Reverse Genetic Analysis of Ourmiaviruses Reveals the Nucleolar Localization of the Coat Protein in Nicotiana benthamiana and Unusual Requirements for Virion Formation

Crivelli, Giulia; Ciuffo, M.; Genre, Andrea; Masenga, V.; Turina, Massimo

doi:10.1128/jvi.02565-10

Cited by 33 publications

(48 citation statements)

References 67 publications

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“…A classical test for revealing silencing suppressor activity was initially performed by co-agroinfiltrating 16C N. benthamiana with pBin-GFP (Margaria et al 2007) and each of the pBin61 derivatives of OuMV RdRp, MP, and CP previously described (Crivelli et al 2011). As positive control, we used the well-characterized silencing suppressor p19 of Tomato bushy stunt virus (TBSV), which exhibited suppression, but we failed to detect suppression activity with the OuMV MP, CP, or RdRp (not shown).…”

Section: Effects Of Mutations In the Kr Region On Host Rangementioning

confidence: 99%

“…As positive control, we used the well-characterized silencing suppressor p19 of Tomato bushy stunt virus (TBSV), which exhibited suppression, but we failed to detect suppression activity with the OuMV MP, CP, or RdRp (not shown). However, in this assay, RNA2 and RNA3 were agroinfiltrated alone using a pBin61 backbone (Crivelli et al 2011) and the corresponding proteins were expressed to much lower levels than those occurring during virus infection. The low accumulation of MP and CP could explain the observed lack of silencing suppression; therefore, for this reason, we repeated the experiment with replicationcompetent constructs expressing RNA2 and RNA3 with RNA1 ( Supplementary Fig.…”

Section: Effects Of Mutations In the Kr Region On Host Rangementioning

confidence: 99%

“…In fact, without the CP, RNA1 and RNA2 move only rarely from inoculated leaves through the phloem and reach the upper noninoculated leaves, and the infection foci emanating from phloem exit sites on upper leaves are very limited (Crivelli et al 2011). In N. benthamiana agroinfiltrated leaves, green fluorescent protein (GFP)-CP localized in the nucleus and preferentially in the nucleoli of epidermal cells (Crivelli et al 2011). Such intracellular targeting seems to be determined by a short stretch of amino acids at the N-terminal region of the protein.…”

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confidence: 99%

“…The OuMV CP is strictly dispensable for cell-to-cell and long-distance movement in N. benthamiana; nevertheless, it is crucial for efficient virus spread and high accumulation in upper noninoculated leaves. In fact, without the CP, RNA1 and RNA2 move only rarely from inoculated leaves through the phloem and reach the upper noninoculated leaves, and the infection foci emanating from phloem exit sites on upper leaves are very limited (Crivelli et al 2011). In N. benthamiana agroinfiltrated leaves, green fluorescent protein (GFP)-CP localized in the nucleus and preferentially in the nucleoli of epidermal cells (Crivelli et al 2011).…”

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confidence: 99%

“…Another peculiarity of the OuMV CP is the unique morphology of the virions among plant viruses: bacilliform particles of three different lengths with pointed ends (Lisa et al 1988). Ectopic expression of RNA3 in Nicotiana benthamiana does not result in virion formation because the active replication of OuMV is necessary for virus particle assembly (Crivelli et al 2011). The OuMV CP is strictly dispensable for cell-to-cell and long-distance movement in N. benthamiana; nevertheless, it is crucial for efficient virus spread and high accumulation in upper noninoculated leaves.…”

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The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense

Rossi¹,

Vallino²,

Abbà³

et al. 2015

MPMI

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The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV 6710 , with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV 6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CP wt , in situ immunolocalization experiments indicated that CP 6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.Ourmia melon virus (OuMV) is the best-characterized species of the Ourmiaviridae family, which also consists of Epirus cherry virus (EpCV) and Cassava virus C (CaVC) species (King et al. 2012). Three plus-strand RNA molecules form the OuMV genome and each segment encodes a single protein: RNA1 directs synthesis of an RNA-dependent RNA polymerase (RdRp), RNA2 encodes a movement protein (MP), and RNA3 specifies the 22-kDa coat protein (CP) (Rastgou et al. 2009). Ourmiaviruses have affinities with other postive single stranded RNA multipartite viruses, such as the members of the family Bromoviridae, but their unique structural and molecular features have resulted in their classification in a novel viral family. In fact, phylogenetic analyses showed that the members of the family likely went through a very unusual evolutionary process based on a possible reassortment between a plant virus and a mycovirus. In particular, OuMV RdRp shares distant similarity only with RdRp of narnaviruses, a group of viruses which mainly infects fungi; however, the MP is distantly related to MP of plant tombusviruses. The CP has limited but significant similarity with CP of plant and mammalian viruses, although its phylogenetic origin remains uncertain (Rastgou et al. 2009). Another peculiarity of the OuMV CP is the unique morphology of the virions among plant viruses: bacilliform particles of three different lengths with pointed ends (Lisa et al. 1988). Ectopic expression of RNA3 in Nicotiana benthamiana does not result in virion formation because the active replication of OuMV is necessary fo...

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Section: Effects Of Mutations In the Kr Region On Host Rangementioning

confidence: 99%

Section: Effects Of Mutations In the Kr Region On Host Rangementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense

Rossi¹,

Vallino²,

Abbà³

et al. 2015

MPMI

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Engineering Maize rayado fino virus for virus‐induced gene silencing

Mlotshwa¹,

Xu²,

Willie

et al. 2020

Plant Direct

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Maize rayado fino virus (MRFV) is the type species of the genus Marafivirus in the family Tymoviridae. It infects maize (Zea mays), its natural host, to which it is transmitted by leafhoppers including Dalbulus maidis and Graminella nigrifrons in a persistent‐propagative manner. The MRFV monopartite RNA genome encodes a precursor polyprotein that is processed into replication‐associated proteins. The genome is encapsidated by two carboxy co‐terminal coat proteins, CP1 and CP2. Cloned MRFV can be readily transmitted to maize by vascular puncture inoculation (VPI), and such virus systems that can be used in maize are valuable to examine plant gene function by gene silencing. However, the efficacy of marafiviruses for virus‐induced gene silencing (VIGS) has not been investigated to date. To this end, MRFV genomic loci were tested for their potential to host foreign insertions without attenuating virus viability. This was done using infectious MRFV clones engineered to carry maize phytoene desaturase (PDS) gene fragments (ZmPDS) at various genomic regions. Several MRFV‐PDS constructs were generated and tested for infectivity and VIGS in maize. This culminated in identification of the helicase/polymerase (HEL/POL) junction as a viable insertion site that preserved virus infectivity, as well as several sites at which sequence insertion caused loss of virus infectivity. Transcripts of viable constructs, carrying PDS inserts in the HEL/POL junction, induced stable local and systemic MRFV symptoms similar to wild‐type infections, and triggered PDS VIGS initiating in veins and spreading into both inoculated and noninoculated leaves. These constructs were remarkably stable, retaining inserted sequences for at least four VPI passages while maintaining transmissibility by D. maidis. Our data thus identify the MRFV HEL/POL junction as an insertion site useful for gene silencing in maize.

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