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First published online 22 December 2004
doi: 10.1242/jcs.01616

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Functional analysis of CLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogen Colletotrichum lindemuthianum

¹ UMR 5546 CNRS-Université Paul Sabatier
² IFR 40 Pôle de Biotechnologie Végétale, 24 Chemin de Borde Rouge, BP17 Auzeville, 31326 Castanet-Tolosan, France

View larger version (48K): [in a new window]   Fig. 1. Transcriptional regulation of the GFP gene fused to the PG2-90 promoter fragment. Total RNA was extracted from a PG2-90::GFP transgenic strain (Herbert et al., 2002) grown on glucose or pectin medium for the time indicated. Northern blot analysis was performed using a CLPG2 or a GFP probe. Equal loading of the RNAs (16S) on to the membrane was checked by staining with methylene blue.

View larger version (91K): [in a new window]   Fig. 2. Characterization of C. lindemuthianum strains transformed with PG2-90::CLPT1 or PG2-90::CLPT1(N123I) constructs. (A) Northern blot analysis of total RNA extracted from the different strains grown on pectin medium and probed with CLPT1 coding sequence. The band from the wild-type gene is indicated by the arrowhead. (B) Detection by western blot analysis of CLPT1 from wild-type and transgenic strains. Protein extracts were prepared from the mycelium grown for 4 days on glucose or pectin medium as indicated. Equal amounts of protein were loaded onto the gel. CLPT1 was revealed using purified anti-MBP-CLPT1 antibodies and anti-rabbit IgG labelled with alkaline phosphatase as secondary antibodies.

View larger version (18K): [in a new window]   Fig. 3. Effect of CLPT1 and CLPT1(N123I) on pectinase secretion. The mycelium was grown for 4 days on glucose medium before being transferred to pectin medium. Extracellular polygalacturonase activity produced by the different strains was measured using polygalacturonic acid as substrate. The data are the mean of three independent experiments. , wild-type, , transgenic control strain (empty vector); , CLPT1.4; x, N123I.6; , N123I. 9.

View larger version (90K): [in a new window]   Fig. 4. Cell wall thickenings induced by CLPT1(N123I). Confocal laser scanning microscopy of Congo Red-stained hyphae from wild type (WT), CLPT1- (CLPT1.9) or CLPT1(N123I) (N123I.6) expressing strains grown on a cellophane membrane placed on pectin medium. Each image is a projection of 10-15 confocal planes acquired in the z dimension. Note the accumulation of numerous Congo Red stained spots (white arrows) along the hyphal cell wall of N123I.6. Bars, 5 µm.

View larger version (106K): [in a new window]   Fig. 5. Effect of CLPT1(N123I) on ultrastructural morphology. Conidia from the wild-type strain or the transgenic strains were allowed to germinate on a cellophane membrane placed on top of a solid medium containing glucose. After 24 hours of growth, the filters were transferred to a fresh medium containing either glucose (A-C,G,H) or pectin (D-F,I,J) as sole carbon sources. Micrographs of ultrathin sections of hyphae stained with (A-F) uranyl acetate and lead citrate or (G-J) PATAg. Arrowheads indicate the vesicle-like material; white arrows, the large spots accumulated within the hyphae of the CLPT1 (N123I)-overexpressing strains grown on pectin. Bars, 1 µm.

View larger version (51K): [in a new window]   Fig. 6. Development of infection structures and pathogenicity of transgenic strains expressing CLPT1(N123I). (A) Conidia (Co) of C. lindemuthianum wild-type (WT) and CLPT1(N123I) strains (N123I.6 and N123I.9) germinating on glass slides to form appressoria (Ap). Note the abnormal development of appressoria in the N123I strains. (B) Bean leaves were inoculated with a suspension of conidia from the wild-type strain (WT) or from the CLPT1(N123I) strains (N123I.6; N123I.9). (C) Details of anthracnose lesions produced by the different strains. (D) Microscopic examination of infected bean tissues. The wild-type strain (WT) develops an appressorium (Ap) that penetrates a hypocotyl epidermal cell and differentiates an infection vesicle (IV) 24 hours after inoculation. The N123I.6 conidia germinate, fail to penetrate host cells and grow saprophytically on the surface of the plant tissue.

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