Molecular Characterization of a Novel Single-stranded RNA Virus, ChRV1, Isolated From the Plant Pathogenic Fungus Colletotrichum Higginsianum

In this study, a novel single-stranded RNA virus was isolated from the plant pathogenic fungus, Colletotrichum higginsianum strain HTC-5, named “Colletotrichum higginsianum ssRNA virus 1” (ChRV1). The complete genome of ChRV1 is 3850 bp in length with a GC content of 52 % and encodes two in-frame open reading frames (ORFs): ORF1 (smaller) and ORF2 (larger). ORF1 encodes a protein with highest similarity to proteins encoded by Phoma matteucciicola RNA virus 1 (PmRV1, 47.99% identity) and Periconia macrospinosa ambiguivirus 1 (PmAV1, 50.73% identity). ORF2 encodes a protein with a conserved RNA-dependent RNA polymerase (RdRp) domain with similarity to RdRps of PmRV1 (61.41% identity) and PmAV1 (60.61% identity), which are unclassied (+)ssRNA mycoviruses reported recently. Phylogenetic analysis of the RdRp domain suggested that ChRV1 grouped together with PmRV1, PmAV1 and other unclassied (+)ssRNA mycoviruses, and had a distant relationship to invertebrate viruses and plant viruses of the family Tombusviridae. This is the rst report of a novel (+)ssRNA virus infecting the phytopathogenic fungus C. higginsianum.


Introduction
Mycoviruses (also called fungal virus) have been identi ed in all major groups of fungal groups [1,2]. The majority of mycoviruses are mainly include four groups: double-stranded RNA (dsRNA), positivesense single-stranded RNA (+ssRNA), negative-sense single-stranded RNA (-ssRNA) and single-stranded DNA (ssDNA) genomes [3]. Due to the rapidly development of high-throughput sequencing technologies, a growing number of +ssRNA mycoviruses have been discovered recently, among which have not been assigned to formal taxa. For example: Trichoderma harzianum ssRNA virus 1 (ThAV1), Setosphaeria turcica ssRNA virus 1 (StAV1), Verticillium longisporum ssRNA virus 1 (VlAV1) and Periconia macrospinosa ssRNA virus 1 (PmAV1) [4][5][6], which were unclassi ed (+)ssRNA mycoviruses. Gilbert KB et al. [6] recently proposed the establishment of the family Ambiguiviridae that contains the above viruses and other discovered related viruses. Most mycoviruses are inconspicuous, causing little phenotypic effects in their fungal hosts. However, a few mycoviruses can induce phenotypic alterations, causing hypovirulence of their fungal host, which have potential as biological control agents of fungal diseases. The best exempli ed example was the successful use of Cryphonectria hypovirus 1 (CHV1) to control chestnut blight disease in Europe [7].
Colletotrichum higginsianum is a hemibiotrophic pathogen and responsible for anthracnose disease on cruciferous plants, such as species of Brassica and Raphanus [8], also including Arabidopsis. The Arabidopsis/C. higginsianum pathosystem has emerged as an attractive model to study plant-pathogen interactions [9]. The discovery and identi cation of mycoviruses in C. higginsianum is an effective way to search potential biological control agents against anthracnose disease. To date, only two mycoviruses have been characterized from this fungus. Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1), identi ed in a C. higginsianum strain IMI349063A in United States of America [10], and Colletotrichum higginsianum mitovirus 1 (ChMV1), which we recently reported from a C. higginsianum strain HTC-5.
In this study, we reported the characterization of a rst novel (+) ssRNA mycovirus from C. higginsianum strain HTC-5, which we have provisionally named Colletotrichum higginsianum ssRNA virus 1 (ChRV1).

Provenance Of The Virus Material
The C. higginsianum strain HTC-5 was isolated from a cabbage leaf with anthracnose disease in Changsha, Hunan Province, China, and identi ed as C. higginsianum according to our previous study. This fungus was cultured on potato dextrose broth at 28°C in the dark for extraction of dsRNA. The dsRNA was extracted as described by Morris and Dodds using the CF-11 cellulose chromatography method [11]. Then the extracted dsRNA was digested with DNase I and S1 nuclease (Takara, Dalian, China) to eliminate DNA and ssRNA prior to gel electrophoresis and visualization. A dsRNA segment of approximately 3.9 kbp in size was observed and puri ed (Fig. 1a) using a FastPure® Gel DNA Extraction Mini Kit (Vazyme Biotech Co.,Ltd, Nanjing, China). A cDNA library was constructed using reverse transcriptase and random hexanucleotide primers (5'-CGA TCG ATC ATG ATG CAA TGCNNNNNN-3'). Sequence gaps were lled by RT-PCR according to sequence-primers based on the cDNA sequences obtained. The 5'-and 3'-terminal sequences were obtained by adapter ligation and PCR ampli cation as described previously [12]. All PCR amplicons were cloned into the pMD19-T vector (Takara, Dalian, China), transformed into Escherichia coli DH5α cells. Positive clones were selected and sequenced, and each clone was sequenced independently at least three times. The full cDNA sequence was assembled and subjected to GenBank database with accession number of MW218984.
Sequence analysis, such as ORF nding, homology search, and conserved domains search were performed using the website of the National Center for Biotechnology Information (NCBI) database.
Multiple alignments were conducted using Clustal X 2.0 program [13] and annotated using the GeneDoc program [14] Phylogenetic tree was constructed using the neighbor-joining method in MEGA 7 Programs [15]. The transmembrane domains were predicted using TMHMM Server v. 2.0.

Sequence Properties
The full genome sequence of ChRV1 is 3850 nt in length, which encodes two noncontiguous ORFs. The 5'-and 3'-untranslated regions (UTRs) of ChRV1 are 633 nt and 559 nt in length, respectively (Fig. 1b).
ORF1 encoded a 347-aa protein with a predicted molecular mass of 38.3 kDa. A BLASTP search showed that the aa sequence of ChRV1 has a maximum identity of 50.73% (E-value: 7e-69; query cover: 78 %) and 47.99% (E-value: 6e-83; query cover: 86 %) to the protein encoded by Periconia macrospinosa ambiguivirus 1 (PmAV1) and Phoma matteucciicola RNA virus 1 (PmRV1), followed by other unclassi ed (+)ssRNA mycoviruses (Table S1), whose function were unknown. In addition, ORF1 and ORF2 were found in the same frame, the amber stop codon 'UAG' was found at the end of the ORF1, suggesting that the two ORFs are likely to produce a fusion protein with the downstream RdRp via a readthrough strategy, as described previously [6,16]. Moreover, four possible transmembrane helixs were predicted at the Nterminus of the ORF1 protein (Fig. S1), suggesting that the transmembrane domains are important for (+) ssRNA viruses and play a role in anchoring viral protein to host membrane, as proposed for MoVA and VdRV [16,17].
To determine further the relationship between ChRV1 and other mycoviruses, a phylogenetic analysis was constructed using the RdRp aa sequences of ChRV1 and other related RNA viruses. The results indicated that ChRV1 cluster together with PmRV1, PmAV1 and other other unclassi ed (+)ssRNA mycoviruses, but distinct from the invertebrate viruses and plant viruses (Fig. 2). All these selected unclassi ed (+)ssRNA mycoviruses and ChRV1 contain two non-overlapping ORFs and the RdRp domain is encoded by ORF2.
Moreover, these unclassi ed (+)ssRNA mycoviruses have more similar genome organization with invertebrate viruses, presenting two in-frame ORFs [17], and differ from members of the family Tombusviridae, suggesting that these unclassi ed (+)ssRNA mycoviruses are more related to invertebrate viruses than plant viruses of the family Tombusviridae. Additionally, the ChRV1 RdRp contains the amino acid triplet GDN in motif , which differ from the GDD motif in + ssRNA viruses (Fig. 1c). The same triplet was found in previous studies [16,19,20]. Taken together, we concluded that ChRV1 is a novel unclassi ed (+)ssRNA virus, with closer relationship to members of the recently proposed family "Ambiguiviridae". Compliance with ethical standards