Publications

Rice blast is a detrimental rice disease caused by the fungus Magnaporthe oryzae. Here, we identified a resistance gene from the rice cultivar Fuhui 2663 which is resistant to the rice blast isolate KJ201. Through isolated population analyses and sequencing approaches, the candidate gene was traced to chromosome 12. With the use of a map-based cloning strategy, the resistance gene was ultimately mapped to an 80-kb resistance locus region containing the Pita gene. Candidate gene prediction and cDNA sequencing indicated that the target resistance gene in Fuhui 2663 was allelic to Pita, thus being referred to as Pita-Fuhui2663 hereafter. Further analysis showed that the Fuhui 2663 protein had one amino acid change: Ala (A) residue 918 in Pita-Fuhui2663 was replaced by Ser (S) in Pita-S, leading to a significant change in the 3D structure of the Pita-S protein. CRISPR/Cas9 knockout experiments confirmed that Pita-Fuhui2663 is responsible for the resistance phenotype of Fuhui 2663. Importantly, Pita-Fuhui2663 did not affect the main agronomic traits of the variety compared to the Pita gene as verified by knockout experiments, indicative of potential applications of Pita-Fuhui2663 in broader breeding programs. Furthermore, a Pita-Fuhui2663-dCAPS molecular marker with good specificity and high efficiency was developed to facilitate rice breeding for resistance to this devastating disease.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis.

RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage.

CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

Vivipary is a rare sexual reproduction phenomenon where embryos germinate directly on the maternal plants. However, it is a common genetic event of woody mangroves in the Rhizophoraceae family. The ecological benefits of vivipary in mangroves include the nurturing of seedlings in harsh coastal and saline environments, but the genetic and molecular mechanisms of vivipary remain unclear. Here we investigate the viviparous embryo development and germination processes in mangrove Kandelia obovata by a transcriptomic approach. Many key biological pathways and functional genes were enriched in different tissues and stages, contributing to vivipary. Reduced production of abscisic acid set a non-dormant condition for the embryo to germinate directly. Genes involved in the metabolism of and response to other phytohormones (gibberellic acid, brassinosteroids, cytokinin, and auxin) are expressed precociously in the axis of non-vivipary stages, thus promoting the embryo to grow through the seed coat. Network analysis of these genes identified the central regulatory roles of LEC1 and FUS3, which maintain embryo identity in Arabidopsis. Moreover, photosynthesis related pathways were significantly up-regulated in viviparous embryos, and substance transporter genes were highly expressed in the seed coat, suggesting a partial self-provision and maternal nursing. We conclude that the viviparous phenomenon is a combinatorial result of precocious loss of dormancy and enhanced germination potential during viviparous seed development. These results shed light on the relationship between seed development and germination, where the continual growth of the embryo replaces a biphasic phenomenon until a mature propagule is established.

Alternative polyadenylation (APA) is an essential regulatory mechanism for gene expression. The next generation sequencing provides ample opportunity to precisely delineate APA sites genome-wide. Various methods for profiling transcriptome-wide poly(A) sites were developed. By comparing available methods, the ways for adding sequencing adaptors to fit with the Illumina sequencing platform are different. These methods have identified more than 50% genes that undergo APA in eukaryotes. However, due to the unbalanced PCR during library preparation, accurate quantification of poly(A) sites is still a challenge. Here, we describe an updated poly(A) tag sequencing method that incorporates unique molecular identifier (UMI) into the adaptor for removing quantification bias induced by PCR duplicates. Hence, quantification of poly(A) site usages can be achieved by counting UMIs. This protocol, quantifying poly(A) tag sequencing (QPAT-seq), can be finished in 1 day with reduced cost, and is particularly useful for application with a large number of samples.

Heterochromatin is widespread in eukaryotic genomes and has diverse impacts depending on its genomic context. Previous studies have shown that a protein complex, the ASI1-AIPP1-EDM2 (AAE) complex, participates in polyadenylation regulation of several intronic heterochromatin-containing genes. However, the genome-wide functions of AAE are still unknown. Here, we show that the ASI1 and EDM2 mostly target the common genomic regions on a genome-wide level and preferentially interacts with genetic heterochromatin. Polyadenylation (poly(A) sequencing reveals that AAE complex has a substantial influence on poly(A) site usage of heterochromatin-containing genes, including not only intronic heterochromatin-containing genes but also the genes showing overlap with heterochromatin. Intriguingly, AAE is also involved in the alternative splicing regulation of a number of heterochromatin-overlapping genes, such as the disease resistance gene RPP4. We provided evidence that genic heterochromatin is indispensable for the recruitment of AAE in polyadenylation and splicing regulation. In addition to conferring RNA processing regulation at genic heterochromatin-containing genes, AAE also targets some transposable elements (TEs) outside of genes (including TEs sandwiched by genes and island TEs) for epigenetic silencing. Our results reveal new functions of AAE in RNA processing and epigenetic silencing, and thus represent important advances in epigenetic regulation.

The Avicennia marina is a mangrove species widely distributed throughout the tropical and subtropical intertidal wetlands. To adapt to adverse tidal waves and hypoxia environments, A. marina has evolved a sophisticated root system to better secure itself on the muddy soil with downward-grown anchor roots and upward-grown aerial roots, called pneumatophores. However, the process behind the development of a negative-gravitropic pneumatophore is not understood. Paraffin sections reveal anatomical differences among the shoots, anchor roots, and gas exchanging pneumatophores, clearly reflecting their functional diversions. The pneumatophore, in particular, contains abundant aerenchyma tissues and a thin cap structure at the tip. Transcriptomic analyses of both anchor roots and pneumatophores were performed to elucidate gene expression dynamics during the formation of pneumatophores. The results show that the plant hormone auxin regulates multiple different root initiations. The auxin related gene IAA19 plays a key role in pneumatophore development while the interaction of ethylene and abscisic acid is important for aerenchyma formation. Moreover, the molecular mechanisms behind pneumatophore anti-gravitropic growth may be regulated by the reduced strength of the statolith formation signaling pathway. These results shed light on the mechanistic understanding of pneumatophore formation in mangrove plants.

Despite a much higher proportion of intragenic heterochromatin-containing genes in crop genomes, the importance of intragenic heterochromatin in crop development remains unclear. Intragenic heterochromatin can be recognised by a protein complex, ASI1-AIPP1-EDM2 (AAE) complex, to regulate alternative polyadenylation. Here, we investigated the impact of rice ASI1 on global poly(A) site usage through poly(A) sequencing and ASI1-dependent regulation on rice development. We found that OsASI1 is essential for rice pollen development and flowering. OsASI1 dysfunction has an important impact on global poly(A) site usage, which is closely related to heterochromatin marks. Intriguingly, OsASI1 interacts with the intronic heterochromatin of OsXRNL, a nuclear XRN family exonuclease gene involved in the processing of an miRNA precursor, to promote the processing of full-length OsXRNL and regulate miRNA abundance. We found that OsASI1-mediated regulation of pollen development partially depends on OsXRNL. Finally, we characterised the rice AAE complex and its involvement in alternative polyadenylation and pollen development. Our findings help to elucidate an epigenetic mechanism governing miRNA abundance and rice development, and provide a valuable resource for studying the epigenetic mechanisms of many important processes in crops.

Silver nanoparticles (AgNPs) are widely used in medical and commercial products for their unique antibacterial functions. However, the impact of AgNPs on human neural development is not well understood. To investigate the effect of AgNPs on human neural development, various doses of 20 nm citrate-coated AgNP (AgSC) were administered to human embryonic stem cell derived neural progenitors during the neuronal differentiation. Immunofluorescence staining with neuronal progenitor markers SOX2 (sex determining region Y-box 2) and Nestin (VI intermediate filament protein) showed that AgSC inhibited rosette formation, neuronal progenitor proliferation, and neurite outgrowth. Furthermore, AgSC promoted astrocyte activation and neuronal apoptosis. These adverse effects can be partially recovered with ascorbic acid. A genome-wide transcriptome analysis of both AgSC treated and untreated samples indicated that the most up-graduated genes were a group of Metallothionein (1F, 1E, 2A) proteins, a metal-binding protein that plays an essential role in metal homeostasis, heavy metal detoxification, and cellular anti-oxidative defence. The most significantly down-regulated genes were neuronal differentiation 6 (NEUROD6) and fork head box G1 (FOXG1). GO analyse indicated that the regulation of cholesterol biosynthetic process, neuron differentiation, synapse organization and pattern specification, oliogenesis, and neuronal apoptosis were the most impacted biological processes. KEGG pathway analyse showed that the most significantly impacted pathways were C5 isoprenoid, axon guidance, Notch, WNT, RAS-MAPK signalling pathways, lysosome, and apoptosis. Our data suggests that AgSCs interfered with metal homeostasis and cholesterol biosynthesis which induced oxidative stress, inhibited neurogenesis, axon guidance, and promoted apoptosis. Supplementation with ascorbic acid could act as an antioxidant to prevent AgSC-mediated neurotoxicity.

The dynamic choice of different polyadenylation sites in a gene is referred to as alternative polyadenylation, which functions in many important biological processes. Large-scale messenger RNA 3' end sequencing has revealed that cleavage sites for polyadenylation are presented with microheterogeneity. To date, the conventional determination of polyadenylation site clusters is subjective and arbitrary, leading to inaccurate annotations. Here, we present a weighted density peak clustering method, QuantifyPoly(A), to accurately quantify genome-wide polyadenylation choices. Applying QuantifyPoly(A) on published 3' end sequencing datasets from both animals and plants, their polyadenylation profiles are reshaped into myriads of novel polyadenylation site clusters. Most of these novel polyadenylation site clusters show significantly dynamic usage across different biological samples or associate with binding sites of trans-acting factors. Upstream sequences of these clusters are enriched with polyadenylation signals UGUA, UAAA and/or AAUAAA in a species-dependent manner. Polyadenylation site clusters also exhibit species specificity, while plants ones generally show higher microheterogeneity than that of animals. QuantifyPoly(A) is broadly applicable to any types of 3' end sequencing data and species for accurate quantification and construction of the complex and dynamic polyadenylation landscape and enables us to decode alternative polyadenylation events invisible to conventional methods at a much higher resolution.

Alternative polyadenylation (APA) is a widespread post-transcriptional modification method that changes the 3' ends of transcripts by altering poly(A) site usage. However, the longitudinal transcriptomic 3' end profile and its mechanism of action are poorly understood. We applied diurnal time-course poly(A) tag sequencing (PAT-seq) for Arabidopsis and identified 3284 genes that generated both rhythmic and arrhythmic transcripts. These two classes of transcripts appear to exhibit dramatic differences in expression and translation activisty. The asynchronized transcripts derived by APA are embedded with different poly(A) signals, especially for rhythmic transcripts, which contain higher AAUAAA and UGUA signal proportions. The Pol II occupancy maximum is reached upstream of rhythmic poly(A) sites, while it is present directly at arrhythmic poly(A) sites. Integrating H3K9ac and H3K4me3 time-course data analyses revealed that transcriptional activation of histone markers may be involved in the differentiation of rhythmic and arrhythmic APA transcripts. These results implicate an interplay between histone modification and RNA 3'-end processing, shedding light on the mechanism of transcription rhythm and alternative polyadenylation.