Melon RNA interference (RNAi) lines silenced for <i>Cm‐eIF4E</i> show broad virus resistance

Rodríguez-Hernández, Ana Margarita; Gosálvez, Blanca; Sempere, Raquel Navarro; Burgos, L.; Aranda, Miguel A.; Truniger, Verónica

doi:10.1111/j.1364-3703.2012.00785.x

Cited by 89 publications

(81 citation statements)

References 48 publications

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“…The specificity allows the silencing of only one sequence among closely related sequences, as seen in this study, where only one isoform of eIF4E gene was effectively silenced (Figure 4B). Similar specific silencing of one isoform of eIF4E was also recently described in melon and tomato [64], [65]. The use of intron-containing self-complementary hpRNA (ihp-RNA) constructs was reported to dramatically enhance the RNA silencing efficiency up to 100% [66], [67].…”

Section: Discussionsupporting

confidence: 60%

Silencing of the Host Factor eIF(iso)4E Gene Confers Plum Pox Virus Resistance in Plum

et al. 2013

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Plum pox virus (PPV) causes the most economically-devastating viral disease in Prunus species. Unfortunately, few natural resistance genes are available for the control of PPV. Recessive resistance to some potyviruses is associated with mutations of eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. In this study, we used an RNA silencing approach to manipulate the expression of eIF4E and eIF(iso)4E towards the development of PPV resistance in Prunus species. The eIF4E and eIF(iso)4E genes were cloned from plum (Prunus domestica L.). The sequence identity between plum eIF4E and eIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Quantitative real-time RT-PCR analysis showed that these two genes have a similar expression pattern in different tissues. Transgenes allowing the production of hairpin RNAs of plum eIF4E or eIF(iso)4E were introduced into plum via Agrobacterium-mediated transformation. Gene expression analysis confirmed specific reduced expression of eIF4E or eIF(iso)4E in the transgenic lines and this was associated with the accumulation of siRNAs. Transgenic plants were challenged with PPV-D strain and resistance was evaluated by measuring the concentration of viral RNA. Eighty-two percent of the eIF(iso)4E silenced transgenic plants were resistant to PPV, while eIF4E silenced transgenic plants did not show PPV resistance. Physical interaction between PPV-VPg and plum eIF(iso)4E was confirmed. In contrast, no PPV-VPg/eIF4E interaction was observed. These results indicate that eIF(iso)4E is involved in PPV infection in plum, and that silencing of eIF(iso)4E expression can lead to PPV resistance in Prunus species.

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Section: Discussionsupporting

confidence: 60%

Silencing of the Host Factor eIF(iso)4E Gene Confers Plum Pox Virus Resistance in Plum

et al. 2013

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“…Data on resistance breaking suggested that the new 3′‐CITE could function either with the different eIF4E variants expressed by susceptible or resistant melon, or independently of eIF4E. To learn if this new 3′‐CITE was eIF4E‐dependent, its activity in melon protoplasts of a previously described eIF4E knock‐down line was studied (Rodríguez‐Hernández et al ., ). Through expression of a hairpin construct targeting and thus silencing melon eIF4E , its expression had been shown to be reduced more than six‐fold.…”

Section: Resultsmentioning

confidence: 99%

Interfamilial recombination between viruses led to acquisition of a novel translation‐enhancing RNA element that allows resistance breaking

et al. 2013

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Summary Many plant viruses depend on functional RNA elements, called 3′-UTR cap-independent translation enhancers (3′-CITEs), for translation of their RNAs. In this manuscript we provide direct proof for the existing hypothesis that 3′-CITEs are modular and transferable by recombination in nature, and that this is associated with an advantage for the created virus. By characterizing a newly identified Melon necrotic spot virus (MNSV; Tombusviridae) isolate, which is able to overcome eukaryotic translation initiation factor 4E (eIF4E)-mediated resistance, we found that it contains a 55 nucleotide insertion in its 3′-UTR. We provide strong evidence that this insertion was acquired by interfamilial recombination with the 3′-UTR of an Asiatic Cucurbit aphid-borne yellows virus (CABYV; Luteoviridae). By constructing chimeric viruses, we showed that this recombined sequence is responsible for resistance breaking. Analysis of the translational efficiency of reporter constructs showed that this sequence functions as a novel 3′-CITE in both resistant and susceptible plants, being essential for translation control in resistant plants. In conclusion, we showed that a recombination event between two clearly identified viruses from different families led to the transfer of exactly the sequence corresponding to a functional RNA element, giving rise to a new isolate with the capacity to infect an otherwise non-susceptible host.

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“…drive cap-independent translation of the messenger RNAs bearing it (Nieto et al, 2006;Truniger et al, 2008;Rodríguez-Hernández et al, 2012). Briefly, this assay is based on the capacity of Cm eIF4E-WT to complement the Ma5TE-controlled translation of RNAs when it is transiently expressed in melons that carry the Cm eIF4E H228L allele in homozygosity, which does not support Ma5TE-driven translation (Nieto et al, 2006;Miras et al, 2017b).…”

Section: Testing Substitutions In CM Eif4e-binding Residues In a Tranmentioning

confidence: 99%

“…In vivo translation assays were performed as previously described (Truniger et al, 2008) by electroporating separately with two 59-UTR-luc-39-UTR RNA constructs, differing in the UTRs flanking the luciferase gene that were either from isolate MNSV-Ma5 (eIF4E-dependent) or from the eIF4E-independent isolate MNSV-264. Translation of MNSV-264 can take place in protoplasts from eIF4E knocked-down lines, so it can function in the absence of eIF4E (Rodríguez-Hernández et al, 2012) and therefore serves for normalization of the different protoplast preparations. After 5 to 6 h incubation in the dark at 25°C, protoplasts were lysed in 1x Passive lysis buffer (Promega).…”

Section: Translation Complementation By Transiently Expressed CM Eif4ementioning

confidence: 99%

Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation

et al. 2017

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The association-dissociation of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) with eIF4G is a key control step in eukaryotic translation. The paradigm on the eIF4E-eIF4G interaction states that eIF4G binds to the dorsal surface of eIF4E through a single canonical alpha-helical motif, while metazoan eIF4E-binding proteins (m4E-BPs) advantageously compete against eIF4G via bimodal interactions involving this canonical motif and a second noncanonical motif of the eIF4E surface. Metazoan eIF4Gs share this extended binding interface with m4E-BPs, with significant implications on the understanding of translation regulation and the design of therapeutic molecules. Here we show the high-resolution structure of melon (Cucumis melo) eIF4E in complex with a melon eIF4G peptide and propose the first eIF4E-eIF4G structural model for plants. Our structural data together with functional analyses demonstrate that plant eIF4G binds to eIF4E through both the canonical and noncanonical motifs, similarly to metazoan eIF4E-eIF4G complexes. As in the case of metazoan eIF4E-eIF4G, this may have very important practical implications, as plant eIF4E-eIF4G is also involved in a significant number of plant diseases. In light of our results, a universal eukaryotic bipartite mode of binding to eIF4E is proposed.The initiation of protein synthesis is a key control and highly regulated step in eukaryotic gene expression (Sonenberg and Hinnebusch, 2009). Translation initiation leads to the assembly of the large (60S) and small (40S) ribosomal subunits into an active 80S ribosome that is able to locate the correct start codon of the mRNA molecule. This is facilitated by the coordinated actions of at least 12 protein initiation factors (Browning, 2004;Jackson et al., 2010;Hinnebusch, 2011;Hinnebusch and Lorsch, 2012; Browning and BaileySerres, 2015). In cap-dependent translation, binding of the eukaryotic translation initiation factor 4F (eIF4F) to the 7-methyl guanosine cap (m 7 G cap) at the 59 end of mRNA drives the attachment of the mRNA to the ribosomal 43S preinitiation complex. In mammals, eIF4F is built by three proteins: the mRNA 59 cap binding protein eIF4E, the RNA helicase eIF4A, and the scaffolding protein eIF4G, which contains binding domains for eIF4E, eIF4A, eIF3, and poly(A)-binding protein (PABP; Marcotrigiano et al., 1997;Pestova et al., 2001;Gross et al., 2003). In plants, purified eIF4F heterodimers contain eIF4E and eIF4G (Hinnebusch and Lorsch, 2012); plant eIF4A appears to be loosely associated and is therefore easily lost during purification (Lax et al., 1986). EIF4G associates with eIF3, recruiting the 40S subunit of the ribosome and initiating scanning of the mRNA in the 59 to 39 direction (Jackson et al., 2010;Park et al., 2011). EIF4G additionally binds simultaneously to eIF4E and PABP, the latter being able to interact with the mRNA poly(A) tail (Aitken and Lorsch, 2012; Browning and BaileySerres, 2015), resulting in a transient circularization of the mRNA.Higher plants contain an addit...

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Melon RNA interference (RNAi) lines silenced for Cm‐eIF4E show broad virus resistance

Cited by 89 publications

References 48 publications

Silencing of the Host Factor eIF(iso)4E Gene Confers Plum Pox Virus Resistance in Plum

Silencing of the Host Factor eIF(iso)4E Gene Confers Plum Pox Virus Resistance in Plum

Interfamilial recombination between viruses led to acquisition of a novel translation‐enhancing RNA element that allows resistance breaking

Structure of eIF4E in Complex with an eIF4G Peptide Supports a Universal Bipartite Binding Mode for Protein Translation

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