Regulation of Root-Knot Nematode Behavior by Seed-Coat Mucilage-Derived Attractants.

Seed exudates influence the behavior of soil organisms, but how this occurs remains unclear, particularly for multicellular animals. Here we show that compounds associated with Arabidopsis seed-coat mucilage regulate the behavior of soil-borne animals, specifically root-knot nematodes (RKNs). Infective RKN J2 larvae actively travel toward Arabidopsis seeds through chemotaxis. Analysis of Arabidopsis mucilage mutants demonstrated that the attraction of RKNs to Arabidopsis seeds requires the synthesis and extrusion of seed-coat mucilage. Extracted mucilage alone is not sufficient to attract RKNs, but seed-surface carbohydrates and proteins are required for this process. These findings suggest that the RKN chemoattractant is synthesized de novo upon mucilage extrusion but may be highly unstable. RKNs attracted by this mucilage-dependent mechanism can infect the emerging seedling. However, the attraction signal from seedling roots likely acts independently of the seed-coat signal and may mask the attraction to seed-coat mucilage after germination. Multiple RKN species are attracted by Arabidopsis seeds, suggesting that this mechanism is conserved in RKNs. These findings indicate that seed exudate can regulate the behavior of multicellular animals and highlight the potential roles of seed-coat mucilage in biotic interactions with soil microorganisms.

In situ Hybridization (ISH) in Preparasitic and Parasitic Stages of the Plant-parasitic Nematode Meloidogyne spp.

The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the plant parasite Meloidogyne spp. (root-knot nematode) to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues. We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure.

A root-knot nematode small glycine and cysteine-rich secreted effector, MiSGCR1, is involved in plant parasitism.

Root-knot nematodes, Meloidogyne spp., are obligate endoparasites that maintain a biotrophic relationship with their hosts. They infect roots as microscopic vermiform second-stage juveniles, and establish specialized feeding structures called 'giant-cells', from which they withdraw water and nutrients. The nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we compared Illumina RNA-seq transcriptomes for M. incognita obtained at various points in the lifecycle, and identified 31 genes more strongly expressed in parasitic stages than in preparasitic juveniles. We then selected candidate effectors for functional characterization. Quantitative real-time PCR and in situ hybridizations showed that the validated differentially expressed genes are predominantly specifically expressed in oesophageal glands of the nematode. We also soaked the nematodes in siRNA to silence these genes and to determine their role in pathogenicity. The silencing of the dorsal gland specific-Minc18876 and its paralogues resulted in a significant, reproducible decrease in the number of mature females with egg masses, demonstrating a potentially important role for the small glycine- and cysteine-rich effector MiSGCR1 in early stages of plant-nematode interaction. Finally, we report that MiSGCR1 suppresses plant cell death induced by bacterial or oomycete triggers of plant defense.