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.

Complexity and Local Specificity of the Virome Associated with Tospovirus-Transmitting Thrips Species.

Frankliniella occidentalis (western flower thrips [WFT]) and Thrips tabaci (onion thrips [OT]) are insect species that greatly impact horticultural crops through direct damage and their efficient vectoring of tomato spotted wilt virus and iris yellow spot virus. In this study, we collected thrips of these species from 12 field populations in various regions in Italy. We also included one field population of Neohydatothrips variabilis (soybean thrips [ST]) from the United States. Total RNA data from high-throughput sequencing (HTS) were used to assemble the virome, and then we assigned putative viral contigs to each thrips sample by real-time reverse transcription-quantitative PCR (qRT-PCR). Excluding plant and fungal viruses, we were able to identify 61 viral segments, corresponding to 41 viruses: 14 were assigned to WFT, 17 to OT, and 1 to ST; 9 viruses could not be assigned to any species based on our stringent criteria. All these viruses are putative representative of new species (with only the exception of a sobemo-like virus that is 100% identical to a virus recently characterized in ST) and some belong to new higher-ranking taxa. These additions to the viral phylogeny suggest previously undescribed evolutionary niches. Most of Baltimore's classes of RNA viruses were present (positive- and minus-strand and double-stranded RNA viruses), but only one DNA virus was identified in our collection. Repeated sampling in a subset of locations in 2019 and 2020 and further virus characterization in a subset of four thrips populations maintained in the laboratory allowed us to provide evidence of a locally persistent thrips core virome that characterizes each population. IMPORTANCE Harnessing the insect microbiome can result in new approaches to contain their populations or the damage they cause vectoring viruses of medical, veterinary, or agricultural importance. Persistent insect viruses are a neglected component of their microbiota. In this study, for the first time, we characterize the virome associated with the two model systems for tospovirus-transmitting thrips species, of utmost importance for the direct and indirect damage they cause to a number of different crops. The thrips virome characterized includes several novel viruses, which in some cases reveal previously undescribed clades. More importantly, some of the viruses we describe are part of a core virome that is specific and consistently present in distinct geographical locations monitored over the years, hinting at a possible mutualistic symbiotic relationship with their host.

Analysis of the virome associated to grapevine downy mildew lesions reveals new mycovirus lineages.

The obligatory biotrophic oomycetes Plasmopara viticola is the causal agent of downy mildew, a destructive disease of grapevine worldwide. So far, chemical fungicides are widely employed to limit this pathogen, but their adverse effects are stimulating the quest for environmentally friendly alternative approaches. Here, we report on the search for mycoviruses that might be later developed as biocontrol agents for this pathogen. Symptomatic leaves were collected from various regions in Spain and Italy and mycelia associated to leaf lesions was harvested. Total RNA extractions were depleted of rRNA and metatranscriptomes were generated using a high-throughput sequencing approach. The virome associated to leaf lesions was then characterized through a bioinformatic pipeline relying on blast searches against current viral databases. Here, we present an inventory of 283 new RNA viruses: 222 positive strand RNA viruses, 29 negative strand RNA viruses, 27 double-stranded RNA viruses and 5 ORFan virus RdRP segments, which could not be reliably assigned to any existing group in the Riboviria. In addition to ORFan viruses, we found other surprising new evolutionary trajectories in this wide inventory of viruses. The most represented viruses in our collection are those in phylum Lenarviricota, and, among them, a group of mycovirus segments distantly related to narnaviruses, but characterized by a polymerase palm domain lacking subdomain C, with the putative GDD catalytic triad. We also provided evidence of a strict association between two RNA segments that form a new mycovirus clade of positive strand RNA in the phylum Kitrinoviricota, order Martellivirales. In the phylum Negarnaviricota, we report for the first time in the order Mononegavirales a clade of viruses that is ambisense, a feature that so far was present only in the order Bunyavirales. Furthermore, in the same phylum we detected the widespread occurrence and abundant accumulation in our libraries of a distinct mycovirus clade distantly related to the Muvirales and Goujanvirales orders, which so far include only viruses infecting invertebrates. Possible new oomycetes-specific virus clades are also described in the phylum Duplornaviricota. These data greatly expand the evolutionary history of mycoviruses adding new layers of diversity to the realm Riboviria.

A complex virome including two distinct emaraviruses associated with virus-like symptoms in Camellia japonica.

Camellia japonica plants manifesting a complex and variable spectrum of viral symptoms like chlorotic ringspots, necrotic rings, yellowing with necrotic rings, yellow mottle, leaves and petals deformations, and flower color-breaking have been studied since 1940, mainly by electron microscopic analyses; however, a strong correlation between the symptoms and one or more well-characterized viruses was never verified. In this work, samples collected from symptomatic plants were analyzed using the next-generation sequencing technique, and a complex virome composed of members of the Betaflexiviridae and Fimoviridae families was identified. In particular, the genomic fragments typical of the emaravirus group were organized in the genomes of two new emaraviruses species, tentatively named Camellia japonica-associated emaravirus 1 and 2. They are the first emaraviruses described in camellia plants and found in symptomatic plants. At the same time, in both symptomatic and asymptomatic plants, five betaflexivirus isolates were detected that, based on amino acid sequence comparisons, can be considered two new isolates of the recently characterized camellia ringspot-associated virus 1 and 2 (CRSaV-1/2). These recently identified betaflexiviruses associated with C. japonica disease show an unusual hyper-conservation of the coat protein at the amino acid level. The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are MN385581, MN532567, MN532565, MN385582, MN532566, MN385573, MN385577, MN385574, MN385578, MN385575, MN385579, MN385576, MN385580, MN557024, MN557025, MN557026, MN557027, and MN557028.

The mycovirome of a fungal collection from the sea cucumber Holothuria polii.

Holothuria polii is a marine animal with an important ecological and economic impact. In the present study we analysed the presence of mycoviruses associated to fungi that were isolated from different H. polii tissues. Among the 48 fungal isolates analysed we identified 10 viruses in 8 strains belonging to 7 fungal species. Five out of nine viruses have a dsRNA genome: three of them belong to the Partitiviridae family, one belongs to a still undefined clade of bipartite viruses and the last one belongs to the Chrysoviridae family. We also identified two viruses belonging to a recently proposed new mycovirus taxon named polymycovirus. Two viruses belong to the positive single stranded RNA clade: one falls into the new Botourmiaviridae family, specifically in the Magoulivirus genus, and the other one falls into a still undefined clade phylogenetically related to tombusviruses. Finally, we also identified a virus with a negative stranded RNA genome showing similarity to a group of viruses recently proposed as a new family of mycoviruses in the order Bunyavirales. A bioinformatics approach comparing two datasets of contigs containing two closely related mycobunyaviruses allowed us to identify putative nucleocapsids (Nc) and non-structural (Ns) associated proteins. The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: PRJNA432529, MG913290, MG913291, MG887747, MG887748, MG887749, MG887750, MG887751, MG887752, MG887753, MG887754, MG887755, MG887756, MG887757, MG887758, MG887759, MG887760, MG887761, MG887762, MG887763, MG887764, MG887765, MG887766, MG887767, MH271211, MN163273, MN163274.

Transcriptome and proteome analysis reveal new insight into proximal and distal responses of wheat to foliar infection by Xanthomonas translucens.

The molecular details of local plant response against Xanthomonas translucens infection is largely unknown. Moreover, there is no knowledge about effects of the pathogen on the root's transcriptome and proteome. Therefore, we investigated the global gene and protein expression changes both in leaves and roots of wheat (Triticum aestivum) 24 h post leaf infection of X. translucens. This simultaneous analysis allowed us to obtain insight into possible metabolic rearrangements in above- and belowground tissues and to identify common responses as well as specific alterations. At the site of infection, we observed the implication of various components of the recognition, signaling, and amplification mechanisms in plant response to the pathogen. Moreover, data indicate a massive down-regulation of photosynthesis and confirm the chloroplast as crucial signaling hub during pathogen attack. Notably, roots responded as well to foliar attack and their response significantly differed from that locally triggered in infected leaves. Data indicate that roots as a site of energy production and synthesis of various secondary metabolites may actively influence the composition and colonisation level of root-associated microbes. Finally, our results emphasize the accumulation of jasmonic acid, pipecolic acid and/or the downstream mediator of hydrogen peroxide as long distal signals from infected leaves to roots.

Common and metal-specific proteomic responses to cadmium and zinc in the metal tolerant ericoid mycorrhizal fungus Oidiodendron maius Zn.

Although adaptive metal tolerance may arise in fungal populations in polluted soils, the mechanisms underlying metal-specific tolerance are poorly understood. Comparative proteomics is a powerful tool to identify variation in protein profiles caused by changing environmental conditions, and was used to investigate protein accumulation in a metal tolerant isolate of the ericoid mycorrhizal fungus Oidiodendron maius exposed to zinc and cadmium. Two-dimensional gel electrophoresis and shotgun proteomics followed by mass spectrometry lead to the identification of common and metal-specific proteins and pathways. Proteins selectively induced by cadmium exposure were molecular chaperons of the Hsp90 family, cytoskeletal proteins and components of the translation machinery. Zinc significantly up-regulated metabolic pathways related to energy production and carbohydrates metabolism, likely mirroring zinc adaptation of this fungal isolate. Common proteins induced by the two metal ions were the antioxidant enzyme Cu/Zn superoxide dismutase and ubiquitin. In mycelia exposed to zinc and cadmium, both proteomic techniques also identified agmatinase, an enzyme involved in polyamine biosynthesis. This novel finding suggests that, like plants, polyamines may have important functions in response to abiotic environmental stress in fungi. Genetic evidence also suggests that the biosynthesis of polyamines via an alternative metabolic pathway may be widespread in fungi.