Silencing the conserved small nuclear ribonucleoprotein SmD1 target gene alters susceptibility to root-knot nematodes in plants

Abstract

Root-knot nematodes (RKNs) are among the most damaging pests of agricultural crops. Meloidogyne is an extremely polyphagous genus of nematodes that can infect thousands of plant species. A few genes for resistance (R-genes) to RKN suitable for use in crop breeding have been identified, but virulent strains and species of RKN have emerged that render these R-genes ineffective. Secretion of RKN effectors targeting plant functions mediates the reprogramming of root cells into specialized feeding cells, the giant cells, essential for RKN development and reproduction. Conserved targets among plant species define the more relevant strategies for controlling nematode infection. The EFFECTOR18 (EFF18) protein from M. incognita interacts with the spliceosomal small nuclear ribonucleoprotein D1 (SmD1) in Arabidopsis (Arabidopsis thaliana), disrupting its function in alternative splicing regulation and modulating the giant cell transcriptome. We show here that EFF18 is a conserved RKN-specific effector that targets this conserved spliceosomal SmD1 protein in Solanaceae. This interaction modulates alternative splicing events produced by tomato (Solanum lycopersicum) in response to M. incognita infection. The alteration of SmD1 expression by virus-induced gene silencing in Solanaceae affects giant cell formation and nematode development. Thus, our work defines a promising conserved SmD1 target gene to develop broad resistance for the control of Meloidogyne spp. in plants.

Figure 1

EFF18 is a conserved effector in RKNs. A, Phylogenetic tree and schematic diagram of RKN EFF18 protein sequences. The tree scale corresponds to the number of substitutions per site based on the amino-acid matrix (JTT). In the schematic diagram of EFF18 proteins, the predicted secretion SP (gray boxes), the aspartic acid and glutamic acid (D-E)-rich region (red boxes), the lysine (K)-rich C-terminal region (blue boxes), and the nuclear mono- (purple boxes) or bi- (orange boxes) partite localization signals (NLS) are shown. EFF18 proteins from the closest group to MiEFF18a carry one mono- and one bipartite NLS, whereas the most divergent copies have only a single monopartite NLS. B, Pairwise sequence identity matrix for RKN EFF18 protein sequences. C, EFF18 localized to the nucleus and nucleolus of plant cells. The MiEFF18s, MaEFF18a, and MeEFF18a sequences were fused to that encoding GFP in a C-terminal position and expressed in N. benthamiana leaves by agroinfiltration. GFP was used as a control and gave fluorescence in the cytoplasm and the nucleus (n), but not the nucleolus (arrowhead). Bars = 10 µm.

Figure 2

RKN EFF18s are specifically expressed in the subventral glands. In situ hybridization, showing EFF18 transcripts in the subventral glands of preparasitic J2s of M. arenaria and M. enterolobii. Sense probes for the MaEFF18 and MeEFF18 transcripts were used as a negative control. SvG, subventral glands. Bars = 40 µm.

Figure 3

Conserved SmD1 proteins are targeted by EFF18. A, MAFFT protein sequence alignment of the S. lycopersicum (Sl), N. benthamiana (Nb), and A. thaliana (At) SmD1 proteins. B, Schematic representation of Sm1 and Sm2 motif in SmD1 proteins. C, GFP-AtSmD1b, GFP-SlSmD1a, and GFP-NbSmD1b accumulate in the nucleus and particularly in the nucleolus when transiently expressed in N. benthamiana epidermal leaf cells. GFP was used as a nucleocytoplasmic control. n, nucleoplasm; white arrowheads show nucleolus. Bars = 5 µm. D, Pairwise Y2H assays showed that the MiEFF18a and MeEFF18 proteins were able to interact with the SmD1 proteins of A. thaliana, S. lycopersicum, and N. benthamiana. We used MiEFF18a and MiEFF16 as a positive and negative control, respectively. Diploid yeasts containing the bait and prey plasmids carrying controls, effectors or SmD1 were serially diluted and spotted on plates. The 10-2 dilution is shown. SD-WL corresponds to the nonselective medium without tryptophan (W) and leucine (L). Only yeasts carrying a protein–protein interaction can survive on the SD-WLH (H, histidine) + 0.5-mM 3-aminotriazole (3-AT) selective medium.

Figure 4

Alternative splicing is triggered in tomato roots upon M. incognita infection. A, Tomato genes with alternative splicing events (IR, exon skipping, alternative 3′-splice site, and alternative 5′-splice site) in galls 7 and 14 dpi with M. incognita, relative to uninfected roots. B, Venn diagram showing the overlap between DSGs in M. incognita-induced galls at 7 and 14 dpi. C, Tomato genes differentially expressed (up or downregulated) in galls 7 and 14 dpi with M. incognita, relative to uninfected roots. D, Venn diagram showing the overlap between DSGs and DEGs in M. incognita-induced galls at either 7 or 14 dpi.

Figure 5

The silencing of SmD1 genes by VIGS affects susceptibility to M. incognita in S. lycopersicum. A, Timeline used for the VIGS experiments in S. lycopersicum. B, RT–qPCR demonstrating the effective silencing of SlSmD1 in TRV-SmD1 line when compared to the control TRV-GFP. RNAs were extracted from one leaf harvested on six randomly selected plants for each condition. SlRPN7 was used for data normalization. C, Infection test on tomato plants in which SlSmD1 genes were silenced (TRV-SmD1) and control tomato plants (TRV-GFP). Females producing egg masses were counted 7 weeks after inoculation with 150 M. incognita J2s per plant. Boxes indicate interquartile range (25–75th percentile). The central lines within the boxes represent medians. Whiskers indicate the minimal and maximum of the usual values present in the data set. The crosses represent average values. The circle outside the box represents outlier. Results of two independent replicates are shown. Statistical significance was determined by Kruskal–Wallis tests, and significant differences were observed between TRV-GFP control and TRV-SmD1 plants (*P ≤ 0.01).

Figure 6

The silencing of SmD1 genes by VIGS affects susceptibility to M. incognita in N. benthamiana. A, Timeline used for VIGS experiment in N. benthamiana. B, N. benthamiana plants with silenced SmD1 genes (TRV-SmD1, right) and TRV2-empty control plants (TRV-empty, left), showing some developmental defects of the leaves (upper) and a shorter root system (lower). Red arrows point to galls on these pictures. Bars = 1 cm. C, RT–qPCR showing that the NbSmD1b gene, sharing 93.6% and 94.2% identity with SlSmD1a and SlSmD1b, respectively, was effectively silenced. The data shown are the normalized relative transcripts quantities calculated from three independent biological replicates using Qbase. NbEF1a and NbACT1 genes were used for data normalization. Error bars represent sd. D, Infection test on N. benthamiana control plants (TRV-empty) and plants in which NbSmD1b was silenced (TRV-SmD1). Galls were counted 2 weeks after inoculation with 200 M. incognita J2s per plant. Boxes indicate interquartile range (25–75th percentile). The central lines within the boxes represent medians. Whiskers indicate the minimal and maximum of the usual values present in the data set. The crosses represent average values. The circle outside the box represents outlier. Results of three independent replicates are shown. Statistical significance was determined by Kruskal–Wallis tests, and significant differences were observed between TRV-empty control and TRV-SmD1 plants (*P ≤ 0.05).

Figure 7

SmD1 plays an important role in the formation of giant cells. A, The filiform stage 2 juveniles/swollen juveniles (J2s/Js) ratio obtained by acid fuchsin staining in the N. benthamiana root system with (TRV-SmD1) and without (TRV-empty) silencing with the TRV-SlSmD1 construct, following infection with M. incognita. Bars = 100 µm. B, Galls of negative control plants and plants with SmD1 silencing collected 2-week post infection for measurement of the area of giant cells (dotted line) by the BABB clearing method (Cabrera et al., 2018). The biggest giant cell measured is shown by a surrounding dotted white line. Bar = 100 µm. C, Box-and-whisker plot of giant cell size (µm²) measurements (n = 32 and 26 galls). Boxes indicate interquartile range (25–75th percentile). The central lines within the boxes represent medians. Whiskers indicate the minimal and maximum of the usual values present in the data set. The crosses represent average values. Data are means of two biological replicates. Statistical significance was determined by a t test (**P ≤ 0.01).