Investigation of Host Range of and Host Defense against a Mitochondrially Replicating Mitovirus

Abstract

Mitoviruses (genus Mitovirus, family Narnaviridae) are mitochondrially replicating viruses that have the simplest positive-sense RNA genomes of 2.2 to 4.4 kb with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase. Cryphonectria parasitica mitovirus 1 (CpMV1) from U.S. strain NB631 of the chestnut blight fungus, Cryphonectria parasitica, was the first virus identified as a mitochondrially replicating virus. Despite subsequent discovery of many other mitoviruses from diverse fungi, no great advances in understanding mitovirus biology have emerged, partly because of the lack of inoculation methods. Here we developed a protoplast fusion-based protocol for horizontal transmission of CpMV1 that entailed fusion of recipient and donor protoplasts, hyphal anastomosis, and single-conidium isolation. This method allowed expansion of the host range to many other C. parasitica strains. Species within and outside the family Cryphonectriaceae, Cryphonectria radicalis and Valsa ceratosperma, also supported the replication of CpMV1 at a level comparable to that in the natural host. No stable maintenance of CpMV1 was observed in Helminthosporium victoriae. PCR-based haplotyping of virus-infected fungal strains confirmed the recipient mitochondrial genetic background. Phenotypic comparison between CpMV1-free and -infected isogenic strains revealed no overt effects of the virus. Taking advantage of the infectivity to the standard strain C. parasitica EP155, accumulation levels were compared among antiviral RNA silencing-proficient and -deficient strains in the EP155 background. Comparable accumulation levels were observed among these strains, suggesting the avoidance of antiviral RNA silencing by CpMV1, which is consistent with its mitochondrial replication. Collectively, the results of study provide a foundation to further explore the biology of mitoviruses. IMPORTANCE Capsidless mitoviruses, which are ubiquitously detected in filamentous fungi, have the simplest RNA genomes of 2.2 to 4.4 kb, encoding only RNA-dependent RNA polymerase. Despite their simple genomes, detailed biological characterization of mitoviruses has been hampered by their mitochondrial location within the cell, posing challenges to their experimental introduction and study. Here we developed a protoplast fusion-based protocol for horizontal transfer of the prototype mitovirus, Cryphonectria parasitica mitovirus 1 (CpMV1), which was isolated from strain NB631 of the chestnut blight fungus (Cryphonectria parasitica), a model filamentous fungus for studying virus-host interactions. The host range of CpMV1 has been expanded to many different strains of C. parasitica and different fungal species within and outside the Cryphonectriaceae. Comparison of CpMV1 accumulation among various RNA silencing-deficient and -competent strains showed clearly that the virus was unaffected by RNA silencing. This study provides a solid foundation for further exploration of mitovirus-host interactions.

Different Genetic Sources Contribute to the Small RNA Population in the Arbuscular Mycorrhizal Fungus Gigaspora margarita

“…In another study the sRNA response to a mitovirus in F. circinatum also pointed to a protection from the cytoplasmic antiviral RNAi since, also in this case, a relatively low accumulation of sRNA had the same length distribution of mitochondrial sRNAs (Muñoz-Adalia et al, 2018). A similar situation has been observed for the mitovirus infecting the ascomycete C. parasitica (Shahi et al, 2019). The analysis of mitoviral sRNA in G. margarita did not allow us to discern whether mitoviruses replicate in mitochondria or cytosol since, contrary to what happens in plants, we do not detect differences in the nucleotide length profiles for sRNAs with nuclear and mitochondrial origins.…”

Establishment of Neurospora crassa as a model organism for fungal virology

“…For capsidless viruses, we previously established protoplast fusion techniques for virus introduction 20 , because virion transfection was not applicable. This method was tested for the putative capsidless RNA virus NiFV1 (a fusarivirus) originally hosted by N. intermedia (Fig.…”

“…For several capsidless (+)ssRNA viruses, transfection with in vitro-synthesized full-length genomic RNA 15,16 or transformation with full-length cDNA 17,18 are available. Protoplast fusion-based virus horizontal transmission was shown to be useful between virus-infected donors and many recipients for any viruses, i.e., encapsidated and capsidless viruses 19,20 .…”

Detection and Molecular Characterization of Picobirnaviruses (PBVs) in the Mongoose: Identification of a Novel PBV Using an Alternative Genetic Code

“…Based on analysis of RdRp sequences that belong to the clade of PBV-like viruses using the alternative genetic code, and the lack of identifiable PBV-like capsid sequences in the respective metagenomics data, Yinda et al (2018) proposed that PBV-like strains using the invertebrate mitochondrial genetic code might behave like mitoviruses [10]. Mitoviruses are virus-like elements that are omnipresent in the mitochondria of fungi, possess a plus-strand RNA genome that generally employ the mold mitochondrial genetic code to translate RdRp, and lack a capsid [9,28,29].…”