Tingting Zhang

In this study, we isolated two novel mitoviruses co-infecting the fungus Gnomoniopsis sanguisorbae, which were designated as “Gnomoniopsis sanguisorbae mitovirus L” (GsMV-L) and “Gnomoniopsis sanguisorbae mitovirus M” (GsMV-M). The complete genome lengths of GsMV-L and GsMV-M were determined to be 2,785 and 2,529 nucleotides, respectively, with a GC content of 44% and 35%, respectively. Both viruses possess a single open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp) containing conserved catalytic motifs. BLASTp analysis revealed that the RdRps of GsMV-L and GsMV-M exhibited the highest amino acid sequence similarity to those of sunflower phomopsis-associated mitovirus 1 and Phomopsis viticola mitovirus 962_1, with 50.97% and 41.97% identity, respectively. Phylogenetic analysis based on RdRp sequences indicated that GsMV-L clustered with members of the genus Triamitovirus, while GsMV-M clustered with members of the genus Unuamitovirus, both of which are taxonomically classified within the family Mitoviridae.

Parasitic and symbiotic plant–fungus relationships have existed for millions of years, and phylogenetic analyses of certain virus families indicate transmission between plants and fungi. A group of tobamo-like viruses from various fungi has recently been identified. Tobamo-like viruses are homologous to plant viruses of the Tobamovirus genus in the Virgaviridae family, but it was unknown whether they form typical virus particles and can replicate and exhibit cell-to-cell movement in plants. Here, a novel tobamo-like virus, Nigrospora aurantiaca tobamo-like virus 1 (NaTLV1), obtained from the phytopathogenic fungus Nigrospora aurantiaca , was characterized. Its genome (10,301 nucleotides) comprises four open reading frames (ORFs) and a poly(A) tail. ORF1 encodes a methyltransferase and helicase, and ORF2 encodes RNA-dependent RNA polymerase, which are closely related to proteins of Virgaviridae viruses. ORF3 encodes a putative movement protein, and ORF4 encodes a putative coat protein, which are closely related to proteins of Gammaflexiviridae and Betaflexiviridae viruses. NaTLV1 formed filamentous virus particles and could replicate in Nicotiana benthamiana . However, the cell-to-cell movement of NaTLV1 was not observed in N. benthamiana but could not be ruled out. NaTLV1 underwent horizontal and vertical transmissions via hyphal anastomosis and conidia, respectively. There were no significant differences in phenotype or virulence between NaTLV1-infected (A4) and NaTLV1-free (V1) N. aurantiaca strains. In general, our findings may be useful in elucidating the origination and evolution of tobamo-like viruses, plant virus families ( Virgaviridae and Betaflexiviridae ), and a fungal virus family ( Gammaflexiviridae ). IMPORTANCE: Cross-kingdom infections involving plant- and fungal-associated viruses have been directly observed in nature, and some of these viruses share a high degree of genetic similarity. A group of novel tobamo-like viruses with nonsegmented single-stranded RNA genomes was recently isolated from diverse fungal groups. Here, we identified a novel virus, Nigrospora aurantiaca tobamo-like virus 1 (NaTLV1), in the phytopathogenic fungus Nigrospora aurantiaca . NaTLV1 was phylogenetically related to other tobamo-like viruses, plant-associated viruses in the Virgaviridae and Betaflexiviridae families, and fungus-associated viruses in the Gammaflexiviridae family. NaTLV1 formed filamentous virus particles and could replicate in Nicotiana benthamiana, but the cell-to-cell movement of NaTLV1 was not observed. This study provides potential insights into the origins and evolution of mycoviruses originating from plant viruses and indicates that tobamo-like viruses may have adapted to an intracellular lifestyle during evolution. The identification of novel tobamo-like viruses is crucial for understanding fungal and plant virus evolution.

To date, complete genome sequences of 14 double-stranded RNA (dsRNA) phages are available, and studies have shown that the host range of dsRNA phages is limited. The hosts of most dsRNA phages belong to the genus Pseudomonas . However, the dsRNA phage phiNY, which has a non-lytic life cycle, was isolated from Microvirgula aerodenitrificans . Currently, the interaction between dsRNA phage phiNY and its host bacteria is unclear, which is not beneficial to a comprehensive understanding of dsRNA phage biology and the exploitation of dsRNA phage with non-lytic life cycle for biomedical applications and others. Phage adsorption is a crucial step through the interactions between receptor-binding protein (RBP) of the phage and its receptors to initiate the infection process, which dictates host range specificity. Thus, we identified the RBP and host receptor of phiNY. Through homology alignment, amino acid sequence similarity analysis, and the phylogenetic tree analysis, orf11, located in the M-segment of dsRNA phage phiNY, encodes a putative RBP. We further performed the whole-cell enzyme-linked immunosorbent assay (ELISA), western blotting assay, and indirect immunofluorescence assay and demonstrated that this orf11 is an RBP. Finally, using affinity chromatography, ELISA, and dynamic light scattering, we identified lipopolysaccharides (LPSs) on the surface of the host M. aerodenitrificans strain LH9 as host receptors involved in the adsorption of the dsRNA bacteriophage phiNY and observed the state of phiNY RBP after combining with LPS by atomic force microscopy. These results will guide future studies on phage-host interaction in a dsRNA phage with a non-lytic life cycle. IMPORTANCE: The interactions between the lytic dsRNA phages and their host receptors have been clarified in previous studies. However, the interaction between the dsRNA phage phiNY (which has a non-lytic life cycle) and its host receptors during the dsRNA phage adsorption process was unknown. Here, we found that phiNY uses the orf11 protein as a receptor-binding protein (RBP). In addition, we found that this orf11 recognizes lipopolysaccharide from the host bacterium Microvirgula aerodenitrificans strain LH9 as a specific receptor. These results suggest that phiNY, like lytic dsRNA phages, uses an RBP to bind to a similar host receptor (i.e., lipopolysaccharide). Determining the interaction between the dsRNA phage phiNY and its host receptors will help to elucidate the mechanisms underlying the phiNY non-lytic life cycle and enhance our understanding of its infection mechanism.

Phages and phage-encoded lytic enzymes are promising antimicrobial agents. In this study, we report the isolation and identification of bacteriophage KP2025 from Klebsiella pneumoniae. Bioinformatics analysis of KP2025 revealed a putative endolysin, LysKP213, containing a T4-like_lys domain. Purified LysKP213 was found to be highly thermostable, retaining approximately 44.4% of its lytic activity after 20 h of incubation at 95°C, and approximately 57.5% residual activity after 30 min at 121°C. Furthermore, when administered in combination with polymyxin B or fused at the N-terminus with the antimicrobial peptide cecropin A (CecA), LysKP213 exhibited increased antibacterial activity against Gram-negative pathogens, including K. pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli, both in vitro and in vivo. These results indicated that LysKP213 is a highly thermostable endolysin that, when combined with or fused with an outer membrane permeabilizer, has enhanced antibacterial activity and is a candidate agent for the control of infections by Gram-negative pathogens.

Members of the fungal genus Hypoxylon of the family Xylariaceae are known to produce secondary metabolites with significant chemical diversity. There are more than 200 species in the genus, including the filamentous fungus Hypoxylon fendleri. To the best of our knowledge, there have been no reports of mycoviruses in H. fendleri. In this study, a novel mycovirus, designated “Hypoxylon fendleri mitovirus 1” (HfMV1), was isolated from this fungus. The genome of HfMV1 is 2850 nt in length with a G + C content of 36% and contains a large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). BLASTp analysis revealed that the RdRp domain of HfMV1 had 28.30–50.90% sequence identity to those of members of the genus Duamitovirus and had the highest identity (50.90%) to Fusarium graminearum mitovirus 2-2 (FgMV2-2). Phylogenetic analysis further indicated that HfMV1 is a member of the genus Duamitovirus of the family Mitoviridae. This is the first report of a mycovirus in H. fendleri.

Talaromyces amestolkiae is an important fungal species owing to its ubiquity in soils, plants, air, and food. In this study, we identified a novel six-segmented polymycovirus, Talaromyces amestolkiae polymycovirus 1 (TaPmV-1). Each of the double-stranded (ds) RNA segments of TaPmV-1 contained a single open reading frame, and the proteins encoded by dsRNA1, dsRNA2, dsRNA3, and dsRNA 5 shared significant amino acid identities of 56, 40, 47, and 43%, respectively, with the corresponding proteins of Aspergillus fumigatus polymycovirus-1(AfuPmV-1). DsRNA1, dsRNA3, and dsRNA5 of TaPmV-1 encoded an RNA-dependent RNA polymerase (RdRp), a viral methyltransferase, and a PAS-rich protein, respectively. The functions of the proteins encoded by dsRNA2, dsRNA4, and dsRNA6 have not been elucidated. Comparison of the virus-infected strain LSH3 with virus-cured strain LSHVF revealed that infection with TaPmV-l may reduce the production of red pigments and induce the clustering of fungal sclerotia. Furthermore, transcriptomic analyses demonstrated that infection with TaPmV-l downregulated the expression of transcripts related to metabolism, and may correlate with the reduced production of red pigments and clustering of sclerotia in T. amestolkiae. These results of this study provide novel insights into the mechanism of fungal gene regulation by polymycovirus infections at the transcriptome level, and this study is the first to report a novel polymycovirus of T. amestolkiae.

The plant-pathogenic fungus Exobasidium gracile belongs to the basidiomycetous genus Exobasidium and causes leaf abnormalities in Camellia oleifera. Here, we report a novel mycovirus from E. gracile strain Z-2, called “Exobasidium gracile zybavirus 2” (EgZV2), which is related phylogenetically to a member of the genus Zybavirus in the family Amalgaviridae. Like those of other zybaviruses, the EgZV2 genome has two large open reading frames (ORFs) encoding an unknown protein and an RNA-dependent RNA polymerase (RdRp), respectively. In addition, we found a conserved +1 programmed ribosomal frameshifting (PRF) motif (UUUCGG) between ORF1 and ORF2 in EgZV2.

Viruses with dsRNA genomes are quite diverse and infect organisms in all three domains of life. Although dsRNA viruses that infect humans, plants, and fungi are quite common, dsRNA viruses that infect bacteria, known as bacteriophages, are quite understudied, and only seven dsRNA phages have been sequenced so far.

To our knowledge, there have been no reports of mycoviruses infecting Rhodosporidiobolus odoratus. Here, we describe the sequence of a novel mycovirus isolated from R. odoratus, which was designated "Rhodosporidiobolus odoratus RNA virus 1" (RoRV1). Sequence analysis revealed that RoRV1 has two discontinuous open reading frames (ORFs), ORF1 and ORF2, potentially encoding a hypothetical protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis based on RdRp sequences clearly placed RoRV1 in the genus Totivirus, family Totiviridae. The fungus also contains two additional, smaller dsRNAs, which might represent RoRV1 satellite RNAs.