First detection of mycoviruses in Gnomoniopsis castaneae suggests a putative horizontal gene transfer event between negative-sense and double-strand RNA viruses.

Gnomoniopsis castaneae is an ascomycetous fungus mainly known as a major pathogen of chestnut causing nut rots, although it is often found as an endophyte in chestnut tissues. To date, no virus has been reported as associated with to this fungus. Here, a collection of G. castaneae isolates from several European countries was screened to detect mycoviruses infecting the fungus: for the first time we report the identification and prevalence of mitovirus Gnomoniopsis castaneae mitovirus 1 (GcMV1) and the chrysovirus Gnomoniopsis castaneae chrysovirus 1 (GcCV1). Interestingly, we provide evidence supporting a putative horizontal gene transfer between members of the phyla Negarnaviricota and Duplornaviricota: a small putative protein of unknown function encoded on the RNA3 of GcCV1 (Chrysoviridae) has homologs in the genome of viruses of the family Mymonaviridae.

New clades of viruses infecting the obligatory biotroph Bremia lactucae representing distinct evolutionary trajectory for viruses infecting oomycetes.

Recent advances in high throughput sequencing (HTS) approaches allowed a broad exploration of viromes from different fungal hosts, unveiling a great diversity of mycoviruses with interesting evolutionary features. The word mycovirus historically applies also to viruses infecting oomycetes but most studies are on viruses infecting fungi, with less mycoviruses found and characterized in oomycetes, particularly in the obligatory biotrophs. We, here, describe the first virome associated to Bremia lactucae, the causal agent of lettuce downy mildew, which is an important biotrophic pathogen for lettuce production and a model system for the molecular aspects of the plant-oomycetes interactions. Among the identified viruses, we could detect (1) two new negative sense ssRNA viruses related to the yueviruses, (2) the first example of permuted RdRp in a virus infecting fungi/oomycetes, (3) a new group of bipartite dsRNA viruses showing evidence of recent bi-segmentation and concomitantly, a possible duplication event bringing a bipartite genome to tripartite, (4) a first representative of a clade of viruses with evidence of recombination between distantly related viruses, (5) a new open reading frame (ORF)an virus encoding for an RdRp with low homology to known RNA viruses, and (6) a new virus, belonging to riboviria but not conserved enough to provide a conclusive phylogenetic placement that shows evidence of a recombination event between a kitrinoviricota-like and a pisuviricota-like sequence. The results obtained show a great diversity of viruses and evolutionary mechanisms previously unreported for oomycetes-infecting viruses, supporting the existence of a large diversity of oomycetes-specific viral clades ancestral of many fungal and insect virus clades.

A collection of Trichoderma isolates from natural environments in Sardinia reveals a complex virome that includes negative-sense fungal viruses with unprecedented genome organizations.

Trichoderma genus includes soil-inhabiting fungi that provide important ecosystem services in their interaction with plants and other fungi, as well as biocontrol of fungal plant diseases. A collection of Trichoderma isolates from Sardinia has been previously characterized, but here we selected 113 isolates, representatives of the collection, and characterized their viral components. We carried out high-throughput sequencing of ribosome-depleted total RNA following a bioinformatics pipeline that detects virus-derived RNA-directed RNA polymerases (RdRps) and other conserved viral protein sequences. This pipeline detected seventeen viral RdRps with two of them corresponding to viruses already detected in other regions of the world and the remaining fifteen representing isolates of new putative virus species. Surprisingly, eight of them are from new negative-sense RNA viruses, a first in the genus Trichoderma. Among them is a cogu-like virus, closely related to plant-infecting viruses. Regarding the positive-sense viruses, we report the presence of an 'ormycovirus' belonging to a recently characterized group of bisegmented single-stranded RNA viruses with uncertain phylogenetic assignment. Finally, for the first time, we report a bisegmented member of Mononegavirales which infects fungi. The proteins encoded by the second genomic RNA of this virus were used to re-evaluate several viruses in the Penicillimonavirus and Plasmopamonavirus genera, here shown to be bisegmented and encoding a conserved polypeptide that has structural conservation with the nucleocapsid domain of rhabdoviruses.

Hybrids of RNA viruses and viroid-like elements replicate in fungi.

Earth's life may have originated as self-replicating RNA, and it has been argued that RNA viruses and viroid-like elements are remnants of such pre-cellular RNA world. RNA viruses are defined by linear RNA genomes encoding an RNA-dependent RNA polymerase (RdRp), whereas viroid-like elements consist of small, single-stranded, circular RNA genomes that, in some cases, encode paired self-cleaving ribozymes. Here we show that the number of candidate viroid-like elements occurring in geographically and ecologically diverse niches is much higher than previously thought. We report that, amongst these circular genomes, fungal ambiviruses are viroid-like elements that undergo rolling circle replication and encode their own viral RdRp. Thus, ambiviruses are distinct infectious RNAs showing hybrid features of viroid-like RNAs and viruses. We also detected similar circular RNAs, containing active ribozymes and encoding RdRps, related to mitochondrial-like fungal viruses, highlighting fungi as an evolutionary hub for RNA viruses and viroid-like elements. Our findings point to a deep co-evolutionary history between RNA viruses and subviral elements and offer new perspectives in the origin and evolution of primordial infectious agents, and RNA life.

Overexpression of the C4 protein of tomato yellow leaf curl Sardinia virus increases tomato resistance to powdery mildew.

Powdery mildew (PM) is one of the most important diseases of greenhouse and field-grown tomatoes. Viruses can intervene beneficially on plant performance in coping with biotic and abiotic stresses. Tomato yellow leaf curl Sardinia virus (TYLCSV) has been reported recently to induce tolerance against drought stress in tomato, and its C4 protein acts as the main causal factor of tolerance. However, its role in response to biotic stresses is still unknown. In this study, transgenic tomato plants carrying the TYLCSV C4 protein were exposed to biotic stress following the inoculation with Oidium neolycopersici, the causal agent of tomato PM. Phytopathological, anatomic, molecular, and physiological parameters were evaluated in this plant pathosystem. Heterologous TYLCSV C4 expression increased the tolerance of transgenic tomato plants to PM, not only reducing symptom occurrence, but also counteracting conidia adhesion and secondary hyphae elongation. Pathogenesis-related gene expression and salicylic acid production were found to be higher in tomato transgenic plants able to cope with PM compared to infected wild-type tomato plants. Our study contributes to unraveling the mechanism leading to PM tolerance in TYLCSV C4-expressing tomato plants. In a larger context, the findings of TYLCSV C4 as a novel PM defense inducer could have important implications in deepening the mechanisms regulating the management of this kind of protein to both biotic and abiotic stresses.

Group VII ethylene response factors, MtERF74 and MtERF75, sustain nitrogen fixation in Medicago truncatula microoxic nodules.

Group VII ethylene response factors (ERF-VII) are plant-specific transcription factors (TFs) known for their role in the activation of hypoxia-responsive genes under low oxygen stress but also in plant endogenous hypoxic niches. However, their function in the microaerophilic nitrogen-fixing nodules of legumes has not yet been investigated. We investigated regulation and the function of the two Medicago truncatula ERF-VII TFs (MtERF74 and MtERF75) in roots and nodules, MtERF74 and MtERF75 in response to hypoxia stress and during the nodulation process using an RNA interference strategy and targeted proteolysis of MtERF75. Knockdown of MtERF74 and MtERF75 partially blocked the induction of hypoxia-responsive genes in roots exposed to hypoxia stress. In addition, a significant reduction in nodulation capacity and nitrogen fixation activity was observed in mature nodules of double knockdown transgenic roots. Overall, the results indicate that MtERF74 and MtERF75 are involved in the induction of MtNR1 and Pgb1.1 expression for efficient Phytogb-nitric oxide respiration in the nodule.

Three new clades of putative viral RNA-dependent RNA polymerases with rare or unique catalytic triads discovered in libraries of ORFans from powdery mildews and the yeast of oenological interest Starmerella bacillaris.

High throughput sequencing allowed the discovery of many new viruses and viral organizations increasing our comprehension of virus origin and evolution. Most RNA viruses are currently characterized through similarity searches of annotated virus databases. This approach limits the possibility to detect completely new virus-encoded proteins with no detectable similarities to existing ones, i.e. ORFan proteins. A strong indication of the ORFan viral origin in a metatranscriptome is the lack of DNA corresponding to an assembled RNA sequence in the biological sample. Furthermore, sequence homology among ORFans and evidence of co-occurrence of these ORFans in specific host individuals provides further indication of a viral origin. Here, we use this theoretical framework to report the finding of three conserved clades of protein-coding RNA segments without a corresponding DNA in fungi. Protein sequence and structural alignment suggest these proteins are distantly related to viral RNA-dependent RNA polymerases (RdRP). In these new putative viral RdRP clades, no GDD catalytic triad is present, but the most common putative catalytic triad is NDD and a clade with GDQ, a triad previously unreported at that site. SDD, HDD, and ADD are also represented. For most members of these three clades, we were able to associate a second genomic segment, coding for a protein of unknown function. We provisionally named this new group of viruses ormycovirus. Interestingly, all the members of one of these sub-clades (gammaormycovirus) accumulate more minus sense RNA than plus sense RNA during infection.

A structural homologue of the plant receptor D14 mediates responses to strigolactones in the fungal phytopathogen Cryphonectria parasitica.

Strigolactones (SLs) are plant hormones and important signalling molecules required to promote arbuscular mycorrhizal (AM) symbiosis. While in plants an α/ß-hydrolase, DWARF14 (D14), was shown to act as a receptor that binds and cleaves SLs, the fungal receptor for SLs is unknown. Since AM fungi are currently not genetically tractable, in this study, we used the fungal pathogen Cryphonectria parasitica, for which gene deletion protocols exist, as a model, as we have previously shown that it responds to SLs. By means of computational, biochemical and genetic analyses, we identified a D14 structural homologue, CpD14. Molecular homology modelling and docking support the prediction that CpD14 interacts with and hydrolyses SLs. The recombinant CpD14 protein shows α/ß hydrolytic activity in vitro against the SLs synthetic analogue GR24; its enzymatic activity requires an intact Ser/His/Asp catalytic triad. CpD14 expression in the d14-1 loss-of-function Arabidopsis thaliana line did not rescue the plant mutant phenotype. However, gene inactivation by knockout homologous recombination reduced fungal sensitivity to SLs. These results indicate that CpD14 is involved in SLs responses in C. parasitica and strengthen the role of SLs as multifunctional molecules acting in plant-microbe interactions.

Aspergillus Goes Viral: Ecological Insights from the Geographical Distribution of the Mycovirome within an Aspergillus flavus Population and Its Possible Correlation with Aflatoxin Biosynthesis.

Microbial multi-level interactions are essential to control the success of spreading and survival of most microbes in natural environments. Phytopathogenic mycotoxigenic fungal species, such as Aspergillus flavus, represent an important issue in food safety. Usually, non-toxigenic strains are exploited for biocontrol strategies to mitigate infections by toxigenic strains. To comprehend all the biological variables involved in the aflatoxin biosynthesis, and to possibly evaluate the interplay between A. flavus toxigenic and non-toxigenic strains during intraspecific biocompetition, the "virological" perspective should be considered. For these reasons, investigations on mycoviruses associated to A. flavus populations inhabiting specific agroecosystems are highly desirable. Here, we provide the first accurate characterization of the novel mycovirome identified within an A. flavus wild population colonizing the maize fields of northern Italy: a selection of A. flavus strains was biologically characterized and subjected to RNAseq analysis, revealing new mycoviruses and a peculiar geographic pattern distribution in addition to a 20% rate of infection. More interestingly, a negative correlation between viral infection and aflatoxin production was found. Results significantly expanded the limited existent data about mycoviruses in wild A. flavus, opening new and intriguing hypotheses about the ecological significance of mycoviruses.

Identification and Molecular Characterization of Novel Mycoviruses in Saccharomyces and Non-Saccharomyces Yeasts of Oenological Interest.

Wine yeasts can be natural hosts for dsRNA, ssRNA viruses and retrotransposon elements. In this study, high-throughput RNA sequencing combined with bioinformatic analyses unveiled the virome associated to 16 Saccharomyces cerevisiae and 8 non-Saccharomyces strains of oenological interest. Results showed the presence of six viruses and two satellite dsRNAs from four different families, two of which-Partitiviridae and Mitoviridae-were not reported before in yeasts, as well as two ORFan contigs of viral origin. According to phylogenetic analysis, four new putative mycoviruses distributed in Totivirus, Cryspovirus, and Mitovirus genera were identified. The majority of commercial S. cerevisiae strains were confirmed to be the host for helper L-A type totiviruses and satellite M dsRNAs associated with the killer phenotype, both in single and mixed infections with L-BC totiviruses, and two viral sequences belonging to a new cryspovirus putative species discovered here for the first time. Moreover, single infection by a narnavirus 20S-related sequence was also found in one S. cerevisiae strain. Considering the non-Saccharomyces yeasts, Starmerella bacillaris hosted four RNAs of viral origin-two clustering in Totivirus and Mitovirus genera, and two ORFans with putative satellite behavior. This study confirmed the infection of wine yeasts by viruses associated with useful technological characteristics and demonstrated the presence of complex mixed infections with unpredictable biological effects.

The virome from a collection of endomycorrhizal fungi reveals new viral taxa with unprecedented genome organization.

Mutualistic plant-associated fungi are recognized as important drivers in plant evolution, diversity, and health. The discovery that mycoviruses can take part and play important roles in symbiotic tripartite interactions has prompted us to study the viromes associated with a collection of ericoid and orchid mycorrhizal (ERM and ORM, respectively) fungi. Our study, based on high-throughput sequencing of transcriptomes (RNAseq) from fungal isolates grown in axenic cultures, revealed in both ERM and ORM fungi the presence of new mycoviruses closely related to already classified virus taxa, but also new viruses that expand the boundaries of characterized RNA virus diversity to previously undescribed evolutionary trajectories. In ERM fungi, we provide first evidence of a bipartite virus, distantly related to narnaviruses, that splits the RNA-dependent RNA polymerase (RdRP) palm domain into two distinct proteins, encoded by each of the two segments. Furthermore, in one isolate of the ORM fungus Tulasnella spp. we detected a 12 kb genomic fragment coding for an RdRP with features of bunyavirus-like RdRPs. However, this 12 kb genomic RNA has the unique features, for Bunyavirales members, of being tri-cistronic and carrying ORFs for the putative RdRP and putative nucleocapsid in ambisense orientation on the same genomic RNA. Finally, a number of ORM fungal isolates harbored a group of ambisense bicistronic viruses with a genomic size of around 5 kb, where we could identify a putative RdRP palm domain that has some features of plus strand RNA viruses; these new viruses may represent a new lineage in the Riboviria, as they could not be reliably assigned to any of the branches in the recently derived monophyletic tree that includes most viruses with an RNA genome.

Extreme Diversity of Mycoviruses Present in Isolates of Rhizoctonia solani AG2-2 LP From Zoysia japonica From Brazil.

Zoysia japonica, in Brazil, is commonly infected by Rhizoctonia solani (R. solani) in humid and cool weather conditions. Eight isolates of R. solani, previously identified as belonging to the AG2-2 LP anastomosis group, isolated from samples from large path symptoms, were collected from three counties in São Paulo state (Brazil) and investigated for the presence of mycoviruses. After detection of double-strand RNA (dsRNA) in all samples, RNA_Seq analysis of ribosomal RNA-depleted total RNA from in vitro cultivated mycelia was performed. Forty-seven partial or complete viral unique RNA dependent-RNA polymerase (RdRp) sequences were obtained with a high prevalence of positive sense ssRNA viruses. Sequences were sufficiently different from the first match in BLAST searches suggesting that they all qualify as possible new viral species, except for one sequence showing an almost complete match with Rhizoctonia solani dsRNA virus 2, an alphapartitivirus. Surprisingly four large contigs of putative viral RNA could not be assigned to any existing clade of viruses present in the databases, but no DNA was detected corresponding to these fragments confirming their viral replicative nature. This is the first report on the occurrence of mycoviruses in R. solani AG2-2 LP in South America.