Highlights• First report of mycoviruses isolated from fungi from marine environment.• Survey of mycoviruses using multiple approaches for both DNA and RNA genomes.• RNAseq analysis is superior to sRNA for de novo assembly of mycoviruses.• Twelve new virus species were characterized molecularly.• Expression of a viral RNA from an endogenized cDNA segment. AbstractThe number of reported mycoviruses is increasing exponentially due to the current ability to detect mycoviruses using next-generation sequencing (NGS) approaches, with a large number of viral genomes built in-silico using data from fungal transcriptome projects. We decided to screen a collection of fungi originating from a specific marine environment (associated with the seagrass Posidonia oceanica) for the presence of mycoviruses: our findings reveal a wealth of diversity among these symbionts and this complexity will require further studies to address their specific role in this ecological niche. In specific, we identified twelve new virus species belonging to nine distinct lineages: they are members of megabirnavirus, totivirus, chrysovirus, partitivirus and five still undefined clades. We showed evidence of an endogenized virus ORF, and evidence of accumulation of dsRNA from metaviridae retroviral elements. We applied different techniques for detecting the presence of mycoviruses including (i) dsRNA extraction and cDNA cloning, (ii) small and total RNA sequencing through NGS techniques, (iii) rolling circle amplification (RCA) and total DNA extraction analyses, (iv) virus purifications and electron microscopy. We tried also to critically evaluate the intrinsic value and limitations of each of these techniques. Based on the samples we could compare directly, RNAseq analysis is superior to sRNA for de novo assembly of mycoviruses. To our knowledge this is the first report on the virome of fungi isolated from marine environment.

All known pepper cultivars resistant to Tomato spotted wilt virus (TSWV) possess a single dominant resistance gene, Tsw. Recently, naturally occurring resistance-breaking (RB) TSWV strains have been identified, causing major concerns. We used a collection of such strains to identify the specific genetic determinant that allows the virus to overcome the Tsw gene in Capsicum spp. A reverse genetic approach is still not feasible for TSWV; therefore, we analyzed reassortants between wild-type (WT) and RB strains. Our results confirmed that the S RNA, which encodes both the nucleocapsid protein (N) and a nonstructural protein (NSs), carries the genetic determinant responsible for Tsw resistance breakdown. We then used full-length S RNA segments or the proteins they encode to compare the sequences of WT and related RB strains, and obtained indirect evidence that the NSs protein is the avirulence factor in question. Transient expression of NSs protein from WT and RB strains showed that they both can equally suppress post-transcriptional gene silencing (PTGS). Moreover, biological characterization of two RB strains carrying deletions in the NSs protein showed that NSs is important in maintaining TSWV infection in newly emerging leaves over time, preventing recovery. Analysis of another RB strain phenotype allowed us to conclude that local necrotic response is not sufficient for resistance in Capsicum spp. carrying the Tsw gene.

Flavescence dorée phytoplasma (FDp) infections seriously affect production and survival of grapevine. We analysed the changes in the flavonoid pathway occurring in two red cultivars, the highly susceptible Barbera and the less susceptible Nebbiolo, following FDp infection. A combination of metabolic and transcript analyses was used to quantify flavonoid compounds and expression of a set of genes involved in their biosynthesis. Quantification of anthocyanins, flavonols, proanthocyanidins and related biosynthetic enzymes was performed over the vegetative season, at four time points, on healthy, infected and recovered plants. A strong activation of anthocyanin accumulation was observed in infected Barbera leaves, while the response was less marked in Nebbiolo. Proanthocyanidins also accumulated mainly in infected Barbera leaves, even if basal proanthocyanidin concentration was higher in healthy and recovered Nebbiolo. Biochemical data were supported by transcript analysis: genes of the stem flavonoid pathway and of the anthocyanin and proanthocyanidin branches were expressed at a higher level in infected than in healthy plants, with a different magnitude between the two cultivars. Based on our results, we hypothesize that flavonoid accumulation is a physiological consequence of FD infection without affecting phytoplasma multiplication, although proanthocyanidin accumulation could help repel further infection by the insect vector.

Tomato spotted wilt virus (TSWV) is the type member of tospoviruses (genus Tospovirus), plant-infecting viruses that cause severe damage to ornamental and vegetable crops. Tospoviruses are transmitted by thrips in the circulative propagative mode. We generated a collection of NSs-defective TSWV isolates and showed that TSWV coding for truncated NSs protein could not be transmitted by Frankliniella occidentalis. Quantitative reverse transcription (RT)-PCR and immunostaining of individual insects detected the mutant virus in second-instar larvae and adult insects, demonstrating that insects could acquire and accumulate the NSs-defective virus. Nevertheless, adults carried a significantly lower viral load, resulting in the absence of transmission. Genome sequencing and analyses of reassortant isolates showed genetic evidence of the association between the loss of competence in transmission and the mutation in the NSs coding sequence. Our findings offer new insight into the TSWV-thrips interaction and Tospovirus pathogenesis and highlight, for the first time in the Bunyaviridae family, a major role for the S segment, and specifically for the NSs protein, in virulence and efficient infection in insect vector individuals. IMPORTANCEOur work is the first to show a role for the NSs protein in virus accumulation in the insect vector in the Bunyaviridae family: demonstration was obtained for the system TSWV-F. occidentalis, arguably one of the most damaging combination for vegetable crops. Genetic evidence of the involvement of the NSs protein in vector transmission was provided with multiple approaches.

Flavescence dorée is a serious phytoplasma disease affecting grapevine in several European countries. We studied the interaction of Flavescence dorée phytoplasma with its natural plant host by monitoring the effects of infection on the protein expression profile. Among the 576 analyzed spots, 33 proteins were differentially regulated in infected grapevines. Grouping into MIPS functional categories showed proteins involved in metabolism (21%), energy processes (9%), protein synthesis (3%), protein fate (18%), cellular transport and transport routes (6%), cell defense and virulence (42%). Among the differentially regulated proteins, we selected six targets (thaumatin I, thaumatin II, osmotin-like protein, plant basic secretory protein, AAA(+) Rubisco activase and proteasome α5 subunit) and we analyzed their expression by quantitative RT-PCR on samples collected in 2008 and 2009 in several vineyards in Piedmont region, Italy. There was a positive correlation between mRNA and protein expression for most of the genes in both the years. We discuss the involvement of these proteins in the specific response to phytoplasma infection. To our knowledge, this work is the first to investigate the response of the grapevine proteome to Flavescence dorée phytoplasma infection, and provides reference protein profiles for future comparative proteomic and genomic studies.