Small-RNA analysis of pre-basic mother plants and conserved accessions of plant genetic resources for the presence of viruses.

Pathogen-free stocks of vegetatively propagated plants are crucial in certified plant production. They require regular monitoring of the plant germplasm for pathogens, especially of the stocks maintained in the field. Here we tested pre-basic mother plants of Fragaria, Rubus and Ribes spp., and conserved accessions of the plant genetic resources of Rubus spp. maintained at research stations in Finland, for the presence of viruses using small interfering RNA (siRNA) -based diagnostics (VirusDetect). The advance of the method is that unrelated viruses can be detected simultaneously without resumptions of the viruses present. While no virus was detected in pre-basic mother plants of Fragaria and Ribes species, rubus yellow net virus (RYNV) was detected in pre-basic mother plants of Rubus. Raspberry bushy dwarf virus (RBDV), black raspberry necrosis virus (BRNV), raspberry vein chlorosis virus (RVCV) and RYNV were detected in the Rubus genetic resource collection. The L polymerase encoding sequence characterized from seven RVCV isolates showed considerable genetic variation. The data provide the first molecular biological evidence for the presence of RYNV in Finland. RYNV was not revealed in virus indexing by indicator plants, which suggests that it may be endogenously present in some raspberry cultivars. In addition, a putative new RYNV-like badnavirus was detected in Rubus spp. Blackcurrant reversion virus (BRV) and gooseberry vein banding associated virus (GVBaV) were detected in symptomatic Ribes plants grown in the field. Results were consistent with those obtained using PCR or reverse transcription PCR and suggest that the current virus indexing methods of pre-basic mother plants work as expected. Furthermore, many new viruses were identified in the collections of plant genetic resources not previously tested for viruses. In the future, siRNA-based diagnostics could be a useful supplement for the currently used virus detection methods in certified plant production and thus rationalize and simplify the current testing system.

Viral RNase3 Co-Localizes and Interacts with the Antiviral Defense Protein SGS3 in Plant Cells.

Sweet potato chlorotic stunt virus (SPCSV; family Closteroviridae) encodes a Class 1 RNase III endoribonuclease (RNase3) that suppresses post-transcriptional RNA interference (RNAi) and eliminates antiviral defense in sweetpotato plants (Ipomoea batatas). For RNAi suppression, RNase3 cleaves double-stranded small interfering RNAs (ds-siRNA) and long dsRNA to fragments that are too short to be utilized in RNAi. However, RNase3 can suppress only RNAi induced by sense RNA. Sense-mediated RNAi involves host suppressor of gene silencing 3 (SGS3) and RNA-dependent RNA polymerase 6 (RDR6). In this study, subcellular localization and host interactions of RNase3 were studied in plant cells. RNase3 was found to interact with SGS3 of sweetpotato and Arabidopsis thaliana when expressed in leaves, and it localized to SGS3/RDR6 bodies in the cytoplasm of leaf cells and protoplasts. RNase3 was also detected in the nucleus. Co-expression of RNase3 and SGS3 in leaf tissue enhanced the suppression of RNAi, as compared with expression of RNase3 alone. These results suggest additional mechanisms needed for efficient RNase3-mediated suppression of RNAi and provide new information about the subcellular context and phase of the RNAi pathway in which RNase3 realizes RNAi suppression.

Diagnosis and discovery of fungal viruses using deep sequencing of small RNAs.

Analysis of virus-derived small RNAs with high-throughput sequencing has been successful for detecting novel viruses in plants and invertebrates. However, the applicability of this method has not been demonstrated in fungi, although fungi were among the first organisms reported to utilize RNA silencing. Here, we used virus-infected isolates of the fungal species complex Heterobasidion annosum sensu lato as a model system to test whether mycovirus genome segments can be detected with small RNA deep sequencing. Species of the genus Heterobasidion are some of the most devastating forest pathogens in boreal forests. These fungi cause wood decay and are commonly infected with species of the family Partitiviridae and the unassigned virus species Heterobasidion RNA virus 6. Small RNA deep sequencing allowed the simultaneous detection of all eight double-stranded RNA virus strains known to be present in the tested samples and one putative mitovirus species (family Narnaviridae) with a single-stranded RNA genome, designated here as Heterobasidion mitovirus 1. Prior to this study, no members of the family Narnaviridae had been described as infecting species of Heterobasidion. Quantification of viral double- and single-stranded RNA with quantitative PCR indicated that co-infecting viral species and viruses with segmented genomes can be detected with small RNA deep sequencing despite vast differences in the amount of RNA. This is the first study demonstrating the usefulness of this method for detecting fungal viruses. Moreover, the results suggest that viral genomes are processed into small RNAs by different species of Heterobasidion.

Silencing suppressor protein VPg of a potyvirus interacts with the plant silencing-related protein SGS3.

Viral genome-linked protein (VPg) of potyviruses is involved in multiple steps of the potyvirus infection cycle, including viral multiplication and movement in plants. Recently, we showed that VPg of Potato virus A (PVA; genus Potyvirus) suppresses sense-mediated RNA silencing, which is linked to one or both nuclear or nucleolar localization. Here, we studied interactions between VPg and components of the plant RNA silencing pathway. Results showed that VPg interacts with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana, as shown by yeast two-hybrid analysis and bimolecular fluorescence complementation assays. VPg-SGS3 interactions co-localized with small cytoplasmic bodies that contained plant RNA-dependent RNA polymerase 6 (RDR6) (likely SGS3/RDR6 bodies). The N-terminal zinc finger (ZF) domain of SGS3 was the main determinant of the VPg interaction. Our data also suggest that the ZF domain controls SGS3 localization. SGS3 homodimerization was controlled by multiple protein regions. The VPg-SGS3 interaction appeared beneficial for PVA, as viral RNA levels correlated positively with sgs3 mRNA levels in the SGS3-silenced and SGS3-overexpressing leaves of Nicotiana benthamiana. The data support the idea that VPg acts as a suppressor of RNA silencing and suggest that an interaction with SGS3 may be important, especially in suppression of sense-mediated RNA silencing.