ABSTRACT
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.
ABSTRACT
Cleavage factor I (CFI) proteins are core components of the polyadenylation machinery that can regulate several steps of mRNA life cycle, including alternative polyadenylation, splicing, export and decay. Here, we describe the regulatory mechanisms that control two fungal CFI protein classes in Magnaporthe oryzae: Rbp35/CfI25 complex and Hrp1. Using mutational, genetic and biochemical studies we demonstrate that cellular concentration of CFI mRNAs is a limited indicator of their protein abundance. Our results suggest that several post-transcriptional mechanisms regulate Rbp35/CfI25 complex and Hrp1 in the rice blast fungus, some of which are also conserved in other ascomycetes. With respect to Rbp35, these include C-terminal processing, RGG-dependent localization and cleavage, C-terminal autoregulatory domain and regulation by an upstream open reading frame of Rbp35-dependent TOR signalling pathway. Our proteomic analyses suggest that Rbp35 regulates the levels of proteins involved in melanin and phenylpropanoids synthesis, among others. The drastic reduction of fungal CFI proteins in carbon-starved cells suggests that the pre-mRNA processing pathway is altered. Our findings uncover broad and multilayer regulatory mechanisms controlling fungal polyadenylation factors, which have profound implications in pre-mRNA maturation. This area of research offers new avenues for fungicide design by targeting fungal-specific proteins that globally affect thousands of mRNAs.
Subject(s)
Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Polyadenylation , mRNA Cleavage and Polyadenylation Factors/metabolism , 5' Untranslated Regions , Fungal Proteins/chemistry , Fungal Proteins/genetics , Magnaporthe/genetics , Magnaporthe/growth & development , Magnaporthe/metabolism , Open Reading Frames , Plant Diseases/microbiology , Protein Structure, Tertiary , Proteolysis , TOR Serine-Threonine Kinases/metabolism , mRNA Cleavage and Polyadenylation Factors/chemistry , mRNA Cleavage and Polyadenylation Factors/geneticsABSTRACT
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