Complete genome sequence of a novel mitovirus isolated from Paris polyphylla var. yunnanensis

Paris mitovirus 1 (ParMV1) is a positive-sense RNA virus isolated from diseased Paris polyphylla var. yunnanensis plants in Wenshan, Yunnan. The complete genome sequence of ParMV1 consists of 2,751 nucleotides with a genome structure typical of the mitoviruses. ParMV1 genome has a single open reading frame (ORF: 358-2,637 nt) that encodes RNA-dependent RNA polymerase (RdRp) with a molecular mass of 86.42 kDa. ParMV1 contains six conserved motifs (Ι-VΙ) that are unique to mitoviruses. In addition, the 5′and 3′ terminals of the genome have a stable secondary structure, and the reverse complementary sequence forms a panhandle structure. Comparative genome analysis revealed that ParMV1 shares 23.1–40.6% amino acid (aa) and 32.3–45.7% nucleotide (nt) sequence identities with the RdRp of other mitoviruses. The phylogenetic tree inferred from RdRp aa sequence showed that ParMV1 clusters with mitoviruses, and hence should be considered as a new member of the genus Mitovirus in the family Motiviridae. This is the rst report of a novel mitovirus infecting Paris polyphylla var. yunnanensis.


Introduction
Paris polyphylla var. yunnanensis is a perennial herb, which has important medicinal and economical values with a huge market [1,2] . With the rapid development of the Chinese traditional medicine industry, the demand for P. polyphylla var. yunnanensis as a raw material increased greatly. With the aim of meeting market demand, the practice of single planting structure at large-scale arti cial planting bases in Qujing, Wenshan, Pu'er and other places in Yunnan has led to the emergence of various viral diseases in P. polyphylla var. yunnanensis. Currently, Paris mosaic necrosis virus (PMNV), Paris polyphylla virus X (PPVX), pepper mild mottle virus (PMMoV), Paris virus 1 (ParV1), Yunnan paris negative-stranded virus (YPNSV), and Paris virus 2 (ParV2) [3][4][5][6][7][8] have been reported to infect P. polyphylla var. yunnanensis.
Mitoviruses have been reported mostly in phytopathogenic fungal genera, such as Sclerotinia, Ophiostoma, Puccinia, Rhizoctonia, Cryphonectria, and Alternaria and Botrytis, with a few from Thielaviopsis, Thanatephorus, Helicobasidium, Gremmeniella, arbuscular mycorrhizae glomus and ectomycorrhizal fungi [19][20][21][22] . Plant mitoviruses, like fungal ones, lack coat protein (CP) and virions. They are usually considered to exist in the host mitochondria in the form of intracellular ribonucleoprotein complexes (RNP), which are transmitted during cell division and fusion without being exposed to the extracellular environment [15,23−24] . This means that they cannot be spread by mechanical inoculation and grafting, but can only rely on seeds to be transmitted vertically [25][26] .
Here, we report a new plant mitovirus infecting P. polyphylla var. yunnanensis. This new mitovirus is tentatively named as "Paris mitovirus 1 (ParMV1)". This study enhances our knowledge on the evolution and classi cation of plant mitoviruses, and further research on P. polyphylla var. yunnanensis viral disease.

Provenance Of The Virus Material
In July 2019, diseased P. polyphylla var. yunnanensis plants with foliar chlorotic were observed during a survey. To determine the virus species responsible for the infection, samples of the diseased plants were collected in Wenshan, Yunnan and sent to OE biotech (Shanghai, China) for high throughput sequencing (HTS) on the Illumina HiSeq-2500 platform. The raw reads obtained by HTS were ltered and reassembled into contigs using Trinity algorithm. The assembled contigs were then used to performed online BLASTx (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the virus sequence. To con rm the presence of the virus, total RNA was extracted from the symptomatic and asymptomatic leaves using Plant RNA Kit (Omega Bio-tek, USA). The ParMV1-speci c primers (ParMV1-1F/R; ParMV1-2F/R; ParMV1-3F/R; ParMV1-4F/R; ParMV1-5F/R; ParMV1-6F/R; ParMV1-7F/R) (Table S1) designed from the virus sequence. Prime ScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa Bioengineering, Dalian) and 2× Taq PCR Master Mix (BioTeke, Beijing) were used to perform RT-PCR and amplify the complete genome sequence of the virus. The ORF of the virus genome and molecular weight of the protein were predicted by using the NCBI ORF Finder (https:// www.ncbi. nlm. nih. gov/ or nder/) and EditSeq program of DNASTAR 7.1. The stem-loop structures in the 3' and 5' terminal of the virus genome were predicted using the Mfold Web server (http://unafold.rna.albany.edu/?q = mfold) [9,19] . Multiple sequence alignments and determination of conserved motifs were performed using the ClustalW tool in BioEdit 7.0. Phylogenetic trees were constructed with neighboring joining (NJ) method and 1,000 bootstrap replicates using MEGA 7.0.

Sequece Properties
The 24,886,212 raw reads obtained by HTS were processed and reassembled de novo into transcripts using the paired-end splicing method in Trinity to obtain 1,224 contigs. BLASTx analysis revealed that a contig consisting of 2,751 nt had the highest amino acid sequence identity (44.41%) with Cannabis sativa mitovirus 1 (Accession no. BK010438/ DAB41756). The presence ParMV1 in P. polyphylla var. yunnanensis symptomatic leaves was con rmed by RT-PCR (Fig. 1C).
The complete genome sequence of ParMV1 (Accession no. MT269666) consist of 2,751 nt with an A + U content of 56.05%. ParMV1 contains a single ORF (nt:358-2,637) which is predicted to encode an RNAdependent RNA polymerase (RdRp) with a molecular mass of about 86.42 kDa, and 5′ and 3′ untranslated regions (UTRs) of 357 bp and 114 bp respectively (Fig. 1A). The complete genome sequence and the size of the 5'-and 3'-UTR fragments are consistent with previously reported plant mitoviruses. In addition, the BLASTx analysis showed that ParMV1 has high sequence similarity with plant mitoviruses (Score 507-599, Query Cover 76-79% and E-value of 0.0). These results indicate that ParMV1 has a typical mitovirus genome structure.
Mitoviruses were previously thought to infect only fungi, but the detection of complete genome sequences (2.7 to 3.0 kb) of nearly 20 plant mitoviruses in beet, hemp and petunia in the past years indicates that mtoviruses can also infect plants. A number of mitovirus sequences have been identi ed in the transcriptomes of a large collection of invertebrates [31] , but the origin of these viruses still remains unclear. It has been proposed that the mitoviruses either arose from plant genetic elements or by horizontal transfer from fungal mitochondria to plant mitochondria [32][33][34][35] . Subsequent studies have suggested that plant mitoviruses are formed by the integration of fungal mitovirus cDNA fragments in the mitochondrial DNA of vascular plants [36] . Systematic analysis of mitoviruses in 10 plant species revealed that plant mitoviruses did not originate from pathogenic fungi [26] .
In the diseased P. polyphylla var. yunnanensis leaves from which ParMV1 was detected and isolated, no fungal infection was detected. These diseased plants only exhibited viral-like symptoms. Besides, if ParMV1 had evolved from endophytic fungi, ParMV1 should have clustered with fungal mitoviruses in the clade containing fungal mitoviruses, but not with plant mitoviruses as seen in the RdRp-inferred phylogenetic tree (Fig. 2B). These results therefore indicate that ParMV1 is a plant mitovirus rather than a fungal mitovirus.
The presence of this novel mitovirus was con rmed by HTS and RT-PCR, combined with complete genome structure analysis, molecular phylogeny and comparative genome sequence analysis. Although studies have shown that fungal mitoviruses can affect the growth and virulence of fungi, leading to morphological abnormalities in mitochondria and growth defects, it is uncertain whether or not mitoviruses have a direct impact on plant health [34,37−38] . As a new plant mitovirus, it is uncertain whether ParMV1 directly caused the observed viral disease symptoms in the host plant. Since plant mitoviruses rely on hosts mitochondrial for replication, it is plausible that these viruses may have potential effects on plant hosts mitochondria that may be detrimental to the plant. Further studies are therefore required to ascertain the effects of mitoviruses on their plant host.

Declarations
Compliance with ethical standards Funding This study was funded by the National Natural Science Foundation of China (81860774).

Con ict of interest
The authors declare that they have no con ict of interest.

Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.

Data availability
The dataset generated during the current study is available in the GenBank database. (Acession no.