Protein interactions between RNA silencing maintaining proteins and viral silencing suppressors

RNA-hiljennys on sekvenssispesifinen RNA:n hajotusmenetelmä, jota kasvit käyttävät geeniekspression säätelyyn ja puolustukseen virustartuntoja vastaan. Virukset ovat kehittäneet RNA-hiljennystä vastaan RNA-hiljennyksenestäjiä, jotka voivat estää ja häiritä hiljennysreaktion kulkua monin tavoin. Virusproteiinit voivat esimerkiksi sitoutua hiljennysreaktion keskeisiä vaiheita ylläpitäviin proteiineihin tai hiljennyksen signaloinnista vastaaviin molekyyleihin. Tässä työssä tutkittiin crini- ja potyvirusten tunnettujen RNA-hiljennyksenestäjäproteiinien vuorovaikutuksia neljän RNA-hiljennystä ylläpitävän kasviproteiinin kanssa. Proteiinien vuorovaikutuksia testattiin hiivan kaksihybridi-menetelmällä (YTHS) ja kahden molekyylin välistä fluoresenssikomplementaatiota (BiFC) käyttäen. Jälkimmäisen menetelmän avulla voidaan tutkittua proteiinivuorovaikutusta havainnoida soluympäristössä. Hiivavektoreihin kloonattujen geenien proteiinintuotto hiivasoluissa tarkastettiin western blot -menetelmällä. BiFC-menetelmässä keskityttiin pääosin hiivan kaksihybridi-menetelmällä havaittujen proteiinien välisten vuorovaikutusten tarkasteluun. Tutkimuksessa havaittiin kolme aikaisemmin tuntematonta proteiinien välistä vuorovaikutusta. Tämän lisäksi tutkittujen virusproteiinien todettiin ensimmäistä kertaa sitoutuvan hiljennystä ylläpitäviin kasviproteiineihin. Vastaavien kasviproteiinien on aikaisemmin todettu olevan muiden hiljennyksenestäjien kohteena. Koska kyseisten hiljennystä ylläpitävien proteiinien toiminnot tiedetään varsin tarkasti, voidaan kolmen havaitun proteiinivuorovaikutuksen ajatella häiritsevän RNA-hiljennystä kyseisten kasviproteiinien toimintaa estämällä.RNA silencing is a sequence specific RNA degradation mechanism which is used by plants to regulate gene expression and to combat virus infections. However, viruses have developed so called silencing suppressors, which can prevent and interfere silencing reaction by many ways. For example, virus proteins can bind to maintaining proteins of the silencing reaction or to molecules which are responsible for signaling of the silencing reaction. This thesis focused on the study of protein-protein-interactions between known silencing suppressors of crini- and potyviruses and four maintaining plant proteins of RNA silencing. Protein-protein-interactions were studied using the yeast two-hybrid system (YTHS) and the bimolecular fluorescence complementation assay (BiFC). The latter method enables visualization of the studied protein interactions in plant cells. Protein expression of the cloned genes in yeast vectors were studied by using western blot. BiFC analysis was focused on protein interactions which were found by YTHS. This study detected three previously unknown protein interactions. Two virus proteins were found for the first time to bind directly to silencing maintaining proteins that are known to be targets of other silencing suppressors. Because the functions of these silencing maintaining proteins are known, it is possible that the three interactions described in this study interfere RNA silencing by impeding the functions of the plant proteins

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.Peer reviewe

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.Peer reviewe

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

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Co-expression of RNase3 and IbSGS3 enhances suppression of sense-mediated gene silencing (gene co-suppression).

<p>(a) and (b) Four sectors (1 to 4) of a leaf of <i>N</i>. <i>benthamiana</i> 16c constitutively expressing <i>gfp</i> were agroinfiltrated to co-express GFP and (1) GUS (negative control), (2) IbSGS3 and RNase3, (3) RNase3, or (4) IbSGS3. If RNase3 was not used, the corresponding <i>Agrobacterium</i> strain was replaced with a strain expressing β-glucuronidase (GUS, negative control). IbSGS3 was expressed with the N-proximal part of YFP fused to the C-terminus. If it was not used, it was replaced with an <i>Agrobacterium</i> strain expressing YN (N-proximal half of <i>yfp</i>) to maintain similar sense-mediated silencing pressure. The treatments are positioned differently in the two leaves in terms of the younger (basal) and older (tip) part of the leaf. Silencing of <i>gfp</i> was observed by the disappearance of GFP fluorescence (sectors 1 and 4), whereas GFP fluorescence above the background level indicated suppression of <i>gfp</i> silencing (sectors 2 and 3). The leaf was photographed under UV light at 6 dpi. Similar results were obtained in five independent experiments. (c) Northern analysis of <i>gfp</i> mRNA and <i>gfp</i> mRNA-derived siRNA in the agroinfiltrated leaf tissues. Co-expression of GUS or IbSGS3 with GFP by agroinfiltration in <i>gfp</i>-transgenic leaves resulted in <i>gfp</i> silencing, as shown by the readily detectable accumulation of <i>gfp</i>-derived siRNA (Fig 5C). In contrast, co-expression of GFP and RNase3 resulted only in low accumulation of <i>gfp</i> siRNA, and no <i>gfp</i> siRNA could be detected following co-expression of GFP, RNase3 and SGS3; however, accumulation of <i>gfp</i> mRNA was enhanced (Fig 5C). Co-expression of the RNase3-Ala mutant (disabled from catalytic activity on dsRNA) with GFP resulted in readily detectable accumulation of <i>gfp</i> siRNA, whereas co-expression of RNase3-Ala, SGS3 and GFP resulted in low accumulation of <i>gfp</i> siRNA (Fig 5C). 25S and 5S ribosomal RNA is shown as a loading control, respectively. (d) Western analysis of RNase3 and IbSGS3-YN in the agroinfiltrated leaf sectors illustrated in (a) by immunoblotting using anti-RNase3 and anti-GFP antibodies, respectively. Molecular masses of the detected proteins (kDa) were estimated by comparison with the protein marker run in the gel.</p

Co-localization of RNase3 and RDR6 in epidermal cells of <i>N</i>. <i>benthamiana</i> following co-expression by agroinfiltration.

<p>The red signals of RNase3-dsRED and green signals of (a) AtRDR6-GFP (<i>A</i>. <i>thaliana</i>) and (b) NtRDR6-GFP (<i>N</i>. <i>tabacum</i>) co-localized in cytoplasmic, punctate bodies detected by confocal microscopy at 2 dpi. Images in (a) and (b) illustrate optical planes in which many RDR6-containing bodies were observed. Scale bars, 10 μm.</p

Interactions of RNase3 and AtSGS3, as assessed with BiFC and confocal microscopy, co-localize with cytoplasmic punctate bodies containing AtRDR6 in epidermal cells of <i>N</i>. <i>benthamiana</i>.

<p>The three proteins were co-expressed by agroinfiltration. Images were acquired at an optical plane in which many RDR6-containing bodies were observed at 2 dpi. The dashed circles point out some of the cytoplasmic punctate bodies that show signals of the RNase3-SGS3 interaction and co-localize with RDR6. Scale bar, 10 μm.</p

Subcellular localization of RNase3 in epidermal cells of <i>Nicotiana benthamiana</i> following expression by agroinfiltration.

<p>(a) RNase3-dsRED (red signals) was detected in cytoplasmic punctate bodies by confocal microscopy at 2 dpi. A few of the many bodies are pointed out with arrowheads. It was also present in the nucleus and subnuclear bodies (arrow). (b) Fibrillarin of <i>A</i>. <i>thaliana</i> (Fib2) was expressed as a fusion with GFP (green) and used as a marker for nucleolus (N) and Cajal bodies (C). (c) Merged image revealed localization of RNase3 in subnuclear bodies (arrow) other than nucleolus or Cajal bodies. Scale bars, 10 μm.</p