Draft Genome Sequence Resource of ‘ Fragaria × ananassa ’ Phyllody Phytoplasma Strain StrPh-CL from Chilean Strawberry
2022; American Phytopathological Society; Volume: 106; Issue: 3 Linguagem: Inglês
10.1094/pdis-09-21-1959-a
ISSN1943-7692
AutoresWeier Cui, Nicola Fiore, Alan Zamorano,
Tópico(s)Plant Pathogens and Fungal Diseases
ResumoHomePlant DiseaseVol. 106, No. 3Draft Genome Sequence Resource of 'Fragaria × ananassa' Phyllody Phytoplasma Strain StrPh-CL from Chilean Strawberry PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseDraft Genome Sequence Resource of 'Fragaria × ananassa' Phyllody Phytoplasma Strain StrPh-CL from Chilean StrawberryWeier Cui, Nicola Fiore, and Alan ZamoranoWeier CuiDepartment of Plant Health, Faculty of Agricultural Sciences, University of Chile, Santiago, Chile, Nicola Fiorehttps://orcid.org/0000-0001-6494-2257Department of Plant Health, Faculty of Agricultural Sciences, University of Chile, Santiago, Chile, and Alan Zamorano†Corresponding author: A. Zamorano; E-mail Address: agezac@uchile.clhttps://orcid.org/0000-0003-2770-991XDepartment of Plant Health, Faculty of Agricultural Sciences, University of Chile, Santiago, Chile AffiliationsAuthors and Affiliations Weier Cui Nicola Fiore Alan Zamorano † Department of Plant Health, Faculty of Agricultural Sciences, University of Chile, Santiago, Chile Published Online:8 Mar 2022https://doi.org/10.1094/PDIS-09-21-1959-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Genome AnnouncementStrawberry phyllody has become a common disease on Chilean strawberry (Fragaria × ananassa Duch.) crops in recent years. The pathogen associated with this disease is a phytoplasma belonging to 16SrXIII ribosomal group (Mexican periwinkle virescence group). In this study, we obtained the draft genome sequence of 'Fragaria × ananassa' phyllody phytoplasma strain StrPh-CL from infected periwinkle plants. The assembly consists of 33 contigs of 627,584 bp with an average sequencing coverage of 55.3 and a GC content of 25.4%. The genome is estimated to be 90% complete, with the possible presence of a plasmid of 4,173 bp. Among the 591 predicted protein-coding sequences (CDS), there are 26 putative secreted proteins, including the homologs of two better known pathogenic effectors, SAP54 (phyllogen) and TENGU. This genome resource serves the future research in pathogen-host interaction, evolution, epidemiology, and other aspects of this phytoplasma group.Phytoplasmas are phloem-limited phytopathogenic bacteria naturally transmitted by insect vectors, causing plant diseases and significant agricultural losses. In Chile, phytoplasmas have been detected in economically important crops and ornamental plants including grapevine, cherry, pear, sugar beet, lettuce, Swiss chard, and peony (Arismendi et al. 2011; Facundo et al. 2017; Fiore et al. 2015a, b; González et al. 2011; Quiroga et al. 2017). Since 2015, strawberry crops showing severe phyllody symptoms have been reported in the central production regions, causing loss of entire plants and reducing fruit yields. Phytoplasmas belonging to the 16SrXIII ribosomal group (Mexican periwinkle virescence group) have been detected from the symptomatic strawberry plants (Cui et al. 2019).Phytoplasmas of 16SrXIII group are exclusively found in the Americas, with the representative strain 'Candidatus Phytoplasma (Ca. P.) hispanicum' (Davis et al. 2016). All the 'Fragaria × ananassa' phyllody phytoplasma strains share >97.5% identity of the 16S ribosomal RNA sequence with 'Ca. P. hispanicum,' suggesting that they are related to this reference strain (IRPCM 2004). Based on 16S rRNA sequence, 16SrXIII group is phylogenetically close to 16SrI and 16SrXII groups, both having been better studied with several genome sequences available (Davis et al. 2016). By contrast, studies of 16SrXIII group have stayed on detection and classification, and no genome sequence of 'Ca. P. hispanicum'-related strain has been reported. In this study, we report the first draft genome sequence of 'Fragaria × ananassa' phyllody phytoplasma strain StrPh-CL.Genome sequencing and assembly.Strawberry (Fragaria × ananassa var. Monterrey) plants showing phyllody symptoms were collected from fields located in Litueche, O'Higgins Region of Chile. Periwinkle (Catharanthus roseus) plants were obtained by seeds. A strawberry fruit showing severe phyllody was grafted onto a healthy periwinkle approximately 2 months old to obtain infected periwinkles. Three months post grafting, newly emerged branches showing symptoms of virescence and phyllody were examined with nested PCR of 16S rRNA gene, and the amplicon was sequenced for phytoplasma identification. The periwinkle plant confirmed infected by 'Fragaria × ananassa' phyllody phytoplasma was used for DNA extraction. Total DNA was extracted from midribs, petioles, virescent flowers, and young shoots showing phyllody, according to Cui et al. (2019).The total DNA was sent to Macrogen (Maryland, U.S.A.) for library preparation and sequencing. The library was prepared using TruSeq Nano DNA Kit, and the sequencing was performed with the Illumina platform, generating 83,467,822 paired-end reads with an average length of 151 bp. Quality trimming of the raw sequences was performed with Trimmomatic v0.39 (Bolger et al. 2014) to remove the adaptors, the 3′ and 5′ nucleotides with a Phred quality score below 20, and the reads shorter than 30 nt. The trimmed reads were aligned to the periwinkle genome (GenBank accession no. GCA_000949345) with Bowtie2 v2.4.2 (Langmead and Salzberg 2012). The unmapped reads were assembled into contigs with SPAdes v3.14.1 (Nurk et al. 2013). The assembled contigs were queried against the NCBI nucleotide database using BLAST+ v2.11.0 (Camacho et al. 2009), and only the contigs with hits to phytoplasma sequences were retained. The retained contigs were manually curated and ordered according to their lengths.The draft genome of StrPh-CL consists of 33 contigs with a total size of 627,584 bp and an average coverage of 55.3, with an N50 value of 82,058 (Table 1). Based on a preliminary PFGE electrophoresis experiment, the completeness of this draft genome is approximately 90% (unpublished data). The GC content of the draft genome is 25.4%. RNAmmer web server v1.2 (Lagesen et al. 2007) identified two complete rRNA operons, and tRNAscan-SE web server v2.0 (Lowe and Chan 2016) identified 32 tRNA genes (Table 1). The two copies of 16S rRNA genes (5,286–6,529 on Contig_2 and 34,106–35,349 on Contig_3) share 98.7% and 98.8% identity with 'Ca. P. hispanicum' (sequence ID: AF248960.1), respectively, supporting that StrPh-CL is related to this reference strain.Table 1. General features of the draft genome of 'Fragaria × ananassa' phyllody phytoplasma strain StrPh-CLFeaturesStrPh-CL16Sr group assignment16SrXIII-FGenBank accessionJAGVRH000000000No. of contigs33Genome size (bp)627,584N50 (bp)82,058G+C content (%)25.4Number of predicted genes635Complete protein-coding sequences (CDS)559Partial CDS32rRNA operons2tRNA genes32Table 1. General features of the draft genome of 'Fragaria × ananassa' phyllody phytoplasma strain StrPh-CLView as image HTML Gene annotation and pathogenic effector candidates.Protein-coding gene prediction was performed with Prodigal v2.6.3 (Hyatt et al. 2010). Gene annotation was performed with KAAS (Moriya et al. 2007), eggNOG-mapper (Huerta-Cepas et al. 2019), and WebMGA (Altschul et al. 1990), and the results were integrated. A total of 591 CDS were predicted (Table 1), including 559 complete CDS and 32 partial ones. Among the 591 genes, 440 were annotated with specific COG (cluster of orthologous genes) numbers. Six genes were predicted to encode replication initiator protein (Rep) similar to that of geminiviruses. Geminivirus-like Rep-coding genes have been observed on extrachromosomal DNA molecules of several phytoplasmas (Saccardo et al. 2011), and the presence of these genes suggests the existence of plasmids. All six genes in StrPh-CL are located on short contigs of 1,488 to 9,333 bp, and on one of these contigs, Contig_14, gene arrangement suggests that this contig could be joined into a plasmid, which would be 4,173 bp in size and contain five CDS.According to the current consensus, pathogenic effectors of phytoplasmas are secreted into the host cytoplasm via the Sec-dependent protein translocation system (Sugio et al. 2011). To identify effector candidates, the predicted proteins were first screened with SignalP web server v5.0 (Almagro Armenteros et al. 2019) to select those with a signal peptide. The selected proteins were then loaded onto TMHMM web server v2.0 (Krogh et al. 2001) to remove those with more than one transmembrane domain, which would be integrated into the plasma membrane. Finally, the remaining candidates were examined with BLASTp and the MOTIF Search web tool (Kanehisa et al. 2010) to exclude those with well annotated functions other than secreted effectors. These analyses resulted in a total of 26 pathogenic effector candidates (Table 2), homologs of 24 of them having been found in other phytoplasma genomes.Table 2. Pathogenic effector candidates of StrPh-CLCDS IDaContig no.Length (aa)BLASTp result and known homologsSequence IDFRU_045Contig_1102SVM family proteinsWP_212330693.1FRU_059Contig_1271Hypothetical proteinsWP_212330729.1FRU_078Contig_1194SAP61WP_212330782.1FRU_080Contig_1105TENGUWP_212330783.1FRU_107Contig_1284No homologs foundWP_212330856.1FRU_180Contig_2160SAP67WP_212331043.1FRU_181Contig_2102SAP42WP_212331045.1FRU_183Contig_285No homologs foundWP_212331047.1FRU_199Contig_2169SAP42WP_212331080.1FRU_284Contig_4109SVM family proteinsWP_212331392.1FRU_287Contig_4268Hypothetical proteinsWP_212331397.1FRU_288Contig_481SVM family proteinsWP_212331400.1FRU_383Contig_6162SAP67WP_212331757.1FRU_384Contig_6109SVM family proteinsWP_212331760.1FRU_442Contig_9372SAP49WP_212331922.1FRU_462Contig_10131SVM familly proteinsWP_212331973.1FRU_467Contig_10191SAP40WP_212331981.1FRU_506Contig_15261SVM family proteinsWP_212332077.1FRU_507Contig_15361SAP49WP_212332080.1FRU_525Contig_17117SAP54 / phyllogenWP_212332119.1FRU_526Contig_17199Hypothetical proteinWP_225885840.1FRU_536Contig_18166SAP42WP_212332161.1FRU_551Contig_20117SVM family proteinsWP_212332188.1FRU_564Contig_22114Hypothetical proteinsWP_212332212.1FRU_568Contig_23151SAP08MBS2126629.1aCDS = protein-coding sequences.Table 2. Pathogenic effector candidates of StrPh-CLView as image HTML Among these candidates, homologs of two better studied effectors, SAP54 (phyllogen) and TENGU, were identified, whereas homologs of another two effectors, SAP11 and SAP05, were not found (Table 2). Members of the phyllogen family induce phyllody in host plants and promote insect colonization (MacLean et al. 2014). Interestingly, the phyllogen homolog of StrPh-CL, FRU_525, shares 95.73% identity with the homolog of Bellis virescence phytoplasma from 16SrIII group but only 50.45% identity with that of aster yellows witches'-broom phytoplasma from 16SrI group, which is phylogenetically closer to 16SrXIII group. Phytoplasmas from group 16SrIII have been detected in various host plants in Chile, and two insect vectors have been identified (Fiore et al. 2015a, b; González et al. 2011; Quiroga et al. 2019). These data suggest that this effector might have undergone horizontal transfer among phylogenetically distant but geographically overlapping strains.TENGU, SAP05, and SAP11 enhance the proliferation of axillary meristems, inducing dwarfism and witches' broom in host plants (Chang et al. 2018; Hoshi et al. 2009; Huang et al. 2021). To date, TENGU homologs have been found exclusively in phytoplasmas from 16SrI group (Sugawara et al. 2013). The TENGU homolog of StrPh-CL shares <42% identity with the other homologs and includes an additional 35 aa at the C terminus (Fig. 1), which could affect its function as a pathogenic effector. The possible altered function of TENGU, in addition to missing SAP11 and SAP05 homologs, might explain the lack of symptoms of dwarfism and witches' broom on the strawberry plants infected by StrPh-CL.Fig. 1. Protein sequence alignment of three TENGU homologs.Download as PowerPointThe draft genome of 'Fragaria × ananassa' phyllody phytoplasma strain StrPh-CL is the first sequence of a strain related to 'Candidatus Phytoplasma hispanicum.' It provides a valuable resource for future research of phytoplasmas from 16SrXIII group and will serve as a reference for sequencing approaches and comparative study. The identification of the homologs of the two pathogenic effectors, phyllogen and TENGU, provides intriguing topics on pathogenicity, epidemiology, and the evolutionary history of phytoplasmas in the Americas.Data AvailabilityThe whole-genome shotgun project of 'Fragaria × ananassa' phyllody phytoplasma strain StrPh-CL has been deposited in GenBank under the accession number JAGVRH000000000.The author(s) declare no conflict of interest.Literature CitedAlmagro Armenteros, J. J., Tsirigos, K. D., Sønderby, C. K., Petersen, T. N., Winther, O., Brunak, S., von Heijne, G., and Nielsen, H. 2019. 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Phytopathol. 49:175-195. https://doi.org/10.1146/annurev-phyto-072910-095323 Crossref, ISI, Google ScholarFunding: This research was supported by the National Fund for Scientific and Technological Development (FONDECYT) of Chile, Proyecto Postdoctoral 2017 no. 3170120 and Proyecto Iniciación 2020 no. 11200576.The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 3 March 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Downloaded 540 times Article History Issue Date: 30 Mar 2022Published: 8 Mar 2022Accepted: 22 Nov 2021 Pages: 1031-1034 Information© 2022 The American Phytopathological SocietyFundingNational Fund for Scientific and Technological Development (FONDECYT)Grant/Award Number: 3170120Grant/Award Number: 11200576Keywordsstrawberry phyllodyphytoplasmadraft genomesmall fruitspathogen diversityThe author(s) declare no conflict of interest.PDF download
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