Integrated mRNA and small RNA sequencing for analyzing tea leaf spot pathogen Lasiodiplodia theobromae, under in vitro conditions and the course of infection

Lasiodiplodia theobromae (Pat.) Griffon & Maubl. is a phytopathogenic fungus, which can cause many different diseases on different crops. The pathogen can cause leaf spot on tea plants (Camellia sinensis), which negatively affects the productivity and quality of tea leaves in tea plantations in Guizhou Province, China. Although the genome sequence of L. theobromae has been published, no data on the transcriptome or small RNA sequences of L. theobromae under in vitro conditions and the course of infection of tea leaf are available. Here, we report the high-quality transcriptome and small RNA sequences of L. theobromae under in vitro conditions and the course of infection of tea leaf using the platform of Illumina HiSeq. This comprehensive expression profiling of the fungal pathogen will provide a valuable resource for future research on trait-specific genes of the pathogen, host-pathogen interactions and on disease resistance in the host.

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Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽

10.1017/s0031182019001689 ◽

2019 ◽

Vol 147 (8) ◽

pp. 855-864

Author(s):

Collette Britton ◽

Roz Laing ◽

Eileen Devaney

Keyword(s):

Gene Expression ◽

Small Rna ◽

Small Rnas ◽

Target Genes ◽

Parasitic Nematodes ◽

Small Rna Sequencing ◽

Small Interfering Rnas ◽

Rna Sequences ◽

C Elegans

AbstractSmall RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface.

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Whole genome sequence and gene annotation resource for Didymella bellidis, associated with tea leaf spot

Plant Disease ◽

10.1094/pdis-05-20-0939-a ◽

2020 ◽

Author(s):

Xue Wang ◽

Xian Wu ◽

Shilong Jiang ◽

Qiaoxiu Yin ◽

Dongxue Li ◽

...

Keyword(s):

Genome Sequence ◽

Leaf Spot ◽

Sequence Data ◽

Gene Annotation ◽

Gene Prediction ◽

Guizhou Province ◽

Whole Genome Sequence ◽

Phytopathogenic Fungus ◽

Future Research ◽

Whole Genome

Didymella bellidis is a phytopathogenic fungus that causes leaf spot on tea plants (Camellia sinensis), which negatively affects the productivity and quality of tea leaves in Guizhou Province, China. D. bellidis isolate GZYQYQX2B was sequenced using Pacific Biosciences and Illumina technologies, and assembled into a whole genome of 35.5 Mbp. Transcripts of D. bellidis isolate GZYQYQX2B were predicted from the assembled genome and were further validated by RNA sequence data. In total, 10,731 genes were predicted by integrating three approaches, namely ab initio and homology-based gene prediction, as well as transcriptomics data. The whole-genome sequence of D. bellidis will provide a resource for future research on trait-specific genes of the pathogen and host-pathogen interactions.

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Integrated mRNA and Small RNA Sequencing for Analyzing Leaf Spot Pathogen Didymella segeticola and Its Host, Tea (Camellia sinensis), During Infection

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-20-0207-a ◽

2020 ◽

pp. MPMI-07-20-0207

Author(s):

Rui Yang ◽

Silong Jiang ◽

Dongxue Li ◽

Qiaoxiu Yin ◽

Xian Wu ◽

...

Keyword(s):

Camellia Sinensis ◽

Leaf Spot ◽

Resistance Mechanisms ◽

Guizhou Province ◽

Future Research ◽

Small Rna Sequencing ◽

Illumina Hiseq ◽

Tea Plants ◽

Open Access Article

Leaf spot on tea plants (Camellia sinensis [L.] Kuntze), caused by the fungus Didymella segeticola (Q. Chen) Q. Chen, Crous & L. Cai (syn. Phoma segeticola), negatively affects the productivity and quality of tea leaves in Guizhou Province, China. Although the genome sequence of D. segeticola has been published, no data on the transcriptome or microRNAs (miRNAs) of the pathogen or host during infection are available. Here, we report on the high-quality transcriptome and miRNA sequences of both D. segeticola and tea during infection, using the Illumina HiSeq 4000 or HiSeq 2500 platforms. Comprehensive expression profiling of the fungal pathogen and its host will provide a resource for future research into trait-specific genes of the pathogen and the host as well as on host-pathogen interactions and on disease resistance mechanisms. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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Whole Genome Sequences of the Tea Leaf Spot Pathogen Didymella segeticola

Phytopathology ◽

10.1094/phyto-02-19-0050-a ◽

2019 ◽

Vol 109 (10) ◽

pp. 1676-1678 ◽

Cited By ~ 1

Author(s):

Yafeng Ren ◽

Dongxue Li ◽

Xiaozhen Zhao ◽

Yong Wang ◽

Xingtao Bao ◽

...

Keyword(s):

Leaf Spot ◽

Guizhou Province ◽

Whole Genome Sequence ◽

Future Research ◽

Whole Genome ◽

Plant Host ◽

Illumina Hiseq ◽

Leaf Production ◽

Clusters Of Orthologous Groups ◽

Tea Leaf

The fungal pathogen Didymella segeticola (basionym Phoma segeticola) causes leaf spot on tea (Camellia sinensis), which leads to a loss in tea leaf production in Guizhou Province, China. D. segeticola isolate GZSQ-4 was sequenced using Illumina HiSeq and Pacific Biosciences (PacBio) RS technologies, and then assembled to approximately 33.4 Mbp with a scaffold N50 value of approximately 2.3 Mbp. In total, 10,893 genes were predicted using the Nonredundant, Gene Ontology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and SWISS-PROT databases. The whole-genome sequence of D. segeticola will provide a resource for future research on host−pathogen interactions, determination of trait-specific genes, pathogen evolution, and plant−host adaptation mechanisms.

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xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

10.1101/333658 ◽

2018 ◽

Author(s):

Avi Z. Rosenberg ◽

Carrie Wright ◽

Karen Fox-Talbot ◽

Anandita Rajpurohit ◽

Courtney Williams ◽

...

Keyword(s):

Epithelial Cells ◽

Small Rna ◽

Mirna Expression ◽

Low Cost ◽

Expression Patterns ◽

Small Rna Sequencing ◽

Rna Seq ◽

Cell Type

AbstractAccurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

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Analysis of conserved miRNAs in cynomolgus macaque genome using small RNA sequencing and homology searching

PeerJ ◽

10.7717/peerj.9347 ◽

2020 ◽

Vol 8 ◽

pp. e9347

Author(s):

Xia Huang ◽

Shijia Li ◽

Xiaoming Liu ◽

Shuting Huang ◽

Shuang Li ◽

...

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Mirna Gene ◽

Cynomolgus Macaque ◽

Future Research ◽

Small Rna Sequencing ◽

Homology Searching ◽

Macaque Genome ◽

Cynomolgus Macaques ◽

Mirna Clusters

MicroRNAs (miRNAs) are important regulators that fine-tune diverse cellular activities. Cynomolgus macaques (Macaca fascicularis) are used extensively in biomedical and pharmaceutical research; however, substantially fewer miRNAs have been identified in this species than in humans. Consequently, we investigated conserved miRNA profiles in cynomolgus macaques by homology searching and small RNA sequencing. In total, 1,455 high-confidence miRNA gene loci were identified, 408 of which were also confirmed by RNA sequencing, including 73 new miRNA loci reported in cynomolgus macaques for the first time. Comparing miRNA expression with age, we found a positive correlation between sequence conservation and expression levels during miRNA evolution. Additionally, we found that the miRNA gene locations in cynomolgus macaque genome were very flexible. Most were embedded in intergenic spaces or introns and clustered together. Several miRNAs were found in certain gene locations, including 64 exon-resident miRNAs, six splice-site-overlapping miRNAs (SO-miRNAs), and two pairs of distinct mirror miRNAs. We also identified 78 miRNA clusters, 68 of which were conserved in the human genome, including 10 large miRNA clusters predicted to regulate diverse developmental and cellular processes in cynomolgus macaque. Thus, this study not only expands the number of identified miRNAs in cynomolgus macaques but also provides clues for future research on the differences in miRNA repertoire between macaques and humans.

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The antifungal mechanism of the biogenic antimicrobial Ningnanmycin on Didymella segeticola involves binding to tryptophanyl-tRNA synthetase, inhibiting translation

10.21203/rs.3.rs-112823/v1 ◽

2020 ◽

Author(s):

Li Dongxue ◽

Qiaoxiu Yin ◽

Xue Wang ◽

Shilong Jiang ◽

Dissanayake Saman Pradeep Dharmasena ◽

...

Keyword(s):

Leaf Spot ◽

Mycelial Growth ◽

Morphological Changes ◽

Binding Free Energy ◽

Trna Synthetase ◽

Guizhou Province ◽

Leaf Spot Disease ◽

Mrna Levels ◽

Action Mechanism ◽

Tea Leaf

Abstract BackgroundTea [Camellia sinensis (L.) Kuntze] has been recently cultivated in Guizhou Province, China, where the cultivated area has reached 350,000 hectares, making it the major tea-growing region in world. Tea leaf spot caused by Didymella segeticola can induce the decreases in quality and quantity of tea leaves, which is an important disease in tea plantations at higher altitude, where cold spells occur in late spring. As a promising biogenic antimicrobial agent against crop diseases, Ningnanmycin (NNM) was produced from Streptomyces noursei var. xichangensisn, represented higher field efficiency against fungal, bacterial and viral phytopathogens, lower toxicity and lower residue. However, the action mechanism of NNM against phytopathogens just stays on the stage of anti-viral mechanism, which limits the application of NNM in the management of plant fungal diseases. Here, we studied the action mechanism of NNM against D. segeticola using many methods of transcriptome, ultrastructure, molecular biology and molecular docking.ResultsNNM strongly inhibited the mycelial growth of D. segeticola with the half-maximal effective concentration of 1287.54 U/mL. Optical, fluorescence, scanning and transmission electron microscopy were applied to observe morphological changes of cellular, organelle for D. segeticola treated by NNM. A great number of morphological changes of D. segeticola indicated that NNM could affect the biosynthesis of the phytopathogen. For further, RNA-Seq results showed that NNM treated D. segeticola induced 1,363 significantly differentially expressed genes (DEGs) comparing with the control (P < 0.05). The DEGs were highly enriched in structural component of ribosome, ribosome and translation by Gene Ontology, as well as in ribosome pathway at Kyoto Encyclopedia of Genes and Genomes. NNM regulated the mRNA levels of RPS7, RPS9, RPS10b, RPL9, RPL11 and TrpRS, and represent the different regulation mode by the comparative analysis with a classical translation extension inhibitor, cycloheximide. The molecular docking indicated that NNM possessed a marked affinity with TrpRS, with the binding free energy is -101.55 kcal/mol.ConclusionsNNM could potentially affect translation by binding to tryptophanyl-tRNA synthetase, thus inhibiting mycelial growth. This study will provide insights for anti-fungal mechanism of NNM and contribute to the control and prevention of tea leaf spot disease.

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Integration of Transcriptomic and Proteomic Data Reveals the Possible Action Mechanism of the Antimicrobial Zhongshengmycin against Didymella segeticola, the Causal Agent of Tea Leaf Spot

Phytopathology ◽

10.1094/phyto-02-21-0073-r ◽

2021 ◽

Author(s):

Yafeng Ren ◽

Dongxue Li ◽

Shilong Jiang ◽

Yu Wang ◽

Qin Tang ◽

...

Keyword(s):

Leaf Spot ◽

Mode Of Action ◽

Morphological Changes ◽

Extracellular Polysaccharides ◽

Proteomics Data ◽

Protein Levels ◽

Proteomic Data ◽

Half Maximal Effective Concentration ◽

Tea Leaf

Tea leaf spot, caused by the fungal phytopathogen Didymella segeticola (Q. Chen) Q. Chen, Crous & L. Cai (syn. Phoma segeticola, is an important foliar disease, which can cause a huge loss in terms of the production and quality of tea for no effective management measures to control the disease. This study screened a natural antimicrobial chemical for its activity against D. segeticola and studied its mode of action. Antifungal activity of the Streptomyces-derived antimicrobial Zhongshengmycin (ZSM) against D. segeticola strain GZSQ-4 was assayed in vitro by the mycelial growth rate method. Optical microscopy, scanning and transmission electron microscopy were used to observe the morphological effects on hyphae treated with ZSM, with these studies being complemented by transcriptomics, proteomics, and bioinformatics studies to identify the differentially expressed genes (DEGs) or the differentially expression proteins (DEPs) in hyphae treated with ZSM. Correlation analysis of transcriptomics and proteomics data were used to reveal the mode of action. The results indicated that ZSM could inhibit the growth of hyphae in vitro with a half-maximal effective concentration (EC50) of 5.9 μg mL-1, inducing some morphological changes in organelles, septa, and extracellular polysaccharides, targeting ribosomes to disturb translation, affecting the biosynthesis of some hyphal proteins at the mRNA and protein levels, and revealing correlation between findings from transcriptomes and proteomes.

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DEUS: an R package for accurate small RNA profiling based on differential expression of unique sequences

Bioinformatics ◽

10.1093/bioinformatics/btz495 ◽

2019 ◽

Vol 35 (22) ◽

pp. 4834-4836

Author(s):

Tim Jeske ◽

Peter Huypens ◽

Laura Stirm ◽

Selina Höckele ◽

Christine M Wurmser ◽

...

Keyword(s):

Differential Expression ◽

Small Rna ◽

Sequence Similarity ◽

Differential Expression Analysis ◽

R Package ◽

Supplementary Information ◽

Small Rna Sequencing ◽

Sequencing Data ◽

Rna Sequences ◽

Rna Profiling

Abstract Summary Despite their fundamental role in various biological processes, the analysis of small RNA sequencing data remains a challenging task. Major obstacles arise when short RNA sequences map to multiple locations in the genome, align to regions that are not annotated or underwent post-transcriptional changes which hamper accurate mapping. In order to tackle these issues, we present a novel profiling strategy that circumvents the need for read mapping to a reference genome by utilizing the actual read sequences to determine expression intensities. After differential expression analysis of individual sequence counts, significant sequences are annotated against user defined feature databases and clustered by sequence similarity. This strategy enables a more comprehensive and concise representation of small RNA populations without any data loss or data distortion. Availability and implementation Code and documentation of our R package at http://ibis.helmholtz-muenchen.de/deus/. Supplementary information Supplementary data are available at Bioinformatics online.

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Functional Annotation of circRNAs in Tea Leaves after Infection by the Tea Leaf Spot Pathogen, Lasiodiplodia theobromae

Phytopathology ◽

10.1094/phyto-05-21-0184-a ◽

2021 ◽

Author(s):

Yuanyou Yang ◽

Xinyue Jiang ◽

Jiayan Shi ◽

Yu Wang ◽

Honglin Huang ◽

...

Keyword(s):

Transcription Factor ◽

Protein Kinase ◽

Leaf Spot ◽

Functional Annotation ◽

Wrky Transcription Factor ◽

Differentially Expressed ◽

Tea Leaves ◽

Lasiodiplodia Theobromae ◽

Host Pathogen ◽

Tea Leaf

Tea leaf spot, caused by Lasiodiplodia theobromae, is an important disease that can seriously decrease the production and quality of tea (Camellia sinensis [L.] Kuntze) leaves. The analysis of circular RNA (circRNA) in tea leaves after infection by the pathogen could improve understanding about the mechanism of host-pathogen interactions. In this study, high-performance sequencing of circRNA from C. sinensis Fuding-dabaicha leaves that had been infected with L. theobromae was conducted using the Illumina HiSeq 4000 platform. A total of 192 and 153 differentially-expressed circRNAs from tea leaves were significantly up- and down-regulated, respectively, after infection with L. theobromae. A GO analysis indicated that the differentially expressed circRNA-hosting-genes for DNA binding were significantly enriched. The genes with significantly differential expressions that were annotated in the specified database (S genes) were Sigma factor E isoform 1, Triacylglycerol lipase SDP1, DNA-directed RNA polymerase III subunit 2, WRKY transcription factor WRKY24, and regulator of nonsense transcripts 1 homolog. A KEGG analysis indicated that the significantly enriched circRNA-hosting-genes involved in the plant-pathogen interaction pathway were Calmodulin-domain protein kinase 5 isoform 1, probable WRKY transcription factor 33, U-box domain-containing protein 35, probable inactive receptor-like protein kinase At3g56050, WRKY transcription factor WRKY24, mitogen-activated protein kinase kinase kinase YODA, SGT1, and protein DGS1, etc. Functional annotation of circRNAs in tea leaves infected by L. theobromae will provide a valuable resource for future research on host-pathogen interactions.

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