The root-knot nematode Meloidogyne incognita produces a functional mimic of the Arabidopsis INFLORESCENCE DEFICIENT IN ABSCISSION signaling peptide

Abstract

INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is a signaling peptide that regulates cell separation in Arabidopsis including floral organ abscission and lateral root emergence. IDA is highly conserved in dicotyledonous flowering plant genomes. IDA-like sequences were also found in the genomic sequences of root-knot nematodes, Meloidogyne spp., which are globally deleterious pathogens of agriculturally important plants, but the role of these genes is unknown. Exogenous treatment of the Arabidopsis ida mutant with synthetic peptide identical to the M. incognita IDA-like 1 (MiIDL1) protein sequence minus its N-terminal signal peptide recovered both the abscission and root architecture defects. Constitutive expression of the full-length MiIDL1 open reading frame in the ida mutant substantially recovered the delayed floral organ abscission phenotype whereas transformants expressing a construct missing the MiIDL1 signal peptide retained the delayed abscission phenotype. Importantly, wild-type Arabidopsis plants harboring an MiIDL1-RNAi construct and infected with nematodes had approximately 40% fewer galls per root than control plants. Thus, the MiIDL1 gene produces a functional IDA mimic that appears to play a role in successful gall development on Arabidopsis roots.

Fig. 1.

Dendrogram and alignment of conserved C-terminal amino acid sequences for a selection of representative plant and nematode genes with similarity to the AtIDA EPIP domain: Arabidopsis (Arabidopsis thaliana, At), soybean (Glycine max, Gm), tomato (Solanum lycopersicum, Sl), soybean cyst nematode (Heterodera glycines, Hg), sugar beet nematode (H. schachtii, Hs), southern root-knot nematode (Meloidogyne incognita, Mi), northern root-knot nematode (M. hapla, Mh) and peach root-knot nematode (M. floridensis). Sequence names marked with a single asterisk are from cyst nematodes (Hs and Hg), those with two asterisks are from root-knot nematodes, and the MiIDL1 peptide is highlighted in bold and underscored. The numbers under the heading of C-term SP indicate the amino acid position C-terminal from the end of the predicted signal peptide.

Fig. 2.

Percentage abscission of individual flowers cut from wild-type and mutant Arabidopsis plants and then treated in agar with 10 μM of the indicated peptide (listed in Table 1). Columbia (Col-0) and C24 are ecotypes of Arabidopsis that were used as controls. The mutant ida-1 is in the C24 background (lighter gray bars), whereas, the ida-2 and hh33 mutants are in the Columbia background (darker gray bars). Standard error bars indicate the variance of the means for between three and five experiments and each experiment included between 10 and 30 flowers for each treatment. Letters above standard-error bars indicate means that are statistically grouped using a Tukey–Kramer test in SAS.

Fig. 3.

Arabidopsis wild-type Columbia (A, B) and ida-2 mutant seeds (C, D) were germinated and grown for 2 weeks in two separate experiments on agar containing 0.0, 0.1, and 0.5 (A, C) or 0.0 and 1.0 μM MiIDL1p peptide (B, D).

Fig. 4.

Arabidopsis ida-2 mutant plants transformed with CaMV 35S promoter-driven full-length MiIDL1 open reading frame (ORF), MiIDL1 ORF minus the predicted signal peptide, or non-transformed wild-type and ida-2 mutant plants for comparison. Two transformation events (MG4-8 and MG7-3; MS2-4 and MS7-1) are included for each of the different constructs. The boxed insets are a 4-fold zoom of the indicated silique base.

Fig. 5.

Gall number and size on M. incognita infected roots of wild-type Arabidopsis transformed with GUS and MiIDL1-RNAi. (A) Mean number of galls per root system of 10–12 independently grown plants for multiple transgenic lines for GUS (DG) and MiIDL1 (DM) at 35 dpi (light gray bars) and 42 dpi (darker gray bars). (B) Photos showing the relative size of galls at 35 dpi on GUS and MiIDL1-RNAi plants. (C) Box chart of gall sizes (pixels counted using ImageJ software) from multiple photos of different plants from the lines indicated (the area of 50–100 galls was measured per transgenic line). In (A), DG and DM indicate GUS or MiIDL1-RNAi, respectively, and the first number after DG or DM indicate the transformation event from T0 plants and the second number the seed collection (line) from T1 plants. hh33 is the hae-3/hsl2-3 double mutant. Galls were counted on T3 plants. The standard error bars in (A) reflect gall counts for 9–14 plants for each line. Letters above or next to the bars in (A, C) indicate means that are statistically grouped using a Tukey–Kramer test in SAS. The P value (two-tailed t-test) for the significance of the difference between the means for the GUS lines compared with the MiDL1 lines in (A) is 4.1 × 10⁻⁷. Arrows in (B) point to representative galls. Points in (C) are outliers.

Fig. 6.

Gene expression profile for MiIDL1 (diamonds) and MiIDL2 (circles) normalized to the nematode elongation factor 1b (MiEF1b) in wild-type Arabidopsis infected with root-knot nematodes (M. incognita).