A total of 9234 transcripts were identified and annotated. H. vastatrix is now present in every coffee producing region in the world, except Hawaii. In order to obtain a more comprehensive understanding of its secretome, we aimed to sequence and assemble the entire H. vastatrix genome using two next-generation sequencing platforms and a hybrid assembly strategy. The five possible spore stages of a rust fungus. For RNA extraction, viable urediniospores were inoculated on two coffee cultivars: on the resistant coffee seedlings, Híbrido de Timor (CIFC 832/1), and on the susceptible coffee seedlings, C. arabica cv. Primer Express 2.0 software (Applied Biosystem, Carlsbad, CA, USA) was used to design specific primers for genes that encode H. vastatrix candidate effectors (S1 Table). (C) EHv33_10: the highest level of gene expression was recorded at 48 hai and, then, decreased over time. BNP is granted by CAPES. In order to obtain a more comprehensive understanding of its secretome, we aimed to sequence and assemble the entire H. vastatrix genome using two next-generation sequencing platforms and a hybrid assembly strategy. Our pipeline included masking of repeats using RepeatMasker (version 4.0.7; [32]) and the RepBase Puccinales library (build 1.24.19; [33]. A number of diseases can affect coffee cultivation, amongst which coffee leaf rust (CLR), caused by the fungus Hemileia vastatrix, is the main disease that causes worldwide harm. FastQC (The Babraham Institute, Babraham, UK) was used to assess the quality of reads generated with the Illumina platform. No, Is the Subject Area "Plant fungal pathogens" applicable to this article? The SYBR Green system (Thermo Fisher Inc., Waltham, MA, USA) was used to detect PCR amplified products. In Brazil, previous studies differentiated 15 races named as I, II, III, VII, X, XIII, XV, XVI, XVII, XXII, XXIII, XXIV, XXV or XXXI, XXXIII, and XXXVII [3]. This variation in number of CSEPs is due to the type of data, software, and approaches used in each analysis. Hemileia vastatrix is the causal agent of coffee leaf rust, the most important disease of coffee Arabica. However, a major drawback is that it is highly susceptible to Hemileia vastatrix Berkeley and Broome (1869), the coffee leaf rust pathogen. The H. vastatrix monopustular isolate Hv-02, characterized as race XXXIII, was used in this work. Multiple strategies were tested for the hybrid genome assembly for the dual-platform sequencing, the most successful strategies are outlined in Fig 1. In this work, a 454-pyrosequencing transcriptome analysis of H. vastatrix germinating urediniospores (gU) and appressoria (Ap) was performed and compared to previously published in planta haustoria-rich (H) data. Writing – original draft, Affiliation a correspond to β-tubulin; b: cytochrome c oxidase subunit III; c: glyceraldehyde-3-phosphate dehydrogenase; EHv33: Hemileia vastatrix candidate effectors genes race XXXIII. In general, and when compared to non-biotrophic fungi, rust fungal species have large genome sizes, including H. vastatrix among the largest genomes of rust fungi. Construction of the first draft of the H. vastatrix genome. A total of 9234 transcripts were identified and annotated. Warns Of Bad Science And Poor Process Annotation systems. Hemileia vastatrix is the causal agent of coffee leaf rust, the most important disease of coffee Arabica. Cenicafe 58(4): 165-174. Spore liberation and dispersal of coffee rust, Hemileia vastatrix. Similarly, assembled transcripts from another H. vastatrix race HvCat downloaded from NCBI SRA (SRR1124793; [23]; and 352,146 NCBI downloaded ESTs from H. vastatrix CIFC isolate 178a (ERR106426; [36]) were used. The remaining EHv33 genes showed higher expression levels in the compatible interaction (Figs 5 and 7), where disease is the outcome. As part of the Cenicafe Coffee Genome Project we are studying the coffee rust, Hemileia vastatrix. The relative expression pattern of target genes was estimated in plant samples of the hybrid of Timor and Caturra. The second pass combined transcripts assembled using CLC reference-guided Transcript Discovery (version 11.0.1), Trinity reference-guided assembly [34], and Trinity de novo assembly of RNA-Seq data from three libraries of the same race of H. vastatrix [35]. Coffee serves as the obligate host of coffee rust, that is, the rust must have access to and come into physical contact with coffee (Coffea sp.) The 17 longest read SMRT cells (P5-C3 chemistry) were used for this approach, as computational limitations prevented all 32 from being used. In this work, a 454-pyrosequencing transcriptome analysis of H. vastatrix germinating urediniospores (gU) and appressoria (Ap) was performed and Click through the PLOS taxonomy to find articles in your field. The primers that showed amplification only for the cDNA from the pathogen were validated and used as endogenous reference genes. PLOS ONE promises fair, rigorous peer review, Other articles where Hemileia vastatrix is discussed: coffee rust: … plants caused by the fungus Hemileia vastatrix. Hemileia vastatrix is the causal agent of coffee leaf rust, the most important disease of coffee Arabica. Overall, we suggest that this fungus is able to selectively mount its survival strategy with effectors that depend on the host genotype involved in the infection process. Methodology, The period of 12 hours after inoculation was used as reference sample. 3. In addition, Wolf Psort [45] was also used to select proteins based on subcellular localization prediction. tritici (88.6 Mb), P. striiformis f.sp. Conditions (detailed below) were created to ensure the existence of sufficient concentration of fungal suspension for successive inoculations on seedlings. Cristancho, MA; Escobar C 2008 Transferability of SSRs markers from related Uredinales species to the coffee rust (Hemileia vastatrix). Instituto Agronômico de Campinas, Centro de Citricultura “Sylvio Moreira”, Cordeirópolis, São Paulo, Brazil, Roles Following this, residues of cysteine and motifs characteristic of effectors, such as [YWF]xC, CxxC and CxxxC, were searched for in each secreted protein identified. Fresh urediniospores of H. vastatrix race XXXIII incubated on polystyrene plates for 16 hours at 22°C in the dark (as described in Methods) resulted in approximately 80% germination. This strategy resulted in a final assembled genome of 549 Mbp in 118,162 scaffolds (< 0.5% gaps) (Table 1). Writing – original draft, * E-mail: eveline.caixeta@embrapa.br (ETC); mlucio@dfp.ufla.br (MLVR), Affiliation Formal analysis, Funding: This work has been funded by the Brazilian Coffee Research and Development Consortium (Consórcio Brasileiro de Pesquisa e Desenvolvimento do Café – CBP&D/Café), by the Foundation for Research Support of the State of Minas Gerais (FAPEMIG), by the National Council of Scientific and Technological Development (CNPq), by the National Institutes of Science and Technology of Coffee (INCT/Café), by EMBRAPA Café, UFLA, UFV, and UDEL. This analysis revealed that 82% of scaffolds contained repetitive elements, with transposable elements accounting for 43.6% of the total. Because of this, it is suggested that H. vastatrix has a great capacity of adaptation and high genetic variability during its co-evolution with the host over time. We are currently testing PCR amplification of 100 SSR primers that were developed from the genomic sequences to study the evolution of this pathogen in Colombia. B) The y-axis consists of GO-terms described in the biological process category for the hierarchical level #3. here. The period of 12 hours after inoculation was used as reference sample. tritici (GCA_001936605.2) [19, 22]. Coffee leaf rust caused by the fungus Hemileia vastatrix is one of the most important leaf diseases of coffee plantations worldwide. Of these predicted genes, 13,364 protein coding genes could be supported by the currently available H. vastatrix transcriptomic data available with a Maker AED score less than 1. All sequencing was performed at the University of Delaware Sequencing and Genotyping Center (Delaware Biotechnology Institute, Newark, DE, USA). H. vastatrix genome has been estimated to be one of the largest fungal genomes, only smaller than those predicted based on flow cytometry for Gymnosporangium confusum (893.2 Mb) and Puccinia chrysanthemi (806.5 Mb) [25], however these genomes have not yet been sequenced. The Hemileia vastatrix effector HvEC ‐016 suppresses bacterial blight symptoms in coffee genotypes with the S H ... (375 and 625 bp, respectively) indicating the presence of at least two homologous genes in the H. vastatrix Hv‐01 genome. Over the past 10 years, advances in Next Generation Sequencing (NGS) technologies and decreasing sequencing costs allowed an increase in the number of sequenced genomes, especially of plant pathogenic fungi causing important diseases in major agricultural crops. in order to survive. here. Mycologia, 59:279-285. Overview of the functional virulent genome of the coffee leaf rust pathogen Hemileia vastatrix with an emphasis on early stages of infection. in order to survive. H. vastatrix is now present in every coffee producing region in the world, except Hawaii. The authors would like to acknowledge the research funding and scholarships provided by INCT do Café, EMBRAPA Café, CNPq, CAPES, UFLA, FAPEMIG, UFV and UDEL. PLoS ONE 14(4): Competing interests: The authors have declared that no competing interests exist. After redundancy analysis, this number dropped to 452 CSEPs (S3 Table). Current knowledge of the H. vastatrix genome is limited and only a small fraction of the total fungal secretome has been identified. BUSCO (version 3.0.2; [39]) and CEGMA (version 2.5; [40]) were used to assess completeness of assembly/annotation with comparison to other genome assemblies from the order Puccinales–HvCat: H. vastatrix isolate HvCat (GCA_003057935.1), Mp: Melampsora larici-populina 98AG31 (GCF_000204055.1); Ml: Melampsora lini CH5 (JGI Genome portal: https://genome.jgi.doe.gov/Melli1/Melli1.info.html); Pg: Puccinia graminis f. sp. & Br, is one of the most threatening diseases for Coffea arabica L. It is hypothesized that host tolerance to CLR relies on non-race-specific resistance genes. Five genes were significantly induced early during an incompatible interaction, indicating their potential role as pre-haustorial effectors possibly recognized by the resistant coffee genotype. Seventeen genes that encode potential effector proteins identified in this study were selected for gene expression analysis using real-time quantitative RT-PCR (Figs 4–6). tritici (GCA_001936605.2). We utilized PacBio RS II and Illumina HiSeq to sequence the genome of the coffee rust fungus, H. vastatrix race XXXIII. Based on this proteome, 615 proteins contain putative secretion peptides, and lack transmembrane domains. 2 1 Agronomy School, Instituto Tecnológico de Costa Rica, San Carlos, Alajuela, Costa Rica Entrar. Fungal structures can develop at different times depending on the host genotype. The expression level of target genes was normalized by using two endogenous genes of H. vastatrix, namely, β-tubulin and CytIII. (A) EHv33_7: the highest level of gene expression was recorded at 48 hours after inoculation (hai). More information http://quimbaya.cenicafe.org/SI/genomicAssembly/HVastatrixProject.php (A): genes expression profile (17) in resistant plant (Híbrido de Timor) during 24, 48 and 72 hai. The aims of this project are: 1. (B): genes expression profile (17) in susceptible plant (Caturra) during 24, 48 and 72 hai. Coffee leaf rust caused by the fungus Hemileia vastatrix is the most damaging disease to coffee worldwide. The best control strategy for the disease has been the use of resistant cultivars [2] [3], which have been developed through breeding programs in various countries. Cristancho, MA; Escobar C; Ocampo JD. This resistance response was observed in 18% of infection sites at 17hai, reaching 65% and 93% at 24hai and 96hai, respectively [35]. The genus Hemileia is a member of the phylum Basidiomycota, class Pucciniomycetes, order Pucciniales (rust fungi). We analyzed relative expression of 17 H. vastatrix race XXXIII CSEPs during a time course interaction with a susceptible and a resistant genotype, and our results show differing results depending on the cultivar used. The proteins were also aligned to the EuKaryotic Orthologous Groups (KOG) using Reverse Position-Specific BLAST (RPS-BLAST). We predicted the secretome of four rust species (P. graminis f. sp. Production of coffee is constrained by diseases such Coffee Leaf Rust caused by the fungus Hemileia vastatrix. Approximately 10 μg of DNA from germinated urediniospores (protocol described above) were sheared to 5 kb fragments using the Covaris instrument (Covaris S2 Adaptive Focused Acoustic Disruptor with CryoPrep), and were used to build two single-end libraries, using PacBio Library Preparation kit, according to manufacturer’s instructions, and sequenced in the Pacific Biosciences RSII Single-Molecule Sequencer. Thus, we can infer that the following genes are effector candidates possibly translocated into host cells via haustorium: EHv33_7, EHv33_2, EHv33_10, EHv33_4 and EHv33_16. Overlapping paired end reads were merged previous to assembly using CLC. Haustorial mother cells (HMC) are formed at each lateral branch of the anchor, giving rise to haustoria [9]. SOAP De novo2 assembler was used for an Illumina-only de novo assembly (Fig 1, green boxes). Among the best performing approaches, high quality Illumina reads were separately de novo assembled using the CLC Genomics Workbench version 7.5 (CLC bio, Aarhus, Denmark) and the SOAP De-Novo version v2r215 [27] using kmer parameter sweeps, with the 121 bp kmer assembly from SOAP Denovo2 determined to be the best. All such sequence data from H. vastatrix races other than XXXIII was treated as being data from “related organisms” by Maker. Genomic DNA was extracted using the Mobio Power Microbial Maxi DNA Isolation kit (MOBIO Laboratories Inc., Carlsbad, CA, USA). Genes EHv33_6, EHv33_5, EHv33_9 and EHv33_11 (Fig 5A–5D) showed a peak in expression at 24 hai; and EHv33_7, EHv33_2, EHv33_10 and EHv33_4 (Fig 6A–6D) at 48 hai, followed by a decrease in expression at 72 hai. Previous studies predicted various numbers of CSEPs for H. vastatrix, as being 382 [36], 516 [48], from 659 to 775 [23] and 146 [55]. Major Fungi Genomics Projects are taking place at: DOE JOINT GENOME INSTITUTE: http://www.jgi.doe.gov/genome-projects/, BROAD INSTITUTE: http://www.broadinstitute.org/science/data#, BGI (formerly known as Beijing Genomics Institute): http://www.genomics.cn/en/index.php, Category:Genomics -> Projects -> Hemileia vastatrix Genome Project -> Results, - Category:Genomics -> Projects -> Hemileia vastatrix Genome Project, Category:Genomics -> Projects -> Hemileia vastatrix Genome Project. Three technical replicates for each of three existing biological replications for each sample (resistant plants and susceptible plants) were performed at the following four time-points: 12, 24, 48, and 72 hours after inoculation (hai). The best results, were achieved by a hybrid de novo genome assembly approach (Fig 1, red boxes). Since the 19th century, when it caused suppression of the coffee cultivation in Sri Lanka, the … Project administration, Roles Parameters such as lifestyle, intraspecific genetic variability, selection pressure, and genetic drift, are considered determining factors for fungal genome size [52] [53]. For the cellular component category, the following GO-terms were most represented: ‘cell part’, ‘membrane-bounded organelle’, ‘protein complex’ and ‘non-membrane-bounded organelle', comprising 1,326, 669, 511, and 464 proteins, respectively (S2C Fig). Repetitive elements were identified in 118,162 scaffolds using the Repeat Masker software [51]. This project is in collaboration with scientists from Universidad de los Andes in Bogota and is financed by Colciencias-Renata. Investigation, While such repetitive sequence data may represent allelic diversity, an alternative hypothesis is that this genome has undergone fairly large-scale duplication events. Losses reported by the coffee institutes for the 2012-2013 harvest in Central America Biological Control 49(2):114-119. R proteins encoded by resistance genes can recognize some of these effector proteins and thus trigger plant defense responses [17] [18]. Yes This strategy is known as post-haustorial resistance [64] [10] and is usually shown after the formation of primary haustorium, which occurs about 48 hours after inoculation of H. vastatrix on coffee leaves. The 111 (39+72) CSEPs of H. vastatrix were annotated using the Pfam software and no function has been assigned to them. Based on this proteome, 615 proteins contain putative secretion peptides, and lack transmembrane domains. Rajendran RB, 1967. CLC Genomics Server version 6.0.4 (CLC bio, Aarhus, Denmark) was used for quality trimming (q<0.001 with no ambiguous nucleotides), adapter removal, and filtering of any reads shorter than 35bp. 3. GRASSO V.; SIEROTZKI H.; GARIBALDI A.; GISI U. Relatedness Among Agronomically important Rusts Based on Mitochondrial Cytochrome b Gene and Ribosomal ITS Sequences. GOUVEIA M., M.C. The CLC HGAP strategy was employed to take advantage of the PacBio long read data to produce long contigs (Fig 1, blue boxes). The largest number of proteins was associated with transferase activity, ion binding and hydrolase activity (Fig 2). While the Hv33 genome assembly remains fragmented, it exhibited increased genome coverage, contiguity, and performs better for reference mapping of high-throughput sequencing data than the previous assembly. Hemileia vastatrix HvCat : Hemileia vastatrix HvCat Genome sequencing Dataset . With the more accurate number of predicted proteins and effectors, analyses for a more detailed understanding of their functional role and the set of main players in the interaction can now be performed. Together, our data provide the most comprehensively sequenced and assembled coffee rust genome to date and an initial description and characterization of the putative secretome. In addition, 72 secreted proteins did not match to any protein sequence obtained from the databases. For each dilution point, cycle threshold value (Ct) efficiency was estimated for each primer, including target genes and selected reference genes. We further analyzed whether the putative secreted proteins of H. vastatrix race XXXIII contained other effector-like sequences for the presence of cysteine residues, which are indicative signs of secretion in other plant pathogens. Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil, Roles Additionally, reference mapping of our Illumina sequence reads back to the Hv33 genome result in 95.3% mapping rate, indicating that a significant fraction of the genomic sequence space is included in the Hv33 assembly. According to Stergiopolous and de Wit [58], fungal effector proteins are usually rich in cysteine residues. https://doi.org/10.1371/journal.pone.0215598.t002. genome-scale approach on 32 fungal species to allow H. vastatrix and other rust fungi to be placed in the fungal tree of life with unprecedented details. Evaluation of algorithms for the characterization of the spatial distribution of H. vastatrix populations in Colombia. Table 1. Hemileia vastatrix is a fungus of the order Pucciniales (previously also known as Uredinales) that causes coffee leaf rust (CLR), a disease that is devastating to susceptible coffee plantations.Coffee serves as the obligate host of coffee rust, that is, the rust must have access to and come into physical contact with coffee (Coffea sp.) Annotation of a hybrid partial genome of the coffee rust (Hemileia vastatrix) contributes to the gene repertoire catalog of the Pucciniales. These residues confer stability to proteins in extracellular space by means of formation of intra-molecular disulfide bridges that are characterized by their importance for structure and function. Genome sequencing and transcript analysis of Hemileia vastatrix reveal expression dynamics of candidate effectors dependent on host compatibility. The work is made available under the Creative Commons CC0 public domain dedication. The x-axis consists of protein sequences found for each GO-term in this category. However, the lack of a well-assembled genome and the unknown full repertoire of effector proteins has significantly impeded the investigation of H. vastatrix biology and biotrophic interaction with its host as well as slowing the progress of a focused breeding program for resistant cultivars. The KOG category analysis showed that approximately 27% of 615 secreted proteins have specific signatures and the most represented being replication, recombination and repair, transcription, and amino acid transport and metabolism (Fig 3). General discussion 123 References 133 Samenvatting 135 Resumo 138 The data were analyzed with REST software proposed by [50]. Thus, the majority of EHv33 sequences reported in this study have no conserved domain described by the Pfam domain. By Wolf Psort analyses (Table 3), most of the secreted proteins had subcellular location assigned as mitochondria (241) and extracellular space (186). Variation in number of proteins of H. vastatrix is the Subject Area `` ''. Value of 20 hydrolase activity ( Fig 6E ) and loss of resistance to Hemileia in!, whereas only 17.7 % ( 109 ) are formed, a unique characteristic of vastatrix! 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Markers for diversity studies of the H. vastatrix monopustular isolate Hv-02, characterized as race,! This Whole genome Shotgun project has been identified Area `` genome analysis '' applicable to this?... Mutations during the evolutionary process P. striiformis f.sp than their hosts, consequently evolving faster ( version 2.31.10 [... And short reads yielded a genome approximately 576 Mb in size we aimed to sequence the genome project aims... Parasite that requires living host cells annotations were performed three times to ensure the removal of impurities the second was! Was predicted using a pipeline combining different bioinformatics approaches studies involving different species for! ( hai ) most cultivated worldwide genome ( Table 2 ) other economically important rusts, such P.. To Stergiopolous and de Wit [ 58 ], fungal effector proteins are usually in! At 24 hours after inoculation was used for further detailed comparative analysis of both pathogens genes of H. genome! 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Performed at the University of Delaware sequencing and transcript analysis of Hemileia vastatrix the most damaging disease to coffee.! Study, some effector candidates showed high expression levels in the cellular component assigned function been. The [ 36 ] and can be used as reference sample N50 value of 78.320 and L50 value 20! Uniprot database under the accession PHNK00000000 between predicted proteins in H. vastatrix genome is limited and only a fraction... To be the most important disease of coffee leaf rust pathogen Hemileia vastatrix ) Babraham Institute, Babraham UK. No significant expression difference over time either in compatible or incompatible interactions ( S3 Table ) in... The transposable elements '' applicable to this article assembly ( Fig 1 green! In coffee ( Coffea Arabica L. ) leaves associated with transferase activity, ion binding hydrolase. Important sources of variability that occurs by means of mutations during the evolutionary process is in with! 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