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. 2002 Sep;130(1):58-67.
doi: 10.1104/pp.003525.

Differential Expression of a Metallothionein Gene During the Presymbiotic Versus the Symbiotic Phase of an Arbuscular Mycorrhizal Fungus

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Differential Expression of a Metallothionein Gene During the Presymbiotic Versus the Symbiotic Phase of an Arbuscular Mycorrhizal Fungus

Luisa Lanfranco et al. Plant Physiol. .
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Abstract

A full-length cDNA encoding a metallothionein (MT)-like polypeptide, designated GmarMT1, was identified in an expressed sequence tag collection from germinated spores of the arbuscular mycorrhizal fungus Gigaspora margarita (BEG34). The GmarMT1 gene is composed of two exons separated by an 81-bp intron. It codes for a 65-amino acid polypeptide comprising a plant type 1 MT-like N-terminal domain and a C-terminal domain that is most closely related to an as-yet-uncharacterized fungal MT. As revealed by heterologous complementation assays in yeast, GmarMT1 encodes a functional polypeptide capable of conferring increased tolerance against Cd and Cu. The GmarMT1 RNA is expressed in both presymbiotic spores and symbiotic mycelia, even in the absence of metal exposure, but is significantly less abundant in the latter stage. An opposite pattern was observed upon Cu exposure, which up-regulated GmarMT1 expression in symbiotic mycelia but not in germinated spores. Together, these data provide the first evidence, to our knowledge, for the occurrence in an arbuscular mycorrhizal fungus of a structurally novel MT that is modulated in a metal and life cycle stage-dependent manner and may afford protection against heavy metals (and other types of stress) to both partners of the endomycorrhizal symbiosis.

Figures

Figure 1
Figure 1
Nucleotide and deduced amino acid sequence of GmarMT1. The intron sequence is shown in lowercase letters; Cys residues are in bold; C-X-C motifs and GmarMT1-specific primers (MT1/MT2) are underlined.
Figure 2
Figure 2
Alignment of GmarMT1 with MT-like polypeptides from other organisms. The polypeptide sequence of GmarMT1 (boxed) was aligned with the partial sequence of a predicted polypeptide from G. rosea (Stoffel et al., 2001) and with seven of the best scoring sequences identified by BLAST analysis: Arabidopsis MT1A (National Center for Biotechnology Information [NCBI] accession no. P43392; Yeh et al., 1995; Zhou and Goldsbrough, 1995); Arabidopsis MT1C (NCBI accession no. Q38804; Zhou and Goldsbrough, 1995) canola (Brassica napus) MT-like (NCBI accession no. P43402; Buchanan-Wollaston, 1994); rice (Oryza sativa) MT1 (NCBI accession no. Q40633; Hsieh et al., 1995); barley (Hordeum vulgare) MT1 (NCBI accession no. P26571; Okumura et al., 1991); Mimulus guttatus MT1 (NCBI accession no. P20238; de Miranda et al., 1990); and A. bisporus MT like (NCBI accession no. CAB85689; Eastwood et al., 2001). Amino acid residues that are identical in at least seven of the nine sequences are shown on a black background, and residues shared by all three fungal sequences are shaded gray; these two types of conserved residues are indicated with uppercase and lowercase letters, respectively, in the consensus pattern shown below the alignment. Gaps introduced to optimize the alignment are indicated by dots.
Figure 3
Figure 3
Increased HM tolerance conferred by GmarMT1 in metal-hypersensitive yeast mutants. Δyap-1 (A) or Δcup1 (B) yeast mutants harboring the pFL61-GmarMT1 plasmid (GmarMT1), the positive control plasmid pFL61-Mt2a (AtMt2a), or the empty pFL61 vector (−) were grown on SD-agar (−uracil) plates with the indicated linear gradients of CdSO4 (A) or CuSO4 (B).
Figure 4
Figure 4
RT-PCR analysis of GmarMT1 mRNA levels in presymbiotic and symbiotic life cycle stages of G. margarita. Balanced amounts of cDNA from quiescent spores (lane 1), germinated spores (lane 2), or mycorrhizal roots (lane 3) were amplified with GmarMT1-specific oligonucleotide primers (MT1). 18S rDNA amplicons, obtained from parallel control reactions and loaded in the same order as above, were used as internal standards (18S). The sizes of GmarMT1 and 18S rDNA amplicons are indicated. No cDNA template was added to reaction mixtures run in lane 4; DNA size markers (HaeIII-digested pUC18) were run in lane M.
Figure 5
Figure 5
GmarMT1 mRNA expression levels after metal exposure. Balanced amounts of total RNA extracted from germinated spores (a) and mycorrhizal roots (b and c), either untreated or exposed to the indicated concentrations of CuSO4 or CdSO4, were reverse-transcribed and amplified with GmarMT1-specific primers (MT1). The 18S rRNA was used as an internal calibration standard for all reactions (18S). The sizes and migration positions of GmarMT1 (MT1) and 18S rDNA (18S) amplicons are indicated. Template RNA was omitted from reaction mixtures shown in lane −; DNA size markers (HaeIII-digested pUC18) were run in lane M.
Figure 6
Figure 6
Scheme of the differential expression and HM-induction of GmarMT1 in two phases of the G. margarita life cycle. The steps illustrated are germinating spores of G. margarita as seen under the stereomicroscope (presymbiotic phase; bar = 300 μm) and an arbuscule visualized by fluorescence microscopy (symbiotic phase; bar = 5 μm). The GmarMT1 mRNA is selectively up-regulated by Cu ions in the symbiotic mycelium.

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