A proposed new virus in the genus Marafivirus detected from Oriental persimmon ‘Kumemaru’

A proposed new virus in the genus Marafivirus detected from Oriental persimmon ‘Kumemaru’ | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A proposed new virus in the genus Marafivirus detected from Oriental persimmon ‘Kumemaru’ Naoko Fujita, Yuka Hagiwara-Komoda This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7522521/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A virus-like sequence was identified in the male persimmon cultivar ‘Kumemaru’ during transcriptome analysis targeting male-promoting factors. The complete genome, 6,397 nucleotides in length, encodes a large polyprotein containing replication-associated domains and putative coat proteins. Phylogenetic analysis based on the coat protein amino acid sequences placed the virus within the genus Marafivirus , closely related to Blackberry virus S and Nectarine virus M. Our data indicate that this virus, for which the name persimmon marafivirus (PerMaV) is proposed, is a hitherto undescribed species of the genus Marafivirus , family Tymoviridae. Figures Figure 1 Figure 2 Figure 3 Text Oriental persimmon ( Diospyros kaki Thunb.) is a deciduous fruit tree that originated in East Asia. Persimmon fruit has gained worldwide popularity due to its appealing flavor and nutritional value. The rich cultural significance and economic importance of Oriental persimmon have driven extensive breeding efforts to improve fruit quality, yield stability, and environmental adaptability. New cultivars by cross-breeding have been developed mostly in Japan which has the longest history for development of new persimmon cultivars since 1959 (Reviewed in [1]). One of the critical factors influencing persimmon cultivation is its complex sexual characteristics, which may vary among cultivars and significantly affect pollination strategies and fruit set. Persimmons exhibit a diverse range of reproductive behaviors, including dioecy (separate male and female plants) and monoecy (both male and female flowers on the same plant) but mainly of females [2], with male trees occurring rarely [3, 4]. ‘Kumemaru’ is a rare male cultivar selected from a chance seedling in the purpose of the collection of stable pollinizers at Toyama prefecture in Japan. To elucidate the molecular mechanism of male expression in cv. Kumemaru, we previously performed RNA-seq analyses using flower buds and identified transcript polymorphisms unique to the cultivar ‘Kumemaru’ that may contribute to male development [5]. Unexpectedly, the analysis revealed abundant viral-like sequences which was not matched any known viruses but most likely to be classified into the genus Marafivirus . In this report, we propose a novel virus in the genus Marafivirus , tentatively named persimmon marafivirus (PerMaV). In addition, we developed an infectious cDNA clone of PerMaV, which may serve as a useful tool for future studies in Oriental persimmon. The full-length genome of PerMaV was obtained by initially assembling Illumina sequencing data [5] (DDBJ Sequence Read Archive under BioProject ID PRJDB9564 (Run ID DRR233540-DRR233569)), followed by partial sequencing using RT-PCR with primers designed based on the de novo assembly. The complete genome sequence was determined and deposited in GenBank (accession no. PX251123). The PerMaV genome sequence contained ‘Marafibox’, a consensus 16-nucleotide sequence of marafivirus, which involves in synthesis of subgenomic RNA [6, 7]. A putative marafibox in the PerMaV genome was identical to the corresponding region in Bermuda grass etched-line virus (BELV), Blackberry virus S (BlVS), Grapevine Syrah virus (GSyV-1), Maize rayado fino virus (MRFV), Nectarine marafivirus M (NeVM), Oat blue dwarf virus (OBDV) [8, 9, 10, 11, 12, 13] (Fig. 1 A). The complete genome of PerMaV consisted of 6,397 nt, excluding the 3’ poly(A) tail, and was characterized by a high overall cytosine content (A, 16.79%; C, 38.5%; G, 22.23%; T 22.48%), similar to genomes of other tymovirids. There is a single large open reading frame (ORF), with a genome organization suggestive of a marafivirus. This ORF started with an AUG codon at position 54 and extended for 6286 nucleotides. Searches for protein similarities revealed conserved signature domains for methyltransferase (MTR) (nucleotide positions 93 to 377), tymovirus endopeptidase (nucleotide positions 795—901), RNA helicase (HEL) (nucleotide positions 986 to 1219), coat protein (CP) (nucleotide positions 1919 to 2094), and RNA dependent RNA polymerase (RdRp) (nucleotide positions 1472 to 1748). To elucidate the taxonomic position of PerMaV, we constructed a phylogenetic tree using the complete genome sequence of PerMaV and representative members of the genus Marafivirus (Fig. 1 B). The resulting phylogeny showed that PerMaV clustered closely with BlVS, forming a sister group to a clade that includes Citrus sudden death-associated virus (CsDaV) [14], Grapevine asteroid mosaic-associated virus (GAMaV) [15], NeVM, and OBDV. A phylogenetic analysis based on the amino acid sequences of CP confirmed that PerMaV was located within the Marafivirus clade and was most closely related to BlVS (Fig. 1 C). The complete genome shared 62.5% nucleotide (nt) sequence identity with BlVS. The coat protein shared 65.02% amino acid (aa) sequence identity with GAMaV (Fig. 1 S). The genome sequencing data suggested that PerMaV is a new species in the genus Marafivirus (family Tymoviridae ). To investigate the tissue localization of PerMaV in the buds, we conducted in situ hybridization using an RNA probe targeting the coat protein (CP) sequence. The RNA probe was prepared using PerMaV CP-specific primers: PerMaV_CP_45F ‘5-TGACACTCTTGTGCCTCAGC-3’ and PerMaV_CP_413R ‘5-AGATGGGTGGTGGTTGACAT-3’. The amplified fragment cloned into pGEM-T Easy vector (Promega K. K., Tokyo, Japan) was used to synthesize DIG-labeled antisense and sense RNA probes using ProbeQuant G-50 Micro Columns (Cytiva, Tokyo, Japan). Tissue sectioning was performed using Kawamoto’s film method [16]. In situ hybridization was conducted according to the manual for a commercial kit ‘In situ Hybridization Reagents (ISHR)’ (Nippon Gene Co., Ltd., Tokyo Japan). The RNA probe (5 µg/mL) was hybridized at 50ºC overnight. Signal detection was carried out using Anti-DIG-FITC (merck KGaA, Darmstadt, Germany). The prepared specimens were stained with SlowFade gold Antifade Mountant with DAPI (Invitrogen, MA, US) and were observed using a microscope Olympus BZ51 (Evident Co., Tokyo, Japan). Strong green fluorescence signals were detected in young leaves surrounding the shoot apical meristem (Fig. 2 E) and weaker but discernible signals in the floral meristem (Fig. 2 D). No significant signals were detected when using the sense probe as a control (Fig. 2 G–I). These resuls suggest that PerMaV is actively replicating in the young leaves and shoot apex within the buds. Next, we generated a cDNA clone of PerMaV and analyzed its infectivity. The full-length PerMaV genome was amplified from cDNA synthesized using SuperScript IV (Invitrogen, MA, US) with primers LIC26-PerMaV_F (5′-CGAGCTAGTTGGAATAGGTTCGATCACCCTTCACCGTTCTCT-3′) and LIC26-PerMaV_R (5′-TGCAGTATGGAGTTGGGTTCGGCCTTTGGATCGGCCTG-3′). The amplified PerMaV fragment was inserted into the HpaI site of the pPLV26 vector [17] using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, MA, USA), according to the manufacturer’s instructions. The DNA template for in vitro transcription was prepared by PCR as pPLV26-PerMaV as a template and primers T7-PMaV-F (5'-CTAATACGACTCACTATAGGATCACCCTTCACCGTTCTCTG-3') and PMaV-polyA-R (5'-TTTTTTTTTTTTTTTGGCCTTTGGATCGGCCTGCT-3'), resulting in production of a DNA fragment containing the T7 promoter sequence. Using this DNA fragment as a template, PerMaV RNA was transcribed in vitro using HiScribe T7 ARCA mRNA Kit (New England Biolabs, MA, USA). 'Fuyu' persimmon seedlings were used for infectivity assay of the PerMaV RNA transcribed from the cDNA clone. The first true leaf of each of the three seedlings (Fig. 3 A) was mechanically inoculated with 5 µl of the transcription reaction solution in the presence of carborumdum. Four weeks later, total RNAs were purified from the inoculated and non-inoculated upper leaves using ISOSPIN Plant RNA (with Assist Buffer) (Nippon Gene). One step RT-PCR was conducted using MegaFi™ One-Step RT-PCR (Applied Biological Materials Inc.) with the purified total RNAs as templates. PerMaV infection was detected using the primers PerMaV_CP_45F and PerMaV_CP_413R, described above. RT-PCR analysis revealed that the bands of PerMaV-specific DNA products were detected in the inoculated leaves of all three seedlings (Fig. 3 B, lanes 2, 4, 6). In addition, the virus-specific band was also detected in the upper leaf sample of #3 seedling (Fig. 3 B, lane 7), implying that systemic infection of PerMaV occurred in this persimmon seedling. These results suggest that infectious PerMaV cDNA clone was successfully generated in this study. In this study, we identified a new species in the genus Marafivirus from the male Oriental persimmon cultivar 'Kumemaru'. Given the previous observation that male-promoting factors in 'Kumemaru' may be stress-induced [5], it is plausible that PerMaV contributes to male flower development indirectly by modulating host stress signaling pathways. Such virus-induced stress signaling could influence key regulators of sex determination through epigenetic mechanisms [18]. On the other hand, persimmon seedlings inoculated with the infectious cDNA clone (hereafter, the PerMaV vector), showed no symptoms, indicating that PerMaV is a latent virus. Given this latent nature, the PerMaV vector may serve as a valuable reverse genetics tool or have potential agricultural applications. For example, the use of the latent virus Apple latent spherical virus (ALSV) in apple trees has been shown to induce precocious flowering [19]. Although the ALSV vector is effective in apple, it was not applicable to persimmon (data not shown). The PerMaV may offer a more suitable viral vector system for functional studies and breeding application in Oriental persimmon. Declarations Conflicts of interest/Competing interests The authors declare no conflicts of interest. Acknowledgments We thank the Grape and Persimmon Research Station, NIFTS, NARO, Hiroshima, Japan, for kindly providing plant materials used in this study. We are also grateful to Dr. Takashi Akagi (Okayama University, at the time of this study) for giving us the opportunity to initiate this research. We also thank Dr. Yuki Furuse (The University of Tokyo) for kindly reviewing the manuscript in part. Data availability The complete genome sequence of persimmon marafivirus (PerMaV) is available in the GenBank database under the accession number PX251123. References Sato A, Yamada M (2016) Persimmon breeding in Japan for pollination-constant non-astringent (PCNA) type with marker-assisted selection. Breed Sci 66:60–68. https://doi.org/10.1270/jsbbs.66.60 Yonemori K, Sugiura A, Tanaka K, Kameda K (1993) Floral ontogeny and sex determination in monoecious-type persimmons. J Am Soc Hortic Sci 118:293–297 https://doi.org/10.21273/JASHS.118.2.293 Yakushiji H, Yamada M, Yonemori K, Sato A, Kimura N (1995) Staminate flower production on shoots of ‘Fuyu’ and ‘Jiro’ persimmon (Diospyros kaki Thunb.). 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Plant Cell 28:2905–2915. https://doi.org/10.1105/tpc.16.00532 Sasaki S, Yamagishi N, Yoshikawa N (2011) Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple latent spherical virus vectors. Plant Methods 7:15 https://doi.org/10.1186/1746-4811-7-15 Supplementary Files PerMaVgenome.txt PerMaVpolyproteintranslation.fasta FigS1.jpg Fig. S1– Pairwise sequence identity matrices of persimmon marafivirus (PerMaV) and representative marafiviruses. (A) Nucleotide sequence identity matrix of complete genomes. (B) Amino acid sequence identity matrix of capsid proteins. Values indicate percentage identities derived from multiple sequence alignments. PerMaV showed 48.44–62.5% nucleotide identity across complete genomes and 55.6–65.02% amino acid identity across capsid proteins, both below the species demarcation thresholds of 80% and 90%, respectively. 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The GenBank accession numbers are placed after the virus names. Viruses used in the phylogenetic analysis are citrus sudden death-associated virus (CsDaV), grapevine asteroid mosaic associated virus (GAMaV), nectarine marafivirus M (NeVM), oat blue dwarf virus (OBDV), blackberry virus S (BlVS), maize rayado fino virus (MRFV), switchgrass mosaic virus (SwMV), peach marifivirus D (PeVD), olive latent virus 3 (OLV-3), grapevine syrah virus (GSyV-1), grapevine rupestris vein feathering virus (GRVFV), alfalfa virus FA (AVF), medicago sativa marafivirus (MsMV1). \u003cstrong\u003eC\u003c/strong\u003ePhylogenetic tree constructed with amino acid sequences of coat proteins of members of the family Tymoviridae. Viruses in the genus Tymovirus are chayote mosaic virus (ChMV), chiltepin yellow mosaic virus (ChiTYMV), tomato blistering mosaic virus (ToBMV), physalis mottle virus (PhMV), nemesia ring necrosis virus (NeRNV), dulcamara mottle virus (DuMV), Asclepias asymptomatic virus (AsAV), turnip yellow mosaic virus (TYMV), cassia yellow mosaic-associated virus (CYMY), passion fruit yellow mosaic virus (PFYMV). Fig fleck-associated virus (GFkV). Shallot virus X (ShVX), genus Allexivirus, family Alphaflexiviridae, was used as an out-group.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7522521/v1/aed9dfbe463580b3fe23024f.jpg"},{"id":92081318,"identity":"42f7efce-b221-4271-bc9b-34617c20ffe4","added_by":"auto","created_at":"2025-09-24 11:55:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":159318,"visible":true,"origin":"","legend":"\u003cp\u003eTissue localization of PerMaV. \u003cstrong\u003eA\u003c/strong\u003e Persimmon bud used for in situ hybridization. B–E Hybridization with PerMaV coat protein antisense probe showing FITC signals (green) in apex (ap) and young leaves (yl). F–I Sense probe control showing no detectable signal. Bars = 2 mm (\u003cem\u003esingle line\u003c/em\u003e), 100 µm (\u003cem\u003edouble line\u003c/em\u003e)\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7522521/v1/fcc205d340f2fb90992bb145.jpg"},{"id":92081314,"identity":"7de02570-d6fe-4486-9d03-2d08b3563f12","added_by":"auto","created_at":"2025-09-24 11:55:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30344,"visible":true,"origin":"","legend":"\u003cp\u003eInfectivity assay of the PerMaV cDNA clone. \u003cstrong\u003eA\u003c/strong\u003e One-month-old persimmon seedlings used in this study. Arrowheads indicate the true leaves inoculated with PerMaV RNA. \u003cstrong\u003eB\u003c/strong\u003eDetection of PerMaV-specific DNA fragments by RT-PCR. The inoculated leaves (IL) and upper uninoculated leaves (UL) were harvested from the persimmon seedlings at four-week post inoculation and used for the analysis. A healthy persimmon seedling was also analyzed as a negative control (lane 1). As a positive control (+), the PCR product obtained using pPLV26-PerMaV was shown (lane 8).\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7522521/v1/b971a692a56227d046d48de2.jpg"},{"id":99796277,"identity":"bddf3a00-3313-47e9-b9db-f9dcdcc4bb0f","added_by":"auto","created_at":"2026-01-08 13:41:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":582352,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7522521/v1/ebd66661-d77b-4778-b0d0-28812fdca09d.pdf"},{"id":92081319,"identity":"63ba12e0-f8d9-4b63-a133-1814ba4e7671","added_by":"auto","created_at":"2025-09-24 11:55:23","extension":"txt","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6422,"visible":true,"origin":"","legend":"","description":"","filename":"PerMaVgenome.txt","url":"https://assets-eu.researchsquare.com/files/rs-7522521/v1/6785bbb6df740924403c08d6.txt"},{"id":92081320,"identity":"89a532b8-1c33-497a-9c2b-5745d24594ff","added_by":"auto","created_at":"2025-09-24 11:55:24","extension":"fasta","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2146,"visible":true,"origin":"","legend":"","description":"","filename":"PerMaVpolyproteintranslation.fasta","url":"https://assets-eu.researchsquare.com/files/rs-7522521/v1/c4f0e5db750048e5b412912e.fasta"},{"id":92081795,"identity":"2229d22f-5ede-486e-bc96-d87639f7ee6a","added_by":"auto","created_at":"2025-09-24 12:03:22","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":114360,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1\u003c/strong\u003e– Pairwise sequence identity matrices of persimmon marafivirus (PerMaV) and representative marafiviruses. (A) Nucleotide sequence identity matrix of complete genomes. (B) Amino acid sequence identity matrix of capsid proteins. Values indicate percentage identities derived from multiple sequence alignments. PerMaV showed 48.44–62.5% nucleotide identity across complete genomes and 55.6–65.02% amino acid identity across capsid proteins, both below the species demarcation thresholds of 80% and 90%, respectively.\u003c/p\u003e","description":"","filename":"FigS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7522521/v1/933d876be85795544c0ccbdf.jpg"}],"financialInterests":"","formattedTitle":"A proposed new virus in the genus Marafivirus detected from Oriental persimmon ‘Kumemaru’","fulltext":[{"header":"Text","content":"\u003cp\u003eOriental persimmon (\u003cem\u003eDiospyros kaki\u003c/em\u003e Thunb.) is a deciduous fruit tree that originated in East Asia. Persimmon fruit has gained worldwide popularity due to its appealing flavor and nutritional value. The rich cultural significance and economic importance of Oriental persimmon have driven extensive breeding efforts to improve fruit quality, yield stability, and environmental adaptability. New cultivars by cross-breeding have been developed mostly in Japan which has the longest history for development of new persimmon cultivars since 1959 (Reviewed in [1]). One of the critical factors influencing persimmon cultivation is its complex sexual characteristics, which may vary among cultivars and significantly affect pollination strategies and fruit set. Persimmons exhibit a diverse range of reproductive behaviors, including dioecy (separate male and female plants) and monoecy (both male and female flowers on the same plant) but mainly of females [2], with male trees occurring rarely [3, 4].\u003c/p\u003e\u003cp\u003e\u0026lsquo;Kumemaru\u0026rsquo; is a rare male cultivar selected from a chance seedling in the purpose of the collection of stable pollinizers at Toyama prefecture in Japan. To elucidate the molecular mechanism of male expression in cv. Kumemaru, we previously performed RNA-seq analyses using flower buds and identified transcript polymorphisms unique to the cultivar \u0026lsquo;Kumemaru\u0026rsquo; that may contribute to male development [5]. Unexpectedly, the analysis revealed abundant viral-like sequences which was not matched any known viruses but most likely to be classified into the genus \u003cem\u003eMarafivirus\u003c/em\u003e. In this report, we propose a novel virus in the genus \u003cem\u003eMarafivirus\u003c/em\u003e, tentatively named persimmon marafivirus (PerMaV). In addition, we developed an infectious cDNA clone of PerMaV, which may serve as a useful tool for future studies in Oriental persimmon.\u003c/p\u003e\u003cp\u003eThe full-length genome of PerMaV was obtained by initially assembling Illumina sequencing data [5] (DDBJ Sequence Read Archive under BioProject ID PRJDB9564 (Run ID DRR233540-DRR233569)), followed by partial sequencing using RT-PCR with primers designed based on the \u003cem\u003ede novo\u003c/em\u003e assembly. The complete genome sequence was determined and deposited in GenBank (accession no. PX251123). The PerMaV genome sequence contained \u0026lsquo;Marafibox\u0026rsquo;, a consensus 16-nucleotide sequence of marafivirus, which involves in synthesis of subgenomic RNA [6, 7]. A putative marafibox in the PerMaV genome was identical to the corresponding region in Bermuda grass etched-line virus (BELV), Blackberry virus S (BlVS), Grapevine Syrah virus (GSyV-1), Maize rayado fino virus (MRFV), Nectarine marafivirus M (NeVM), Oat blue dwarf virus (OBDV) [8, 9, 10, 11, 12, 13] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe complete genome of PerMaV consisted of 6,397 nt, excluding the 3\u0026rsquo; poly(A) tail, and was characterized by a high overall cytosine content (A, 16.79%; C, 38.5%; G, 22.23%; T 22.48%), similar to genomes of other tymovirids. There is a single large open reading frame (ORF), with a genome organization suggestive of a marafivirus. This ORF started with an AUG codon at position 54 and extended for 6286 nucleotides. Searches for protein similarities revealed conserved signature domains for methyltransferase (MTR) (nucleotide positions 93 to 377), tymovirus endopeptidase (nucleotide positions 795\u0026mdash;901), RNA helicase (HEL) (nucleotide positions 986 to 1219), coat protein (CP) (nucleotide positions 1919 to 2094), and RNA dependent RNA polymerase (RdRp) (nucleotide positions 1472 to 1748).\u003c/p\u003e\u003cp\u003eTo elucidate the taxonomic position of PerMaV, we constructed a phylogenetic tree using the complete genome sequence of PerMaV and representative members of the genus \u003cem\u003eMarafivirus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The resulting phylogeny showed that PerMaV clustered closely with BlVS, forming a sister group to a clade that includes Citrus sudden death-associated virus (CsDaV) [14], Grapevine asteroid mosaic-associated virus (GAMaV) [15], NeVM, and OBDV. A phylogenetic analysis based on the amino acid sequences of CP confirmed that PerMaV was located within the \u003cem\u003eMarafivirus\u003c/em\u003e clade and was most closely related to BlVS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The complete genome shared 62.5% nucleotide (nt) sequence identity with BlVS. The coat protein shared 65.02% amino acid (aa) sequence identity with GAMaV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS). The genome sequencing data suggested that PerMaV is a new species in the genus \u003cem\u003eMarafivirus\u003c/em\u003e (family \u003cem\u003eTymoviridae\u003c/em\u003e).\u003c/p\u003e\u003cp\u003eTo investigate the tissue localization of PerMaV in the buds, we conducted \u003cem\u003ein situ\u003c/em\u003e hybridization using an RNA probe targeting the coat protein (CP) sequence. The RNA probe was prepared using PerMaV CP-specific primers: PerMaV_CP_45F \u0026lsquo;5-TGACACTCTTGTGCCTCAGC-3\u0026rsquo; and PerMaV_CP_413R \u0026lsquo;5-AGATGGGTGGTGGTTGACAT-3\u0026rsquo;. The amplified fragment cloned into pGEM-T Easy vector (Promega K. K., Tokyo, Japan) was used to synthesize DIG-labeled antisense and sense RNA probes using ProbeQuant G-50 Micro Columns (Cytiva, Tokyo, Japan). Tissue sectioning was performed using Kawamoto\u0026rsquo;s film method [16]. \u003cem\u003eIn situ\u003c/em\u003e hybridization was conducted according to the manual for a commercial kit \u0026lsquo;In situ Hybridization Reagents (ISHR)\u0026rsquo; (Nippon Gene Co., Ltd., Tokyo Japan). The RNA probe (5 \u0026micro;g/mL) was hybridized at 50\u0026ordm;C overnight. Signal detection was carried out using Anti-DIG-FITC (merck KGaA, Darmstadt, Germany). The prepared specimens were stained with SlowFade gold Antifade Mountant with DAPI (Invitrogen, MA, US) and were observed using a microscope Olympus BZ51 (Evident Co., Tokyo, Japan).\u003c/p\u003e\u003cp\u003eStrong green fluorescence signals were detected in young leaves surrounding the shoot apical meristem (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and weaker but discernible signals in the floral meristem (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). No significant signals were detected when using the sense probe as a control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u0026ndash;I). These resuls suggest that PerMaV is actively replicating in the young leaves and shoot apex within the buds.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we generated a cDNA clone of PerMaV and analyzed its infectivity. The full-length PerMaV genome was amplified from cDNA synthesized using SuperScript IV (Invitrogen, MA, US) with primers LIC26-PerMaV_F (5\u0026prime;-CGAGCTAGTTGGAATAGGTTCGATCACCCTTCACCGTTCTCT-3\u0026prime;) and LIC26-PerMaV_R (5\u0026prime;-TGCAGTATGGAGTTGGGTTCGGCCTTTGGATCGGCCTG-3\u0026prime;). The amplified PerMaV fragment was inserted into the HpaI site of the pPLV26 vector [17] using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, MA, USA), according to the manufacturer\u0026rsquo;s instructions. The DNA template for in vitro transcription was prepared by PCR as pPLV26-PerMaV as a template and primers T7-PMaV-F (5'-CTAATACGACTCACTATAGGATCACCCTTCACCGTTCTCTG-3') and PMaV-polyA-R (5'-TTTTTTTTTTTTTTTGGCCTTTGGATCGGCCTGCT-3'), resulting in production of a DNA fragment containing the T7 promoter sequence. Using this DNA fragment as a template, PerMaV RNA was transcribed in vitro using HiScribe T7 ARCA mRNA Kit (New England Biolabs, MA, USA).\u003c/p\u003e\u003cp\u003e'Fuyu' persimmon seedlings were used for infectivity assay of the PerMaV RNA transcribed from the cDNA clone. The first true leaf of each of the three seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) was mechanically inoculated with 5 \u0026micro;l of the transcription reaction solution in the presence of carborumdum. Four weeks later, total RNAs were purified from the inoculated and non-inoculated upper leaves using ISOSPIN Plant RNA (with Assist Buffer) (Nippon Gene). One step RT-PCR was conducted using MegaFi\u0026trade; One-Step RT-PCR (Applied Biological Materials Inc.) with the purified total RNAs as templates. PerMaV infection was detected using the primers PerMaV_CP_45F and PerMaV_CP_413R, described above.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRT-PCR analysis revealed that the bands of PerMaV-specific DNA products were detected in the inoculated leaves of all three seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, lanes 2, 4, 6). In addition, the virus-specific band was also detected in the upper leaf sample of #3 seedling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, lane 7), implying that systemic infection of PerMaV occurred in this persimmon seedling. These results suggest that infectious PerMaV cDNA clone was successfully generated in this study.\u003c/p\u003e\u003cp\u003eIn this study, we identified a new species in the genus \u003cem\u003eMarafivirus\u003c/em\u003e from the male Oriental persimmon cultivar 'Kumemaru'. Given the previous observation that male-promoting factors in 'Kumemaru' may be stress-induced [5], it is plausible that PerMaV contributes to male flower development indirectly by modulating host stress signaling pathways. Such virus-induced stress signaling could influence key regulators of sex determination through epigenetic mechanisms [18]. On the other hand, persimmon seedlings inoculated with the infectious cDNA clone (hereafter, the PerMaV vector), showed no symptoms, indicating that PerMaV is a latent virus. Given this latent nature, the PerMaV vector may serve as a valuable reverse genetics tool or have potential agricultural applications. For example, the use of the latent virus \u003cem\u003eApple latent spherical virus\u003c/em\u003e (ALSV) in apple trees has been shown to induce precocious flowering [19]. Although the ALSV vector is effective in apple, it was not applicable to persimmon (data not shown). The PerMaV may offer a more suitable viral vector system for functional studies and breeding application in Oriental persimmon.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of interest/Competing interests\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eWe thank the Grape and Persimmon Research Station, NIFTS, NARO, Hiroshima, Japan, for kindly providing plant materials used in this study. We are also grateful to Dr. Takashi Akagi (Okayama University, at the time of this study) for giving us the opportunity to initiate this research. We also thank Dr. Yuki Furuse (The University of Tokyo) for kindly reviewing the manuscript in part.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe complete genome sequence of persimmon marafivirus (PerMaV) is available in the GenBank database under the accession number PX251123.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSato A, Yamada M (2016) Persimmon breeding in Japan for pollination-constant non-astringent (PCNA) type with marker-assisted selection.\u0026nbsp;\u003cem\u003eBreed Sci\u003c/em\u003e 66:60\u0026ndash;68.\u003cbr\u003e\u0026nbsp;https://doi.org/10.1270/jsbbs.66.60\u003c/li\u003e\n \u003cli\u003eYonemori K, Sugiura A, Tanaka K, Kameda K (1993) Floral ontogeny and sex determination in monoecious-type persimmons.\u0026nbsp;\u003cem\u003eJ Am Soc Hortic Sci\u003c/em\u003e 118:293\u0026ndash;297\u003cbr\u003e\u0026nbsp;https://doi.org/10.21273/JASHS.118.2.293\u003c/li\u003e\n \u003cli\u003eYakushiji H, Yamada M, Yonemori K, Sato A, Kimura N (1995) Staminate flower production on shoots of \u0026lsquo;Fuyu\u0026rsquo; and \u0026lsquo;Jiro\u0026rsquo; persimmon (Diospyros kaki Thunb.).\u0026nbsp;\u003cem\u003eJ Jpn Soc Hortic Sci\u003c/em\u003e 64:41\u0026ndash;46.\u003cbr\u003e\u0026nbsp;https://doi.org/10.2503/jjshs.64.41\u003c/li\u003e\n \u003cli\u003eXu LQ, Zhang QL, Luo ZR (2008) Occurrence and cytological mechanism of 2n pollen formation in Chinese Diospyros spp. (Ebenaceae) staminate germplasm.\u0026nbsp;\u003cem\u003eJ Hortic Sci Biotechnol\u003c/em\u003e 83:668\u0026ndash;672.\u003cbr\u003e\u0026nbsp;https://doi.org/10.1080/14620316.2008.11512446\u003c/li\u003e\n \u003cli\u003eMasuda K, Fujita N, Yang HW, Ushijima K, Kubo Y, Tao R, Akagi T (2020) Molecular mechanism underlying derepressed male production in hexaploid persimmon.\u0026nbsp;\u003cem\u003eFront Plant Sci\u003c/em\u003e 11:567249\u003cbr\u003e\u0026nbsp;https://doi.org/10.3389/fpls.2020.567249\u003c/li\u003e\n \u003cli\u003eDing SW, Howe J, Keese P, Mackenzie A, Meek D, Osorio-Keese M, Skotnicki M, Srifah P, Torronen M, Gibbs AJ (1990) The tymobox, a sequence shared by most tymoviruses: its use in molecular studies of tymoviruses.\u0026nbsp;\u003cem\u003eNucleic Acids Res\u003c/em\u003e 18:1181\u0026ndash;1187.\u003cbr\u003e\u0026nbsp;https://doi.org/10.1093/nar/18.5.1181\u003c/li\u003e\n \u003cli\u003eSchirawski J, Voyatzakis A, Zaccomer B, Bernardi F, Haenni AL (2000) Identification and functional analysis of the turnip yellow mosaic tymovirus subgenomic promoter.\u0026nbsp;\u003cem\u003eJ Virol\u0026nbsp;\u003c/em\u003e74:11073\u0026ndash;11080.\u003cbr\u003e\u0026nbsp;https://doi.org/10.1128/jvi.74.23.11073-11080.2000\u003c/li\u003e\n \u003cli\u003eIzadpanah K, Zhang YP, Daubert S, Masumi M, Rowhani A (2002) Sequence of the coat protein gene of Bermuda grass etched-line virus, and of the adjacent \u0026apos;marafibox\u0026apos; motif.\u0026nbsp;\u003cem\u003eVirus Genes\u003c/em\u003e 24:131\u0026ndash;134.\u003cbr\u003e\u0026nbsp;https://doi.org/10.1023/A:1014516515454\u003c/li\u003e\n \u003cli\u003eSabanadzovic S, Abou Ghanem-Sabanadzovic N (2009) Identification and molecular characterization of a marafivirus in Rubus spp. Arch Virol 154:1729\u0026ndash;1735.\u003cbr\u003e\u0026nbsp;https://doi.org/10.1007/s00705-009-0510-x\u003c/li\u003e\n \u003cli\u003eGlasa M, Predajňa L, \u0026Scaron;oltys K, Sabanadzovic S, Olmos A (2015) Detection and molecular characterisation of Grapevine Syrah virus-1 isolates from Central Europe. 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Methods Mol Biol 1130:149\u0026ndash;164.\u003cbr\u003e\u0026nbsp;https://doi.org/10.1007/978-1-62703-989-5_11\u003c/li\u003e\n \u003cli\u003eDe Rybel B, van den Berg W, Lokerse AS, Liao CY, van Mourik H, M\u0026ouml;ller B, Llavata-Peris CI, Weijers D (2011) A versatile set of ligation-independent cloning vectors for functional studies in plants. Plant Physiol 156:1292\u0026ndash;1299.\u003cbr\u003e\u0026nbsp;https://doi.org/10.1104/pp.111.177337\u003c/li\u003e\n \u003cli\u003eAkagi T, Henry IM, Kawai T, Comai L, Tao R (2016) Epigenetic regulation of the sex determination gene MeGI in polyploid persimmon. Plant Cell 28:2905\u0026ndash;2915.\u003cbr\u003e\u0026nbsp;https://doi.org/10.1105/tpc.16.00532\u003c/li\u003e\n \u003cli\u003eSasaki S, Yamagishi N, Yoshikawa N (2011) Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple latent spherical virus vectors. Plant Methods 7:15 https://doi.org/10.1186/1746-4811-7-15\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7522521/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7522521/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA virus-like sequence was identified in the male persimmon cultivar \u0026lsquo;Kumemaru\u0026rsquo; during transcriptome analysis targeting male-promoting factors. The complete genome, 6,397 nucleotides in length, encodes a large polyprotein containing replication-associated domains and putative coat proteins. Phylogenetic analysis based on the coat protein amino acid sequences placed the virus within the genus \u003cem\u003eMarafivirus\u003c/em\u003e, closely related to Blackberry virus S and Nectarine virus M. Our data indicate that this virus, for which the name persimmon marafivirus (PerMaV) is proposed, is a hitherto undescribed species of the genus \u003cem\u003eMarafivirus\u003c/em\u003e, family Tymoviridae.\u003c/p\u003e","manuscriptTitle":"A proposed new virus in the genus Marafivirus detected from Oriental persimmon ‘Kumemaru’","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 11:54:21","doi":"10.21203/rs.3.rs-7522521/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b23616d-0ea7-49d8-a4d8-efa08e7b200c","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-07T09:02:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 11:54:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7522521","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7522521","identity":"rs-7522521","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}