No abstract

High-throughput sequencing of small RNAs allowed the identification of a novel DNA virus in a Chinese mulberry tree affected by a disease showing mosaic and dwarfing symptoms. Rollingcircle amplification and PCR with specific primers, followed by sequencing of eleven independent full-length clones, showed that this virus has a monopartite circular DNA genome (,2.95 kb) containing ORFs in both polarity strands, as reported previously for geminiviruses. A field survey showed the close association of the virus with diseased mulberries, so we tentatively named the virus mulberry mosaic dwarf-associated virus (MMDaV). The MMDaV genome codes for five and two putative proteins in the virion-sense and in the complementarysense strands, respectively. Although three MMDaV virion-sense putative proteins did not share sequence homology with any protein in the databases, functional domains [coiled-coil and transmembrane (TM) domains] were identified in two of them. In addition, the protein containing a TM domain was encoded by an ORF located in a similar genomic position in MMDaV and in several other geminiviruses. As reported for members of the genera Mastrevirus and Becurtovirus, MMDaV replication-associated proteins are expressed through the alternative splicing of an intron, which was shown to be functional in vivo. A similar intron was found in the genome of citrus chlorotic dwarf-associated virus (CCDaV), a divergent geminivirus found recently in citrus. On the basis of pairwise comparisons and phylogenetic analyses, CCDaV and MMDaV appear to be closely related to each other, thus supporting their inclusion in a putative novel genus in the family Geminiviridae.

The velvet family proteins VosA and VelB are involved in growth regulation and differentiation in the model fungus Aspergillus nidulans and other filamentous fungi. In this study, the orthologs of VosA and VelB, AoVosA, and AoVelB, respectively, were characterized in the nematode-trapping fungus Arthrobotrys oligospora , which captures nematodes by producing trapping devices (traps). Deletion of the AovelB gene resulted in growth defects in different media, and the aerial hyphae from the Δ AovelB mutant lines were fewer in number and their colonies were less dense than those from the wild-type (WT) strain. The Δ AovelB mutants each displayed serious sporulation defects, and the transcripts of several sporulation-related genes (e.g., abaA , flbC , rodA , and vosA ) were significantly down-regulated compared to those from the WT strain. Furthermore, the Δ AovelB mutant strains became more sensitive to chemical reagents, including sodium dodecyl sulfate and H 2 O 2 . Importantly, the Δ AovelB mutants were unable to produce nematode-capturing traps. Similarly, extracellular proteolytic activity was also lower in the Δ AovelB mutants than in the WT strain. In contrast, the Δ AovosA mutants displayed no obvious differences from the WT strain in these phenotypic traits, whereas conidial germination was lower in the Δ AovosA mutants, which became more sensitive to heat shock stress. Our results demonstrate that the velvet protein AoVelB is essential for conidiation, trap formation, and pathogenicity in A. oligospora , while AoVosA plays a role in the regulation of conidial germination and heat shock stress.

Metagenomic studies have indicated that the diversity of plant viruses was until recently far underestimated. As important components of ecosystems, there is a need to explore the diversity and richness of the viruses associated with plant populations and to understand the drivers shaping their diversity in space and time. Two viral sequence enrichment approaches, double-stranded RNA (dsRNA) and virion-associated nucleic acids (VANA), have been used and compared here for the description of the virome of complex plant pools representative of the most prevalent plant species in unmanaged and cultivated ecosystems. A novel bioinformatics strategy was used to assess viral richness not only at the family level but also by determining operational taxonomic units (OTU) following the clustering of conserved viral domains. A large viral diversity dominated by novel dsRNA viruses was detected in all sites, while a large between-site variability limited the ability to draw a clear conclusion on the impact of cultivation. A trend for a higher diversity of dsRNA viruses was nevertheless detected in unmanaged sites (118 versus 77 unique OTUs). The dsRNA-based approach consistently revealed a broader and more comprehensive diversity for RNA viruses than the VANA approach, whatever the assessment criterion. In addition, dissimilarity analyses indicated both approaches to be largely reproducible but not necessarily convergent. These findings illustrate features of phytoviromes in various ecosystems and a novel strategy for precise virus richness estimation. These results allow us to reason methodological choices in phytovirome studies and likely in other virome studies where RNA viruses are the focal taxa. IMPORTANCE There are today significant knowledge gaps on phytovirus populations and on the drivers impacting them but also on the comparative performance-methodological approaches for their study. We used and compared two viral sequence enrichment approaches, double-stranded RNAs (dsRNA) and virion-associated nucleic acids (VANA), for phytovirome description in complex pools representative of the most prevalent plant species in unmanaged and cultivated ecosystems. Viral richness was assessed by determining operational taxonomic units (OTU) following the clustering of conserved viral domains. There is some limited evidence of an impact of cultivation on viral populations. These results provide data allowing us to reason the methodological choices in virome studies. For researchers primarily interested in RNA viruses, the dsRNA approach is recommended because it consistently provided a more comprehensive description of the analyzed phytoviromes, but it understandably underrepresented DNA viruses and bacteriophages.