Molecular characterization of a novel mycovirus isolated from Rhizoctonia solani AG-1 IA 9-11

Yang Sun Yunnan Agricultural University Yan qiong Li Kunming University Wen han Dong Yunnan Agricultural University Ai li Sun Yunnan Agricultural University Ning wei Chen Yunnan Agricultural University Zi fang Zhao Yunnan Agricultural University Yong qing Li Zhaotong Plant Protection and Quarantine Station Gen hua Yang (  [email protected] ) Yunnan Agricultural University https://orcid.org/0000-0003-3322-1558 Cheng yun Li Yunnan Agricultural University

Research Article

Keywords: SMC, PRK, RT-like super family, RsRV-HN008, RdRp

Posted Date: May 19th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-520549/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Page 1/9 Abstract

The complete genome of the dsRNA isolated from Rhizoctonia solani AG-1 IA 9–11 (designated as Rhizoctonia solani dsRNA virus 11, RsRV11 ) were determined. The RsRV11 genome was 9,555 bp in length, contained three conserved domains, SMC, PRK and RT-like super family, and encoded two non- overlapping open reading frames (ORFs). ORF1 potentially coded for a 204.12 kDa predicted protein, which shared low but signifcant amino acid sequence identities with the putative protein encoded by Rhizoctonia solani RNA virus HN008 (RsRV-HN008) ORF1. ORF2 potentially coded for a 132.41 kDa protein which contained the conserved motifs of the RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that RsRV11 was clustered with RsRV-HN008 in a separate clade independent of other virus families. It implies that RsRV11, along with RsRV-HN008 possibly a new fungal virus taxa closed to the family , and RsRV11 is a new member of mycoviruses.

Introduction

Mycoviruses (fungal ) are viruses that infect fungi, which are found in mushrooms, yeasts, oomycetes and flamentous fungi, including phytopathogenic fungi [1–3]. In 1962, the frst mycovirus was discovered from mushroom, Agaricus bisporus, which developed abnormal fruit bodies, grew slowly and caused serious economic losses [1, 2]. However, the successful control of chestnut blight through hypovirulence mediated by Cryphonectria 1 (CHV1) [4]. It has also inspired researchers to explore fungal viruses related to hypovirulence and diversity of mycoviruses. According to the International Committee on Taxonomy of Viruses (ICTV) Report in 2020, mycoviruses have genomes composed of single-stranded DNA (ssDNA), double-stranded RNA (dsRNA), positive-sense single-stranded RNA (+ ssRNA), negative-sense ssRNA (-ssRNA) and reverse- single-stranded RNA (ssRNA- RT), which contain 23 families like , , Megabirnaviridae and etc [5]. Therefore, the discovery and classifcation of novel mycoviruses have enriched our understanding of the feld.

Rhizoctonia spp. is a kind of flamentous widely distributed in nature, which was reported by De Candolle in 1815. Rhizoctonia solani (R. solani)was described by Kühn in 1858 [6, 7]. The isolates of R. solani AG-1 cause seed and hypocotyl rot, aerial blights, and web blights of many plant species [6, 8]. According to statistics, there are approximately 30 mycoviruses with complete genomes isolated from Rhizoctonia spp., mycoviruses from AG-1 IA including Rhizoctonia solani dsRNA virus 1 (RsRV1) [9], Rhizoctonia solani partitivirus 2 (RsPV2) [10], Rhizoctonia solani dsRNA virus 3 (RsRV3) [11], Rhizoctonia solani partitivirus 5 (RsPV5) [12] in the family and Rhizoctonia solani endornavirus 1 (RsEV1) in the family [13].

Here, we report the genome structure and phylogenetic analysis of a novel mycovirus in R. solani AG-1 IA 9–11 (designated as Rhizoctonia solani dsRNA virus 11, RsRV11).

Provenance Of The Virus Material

Page 2/9 The R. solani AG-1 IA 9–11 was isolated from maize leaves with typical symptoms of maize sheath blight collected from Lincang city, Yunnan province, China and stored at -20 ºC, which was initially grown on potato dextrose agar (PDA) plates at 28 ºC for 2 days, then grown in potato dextrose broth at 28 ºC with orbital shaking at 120 rpm for 5–7 days. Genomic dsRNA was extracted from 0.2 g freeze-dried mycelium using CF11 cellulose chromatography, and then digested with DNase I and S1 nuclease to eliminate contaminated DNA and single-stranded RNA (ssRNA) [14, 15]. The dsRNA showed an approximately 10 kb genomic fragment by 1 % agarose gel electrophoresis, which was amplifed by random primers [16]. The amplifed cDNA product of the virus was ligated into the pMD18-T vector (Takara) and transformed into Escherichia coli DH5ɑ for sequencing. Sequences were compared to the virus in National Center for Biotechnology Information (NCBI). Hyphae were extracted to total RNA to obtain sequences, which were assembled by UID-mRNA seq (Kangce, Wuhan) and verifed with virus- specifc primers. The 5' and 3' ends of the viral genome were obtained using a modifed procedure for rapid amplifcation of cDNA ends (RACE) [17]. The complete cDNA sequence of the virus was assembled with DNAMAN and deposited in NCBI GenBank database under the accession number MW540499.

The structure of the cDNA was analyzed and predicted the open reading frame (ORF) by using the online program ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/) and the homology search was performed through the NCBI BLAST programs. The ClustalX program was used for multiple alignments of amino acid sequences [18]. The Neighbor Join (NJ) algorithm was used to construct a , and a bootstrap test was performed on 1,000 replicates through MEGA 7 [19].

Sequence Properties

The complete genome of the dsRNA virus isolated from R. solani AG-1 IA 9–11 (designated as Rhizoctonia solani dsRNA virus 11, RsRV11) were determined and annotated (Fig. 1A). The RsRV11 genome was 9,555 bp in length, with a GC content of 48.81 %, which had three conserved domains, SMC (aa 151–990, cl37069, chromosome segregation protein SMC, 6.51e-05), PRK (aa 2125–2430, cl35883, RNA polymerase sigma factor ShbA, 3.01e-03) and RT like (aa 7216–8508, cl02808, RNA-dependent DNA polymerase, 3.63e-15). Sequence analysis demonstrated two non-overlapping ORFs on the genome, ORF1 (nt 160–5751) and ORF2 (nt 6016–9519). The 5’ and 3’ untranslated regions (UTRs) of RsRV11 were 159 bp and 36 bp, respectively.

The ORF1 of RsRV11 putatively encodes a 1,863 amino-acid (aa) protein with a calculated molecular mass of 204.12 kDa. BLASTp search revealed that ORF1 may encode a hypothetical protein whose sequence is only 57.33 % identical to the complete RsRV-HN008 hypothetical protein (YP_009158859.1, 1,179 aa) in the GenBank database. ORF1 contained PRK09648 super family domain and a part of SMC prok B super family domain. SMC was also present in the putative proteins encoded by the ORFs of other mycoviruses in different families, SsNSRV-1 from [20, 21], MiFV1 from Fusariviridae [22, 23] and FgHV2 from Hypoviridae [24]. SMC proteins are found in , archaea, and , which were shown (in Caulobacter crescentus) to be induced early in S phase but present and bound to DNA throughout the cell cycle [25–27]. Previously, SMC domains were found in several mycovirus families, like

Page 3/9 Mymonaviridae, Fusariviridae and Hypoviridae. However, RsRV11 was only found to code for the SMC prok B super family domain among mycoviruses.

ORF2 is predicated to encode a 132.41 kDa protein with 1,167 aa. The ORF2 encoded protein has signifcant sequence similarity to the RNA-dependent RNA polymerases (RdRps) of these unclassifed mycoviruses, with RsRV-HN008 being the best match, with aa 78.07 % sequence identity, followed by Rosellinia necatrix megabirnavirus 3 (RnMBV3) and Rosellinia necatrix megabirnavirus 2 (RnMBV2) with aa 29.86 % and 29.71 % identity. In addition, using a multiple protein sequence alignment are characteristic of the RdRps of dsRNA mycoviruses was detected (Fig. 1B).

To determine the phylogenetic status of RsRV11, the RdRp aa sequence of RsRV11 was compared to those of the related unclassifed mycoviruses as well as other mycoviruses belonging to the families Megabirnaviridae, and Chrysoviridae. After comparing 21 selected viruses by multiple sequence alignment, a phylogenetic tree was generated that revealed that RsRV11 was clustered with RsRV-HN008 in a separate clade independent of other families., then forming a well-supported branch together with the family Megabirnaviridae and unclassifed 1 (Fig. 2).

Previous studies indicated that RsRV-HN008 had monopartite dsRNA genomes and their genome organizations were similar to RsRV11, which had only one dsRNA segment but no ribosomal-1 frameshift sequence. The members of Megabirnaviridae have two dsRNA segments and ribosomal-1 frameshift sequences [28]. Then the viruses of Unclassifed 1 have only one dsRNA segment and ribosomal-1 frameshift sequence [29], which had already been proposed to form a new genus called Phlegivirus in Phlegiviridae including Thelephora terrestris virus 1 (TtV1) [29], Rhizoctonia fumigata virus 1 (RfV1) [30], Phlebiopsis gigantea large virus 1 (PgLV1) [31], Lentinula edodes mycovirus HKA (LeV-HKA) [32] and Lentinula edodes mycovirus HKB (LeV-HKB) [33]. According to phylogenetic analysis, we propose that RsRV11, along with RsRV-HN008, should be classifed into a unclassifed 2. The identifcation of this kind of viruses including RsRV11 and other related mycoviruses might contribute to the establishment of a new virus taxon based on similarities in their genome organization and phylogenetic relationships. The discovery of a novel mycovirus in R. solani has improved our understanding of the viral diversity in this plant-pathogenic fungus.

Declarations Acknowledgements

This research was supported the National Natural Scientifc Foundation of China (31960525,31160352༌31360423) and grant-in-aid from Scientifc Research Project of Kunming University (YJL16006), General Program of University Union in Yunnan Province (2018FH 001–032).

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Page 6/9 Figures

Figure 1

(A) Graphical representation of the genome organization of RsRV11. The open reading frames (ORF1 and 2) and untranslated regions (UTRs) are represented by colored boxes and solid black lines, respectively. (B) Multiple amino acid sequence alignment of the conserved motifs in RdRp encoded by RSRV11 and RsRV-HN008. The alignment was conducted using the ClustalX program and highlighted using the GeneDoc program.

Page 7/9 Figure 2

Phylogenetic analysis of RsRV11. Based on a multiple sequence alignment of RdRp aa sequences, a neighbor-joining phylogenetic tree was generated using MEGA7, with bootstrap values determined using 1000 replicates. RsRV11 is indicated in the phylogenetic tree. The virus names and their GenBank accession numbers are as follows: Fusarium pseudograminearum megabirnavirus 1, AYJ09269.1; Scaphoideus titanus toti-like virus 1, QIJ56906.1; Rosellinia necatrix megabirnavirus 1, YP_003288763.1; Rosellinia necatrix megabirnavirus 2, YP_009227124.1; Rosellinia necatrix megabirnavirus 3, BBB86809.1; Rhizoctonia solani RNA virus HN008, YP_009158860.1; Amasya cherry disease associated chrysovirus, CAH03664.1; Sclerotinia sclerotiorum megabirnavirus 1, YP_009143529.1; Culex vishnui subgroup , BCI50698.1; Murri virus, QHA33714.1; Aedes aegypti toti-like virus, QEM39131.1; Barrymore virus, QED21514.1; Neofusicoccum parvum chrysovirus 1, QDB74975.1; Magnaporthe oryzae chrysovirus 1, YP_008914864.1; Lentinula edodes mycovirus HKA, BAM34028.1; Lentinula edodes mycovirus HKB, BAG71788.2; Rhizoctonia fumigata virus 1, AJE29745.1; Thelephora terrestris virus 1, YP_009209482.1; Cryphonectria nitschkei chrysovirus 1, BBJ21307.1; Macrophomina phaseolina chrysovirus 1, YP_009667008.1; Phlebiopsis gigantea large virus 1, YP_003541123.1;

Page 8/9 Supplementary Files

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RsRV11.seq

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