Editing melon eIF4E associates with virus resistance and male sterility

Abstract

The cap-binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap-dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single-nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non-transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non-mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild-type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild-type. An RNA-Seq analysis identified critical genes in pollen development that were down-regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E-specific mRNA translation initiation is a limiting factor for male gametes formation in melon.

Keywords: cucurbit; pollen; potyvirus resistance; resistance breaking; sexual development; translation initiation.

Figure 1

Gene editing of eIF4E mediated by CRISPR/Cas9 in melon plants. (a) Schematic representation of the melon eIF4E genomic map and the gRNA1 target site (red arrows). The target sequence is shown in red letters, and the protospacer adjacent motif (PAM) is marked in bold underlined letters. The black arrows indicate the primers flanking the target sites used to detect the mutations. (b) eIF4E genomic DNA alignment between WT and mutated T0 lines of fragments corresponding to a DNA fragment containing the gRNA1 sequence representative of the two independent lines obtained. A DNA deletion is highlighted in a red box. (c) eIF4E protein sequence alignments between WT and mutated T0 lines. The protein sequence downstream of the deletion site is shown in green; the target region in red and the red asterisk represents a premature stop codon. (d) Segregation and relative frequencies of the 283 F2 plants (excluding non‐germinated seeds) (N = 283, Ratio 1:2:1, degrees of freedom = 2, X² = 57.6, P < 0.05) and (e) hypothesized segregation and relative frequencies of 350 F2 plants assigning non‐germinated seeds to the homozygous recessive group (N = 350, Ratio 1:2:1, degrees of freedom = 2, X² = 4.3, P > 0.05). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Homozygous eif4e mutant plants exhibited immunity to Moroccan watermelon mosaic virus (MWMV) infection. (a) Disease symptoms (leaves ‐upper panel‐ and plants ‐lower panel‐) of heterozygous (eIF4E/eif4e), homozygous wild type (WT) (eIF4E/eIF4E), and recessive homozygous (eif4e/eif4e) of the F2 edited generation and RNAi (control) plants (Rodríguez‐Hernández et al., 2012) at 14 days post inoculation (dpi). (b) Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analysis of MWMV RNA accumulation at 21 dpi in five individuals heterozygous (eIF4E/eif4e), homozygous WT (eIF4E/eIF4E), recessive homozygous (eif4e/eif4e) and RNAi (control) (Rodríguez‐Hernández et al., 2012) plants. Error bars represent standard deviation. (c) Back‐inoculation assay: samples from an eif4e/eif4e resistant plant that presented late MWMV symptoms were used as a source of inoculum (red spots). MWMV‐SQ10_1.1 are WT plants infected with the original source of inoculum used to test the F2 mutants for resistance (yellow spots). (d) Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analysis of MWMV RNA accumulation at 14 dpi in individual plants. SQ10_1, SQ10_2 and SQ10_3 correspond to WT plants inoculated with MWMV‐V. RB_1, RB_2 and RB_3 are RNAi lines infected with MWMV‐RB. C+ is a F2 susceptible plant infected with MWMV. C‐ is a mock inoculated plant. Error bars represent standard deviation. (e) Multiple alignment of amino acid sequences of VPg from MWMV‐RB and MWMV‐SQ10_1.1. The unique amino acid change between the two variants is underlined in red. (f) Simulated 3D surface model of the interaction between MWMV VPg (green) and eIF4E (blue) complex. Tryptophans W82 and W182 from eIF4E involved in the association with m7GTP Guanosine‐5′‐triphosphate cap analogue are shown in sticks and coloured red. The N163Y substitution responsible for the overcoming of the resistance to MWMV is indicated by a red arrow. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Phenotype association of segregating F2 melon male flowers. Staminate and perfect flowers with viable pollen in the anthers of homozygous WT (eIF4E/eIF4E) and heterozygous (eIF4E/eif4e) F2 plants, small and not dehiscent anthers, without mature pollen grains in homozygous recessive (eif4e/eif4e) F2 plants at 40 days post germination. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Transversal sections of anthers throughout development in wild‐type (WT) and eif4e mutant (eif4e/eif4e) observed by light microscopy. Locules from the WT (a, c, e, g, i) and eif4e mutant (b, d, f, h, j) anthers from stages 9 to 12 of development. BP, bicellular pollen; dMsp, degraded microspores; Ds, dyads; E, epidermis; En, endothecium; ML, middle layer; MP, mature pollen; Ms, microsporocyte; Msp, microspores; T, tapetum; Tds, tetrads. Scale bars = 50 μm (a, b, c, d, e, f), 100 μm (g, h, i, j).

Figure 5

Light microscopy (a, b, e, f) and scanning electron microscopy (c, d, g, h) images of transversal sections throughout anther development in the wild‐type (WT) and eif4e mutant (eif4e/eif4e). (b, d) Locules are filled by large amounts of stained/dark material that could correspond to sporopollenin in late stages 10. (f, h) Locules showing clumping of microspores unstructured as well as other stained/dark material of unknown origin in early stage 12. Scale bars = 25 μm, (a, b, e, f), 10 μm (c, d), 5 μm (g, h). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

RNA‐seq along flower developmental episodes for wild‐type (WT) and eif4e mutant melon plants. Four episodes during floral development were considered: Floral structures formation (FS), gamete initiation (GI), gamete maturation (GM), and anthesis (AN) episodes. (a) Number of expressed genes per floral developmental episode. Expressed genes are considered those with an average value of FPKM (fragments per kilo base of transcript per million mapped fragments) > 1 in the three replicates in at least one episode. (b) Principal Component Analysis (PCA) scores plotted for eif4e mutant and WT floral development episodes. PCA was computed using expressed genes. PC1, principal component 1; PC2, principal component 2. The percentage of variance explained by PC1 and PC2 are 34.4% and 15.5%, respectively. Confidence ellipses were plotted around group mean points. (c) Number of DEG (up‐regulated (blue), down‐regulated (orange) and total (grey) in mutant versus WT in different episodes of flower development (adjusted P value < 0.01 and Log₂ fold change >1 and <−1). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7

Differentially expressed genes along flower developmental episodes for wild‐type (WT) and eif4e mutant melon plants. (a) Mutant versus WT enriched GO terms among different episodes of flower development. The X‐axis indicates the enriched categories, and the Y‐axis indicates the differentially expressed gene number in each category. Enriched categories were considered those with a P‐adjust lower than 0.05, which is represented here in a coloured scale from 0 (blues) to 0.05 (reds). (b) Venn diagrams showing mutant versus WT differently expressed up (left) and down (right) regulated genes at different episodes of flower development. [Colour figure can be viewed at wileyonlinelibrary.com]