Functional characterization of transcripts expressed in early-stage Meloidogyne javanica-induced giant cells isolated by laser microdissection

Abstract

The root-knot nematode Meloidogyne javanica induces giant cells and feeds from them during its development and reproduction. To study the cellular processes underlying the formation of giant cells, laser microdissection was used to isolate the contents of early-stage giant cells 4 and 7 days post-infection (dpi) from tomato, and cDNA libraries from both stages were generated with 87 [250 expressed sequence tag (EST) clones] and 54 (309 EST clones) individual transcripts identified, respectively. These transcripts have roles in metabolism, stress response, protein synthesis, cell division and morphogenesis, transport, signal transduction, protein modification and fate, and regulation of cellular processes. The expression of 25 selected transcripts was studied further by real-time quantitative reverse transcriptase-polymerase chain reaction. Among them, 13 showed continuous up-regulation in giant cells from 4 to 7 dpi. The expression of two transcripts was higher than in controls at 4 dpi and remained at the same level at 7 dpi; a further five transcripts were highly expressed only at 7 dpi. The Phi-1 protein gene, a cell cycle-related homologue in tobacco, was expressed 8.5 times more strongly in giant cells than in control cells at 4 dpi, but was reduced to 6.7 times at 7 dpi. Using in situ hybridization, the expression of the Phi-1 gene was preferentially localized in the cytoplasm of giant cells at 4 dpi, together with a pectinesterase U1 precursor gene. The identification of highly expressed transcripts in developing giant cells adds to the knowledge of the plant genes responsive to nematode infection, and may provide candidate genes for nematode control strategies.

Figure 1

Laser microdissection (LM) of giant cells induced by the root‐knot nematode Meloidogyne javanica on roots of tomato. (A, B) Longitudinal sections through the root showing early‐stage giant cells before dissection (A) and with cell contents removed after LM (B). (C, D) Longitudinal sections through the root of a control plant (C) showing region of cells close to the vasculature (D) captured as control cells to calibrate gene expression of giant cell transcripts. GC, giant cells.

Figure 2

In situ hybridization of Meloidogyne javanica‐infected tomato root tissues at 4 days post‐infection (dpi) with probes generated from a Phi‐1 protein gene (A, B) and a pectinesterase‐like protein gene (C, D). Strong signals are present in giant cells when hybridized with antisense probes (A, C) compared with sense probes (B, D). GC, giant cells.