ETS1, a Target Gene of the EWSR1::FLI1 Fusion Oncoprotein, Regulates the Expression of the Focal Adhesion Protein TENSIN3.

The mechanistic basis for the metastasis of Ewing sarcomas remains poorly understood, as these tumors harbor few mutations beyond the chromosomal translocation that initiates the disease. Instead, the epigenome of Ewing sarcoma cells reflects the regulatory state of genes associated with the DNA-binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG's repression of transcription factor genes, concentrating on those that exhibit a broader range of expression in tumors than in Ewing sarcoma cell lines. Focusing on one of these target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3-repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1's binding of promoter regions, substantially altering the transcriptome of Ewing sarcoma cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. Ewing sarcoma cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared with control cells. Visualization of control Ewing sarcoma cells showed a distributed vinculin signal and a network-like organization of F-actin; in contrast, ETS1-activated Ewing sarcoma cells showed an accumulation of vinculin and F-actin toward the plasma membrane. Interestingly, the phenotype of ETS1-activated Ewing sarcoma cell lines depleted of TNS3 resembled the phenotype of the control cells. Critically, these findings have clinical relevance as TNS3 expression in Ewing sarcoma tumors positively correlates with that of ETS1. Implications: ETS1's transcriptional regulation of the gene encoding the focal adhesion protein TENSIN3 in Ewing sarcoma cells promotes cell movement, a critical step in the evolution of metastasis.

Staufen1 Represses the FOXA1-Regulated Transcriptome by Destabilizing FOXA1 mRNA in Colorectal Cancer Cells.

Transcription factors play key roles in development and disease by controlling gene expression. Forkhead box A1 (FOXA1), is a pioneer transcription factor essential for mouse development and functions as an oncogene in prostate and breast cancer. In colorectal cancer (CRC), FOXA1 is significantly downregulated and high FOXA1 expression is associated with better prognosis, suggesting potential tumor suppressive functions. We therefore investigated the regulation of FOXA1 expression in CRC, focusing on well-differentiated CRC cells, where FOXA1 is robustly expressed. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells. We validated FOXA1 mRNA instability in multiple CRC cell lines and in patient-derived CRC organoids, and found that the FOXA1 3'UTR confers instability to the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. Indeed, STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased FOXA1 mRNA stability. Consistent with these data, RNA-seq following STAU1 knockdown in CRC cells revealed that FOXA1 targets were upregulated upon STAU1 knockdown. Collectively, this study uncovers a molecular mechanism by which FOXA1 is regulated in CRC cells and provides insights into our understanding of the complex mechanisms of gene regulation in cancer.

The peptide GOLVEN10 alters root development and noduletaxis in Medicago truncatula.

The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.

Annotated genome sequence of a fast-growing diploid clone of red alder (Alnus rubra Bong.).

Red alder (Alnus rubra Bong.) is an ecologically significant and important fast-growing commercial tree species native to western coastal and riparian regions of North America, having highly desirable wood, pigment, and medicinal properties. We have sequenced the genome of a rapidly growing clone. The assembly is nearly complete, containing the full complement of expected genes. This supports our objectives of identifying and studying genes and pathways involved in nitrogen-fixing symbiosis and those related to secondary metabolites that underlie red alder's many interesting defense, pigmentation, and wood quality traits. We established that this clone is most likely diploid and identified a set of SNPs that will have utility in future breeding and selection endeavors, as well as in ongoing population studies. We have added a well-characterized genome to others from the order Fagales. In particular, it improves significantly upon the only other published alder genome sequence, that of Alnus glutinosa. Our work initiated a detailed comparative analysis of members of the order Fagales and established some similarities with previous reports in this clade, suggesting a biased retention of certain gene functions in the vestiges of an ancient genome duplication when compared with more recent tandem duplications.

ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3.

The mechanistic basis for the metastasis of Ewing sarcomas remains poorly understood, as these tumors harbor few mutations beyond the chromosomal translocation that initiates the disease. Instead, the epigenome of Ewing sarcoma (EWS) cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG's repression of transcription factor genes, concentrating on those that exhibit a broader range of expression in tumors than in EWS cell lines. Focusing on one of these target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1's binding of promoter regions, substantially altering the transcriptome of EWS cells, including the upregulation of the gene encoding TENSIN3 (TNS3), a focal adhesion protein. EWS cell lines expressing ETS1 (CRISPRa) exhibited increased TNS3 expression and enhanced movement compared to control cells. The cytoskeleton of control cells and ETS1-activated EWS cell lines also differed. Specifically, control cells exhibited a distributed vinculin signal and a network-like organization of F-actin. In contrast, ETS1-activated EWS cells showed an accumulation of vinculin and F-actin towards the plasma membrane. Interestingly, the phenotype of ETS1-activated EWS cell lines depleted of TNS3 resembled the phenotype of the control cells. Critically, these findings have clinical relevance as TNS3 expression in EWS tumors positively correlates with that of ETS1.

Comparative Genome Analyses of Plant Rust Pathogen Genomes Reveal a Confluence of Pathogenicity Factors to Quell Host Plant Defense Responses.

Switchgrass rust caused by Puccinia novopanici (P. novopanici) has the ability to significantly affect the biomass yield of switchgrass, an important biofuel crop in the United States. A comparative genome analysis of P. novopanici with rust pathogen genomes infecting monocot cereal crops wheat, barley, oats, maize and sorghum revealed the presence of larger structural variations contributing to their genome sizes. A comparative alignment of the rust pathogen genomes resulted in the identification of collinear and syntenic relationships between P. novopanici and P. sorghi; P. graminis tritici 21-0 (Pgt 21) and P. graminis tritici Ug99 (Pgt Ug99) and between Pgt 21 and P. triticina (Pt). Repeat element analysis indicated a strong presence of retro elements among different Puccinia genomes, contributing to the genome size variation between ~1 and 3%. A comparative look at the enriched protein families of Puccinia spp. revealed a predominant role of restriction of telomere capping proteins (RTC), disulfide isomerases, polysaccharide deacetylases, glycoside hydrolases, superoxide dismutases and multi-copper oxidases (MCOs). All the proteomes of Puccinia spp. share in common a repertoire of 75 secretory and 24 effector proteins, including glycoside hydrolases cellobiohydrolases, peptidyl-propyl isomerases, polysaccharide deacetylases and protein disulfide-isomerases, that remain central to their pathogenicity. Comparison of the predicted effector proteins from Puccinia spp. genomes to the validated proteins from the Pathogen-Host Interactions database (PHI-base) resulted in the identification of validated effector proteins PgtSR1 (PGTG_09586) from P. graminis and Mlp124478 from Melampsora laricis across all the rust pathogen genomes.

Serendipita Fungi Modulate the Switchgrass Root Transcriptome to Circumvent Host Defenses and Establish a Symbiotic Relationship.

The fungal family Serendipitaceae encompasses root-associated lineages with endophytic, ericoid, orchid, and ectomycorrhizal lifestyles. Switchgrass is an important bioenergy crop for cellulosic ethanol production owing to high biomass production on marginal soils otherwise unfit for food crop cultivation. The aim of this study was to investigate the host plant responses to Serendipita spp. colonization by characterizing the switchgrass root transcriptome during different stages of symbiosis in vitro. For this, we included a native switchgrass strain, Serendipita bescii, and a related strain, S. vermifera, isolated from Australian orchids. Serendipita colonization progresses from thin hyphae that grow between root cells to, finally, the production of large, bulbous hyphae that fill root cells during the later stages of colonization. We report that switchgrass seems to perceive both fungi prior to physical contact, leading to the activation of chemical and structural defense responses and putative host disease resistance genes. Subsequently, the host defense system appears to be quenched and carbohydrate metabolism adjusted, potentially to accommodate the fungal symbiont. In addition, prior to contact, switchgrass exhibited significant increases in root hair density and root surface area. Furthermore, genes involved in phytohormone metabolism such as gibberellin, jasmonic acid, and salicylic acid were activated during different stages of colonization. Both fungal strains induced plant gene expression in a similar manner, indicating a conserved plant response to members of this fungal order. Understanding plant responsiveness to Serendipita spp. will inform our efforts to integrate them into forages and row crops for optimal plant-microbe functioning, thus facilitating low-input, sustainable agricultural practices.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

PIP-SNP: a pipeline for processing SNP data featured as linkage disequilibrium bin mapping, genotype imputing and marker synthesizing.

Genome-wide association study data analyses often face two significant challenges: (i) high dimensionality of single-nucleotide polymorphism (SNP) genotypes and (ii) imputation of missing values. SNPs are not independent due to physical linkage and natural selection. The correlation of nearby SNPs is known as linkage disequilibrium (LD), which can be used for LD conceptual SNP bin mapping, missing genotype inferencing and SNP dimension reduction. We used a stochastic process to describe the SNP signals and proposed two types of autocorrelations to measure nearby SNPs' information redundancy. Based on the calculated autocorrelation coefficients, we constructed LD bins. We adopted a k-nearest neighbors algorithm (kNN) to impute the missing genotypes. We proposed several novel methods to find the optimal synthetic marker to represent the SNP bin. We also proposed methods to evaluate the information loss or information conservation between using the original genome-wide markers and using dimension-reduced synthetic markers. Our performance assessments on the real-life SNP data from a rice recombinant inbred line (RIL) population and a rice HapMap project show that the new methods produce satisfactory results. We implemented these functional modules in C/C++ and streamlined them into a web-based pipeline named PIP-SNP (https://bioinfo.noble.org/PIP_SNP/) for processing SNP data.

Distinguishing HapMap Accessions Through Recursive Set Partitioning in Hierarchical Decision Trees.

The HapMap (haplotype map) projects have produced valuable genetic resources in life science research communities, allowing researchers to investigate sequence variations and conduct genome-wide association study (GWAS) analyses. A typical HapMap project may require sequencing hundreds, even thousands, of individual lines or accessions within a species. Due to limitations in current sequencing technology, the genotype values for some accessions cannot be clearly called. Additionally, allelic heterozygosity can be very high in some lines, causing genetic and sometimes phenotypic segregation in their descendants. Genetic and phenotypic segregation degrades the original accession's specificity and makes it difficult to distinguish one accession from another. Therefore, it is vitally important to determine and validate HapMap accessions before one conducts a GWAS analysis. However, to the best of our knowledge, there are no prior methodologies or tools that can readily distinguish or validate multiple accessions in a HapMap population. We devised a bioinformatics approach to distinguish multiple HapMap accessions using only a minimum number of genetic markers. First, we assign each candidate marker with a distinguishing score (DS), which measures its capability in distinguishing accessions. The DS score prioritizes those markers with higher percentages of homozygous genotypes (allele combinations), as they can be stably passed on to offspring. Next, we apply the "set-partitioning" concept to select optimal markers by recursively partitioning accession sets. Subsequently, we build a hierarchical decision tree in which a specific path represents the selected markers and the homogenous genotypes that can be used to distinguish one accession from others in the HapMap population. Based on these algorithms, we developed a web tool named MAD-HiDTree (Multiple Accession Distinguishment-Hierarchical Decision Tree), designed to analyze a user-input genotype matrix and construct a hierarchical decision tree. Using genetic marker data extracted from the Medicago truncatula HapMap population, we successfully constructed hierarchical decision trees by which the original 262 M. truncatula accessions could be efficiently distinguished. PCR experiments verified our proposed method, confirming that MAD-HiDTree can be used for the identification of a specific accession. MAD-HiDTree was developed in C/C++ in Linux. Both the source code and test data are publicly available at https://bioinfo.noble.org/MAD-HiDTree/.

Genome sequence and evolution of Betula platyphylla.

Betula L. (birch) is a pioneer hardwood tree species with ecological, economic, and evolutionary importance in the Northern Hemisphere. We sequenced the Betula platyphylla genome and assembled the sequences into 14 chromosomes. The Betula genome lacks evidence of recent whole-genome duplication and has the same paleoploidy level as Vitis vinifera and Prunus mume. Phylogenetic analysis of lignin pathway genes coupled with tissue-specific expression patterns provided clues for understanding the formation of higher ratios of syringyl to guaiacyl lignin observed in Betula species. Our transcriptome analysis of leaf tissues under a time-series cold stress experiment revealed the presence of the MEKK1-MKK2-MPK4 cascade and six additional mitogen-activated protein kinases that can be linked to a gene regulatory network involving many transcription factors and cold tolerance genes. Our genomic and transcriptome analyses provide insight into the structures, features, and evolution of the B. platyphylla genome. The chromosome-level genome and gene resources of B. platyphylla obtained in this study will facilitate the identification of important and essential genes governing important traits of trees and genetic improvement of B. platyphylla.

LegumeIP V3: from models to crops-an integrative gene discovery platform for translational genomics in legumes.

Legumes have contributed to human health, sustainable food and feed production worldwide for centuries. The study of model legumes has played vital roles in deciphering key genes, pathways, and networks regulating biological mechanisms and agronomic traits. Along with emerging breeding technology such as genome editing, translation of the knowledge gained from model plants to crops is in high demand. The updated database (V3) was redesigned for translational genomics targeting the discovery of novel key genes in less-studied non-model legume crops by referring to the knowledge gained in model legumes. The database contains genomic data for all 22 included species, and transcriptomic data covering thousands of RNA-seq samples mostly from model species. The rich biological data and analytic tools for gene expression and pathway analyses can be used to decipher critical genes, pathways, and networks in model legumes. The integrated comparative genomic functions further facilitate the translation of this knowledge to legume crops. Therefore, the database will be a valuable resource to identify important genes regulating specific biological mechanisms or agronomic traits in the non-model yet economically significant legume crops. LegumeIP V3 is available free to the public at https://plantgrn.noble.org/LegumeIP. Access to the database does not require login, registration, or password.

Iron-Sulfur Cluster Protein NITROGEN FIXATION S-LIKE1 and Its Interactor FRATAXIN Function in Plant Immunity.

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are present in all kingdoms of life as part of a large number of proteins involved in several cellular processes, including DNA replication and metabolism. In this work, we demonstrate an additional role for two Fe-S cluster genes in biotic stress responses in plants. Eleven Fe-S cluster genes, including the NITROGEN FIXATION S-LIKE1 (NFS1) and its interactor FRATAXIN (FH), when silenced in Nicotiana benthamiana, compromised nonhost resistance to Pseudomonas syringae pv. tomato T1. NbNFS1 expression was induced by pathogens and salicylic acid. Arabidopsis (Arabidopsis thaliana) atnfs and atfh mutants, with reduced AtNFS1 or AtFH gene expression, respectively, showed increased susceptibility to both host and nonhost pathogen infection. Arabidopsis AtNFS1 and AtFH overexpressor lines displayed decreased susceptibility to infection by host pathogen P syringae pv. tomato DC3000. The AtNFS1 overexpression line exhibited constitutive upregulation of several defense-related genes and enrichment of gene ontology terms related to immunity and salicylic acid responses. Our results demonstrate that NFS1 and its interactor FH are involved not only in nonhost resistance but also in basal resistance, suggesting a new role of the Fe-S cluster pathway in plant immunity.

pssRNAit: A Web Server for Designing Effective and Specific Plant siRNAs with Genome-Wide Off-Target Assessment.

We report an advanced web server, the plant-specific small noncoding RNA interference tool pssRNAit, which can be used to design a pool of small interfering RNAs (siRNAs) for highly effective, specific, and nontoxic gene silencing in plants. In developing this tool, we integrated the transcript dataset of plants, several rules governing gene silencing, and a series of computational models of the biological mechanism of the RNA interference (RNAi) pathway. The designed pool of siRNAs can be used to construct a long double-strand RNA and expressed through virus-induced gene silencing (VIGS) or synthetic transacting siRNA vectors for gene silencing. We demonstrated the performance of pssRNAit by designing and expressing the VIGS constructs to silence Phytoene desaturase (PDS) or a ribosomal protein-encoding gene, RPL10 (QM), in Nicotiana benthamiana We analyzed the expression levels of predicted intended-target and off-target genes using reverse transcription quantitative PCR. We further conducted an RNA-sequencing-based transcriptome analysis to assess genome-wide off-target gene silencing triggered by the fragments that were designed by pssRNAit, targeting different homologous regions of the PDS gene. Our analyses confirmed the high accuracy of siRNA constructs designed using pssRNAit The pssRNAit server, freely available at https://plantgrn.noble.org/pssRNAit/, supports the design of highly effective and specific RNAi, VIGS, or synthetic transacting siRNA constructs for high-throughput functional genomics and trait improvement in >160 plant species.

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element.

Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon-based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed-derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1-5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1-based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near-saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses.

The CLE53-SUNN genetic pathway negatively regulates arbuscular mycorrhiza root colonization in Medicago truncatula.

Plants and arbuscular mycorrhizal fungi (AMF) engage in mutually beneficial symbioses based on a reciprocal exchange of nutrients. The beneficial character of the symbiosis is maintained through a mechanism called autoregulation of mycorrhization (AOM). AOM includes root-to-shoot-to-root signaling; however, the molecular details of AOM are poorly understood. AOM shares many features of autoregulation of nodulation (AON) where several genes are known, including the receptor-like kinase SUPER NUMERIC NODULES (SUNN), root-to-shoot mobile CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-RELATED (CLE) peptides, and the hydroxyproline O-arabinosyltransferase ROOT DETERMINED NODULATION1 (RDN1) required for post-translational peptide modification. In this work, CLE53 was identified to negatively regulate AMF symbiosis in a SUNN- and RDN1-dependent manner. CLE53 expression was repressed at low phosphorus, while it was induced by AMF colonization and high phosphorus. CLE53 overexpression reduced AMF colonization in a SUNN- and RDN1 dependent manner, while cle53, rdn1, and sunn mutants were more colonized than the wild type. RNA-sequencing identified 700 genes with SUNN-dependent regulation in AMF-colonized plants, providing a resource for future identification of additional AOM genes. Disruption of AOM genes in crops potentially constitutes a novel route for improving AMF-derived phosphorus uptake in agricultural systems with high phosphorus levels.

MtSSPdb: The Medicago truncatula Small Secreted Peptide Database.

A growing number of small secreted peptides (SSPs) in plants are recognized as important regulatory molecules with roles in processes such as growth, development, reproduction, stress tolerance, and pathogen defense. Recent discoveries further implicate SSPs in regulating root nodule development, which is of particular significance for legumes. SSP-coding genes are frequently overlooked, because genome annotation pipelines generally ignore small open reading frames, which are those most likely to encode SSPs. Also, SSP-coding small open reading frames are often expressed at low levels or only under specific conditions, and thus are underrepresented in non-tissue-targeted or non-condition-optimized RNA-sequencing projects. We previously identified 4,439 SSP-encoding genes in the model legume Medicago truncatula To support systematic characterization and annotation of these putative SSP-encoding genes, we developed the M. truncatula Small Secreted Peptide Database (MtSSPdb; https://mtsspdb.noble.org/). MtSSPdb currently hosts (1) a compendium of M. truncatula SSP candidates with putative function and family annotations; (2) a large-scale M. truncatula RNA-sequencing-based gene expression atlas integrated with various analytical tools, including differential expression, coexpression, and pathway enrichment analyses; (3) an online plant SSP prediction tool capable of analyzing protein sequences at the genome scale using the same protocol as for the identification of SSP genes; and (4) information about a library of synthetic peptides and root and nodule phenotyping data from synthetic peptide screens in planta. These datasets and analytical tools make MtSSPdb a unique and valuable resource for the plant research community. MtSSPdb also has the potential to become the most complete database of SSPs in plants.

GPU empowered pipelines for calculating genome-wide kinship matrices with ultra-high dimensional genetic variants and facilitating 1D and 2D GWAS.

Genome-wide association study (GWAS) is a powerful approach that has revolutionized the field of quantitative genetics. Two-dimensional GWAS that accounts for epistatic genetic effects needs to consider the effects of marker pairs, thus quadratic genetic variants, compared to one-dimensional GWAS that accounts for individual genetic variants. Calculating genome-wide kinship matrices in GWAS that account for relationships among individuals represented by ultra-high dimensional genetic variants is computationally challenging. Fortunately, kinship matrix calculation involves pure matrix operations and the algorithms can be parallelized, particular on graphics processing unit (GPU)-empowered high-performance computing (HPC) architectures. We have devised a new method and two pipelines: KMC1D and KMC2D for kinship matrix calculation with high-dimensional genetic variants, respectively, facilitating 1D and 2D GWAS analyses. We first divide the ultra-high-dimensional markers and marker pairs into successive blocks. We then calculate the kinship matrix for each block and merge together the block-wise kinship matrices to form the genome-wide kinship matrix. All the matrix operations have been parallelized using GPU kernels on our NVIDIA GPU-accelerated server platform. The performance analyses show that the calculation speed of KMC1D and KMC2D can be accelerated by 100-400 times over the conventional CPU-based computing.

Identification and Functional Investigation of Genome-Encoded, Small, Secreted Peptides in Plants.

Hundreds to thousands of small secreted peptides (SSPs) are encoded in plant genomes but have been overlooked, and most remain unannotated and unstudied. Despite their low profile, they have been found to confer dramatic effects on growth and development of plants. With the growing appreciation of their significance, the development of appropriate methods to identify and functionally assess the myriad SSPs encoded in plant genomes has become critical. Here, we provide protocols for the computational and physiological analysis of SSPs in plant genomes. We first describe our methodology successfully used for genome-wide identification and annotation of SSP-coding genes in the model legume Medicago truncatula, which can be readily adapted for other plant species. We then provide protocols for the functional analysis of SSPs using various synthetic peptide screens. Considerations for the design and handling of peptides are included. © 2019 by John Wiley & Sons, Inc.

Draft Genome Sequence Resource of Switchgrass Rust Pathogen, Puccinia novopanici Isolate Ard-01.

Puccinia novopanici is an important biotrophic fungal pathogen that causes rust disease in switchgrass. Lack of genomic resources for P. novopanici has hampered the progress toward developing effective disease resistance against this pathogen. Therefore, we have sequenced the whole genome of P. novopanici and generated a framework to understand pathogenicity mechanisms and identify effectors, repeat element invasion, genome evolution, and comparative genomics among Puccinia spp. in the future. Long- and short-read sequences were generated from P. novopanici genomic DNA by PacBio and Illumina technologies, respectively, and assembled a 99.9-Mb genome. Transcripts of P. novopanici were predicted from assembled genome using MAKER and were further validated by RNAseq data. The genome sequence information of P. novopanici will be a valuable resource for researchers working on monocot rusts and plant disease resistance in general.

A microRNA biogenesis-like pathway for producing phased small interfering RNA from a long non-coding RNA in rice.

Phased small interfering RNAs (phasiRNAs) are a class of non-coding RNAs that perform essential functions in plants. Unlike microRNA biogenesis from a hairpin structure, the production of phasiRNAs usually requires a phase initiator and an RNA-dependent RNA polymerase (RDR) to form double-strand RNAs. By using full-length rice cDNA (KL-cDNA) to identify phasiRNA loci, we found that a putative non-coding sequence with a long hairpin structure generates the phasiRNAs, which we name Long Hairpin-structure containing non-coding RNA (LHR). The biogenesis of LHR-derived phasiRNAs was dependent on rice DCL4, but not on RDR2/6, DCL1, or DCL3. Since all of the LHR-phasiRNAs (-5p from the forward strand and -3p from the reverse strand of the dsRNAs) are mapped to the forward strand of LHR, LHR-phasiRNAs should be derived from its hairpin structure, similar to a microRNA precursor. A degradome-based validation suggested that several thylakoid-related genes were targeted by LHR-phasiRNAs. In addition, the production of LHR-phasiRNAs was completely abolished in the lhr mutant, which also exhibited decreased plant height, leaf size, and grain weight, probably through the regulation of photosynthesis. Based on our results, we propose a microRNA biogenesis-like pathway for producing phased siRNAs that expands our understanding of the current model of phased siRNA biogenesis in plants.