Identification of a caleosin associated with hazelnut (Corylus avellana L.) oil bodies.

Caleosins are involved in several cellular and biological processes that are closely associated with the synthesis, degradation and stability of oil bodies (OB). Because of the importance and the multiple roles of these OB-associated proteins, in silico identification of sequences corresponding to putative caleosins in the hazelnut genome has been performed, and the association with seed OB was verified using a proteomic approach. Five full-length sequences (CavCLO-H1, CavCLO-H2, CavCLO-H3, CavCLO-L1, CavCLO-L2), belonging to the two groups of caleosins (H and L), have been identified in the hazelnut genome. The number of identified caleosins is in agreement with that previously observed in other plant species, confirming that caleosins comprise small gene families in plants. A proteomic approach allowed us to verify only the presence of CavCLO-H1 in hazelnut OB, suggesting that several members inside this family could have different roles during plant growth and development. In silico analysis also suggests that CavCLO-H1 may act as a peroxygenase.

Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum.

Hydrophobins are fungal cell wall proteins involved in aggregation of hyphae. Upon the development of the ectomycorrhizal symbiosis between tree roots and fungal hyphae, the transcripts of hydrophobin genes markedly accumulated. As the precise role of these proteins in symbiosis is not yet known, we develop heterologous expression system of the Pisolithus hydrophobin HYDPt-1. This gene has been introduced in Saccharomyces cerevisiae and in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. Introns were required for hydPt-1 transcript accumulation in the basidiomycete H. cylindrosporum. Heterologous transcript accumulation did not alter the phenotype of either species. The lack of altered phenotype resulted from the absence of HYDPt-1 polypeptide accumulation in transformed strains.

Immunolocalization of hydrophobin HYDPt-1 from the ectomycorrhizal basidiomycete Pisolithus tinctorius during colonization of Eucalyptus globulus roots.

• The immunolocalization of one of the hydrophobins of Pisolithustinctorius (HYDPt-1) is reported. Hydrophobin proteins play key roles in adhesion and aggregation of fungal hyphae, and it is already known that formation of ectomycorrhizas on eucalypt roots enhances the accumulation of hydrophobin mRNAs in the mycelium of Pisolithus tinctorius. • Purification of SDS-insoluble proteins from the mycelium of P. tinctorius showed the presence of a 13 kDa polypeptide with properties of class I hydrophobin. • Polyconal antibodies were raised against a recombinant HYDPt-1 polypeptide, and these were used for immunofluorescence-coupled transmission electron microscopy. • HYDPt-1 is a cell wall protein located at the surface of the hyphae with no preferential accumulation in the fungal cells of the different tissues of the ectomycorrhiza (i.e. extraradical hyphae, mantle or Hartig net).

A novel class of ectomycorrhiza-regulated cell wall polypeptides in Pisolithus tinctorius.

Development of the ectomycorrhizal symbiosis leads to the aggregation of fungal hyphae to form the mantle. To identify cell surface proteins involved in this developmental step, changes in the biosynthesis of fungal cell wall proteins were examined in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhizas by two-dimensional polyacrylamide gel electrophoresis. Enhanced synthesis of several immunologically related fungal 31- and 32-kDa polypeptides, so-called symbiosis-regulated acidic polypeptides (SRAPs), was observed. Peptide sequences of SRAP32d were obtained after trypsin digestion. These peptides were found in the predicted sequence of six closely related fungal cDNAs coding for ectomycorrhiza up-regulated transcripts. The PtSRAP32 cDNAs represented about 10% of the differentially expressed cDNAs in ectomycorrhiza and are predicted to encode alanine-rich proteins of 28.2 kDa. There are no sequence homologies between SRAPs and previously identified proteins, but they contain the Arg-Gly-Asp (RGD) motif found in cell-adhesion proteins. SRAPs were observed on the hyphal surface by immunoelectron microscopy. They were also found in the host cell wall when P. tinctorius attached to the root surface. RNA blot analysis showed that the steady-state level of PtSRAP32 transcripts exhibited a drastic up-regulation when fungal hyphae form the mantle. These results suggest that SRAPs may form part of a cell-cell adhesion system needed for aggregation of hyphae in ectomycorrhizas.

Eucalypt NADP-dependent isocitrate dehydrogenase. cDNA cloning and expression in ectomycorrhizae.

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) activity is increased in roots of Eucalyptus globulus subsp. bicostata ex Maiden Kirkp. during colonization by the ectomycorrhizal fungus Pisolithus tinctorius Coker and Couch. To investigate the regulation of the enzyme expression, a cDNA (EgIcdh) encoding the NADP-ICDH was isolated from a cDNA library of E. globulus-P. tinctorius ectomycorrhizae. The putative polypeptide sequence of EgIcdh showed a high amino acid similarity with plant NADP-ICDHs. Because the deduced EgICDH protein lacks an amino-terminal targeting sequence and shows highest similarity to plant cytosolic ICDHs, it probably represents a cytoplasmic isoform. RNA analysis showed that the steady-state level of EgIcdh transcripts was enhanced nearly 2-fold in ectomycorrhizal roots compared with nonmycorrhizal roots. Increased accumulation of NADP-ICDH transcripts occurred as early as 2 d after contact and likely led to the observed increased enzyme activity. Indirect immunofluorescence microscopy indicated that NADP-ICDH was preferentially accumulated in the epidermis and stele parenchyma of nonmycorrhizal and ectomycorrhizal lateral roots. The putative role of cytosolic NADP-ICDH in ectomycorrhizae is discussed.

Differential Localization of Carbohydrate Epitopes in Plant Cell Walls in the Presence and Absence of Arbuscular Mycorrhizal Fungi.

Two monoclonal antibodies (McAbs) generated against rhamnogalacturonan I and characterized as specific for a terminal [alpha]-(1->2)-linked fucosyl-containing epitope (CCRC-M1) and for an arabinosylated [beta]-(1,6)-galactan epitope (CCRC-M7) were used in immunogold experiments to determine the distribution of the epitopes in four plants. Allium porrum, Zea mays, Trifolium repens, and Nicotiana tabacum plants were chosen as representatives of monocots and dicots with different wall structures. Analyses were performed on root tissues in the presence and absence of arbuscular mycorrhizal fungi. A differential localization of the two cell wall epitopes was found between tissues and between species: for example, in leek, CCRC-M1 labeled epidermal and hypodermal cells, whereas CCRC-M7 labeled cortical cells only. Clover walls were labeled by both McAbs, whereas maize and tobacco were only labeled by CCRC-M7. In the presence of the arbuscular mycorrhizal fungi, labeling was additionally found in an apoplastic compartment typical of the symbiosis (the interface) occurring around the intracellular hyphae. Epitopes binding both McAbs were found in the interfacial material, and their distribution mirrored the pattern found in the host cell wall. These findings demonstrate that the composition of the interface zone in a fungus-plant symbiosis reflects the composition of the wall of the host cell.