Whole mount multiplexed visualization of DNA, mRNA, and protein in plant-parasitic nematodes

Abstract

Background: Plant-parasitic nematodes compromise the agriculture of a wide variety of the most common crops worldwide. Obtaining information on the fundamental biology of these organisms and how they infect the plant has been restricted by the ability to visualize intact nematodes using small molecule stains, antibodies, or in situ hybridization. Consequently, there is limited information available about the internal composition of the nematodes or the biology of the effector molecules they use to reprogram their host plant.

Results: We present the Sperling prep - a whole mount method for nematode preparation that enables staining with small molecules, antibodies, or in situ hybridization chain reaction. This method does not require specialized apparatus and utilizes typical laboratory equipment and materials. By dissociating the strong cuticle and interior muscle layers, we enabled entry of the small molecule stains into the tissue. After permeabilization, small molecule stains can be used to visualize the nuclei with the DNA stain DAPI and the internal structures of the digestive tract and longitudinal musculature with the filamentous actin stain phalloidin. The permeabilization even allows entry of larger antibodies, albeit with lower efficiency. Finally, this method works exceptionally well with in situ HCR. Using this method, we have visualized effector transcripts specific to the dorsal gland and the subventral grand of the sugar beet cyst nematode, Heterodera schachtii, multiplexed in the same nematode.

Conclusion: We were able to visualize the internal structures of the nematode as well as key effector transcripts that are used during plant infection and parasitism. Therefore, this method provides an important toolkit for studying the biology of plant-parasitic nematodes.

Keywords: Antibody; Enzymatic permeabilization; Heterodera schachtii; In situ HCR; Plant-parasitic nematode; Small molecule; Sperling prep.; Whole mount.

Fig. 1

Three-dimensional projections of H. schachtii nematodes visualized with small molecule stains. a-b) Whole mount nematodes. c-d) Severed nematodes. The cuticle visualized with brightfield (white), DNA visualized with DAPI (white), and longitudinal muscles visualized with phalloidin (magenta). Anterior (A)-Posterior (P) and Dorsal (D)-Ventral (V) axes indicated. Scale bars, 20 μm

Fig. 2

Three-dimensional projections of H. schachtii nematodes visualized with a small molecule and antibody stains. a-b) Whole mount nematodes. c-d) Severed nematodes. The cuticle visualized with brightfield (white), DNA visualized with DAPI (white), and longitudinal muscles visualized with an antibody generated against C. elegans myosin (magenta). Anterior (A)-Posterior (P) and Dorsal (D)-Ventral (V) axes indicated. Scale bars, 20 μm

Fig. 3

Three-dimensional projection of a whole mount H. schachtii nematode visualized with a small molecule stain and in situ HCRs. The cuticle visualized with brightfield, DNA visualized with DAPI (white), and the transcript of Hsc_gene_2726 (magenta) visualized in the dorsal gland cell. Anterior (A)-Posterior (P) and Dorsal (D)-Ventral (V) axes indicated. Scale bars, 20 μm

Fig. 4

Three-dimensional projection of a whole mount H. schachtii nematode visualized with a small molecule stain and in situ HCRs. The cuticle visualized with brightfield (white), DNA visualized with DAPI (white), the transcript of Hsc_gene_21727 eng2 (cyan) visualized in the subventral gland cell (SvG), and the transcript of Hsc_gene_2726 (magenta) visualized in the dorsal gland cell (DG). Anterior (A)-Posterior (P) and Dorsal (D)-Ventral (V) axes indicated. Scale bars, 20 μm

Fig. 5

Schematic of the nematode preparation method and different staining and in situ HRC possibilities. Anterior (A)-Posterior (P) and Dorsal (D)-Ventral (V) axes indicated. Scale bar, 20 μm