Massimo Turina

Evidence That the Nonstructural Protein of Tomato spotted wilt virus Is the Avirulence Determinant in the Interaction with Resistant Pepper Carrying the Tsw Gene

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.

Molecular characterization of the plant virus genus Ourmiavirus and evidence of inter-kingdom reassortment of viral genome segments as its possible route of origin

Ourmia melon virus (OuMV), Epirus cherry virus (EpCV) and Cassava virus C (CsVC) are three species placed in the genus Ourmiavirus. We cloned and sequenced their RNA genomes. The sizes of the three genomic RNAs of OuMV, the type member of the genus, were 2814, 1064 and 974 nt and each had one open reading frame. RNA1 potentially encoded a 97.5 kDa protein carrying the GDD motif typical of RNA-dependent RNA polymerases (RdRps). The putative RdRps of ourmiaviruses are distantly related to known viral RdRps, with the closest similarity and phylogenetic affinity observed with fungal viruses of the genus Narnaviridae. RNA2 encoded a 31.6 kDa protein which, expressed in bacteria as a His-tag fusion protein and in plants through agroinfiltration, reacted specifically with antibodies made against tubular structures found in the cytoplasm. The ORF2 product is significantly similar to movement proteins of the genus Tombusviridae, and phylogenetic analysis supported this evolutionary relationship. The product of OuMV ORF3 is a 23.8 kDa protein. This protein was also expressed in bacteria and plants, and reacted specifically with antisera against the OuMV coat protein. The sequence of the ORF3 protein showed limited but significant similarity to capsid proteins of several plant and animal viruses, although phylogenetic analysis failed to reveal its most likely origin. Taken together, these results indicate that ourmiaviruses comprise a unique group of plant viruses that might have evolved by reassortment of genomic segments of RNA viruses infecting hosts belonging to different eukaryotic kingdoms, in particular, fungi and plants.

Tospoviruses in the Mediterranean Area

Tospoviruses are among the most serious threats to vegetable crops in the Mediterranean basin. Tospovirus introduction, spread, and the diseases these viruses cause have been traced by epidemiological case studies. Recent research has centered on the close relationship between tospoviruses and their arthropod vectors (species of the Thripidae family). Here, we review several specific features of tospovirus-thrips associations in the Mediterranean. Since the introduction of Frankliniella occidentalis in Europe, Tomato spotted wilt virus (TSWV) has become one of the limiting factors for vegetable crops such as tomato, pepper, and lettuce. An increasing problem is the emergence of TSWV resistance-breaking strains that overcome the resistance genes in pepper and tomato. F. occidentalis is also a vector of Impatiens necrotic spot virus, which was first observed in the Mediterranean basin in the 1980s. Its importance as a cause of vegetable crop diseases is limited to occasional incidence in pepper and tomato fields. A recent introduction is Iris yellow spot virus, transmitted by the onion thrips Thrips tabaci, in onion and leek crops. Control measures in vegetable crops specific to Mediterranean conditions were examined in the context of their epidemiological features and tospovirus species which could pose a future potential risk for vegetable crops in the Mediterranean were discussed.

Role of the Mf1-1 pheromone precursor gene of the filamentous ascomycete Cryphonectria parasitica

Site-directed recombination was used to obtain a Cryphonectria parasitica strain carrying deletions at the Mf1-1 gene locus. Macroscopic features such as growth rate and conidia production were unaffected by Mf1-1 deletions, but, when a strain containing a complete deletion of Mf1-1 was used as spermatia it was male sterile. The same strain was fully competent as a female parent. Deletion of three of the seven putative pheromone peptide repeats within the gene had no effect on mating. Male fertility of the complete deletion strain was restored when an ectopic copy of the Mf1-1 gene was returned by transformation. Expression of the mating type specific pheromone precursor gene Mf1-1 was stimulated by growth in nutritionally poor liquid media. It was found that age and source of inoculum of liquid cultures influences pheromone precusor gene expression, i.e., conidia did not express Mf1-1 and cultures derived from conidia were significantly delayed in expression of this gene, as were cultures derived from young mycelium. Cultures inoculated with older hyphae, however, expressed Mf1-1 within 1 day after inoculation.

A Hydrophobin of the Chestnut Blight Fungus, Cryphonectria parasitica, Is Required for Stromal Pustule Eruption

ABSTRACT Hydrophobins are abundant small hydrophobic proteins that are present on the surfaces of many filamentous fungi. The chestnut blight pathogen Cryphonectria parasitica was shown to produce a class II hydrophobin, cryparin. Cryparin is the most abundant protein produced by this fungus when grown in liquid culture. When the fungus is growing on chestnut trees, cryparin is found only in the fungal fruiting body walls. Deletion of the gene encoding cryparin resulted in a culture phenotype typical of hydrophobin deletion mutants of other fungi, i.e., easily wettable (nonhydrophobic) hyphae. When grown on the natural substrate of the fungus, however, cryparin-null mutation strains were unable to normally produce its fungal fruiting bodies. Although the stromal pustules showed normal development initially, they were unable to erupt through the bark of the tree. The hydrophobin cryparin thus plays an essential role in the fitness of this important plant pathogen by facilitating the eruption of the fungal fruiting bodies through the bark of its host tree.

A severe disease of tomato in the culiacan area (Sinaloa, Mexico) is caused by a new picorna-like viral species

We were able to mechanically transmit a small isometric virus from field tomato samples showing severe necrotic symptoms, collected in the Culiacan area of Sinaloa state (Mexico). After gradient purification and three rounds of single-lesion passage on Chenopodium quinoa, the virus was back-inoculated to tomato plants and reproduced the original apical necrosis symptoms. The virus could be transmitted to a wide range of experimental hosts, including a number of solanaceous plants. Purified virus was used to produce specific polyclonal rabbit antibodies and serological tests such as enzyme-linked immunosorbent assay, Western blot analysis, and an immunochromatographic lateral flow assay. Such assays confirmed the wide distribution of this virus in symptomatic field plants in the area of the epidemic. Purified particles contained two genomic RNA molecules of ca. 7 kb (RNA1) and 5 kb (RNA2) estimated length. Analysis of clones from a cDNA library provided 6.5 and 3.0 kb of sequence for RNA1 and RNA2, respectively. Sequence analysis of the encoded replicase showed greatest similarity with members of the Sequiviridae family, and indicated that the virus we isolated is a new virus species, provisionally named Tomato apex necrosis virus.

The NSs Protein of Tomato spotted wilt virus Is Required for Persistent Infection and Transmission by Frankliniella occidentalis

ABSTRACT 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. IMPORTANCE Our 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.

Multiple approaches for the detection and characterization of viral and plasmid symbionts from a collection of marine fungi

The 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. The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: KT601099-KT601110; KT601114-KT601120; KT592305; KT950836-KT950841.

Mycovirus Cryphonectria Hypovirus 1 Elements Cofractionate with trans-Golgi Network Membranes of the Fungal Host Cryphonectria parasitica

ABSTRACT The mycovirus cryphonectria hypovirus 1 (CHV1) causes proliferation of vesicles in its host, Cryphonectria parasitica, the causal agent of chestnut blight. These vesicles have previously been shown to contain both CHV1 genomic double-stranded RNA (dsRNA) and RNA polymerase activity. To determine the cellular origins of these virus-induced membrane structures, we compared the fractionation of several cellular and viral markers. Results showed that viral dsRNA, helicase, polymerase, and protease p29 copurify with C. parasitica trans-Golgi network (TGN) markers, suggesting that the virus utilizes the fungal TGN for replication. We also show that the CHV1 protease p29 associates with vesicle membranes and is resistant to treatments that would release peripheral membrane proteins. Thus, p29 behaves as an integral membrane protein of the vesicular fraction derived from the fungal TGN. Protease p29 was also found to be fully susceptible to proteolytic digestion in the absence of detergent and, thus, is wholly or predominantly on the cytoplasmic face of the vesicles. Fractionation analysis of p29 deletion variants showed that sequences in the C terminal of p29 mediate membrane association. In particular, the C-terminal portion of the protein (Met-135-Gly-248) is sufficient for membrane association and is enough to direct p29 to the TGN vesicles in the absence of other viral elements.

A newly identified role for Tomato bushy stunt virus P19 in short distance spread

SUMMARY This study identified a role for the Tomato bushy stunt virus (TBSV) p19 protein (P19) in local lesion expansion on cowpea (Vigna unguiculata), and cell-to-cell movement in pepper (Capsicum annuum). The contribution to short distance spread in both hosts was strongly influenced by a cluster of charged amino acids between positions 72 and 78 on the 172 amino acid P19. Charged amino acids near this region between positions 43 and 85 were required for long distance spread in pepper. These results indicate that the central domain of P19 plays a key role for its activities in TBSV movement and that additional regions on this protein contribute to virus spread in a host-specific manner.