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doi: 10.1242/10.1242/jcs.00138

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The CBEL glycoprotein of Phytophthora parasitica var-nicotianae is involved in cell wall deposition and adhesion to cellulosic substrates

UMR 5546 UPS-CNRS, Pôle de Biotechnologie Végétale, 24 Chemin de Borde-Rouge, BP17, Auzeville, F-31326 Castanet-Tolosan, France

View larger version (29K): [in a new window]   Fig. 1. Characterization of P.p.n. strains transformed with sense or antisense constructs of CBEL cDNA. (A) Plasmids used for transformation. HPH, coding sequences of the hygromycin phosphotransferase HPH gene; Phsp70, promoter of a HSP70 gene from Bremia lactucae; Tham34, terminator of the HAM34 gene from B. lactucae; B, BamHI; E, EcoRI; S, SmaI. (B) Molecular characterization of the transformants. (a) Southern blot analysis. In each lane, EcoR1-fragmented genomic DNA from the untransformed control strain (c), and from twelve strains transformed either with plasmid pTHEX11 (EX11, 1-7) or plasmid pTHEX3 (EX3, 1-5), were analyzed and hybridized with a radiolabeled CBEL cDNA probe. (b) Northern blot analysis. Total RNA from the same strains was electrophoresed, blotted and probed with a CBEL cDNA probe. The size of CBEL transcripts is 1.3 kb. Relative amounts of blotted RNA were determined by hybridization with an 18S rRNA probe. (c) Immunodetection of CBEL protein. Protein extracts from the untransformed control strain (c) and from four strains transformed either with plasmid pTHEX11 (EX11, 1 and 4) or plasmid pTHEX3) (EX3, 5 and 3) were subjected to western blot analysis using an anti-CBEL polyclonal antiserum. The Mr of CBEL is 34x103.

View larger version (17K): [in a new window]   Fig. 2. In vitro growth of CBEL+ and CBEL- strains. (A) EX11-1 (CBEL+) and EX11-4 (CBEL-) strains were grown at 25°C on synthetic liquid medium and the mycelium dry weight was determined. The means of two independent experiments are shown. (B) The untransformed (C) and transgenic (EX11-1, EX11-4, EX3-3, EX3-5) strains were grown at 25°C on a cellophane membrane placed on top of V-8 agar medium. Growth was determined by measuring the diameter of the colony. The data is the mean of three independent experiments±s.d.

View larger version (89K): [in a new window]   Fig. 3. Adhesion properties of mycelium from CBEL+ and CBEL- P.p.n. strains growing on a cellophane membrane. The mycelium was grown on a cellophane membrane placed on top of V-8 agar medium. The membrane together with the mycelium was removed from the medium underneath (A-E), cut in sectors of similar sizes, which were then incubated for 2 hours under shaking in water. The presence of remaining adherent mycelium was checked by light microscopy (F-J). (A,F) Untransformed control strain; (B,G) EX11-1 CBEL+ strain; (C,H) EX11-4 CBEL- strain; (D,I) EX3-5 CBEL+ strain; (E,J) EX3-3 CBEL- strain. Results representative of three independent experiments are shown. The mycelial aggregates are indicated by arrows. Note the absence of residual mycelium in H and J. Bars, 70 µm.

View larger version (159K): [in a new window]   Fig. 4. The differentiation of lobed structures in mycelium from CBEL+ and CBEL- P.p.n. strains in contact with a cellophane membrane. The mycelium was grown on a cellophane sheet placed on top of V-8 agar medium. The membrane with the mycelium was removed from the medium underneath, cut in sectors and observed under bright field microscopy. The whole membrane sector from each sample was screened, and typical data from triplicate samples are shown. (A,B) Lobed granular structures (arrows) from the EX11-1 (A) and EX3-5 (B) CBEL+ strains. The asterisk indicates an aggregate of intricate hyphae on a different focus plane. (C) Leakage of protoplasm (open arrow) from a damaged hyphae in the untransformed strain. (D) Hyphae from the EX11-4 CBEL- strain. Note the absence of lobed structures. Bars, 40 µm.

View larger version (134K): [in a new window]   Fig. 5. Development of germlings from the untransformed P.p.n. strain in contact with solid surfaces. Freshly prepared zoospores were induced to encyst by vortexing and spread onto various surfaces. (A) Development on a cellophane membrane placed on top of water agar medium. After 40 hours, the spherical encysted zoospore (c) has germinated to form a germ-tube (asterisks) and two appressoria (arrowheads). A lobed structure (arrow) has emerged from the distal appressorium and has developed in a plane slightly out of focus. Note that the cytoplasm has migrated out of the cyst. (B) Development on the polystyrene surface of a Petri dish. After 20 hours, the encysted zoospore (c) produced a series of appressoria (arrowheads). No further development of different structures was observed after longer incubation. UC, ungerminated cyst. Bars, 10 µm.

View larger version (168K): [in a new window]   Fig. 6. The differentiation of lobed structures in mycelium from CBEL+ and CBEL- P.p.n. strains in contact with cellulosic substrates. The mycelium was grown on a cellophane membrane (A,B) or on flax cellulose fibres dispersed onto a cellophane membrane (C,D) placed on top of V-8 agar medium. After 7 days of culture, the cellophane sheet was removed from the medium underneath and processed for scanning electron microscopy. (A,C) Aggregates of intricate ramified hyphae (arrows) were observed in the EX11-1 CBEL+ strain grown on cellophane or flax fibres. (B,D) The EX11-4 CBEL- strain formed only smooth hyphae that rarely branched on either substrate. Asterisks indicate the flax cellulose fibres. Bars, 40 µm.

View larger version (83K): [in a new window]   Fig. 7. Cell wall thickenings in mycelium from P.p.n. strains. (A-D) Confocal laser scanning microscopy of Congo Red-stained EX11-1 CBEL+ (A,C) or EX11-4 CBEL- (B,D) hyphae grown either on a cellophane (A,B) or a polycarbonate membrane (C,D) placed on top of V-8 agar medium. Note the presence of patches of Congo Red-stained material (arrows) along the CBEL- EX11-4 hyphae grown on either substrate. Image acquisition parameters were adjusted to optimize contrast in images C and D. Bars, 40 µm. (E-G) Transmission electron microscopy of PATAg-stained hyphae grown on a cellophane membrane placed on top of V-8 agar medium. (E) Transverse section of a hypha from the EX11-1 CBEL+ strain. The PATAg-stained cell wall appears as an electron dense layer (asterisk) and the plasmalemma-cell wall interface is strongly labelled (arrow). (F,G) Oblique sections of EX11-4 (F) or EX3-3 (G) CBEL- hyphae. Note the PATAg-stained papillae-like paramural deposits (P) between the cell wall (asterisk) and the plasma membrane (arrow). Bars, 1.5 µm.

View larger version (62K): [in a new window]   Fig. 8. Hyphal aggregates differentiation of mycelium from CBEL+ and CBEL- P.p.n. strains in contact with susceptible tobacco roots. The untransformed (A) and EX11-4 CBEL- strain (B,C) were observed under bright-field microscopy either after rosazurin staining (A,B) or directly in the inoculation assay Petri dish (C). Arrows, hyphal aggregates; arrowheads, appressoria. Bars, 40 µm.

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