Characterization of a Novel Mitovirus Infecting Melanconiella theae Isolated From Tea Plants

Abstract

A dsRNA segment was identified in the fungus Melanconiella theae isolated from tea plants. The complete dsRNA sequence, determined by random cloning together with RACE protocol, is 2,461 bp in length with an AU-rich content (62.37%) and comprises a single ORF of 2,265-nucleotides encoding an RNA-dependent RNA-polymerase (RdRp, 754 amino acids in size). The terminus sequences can fold into predicted stable stem-loop structures. A BLASTX and phylogenetic analysis revealed the dsRNA genome shows similarities with the RdRp sequences of mitoviruses, with the highest identity of 48% with those of grapevine-associated mitovirus 20 and Colletotrichum fructicola mitovirus 1. Our results reveal a novel member, tentatively named Melanconiella theae mitovirus 1 (MtMV1), belongs to the family Mitoviridae. MtMV1 is capsidless as examined by transmission electron microscope, efficiently transmitted through conidia as 100 conidium-generated colonies were analyzed, and easily eliminated by hyphal tipping method combined with green-leaf tea powder. MtMV1 has a genomic sequence obviously divergent from those of most members in the family Mitoviridae and some unique characteristics unreported in known members. This is the first report of a mycovirus infecting Melanconiella fungi to date.

Keywords: Camellia sinensis; Melanconiella theae; MtMV1; mitochondrial virus; mitovirus; mycovirus.

FIGURE 1

Agarose gel electrophoresis of nucleic acids extracted from Melanconiella theae strains, enzyme digest and RT-PCR identification of MtMV1. (A) Nucleic acids extracted from strains WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, WJB-1-20, and WJB-5. (B) The nucleic acids extracted from strain WJB-5 were untreated (Lane 1) or treated with S1 nuclease (Lane 2). (C) RT-PCR identification of MtMV1 in strain WJB-5. M: DL5000 DNA marker (100; 250; 500; 750; 1,000; 1,500; 2,000; 3,000; and 5,000 bp). The arrows indicate the target bands.

FIGURE 2

Genomic organization and phylogenetic analysis of MtMV1. (A) Genome organization of MtMV1. The start and end positions are labeled for the genome, untranslated regions (UTR), and the open reading frame (ORF). (B) ML phylogenetic tree was constructed using DNAMAN software based on RdRp sequences of MtMV1 and other members in the family Mitoviridae listed in Table 1. The bootstrap values were deduced based on 1,000 replicates and indicated beside the branches. The evolutionary distances were computed using the Poisson correction method and the branch lengths correspond to the genetic distance; the scale bar refers to the genetic distance of 0.05. A tringle indicates MtMV1.

FIGURE 3

Multiple alignments of the conserved motifs of RdRp in MtMV1 and other mitoviruses. Lines above the aligned sequences indicate to the positions of the motifs (I–VI). Numbers between every two motifs correspond to the number of aa residues separating the motifs. Identical, conserved, and semi-conserved aa residues are color-highlighted with homology level as 100% (black), 75% (magenta), 50% (turquoise), and 33% (yellow).

FIGURE 4

Predicted secondary structures of the terminal sequences of MtMV1. (A,B) Stem-loop structures folded for the 5′- and 3′-UTR sequences of the genomic positive-strand, respectively. (C) Panhandle structure formed by the reverse complementary regions of the 5′- and 3′-UTRs ends of the MtMV1 genome. The CLC workbench software was used to predict the secondary structures of the terminal sequences and to calculate the free energy. The solid and open arrows indicate the 5′- and 3′-terminal sequences, respectively, in their initial and ending positions.

FIGURE 5

Agarose gel electrophoresis of dsRNA extracted from the mycelia of Melanconiella theae strains. (A) dsRNA extraction from the mycelia of single-conidium generated colonies derived from strain WJB-5 conidia. (B) dsRNA extraction from six strains (WJB-5, WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, and WJB-1-20) after being treated with hyphal tipping combined with antiviral extracts. (C) Untreated WJB-5 as a control. M: DL5000 DNA marker (100; 250; 500; 750; 1,000; 1,500; 2,000; 3,000; and 5,000 bp). The arrows indicate the target viral dsRNA bands.