showed that the nuclear localization of ERF019 is crucial for its susceptible function. RT-qPCR analyses showed that the expression of marker genes for multiple defence pathways was significantly up-regulated in the mutant compared with the wild type during infection. Flg22-induced hydrogen peroxide accumulation and reactive oxygen species burst were impaired in ERF019 overexpression lines, and flg22-induced MAPK activation was enhanced in erf019 mutants. Moreover, transient overexpression of ERF019 strongly suppressed INF-triggered cell death in N. benthamiana. These results reveal the importance of ERF019 in mediating plant susceptibility to P. parasitica through suppression of pathogen-associated molecular pattern-triggered immunity.

Post-transcriptional splicing of nascent RNA contributes to widespread intron retention in plants.

In eukaryotes, genes are transcribed by RNA polymerase-II (Pol-II) and introns are removed by the spliceosome largely cotranscriptionally1-3; analysis using long-read sequencing revealed that splicing occurs immediately after Pol-II passes introns in yeast4,5. Here, we developed a Nanopore-based method to profile chromatin-bound RNA that enables the simultaneous detection of splicing status, Pol-II position and polyadenylation at the genome-wide scale in Arabidopsis. We found that more than half of the introns remain unspliced after Pol-II transcribes 1 kb past the 3' splice site, which is much slower than the rate of splicing reported in yeast4,5. Many of the full-length chromatin-bound RNA molecules are polyadenylated, yet still contain unspliced introns at specific positions. These introns are nearly absent in the cytoplasm and are resistant to nonsense-mediated decay, suggesting that they are post-transcriptionally spliced before the transcripts are released into the cytoplasm; we therefore termed these introns post-transcriptionally spliced introns (pts introns). Analysis of around 6,500 public RNA-sequencing libraries found that the splicing of pts introns requires the function of splicing-related proteins such as PRMT5 and SKIP, and is also influenced by various environmental signals. The majority of the intron retention events in Arabidopsis are at pts introns, suggesting that chromatin-tethered post-transcriptional splicing is a major contributor to the widespread intron retention that is observed in plants, and could be a mechanism to produce fully spliced functional mRNAs for rapid response.

Identification of cucumber circular RNAs responsive to salt stress.

BACKGROUND: Circular RNAs (circRNAs) are 3'-5' head-to-tail covalently closed non-coding RNA that have been proved to play essential roles in many cellular and developmental processes. However, no information relate to cucumber circRNAs is available currently, especially under salt stress condition. RESULTS: In this study, we sequenced circRNAs in cucumber and a total of 2787 were identified, with 1934 in root and 44 in leaf being differentially regulated under salt stress. Characteristics analysis of these circRNAs revealed following features: most of them are exon circRNAs (79.51%) and they prefer to arise from middle exon(s) of parent genes (2035/2516); moreover, most of circularization events (88.3%) use non-canonical-GT/AG splicing signals; last but not least, pairing-driven circularization is not the major way to generate cucumber circRNAs since very few circRNAs (18) contain sufficient flanking complementary sequences. Annotation and enrichment analysis of both parental genes and target mRNAs were launched to uncover the functions of differentially expressed circRNAs induced by salt stress. The results showed that circRNAs may be paly roles in salt stress response by mediating transcription, signal transcription, cell cycle, metabolism adaptation, and ion homeostasis related pathways. Moreover, circRNAs may function to regulate proline metabolisms through regulating associated biosynthesis and degradation genes. CONCLUSIONS: The present study identified large number of cucumber circRNAs and function annotation revealed their possible biological roles in response to salt stress. Our findings will lay a solid foundation for further structure and function studies of cucumber circRNAs.

An RXLR effector secreted by Phytophthora parasitica is a virulence factor and triggers cell death in various plants.

RXLR effectors encoded by Phytophthora species play a central role in pathogen-plant interactions. An understanding of the biological functions of RXLR effectors is conducive to the illumination of the pathogenic mechanisms and the development of disease control strategies. However, the virulence function of Phytophthora parasitica RXLR effectors is poorly understood. Here, we describe the identification of a P. parasitica RXLR effector gene, PPTG00121 (PpE4), which is highly transcribed during the early stages of infection. Live cell imaging of P. parasitica transformants expressing a full-length PpE4 (E4FL)-mCherry protein indicated that PpE4 is secreted and accumulates around haustoria during plant infection. Silencing of PpE4 in P. parasitica resulted in significantly reduced virulence on Nicotiana benthamiana. Transient expression of PpE4 in N. benthamiana in turn restored the pathogenicity of the PpE4-silenced lines. Furthermore, the expression of PpE4 in both N. benthamiana and Arabidopsis thaliana consistently enhanced plant susceptibility to P. parasitica. These results indicate that PpE4 contributes to pathogen infection. Finally, heterologous expression experiments showed that PpE4 triggers non-specific cell death in a variety of plants, including tobacco, tomato, potato and A. thaliana. Virus-induced gene silencing assays revealed that PpE4-induced cell death is dependent on HSP90, NPK and SGT1, suggesting that PpE4 is recognized by the plant immune system. In conclusion, PpE4 is an important virulence RXLR effector of P. parasitica and recognized by a wide range of host plants.

The 25-26 nt Small RNAs in Phytophthora parasitica Are Associated with Efficient Silencing of Homologous Endogenous Genes.

Small RNAs (sRNAs) are important non-coding RNA regulators, playing key roles in developmental regulation, transposon suppression, environmental response, host-pathogen interaction and other diverse biological processes. However, their roles in oomycetes are poorly understood. Here, we performed sRNA sequencing and RNA sequencing of Phytophthora parasitica at stages of vegetative growth and infection of Arabidopsis roots to examine diversity and function of sRNAs in P. parasitica, a model hemibiotrophic oomycete plant pathogen. Our results indicate that there are two distinct types of sRNA-generating loci in P. parasitica genome, giving rise to clusters of 25-26 nt and 21 nt sRNAs, respectively, with no significant strand-biases. The 25-26 nt sRNA loci lie predominantly in gene-sparse and repeat-rich regions, and overlap with over 7000 endogenous gene loci. These overlapped genes are typically P. parasitica species-specific, with no homologies to the sister species P. infestans. They include approximately 40% RXLR effector genes, 50% CRN effector genes and some elicitor genes. The transcripts of most of these genes could not be detected at both the vegetative mycelium and infection stages as revealed by RNA sequencing, indicating that the 25-26 nt sRNAs are associated with efficient silencing of these genes. The 21 nt sRNA loci typically overlap with the exon regions of highly expressed genes, suggesting that the biogenesis of the 21 nt sRNAs may be dependent on the level of gene transcription and that these sRNAs do not mediate efficient silencing of homologous genes. Analyses of the published P. infestans sRNA and mRNA sequencing data consistently show that the 25-26 nt sRNAs, but not the 21 nt sRNAs, may mediate efficient gene silencing in Phytophthora.

MicroRNA-like viral small RNA from porcine reproductive and respiratory syndrome virus negatively regulates viral replication by targeting the viral nonstructural protein 2.

Many viruses encode microRNAs (miRNAs) that are small non-coding single-stranded RNAs which play critical roles in virus-host interactions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically impactful viruses in the swine industry. The present study sought to determine whether PRRSV encodes miRNAs that could regulate PRRSV replication. Four viral small RNAs (vsRNAs) were mapped to the stem-loop structures in the ORF1a, ORF1b and GP2a regions of the PRRSV genome by bioinformatics prediction and experimental verification. Of these, the structures with the lowest minimum free energy (MFE) values predicted for PRRSV-vsRNA1 corresponded to typical stem-loop, hairpin structures. Inhibition of PRRSV-vsRNA1 function led to significant increases in viral replication. Transfection with PRRSV-vsRNA1 mimics significantly inhibited PRRSV replication in primary porcine alveolar macrophages (PAMs). The time-dependent increase in the abundance of PRRSV-vsRNA1 mirrored the gradual upregulation of PRRSV RNA expression. Knockdown of proteins associated with cellular miRNA biogenesis demonstrated that Drosha and Argonaute (Ago2) are involved in PRRSV-vsRNA1 biogenesis. Moreover, PRRSV-vsRNA1 bound specifically to the nonstructural protein 2 (NSP2)-coding sequence of PRRSV genome RNA. Collectively, the results reveal that PRRSV encodes a functional PRRSV-vsRNA1 which auto-regulates PRRSV replication by directly targeting and suppressing viral NSP2 gene expression. These findings not only provide new insights into the mechanism of the pathogenesis of PRRSV, but also explore a potential avenue for controlling PRRSV infection using viral small RNAs.

Phenotypic and genetic characterization of resistance in Arabidopsis thaliana to the oomycete pathogen Phytophthora parasitica.

The interaction between Arabidopsis thaliana and the oomycete pathogen Phytophthora parasitica emerges as a model for exploring the molecular basis and evolution of recognition and host defense. Phenotypic variation and genetic analysis is essential to dissect the underlying mechanisms in plant-oomycete interaction. In this study, the reaction phenotypes of 28 A. thaliana accessions to P. parasitica strain Pp016 were examined using detached leaf infection assay. The results showed the presence of four distinct groups based on host response and disease development. Of all the accessions examined, Zurich (Zu-1) is highly resistant to P. parasitica. Microscopic characterization showed that rapid and severe hypersensitive response at the primary infection epidermal cells is associated with disease resistance. Furthermore, Zu-1 is resistant to a set of 20 diverse P. parasitica strains, which were collected from different host plants and exhibited differential specificities on a set of tobacco cultivars. However, Zu-1 is susceptible to P. parasitica when the root is inoculated, suggesting differential expression of associated resistance genes in the root and foliar tissues. Genetic analysis by crossing Zu-1 and the susceptible accession Landsberg (Ler) showed that the resistance in Zu-1 to P. parasitica is semi-dominant, as shown by infection assays of F1 progenies, and is likely conferred by a single locus, defined as RPPA1 (Zu-1) (for Resistance to P. parasitica 1), as shown by analysis of F2 segregating populations. By employing specific-locus amplified fragment sequencing (SLAF-seq) strategy to identify molecular markers potentially linked to the locus, the strongest associated region was determined to be located between 7.1 and 11.2 Mb in chromosome IV. The future cloning of RPPA1 (Zu-1) locus will facilitate improved understanding of plant broad-spectrum disease resistance to oomycete pathogens.