Activation of geminivirus V-sense promoters in roots is restricted to nematode feeding sites

Abstract

Obligate sedentary endoparasitic nematodes, such as the root-knot and cyst nematodes, elicit the differentiation of specialized nematode nurse or feeding cells [nematode feeding sites (NFS), giant cells and syncytia, respectively]. During NFS differentiation, marked changes in cell cycle progression occur, partly similar to those induced by some geminiviruses. In this work, we describe the activation of V-sense promoters from the Maize streak virus (MSV) and Wheat dwarf virus (WDV) in NFS formed by root-knot and cyst nematodes. Both promoters were transiently active in microinjection experiments. In tobacco and Arabidopsis transgenic lines carrying promoter-beta-glucuronidase fusions, the MSV V-sense promoter was activated in the vascular tissues of aerial plant parts, primarily leaf and cotyledon phloem tissue and some floral structures. Interestingly, in roots, promoter activation was restricted to syncytia and giant cells tested with four different nematode populations, but undetectable in the rest of the root system. As the activity of the promoter in transgenic rootstocks should be restricted to NFS only, the MSV promoter may have utility in engineering grafted crops for nematode control. Therefore, this study represents a step in the provision of some of the much needed additional data on promoters with restricted activation in NFS useful in biotechnological nematode control strategies.

Figure 1

(A) Microinjected Arabidopsis syncytia, 8–10 days after inoculation with Heterodera schachtii. Plants were microinjected with pWDV4i::GUS from Wheat dwarf virus (WDV) (left) or p208::GUS from Maize streak virus (MSV) (right); negative control injected with water (middle). The blue precipitate depicts promoter region activation. Arrows indicate the positions of syncytia. (B) Histochemical β‐glucuronidase (GUS) assay of transgenic Arabidopsis plants carrying a p208::GUS fusion infected with H. schachtii, 14–20 days post‐inoculation. GUS staining in syncytia (left); semi‐thin sections (7 µm) indicating GUS precipitate localization in syncytial cells (right). Scale bars represent: (A) and (B) left, 1 mm; (B) right, 100 µm.

Figure 2

Histochemical β‐glucuronidase (GUS) assay of transgenic tobacco (A, C, E, G) and Arabidopsis (B, D, F, H) plants carrying the Maize streak virus (MSV) p208::GUS fusion. (A, B) GUS staining in vascular tissue of above‐ground organs: cotyledons, leaves and stem. (C) Flower with GUS staining in sepals. (D, F) GUS precipitate in sepals and a faint signal in the style of fully developed flowers; note the absence of a signal in siliques and seeds. (E) Intense GUS activity in the apex of sepals and in glandular trichomes (left) and in the style (right). (G, H) Semi‐thin sections (7 µm) of tobacco and Arabidopsis stems showing GUS localization in phloem tissue. Some activity was detected in tobacco parenchyma cells associated with xylem tissue. Scale bars represent: (A–F) 1 mm; (G, H) 100 µm.

Figure 3

Histochemical β‐glucuronidase (GUS) assay of transgenic plants carrying a p208::GUS fusion in galls, 12–14 days after infection: tobacco (A–C) and Arabidopsis (E, F). Meloidogyne javanica galls (A, E) and M. incognita galls (C). (B, F) Sections (7 µm) of tobacco and Arabidopsis M. javanica galls, respectively. (D) Table representing the percentage of blue galls obtained with different Meloidogyne species. (G) Graph showing the percentage of blue galls after infection of tobacco and Arabidopsis lines with M. javanica at different infection points. Giant cells (GCs) are shown. Scale bars represent: (A, C, E) 1 mm; (B, F) 100 µm.