Microarray analysis and functional tests suggest the involvement of expansins in the early stages of symbiosis of the arbuscular mycorrhizal fungus Glomus intraradices on tomato (Solanum lycopersicum)

Abstract

Arbuscular mycorrhizal (AM) symbiosis occurs between fungi of the phylum Glomeromycota and most terrestrial plants. However, little is known about the molecular symbiotic signalling between AM fungi (AMFs) and non-leguminous plant species. We sought to further elucidate the molecular events occurring in tomato, a non-leguminous host plant, during the early, pre-symbiotic stage of AM symbiosis, i.e. immediately before and after contact between the AMF (Glomus intraradices) and the host. We adopted a semi-synchronized AMF root infection protocol, followed by genomic-scale, microarray-based, gene expression profiling at several defined time points during pre-symbiotic AM stages. The microarray results suggested differences in the number of differentially expressed genes and in the differential regulation of several functional groups of genes at the different time points examined. The microarray results were validated and one of the genes induced during contact between AMF and tomato, the expansin-like EXLB1, was functionally analysed. Expansins, encoded by a large multigene family, facilitate plant cell expansion. However, no biological or biochemical function has yet been established for plant-originated expansin-like proteins. EXLB1 transcripts were localized early during the association to cells that may perceive the fungal signal, and later during the association in close proximity to sites of AMF hypha-root colonization. Moreover, in transgenic roots, we demonstrated that a reduction in the steady-state level of EXLB1 transcript was correlated with a reduced rate of infection, reduced arbuscule expansion and reduced AMF spore formation.

Figure 1

Illustration of time points examined during arbuscular mycorrhizal fungus (AMF)–tomato interaction. Spores are represented by circles, roots by cylinders. hpi, hours post‐inoculation.

Figure 2

Total number of up‐ and down‐regulated genes as determined from microarray experiments at each time point examined during arbuscular mycorrhizal fungus (AMF)–tomato interaction. hpi, hours post‐inoculation. The number of genes is presented above each column.

Figure 3

Functional groups and number of genes that were up‐ and down‐regulated within each functional group. Genes were determined from microarray experiments at all time points examined during the arbuscular mycorrhizal fungus (AMF)–tomato interaction.

Figure 4

Relative transcription of EXLB1 determined by quantitative polymerase chain reaction in tomato roots inoculated with arbuscular mycorrhizal fungus (AMF) vs. non‐inoculated control. hpi, hours post‐inoculation.

Figure 5

In situ localization of EXLB1 transcripts in longitudinal sections of tomato roots inoculated with arbuscular mycorrhizal fungi (AMF). (a–d, e–h) EXLB1 transcript localization in AMF‐inoculated root tissue at 96 h post‐inoculation (hpi) (a–d) and 4 weeks post‐inoculation (wpi) (e–h). (i–l) AMF‐infected control in which no primers were added to the polymerase chain reaction. (m–p) AMF non‐infected control (with primers). (a, e, i, m) Detection of signal. (b, f, j, n) Detection of autofluorescence. (c, g, k, o) Bright field of the section. (d, h, l, p) Overlay of signal detection, autofluorescence detection and bright field. Green signal in cell cytoplasm indicates EXLB1 transcripts (marked by white arrows); red and yellow signals represent autofluorescence; green signal in nuclei (both plant and fungus cells) represents non‐specific genomic DNA amplification. Black arrows indicate fungal hyphae. Bars represent 50 µm (a–d, i–p); bars represent 20 µm (e–h).

Figure 6

(a) Relative expression of EXLB1 vs. elongation factor in transgenic tomato roots inoculated with arbuscular mycorrhizal fungus (AMF). Transgenic roots contain either expansin antisense (EXP‐anti) or green fluorescent protein (GFP) (control) constructs. All 14 generated transgenic lines were analysed. Average and standard deviations are presented for the eight lines containing EXP‐anti and for the six lines containing GFP (control) constructs. (b) Examples of stained AMF symbiotic structures. Hyphae (H), spores (S) and arbuscules (A) are marked. (c) Number of spores per gram dry weight (DW) of transgenic roots inoculated with AMF. Transgenic roots contain either EXP‐anti or GFP (control) constructs. All 14 generated transgenic lines were analysed. Average and standard deviations are presented for the eight lines containing EXP‐anti and for the six lines containing GFP (control) constructs.