Identification of an RNA silencing suppressor encoded by Grapevine leafroll-associated virus 3

Gouveia, Paulo; Dandlen, Susana; Costa, Ângela A.; Marques, Natália; Nolasco, Gustavo

doi:10.1007/s10658-011-9876-1

Cited by 23 publications

(11 citation statements)

References 24 publications

(35 reference statements)

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“…The PMWaV-2 p20 ORF effectively reduced the accumulation of GFP siRNAs until 5 dpi while the PMWaV-2 CP ORF did not cause such a reduction ( Figure 2 ). Similar to the PMWaV-2 CP ORF, the p20 suppressor identified from Citrus tristeza virus (CTV) [ 47 ], p19.7 suppressor of Grapevine leafroll-associated virus -3 (GLRaV-3) [ 48 ] and the VP53, VP37, and the large capsid protein (LCP) suppressor identified from Broad bean wilt virus 2 also do not prevent siRNA accumulation [ 68 ]. When the effects of PMWaV-2 p20 and CP were analyzed using a hairpin-GFP inducer, no suppression of GFP was found, as evident from the disappearance of GFP fluorescence, reduction of GFP mRNAs, and elevated levels of siRNA accumulation ( Figure 4 ).…”

Section: Discussionmentioning

confidence: 99%

“…The relatively weak suppressor activity that PMWaV-2 p20 displayed at low levels of hairpin-GFP inducer was completely nullified at higher hairpin-GFP inducer levels; this is similar to the suppression that is produced by the 16 K protein of Tobacco rattle virus (TRV) [ 55 ]. However, the p19.7 suppressor of GLRaV-3 is able to overcome strong silencing inducers such as dsRNA-GFP [ 48 ].…”

Section: Discussionmentioning

confidence: 99%

“…The ORFs of these suppressors are all located at the 3’-ends of their virus genomes [ 5 ]. However, many members also possess a single suppressor protein such as p21 of Beet yellows virus (BYV) [ 19 ] and p19.7 suppressor of Grapevine leafroll-associated virus -3 (GLRaV-3) [ 48 ].…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Pineapple Mealybug Wilt Associated Virus -1 and -2 for Potential RNA Silencing Suppressors and Pathogenicity Factors

Dey

Borth

Melzer

et al. 2015

Viruses

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Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. Mealybug wilt of pineapple (MWP), an important disease of pineapple, has been associated with at least three distinct viruses, Pineapple mealybug wilt associated virus -1, -2, and -3 (PMWaV-1, -2, and -3). Selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two proteins, p20 and CP, target both local and systemic silencing in N. benthamiana, while the p22 and CPd proteins target only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins, p61, had only systemic suppressor activity. Of all the proteins tested from both viruses, only the PMWaV-2 p20 protein suppressed local silencing induced by double-stranded RNA (dsRNA), but only when low levels of inducing dsRNA were used. None of the proteins analyzed could interfere with the short distance spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that the PMWaV-2 p20 and p22 proteins enhanced the pathogenicity of Potato virus X in N. benthamiana.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Analysis of Pineapple Mealybug Wilt Associated Virus -1 and -2 for Potential RNA Silencing Suppressors and Pathogenicity Factors

Dey

Borth

Melzer

et al. 2015

Viruses

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“…However, by analogy to similarly located ORFs of other members of the family Closteroviridae , GLRaV-3 ORF 8, 9, and 10-encoded proteins could be involved in suppression of the host RNA interference defense (Reed et al, 2003; Lu et al, 2004; Chiba et al, 2006) and viral long-distance transport (Prokhnevsky et al, 2002). The recent work by Gouveia et al (2012) provided experimental support for the suppressor activity of the ORF10 product p19.7 (p20B) in N. benthamiana . This protein was also proposed to be a viral pathogenicity determinant (Gouveia and Nolasco, 2012), an activity rather typical of viral suppressors of RNA interference (Voinnet, 2005).…”

Section: Glrav-3 Genome Organization and Functions Of Encoded Proteinsmentioning

confidence: 98%

Grapevine leafroll-associated virus 3

Maree¹,

Almeida²,

Bester³

et al. 2013

Front. Microbiol.

203

187

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Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

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“…Additionally, one precoated tube without extract was used as control: each for DAS-ELISA extraction buffer, PBS and Rowhani's extraction buffer. A primer pair specific to complete RNA silencing suppressor gene (ORF 10), after modification in reverse primer, was used for RT-PCR amplification (p19.7FP: 5' ATGGACCT ATCGTTTATTAT 3' and p19.7RP: 5' TTTY TAYAGYGCTCCGCAACA 3') (Gouveia et al 2012). RT-PCR condition for the amplification of p19.7 gene was: RT at 52°C for 1 h followed by 30 cycles of denaturation at 94°C, 45 s at 50°C, 90 s at 72°C continued with a final extension period of 10 min.…”

Section: Preparation Of Virus Extracts and Their Loadingmentioning

confidence: 99%

Development of an immunocapture–reverse transcription–polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3)

Kumar

Rai

2014

Phytoparasitica

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Grapevine leafroll-associated virus 3(GLRaV-3) is the most destructive virus causing leaf roll disease in grapevine. ELISA has been widely used to screen the propagating materials for indexing of this virus at nursery stage. But the uneven distribution of GLRaV-3 in vines, its confinement to phloem tissues and impact of seasonal influences on its concentration limit the scope of ELISA. RT-PCR (reverse transcription-polymerase chain reaction), is a more sensitive technique, but not feasible for large scale screening purpose because of the tedious process of RNA isolation. Furthermore, location of virus particles and the presence of inhibitory compounds in the woody tissues of grapevine make RNA isolation problematic. Immunocapture-RT-PCR (IC-RT-PCR), more sensitive than ELISA and RT-PCR alone, is a technique where the virus can be detected without isolating the RNA. In this study, IC-RT-PCR was performed using different combinations of three virus extraction buffers and two virus nucleic acid releasing buffers along with one virus RNA releasing condition for the detection of GLRaV-3. The modified extraction and RNA release protocol developed in this study was validated for specific detection of the virus in the vines of five infected grapevine cultivars. This protocol can help in complementing the GLRaV-3 specific certification program of the country.

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Identification of an RNA silencing suppressor encoded by Grapevine leafroll-associated virus 3

Cited by 23 publications

References 24 publications

Analysis of Pineapple Mealybug Wilt Associated Virus -1 and -2 for Potential RNA Silencing Suppressors and Pathogenicity Factors

Analysis of Pineapple Mealybug Wilt Associated Virus -1 and -2 for Potential RNA Silencing Suppressors and Pathogenicity Factors

Grapevine leafroll-associated virus 3

Development of an immunocapture–reverse transcription–polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3)

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