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

Abstract

Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.

Figure 1

Cm‐eIF4E silencing in melon is specific and correlates with the appearance of Cm‐eIF4E‐derived small interfering RNAs (siRNAs). (a) Cm‐eIF4E and Cm‐eIF(iso)4E mRNAs were quantified by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Average Cm‐eIF4E (left) and Cm‐eIF(iso)4E (right) expression values of 16 noninoculated transformed (T) and 10 noninoculated nontransformed (NT) plants. NT corresponds to segregating T2 plants that did not carry the transgene. WT corresponds to wild‐type BGV130, used as reference in all RT‐qPCR quantifications. (b) Transgene‐derived siRNAs were detected by Northern blot. Digoxigenin‐labelled RNA and cRNA, complementary to both directions of the 175‐nucleotide Cm‐eIF4E fragment of the transgenic construct, were used as probes. The presence of the transgene was identified by PCR.

Figure 2

Cm‐eIF4E silencing in melon results in resistance to Melon necrotic spot virus (MNSV). (a) Analyses of plants of a segregating T2 generation inoculated with MNSV. The relative amounts of MNSV RNA and Cm‐eIF4E mRNA were quantified by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Transgene‐derived siRNAs were detected by Northern blot. The presence of the transgene was identified by PCR. (b) Inoculated cotyledons at 5 and 12 days post‐inoculation (dpi). WT (wild type), T (transformed).

Figure 3

Cm‐eIF4E silencing in melon results in resistance to Zucchini yellow mosaic virus (ZYMV). (a) Analyses of plants from a segregating T2 generation inoculated with ZYMV. The relative amounts of ZYMV RNA and Cm‐eIF4E mRNA were quantified by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Transgene‐derived siRNAs were detected by Northern blot. The presence of the transgene was detected by PCR. (b) Disease symptoms on the leaves of wild‐type (WT) melon plants at 21 and 30 days post‐inoculation (dpi) and the symptomless leaves of transgenic (T) melon plants at 30 dpi.

Figure 4

Cm‐eIF4E silencing in melon results in resistance to Cucumber vein yellowing virus (CVYV). (a) Analysis of homozygous plants from a T2 generation inoculated with CVYV. The relative levels of CVYV RNA and Cm‐eIF4E mRNA were quantified by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Transgene‐derived siRNAs were detected by Northern blot. The presence of the transgene was determined by PCR. (b) Disease symptoms on the leaves of wild‐type (WT) melon plants at 21 and 30 days post‐inoculation (dpi) and the symptomless leaves of transgenic (T) melon plants at 30 dpi.

Figure 5

Cm‐eIF4E silencing in melon results in resistance to Moroccan watermelon mosaic virus (MWMV). Representative plants of a segregating T2 generation inoculated with MWMV at 5 days post‐inoculation (dpi). The presence of the transgene was determined by polymerase chain reaction (PCR). Susceptible plants died a few days later, whereas resistant plants remained symptomless, as shown at 30 dpi. WT, wild‐type.