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First published online 12 February 2003
doi: 10.1242/jcs.00319

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Control of morphogenesis and actin localization by the Penicillium marneffei RAC homolog

Department of Genetics, University of Melbourne, Victoria 3010, Australia

View larger version (87K): [in a new window]   Fig. 1. The cflB gene is localized to the plasma and internal membranes, and co-localizes with actin. The gpdA(p)gfp::cflB strains were grown for 2 days at 25°C. CflB visualized in live cells (A,B) and in fixed cells by immunocytochemistry (C,D). (A) Apical hyphal cells. The GFP::CflB fusion protein is not present in large amounts at the hyphal apex. (B) Subapical hyphal cells. The GFP::CflB fusion protein is localized at the plasma membrane, at internal membranes and at septation sites. The single white arrows indicate the GFP::CflB fusion protein localized at septa. (C) CflB co-localizes with actin at the hyphal apex. Actin is concentrated at sites of polarized growth, such as the hyphal apex, indicated by the white arrow (anti-actin). The GFP::CflB fusion protein co-localizes with actin at the hyphal apex, shown by the white arrow (anti-GFP). (D) CflB co-localizes with actin at nascent septation sites. Actin is localized at nascent septation sites, indicated by white arrows (anti-actin). The white arrows under anti-GFP and CAL staining indicate the GFP::CflB fusion protein, which also localizes at nascent septation sites, and the corresponding cell walls, respectively. Scale bars, 20 µm.

View larger version (49K): [in a new window]   Fig. 2. The cflB gene is expressed as three transcripts at both 25°C and 37°C. (A) Northern-blot analysis of cflB expression. Total RNA was isolated from vegetatively growing hyphae at 25°C (veg.), asexually developing cultures at 25°C (dev.) and yeast cells (37°C). Northern blots were probed with cflB and a histone H3 probe as a RNA loading control. Arrows indicate the three cflB transcripts, and the histone H3 is marked. (B) Northern-blot analysis using total RNA isolated during the dimorphic switching from yeast to hyphae. Yeast cells produced at 37°C were transferred to 25°C and total RNA isolated at 6 hours, 18 hours and 24 hours after transfer. Northern blots were probed with cflB and a histone H3 probe as an RNA loading control. Arrows indicate the three cflB transcripts, and the histone H3 is marked. The relative levels of the three cflB transcripts are shown. Transcript levels were determined by densitometry and normalized relative to the H3 loading control.

View larger version (75K): [in a new window]   Fig. 3. Colonial morphology of P. marneffei cflB strains. (A) Colonial morphology of the P. marneffei cflB strain compared with the parental SPM4 control strain (cflB+). Strains were grown at 25°C for 14 days. The cflB colonies have a fluffy phenotype with reduced conidiation. At higher magnification (2x), no conidiophore structures were evident in the cflB strain. (B) Colonial morphology of P. marneffei cflB mutant transformants. Strains were grown for 12 days at 25°C on noninducing (Nonind.) or inducing medium (Induced). The alcA(p)::cflBD123A dominant-negative transformants displayed reduced conidiation and an increase in the number of aerial hyphae under inducing conditions and the alcA(p)::cflBG18V dominant-activated transformants displayed patchy conidiation that is more apparent when viewed under higher magnification (2x).

View larger version (61K): [in a new window]   Fig. 4. P. marneffei cflB controls polarity during asexual development at 25°C. Strains were grown for 4 days at 25°C (cflB+ and cflB::pyrG+) or induced by 2 days of growth followed by induction for an additional day [alcA(p)::cflBD123A and alcA(p)::cflBG18V]. The arrowheads indicate conidiophore structures (c, conidia; m, metulae; p, phialides). The cflB strain produces aberrant conidiophores with swollen cells, which prevents the identification of the various conidiophore cell types. The alcA(p)::cflBD123A dominant-negative and alcA(p)::cflBG18V dominant-activated transformants also produce aberrant conidiophores. A singular swollen terminal conidium was observed in which there were multiple nuclei. Scale bar, 20 µm. Images were captured using DIC or DAPI (to observe nuclei).

View larger version (111K): [in a new window]   Fig. 5. The cflB gene affects asexual development in A. nidulans. (A) Colonial morphology of A. nidulans cflB strains. Strains were grown for 2 days at 25°C on noninducing (nonind.) or inducing (induced) medium. The alcA(p)::cflB, alcA(p)::cflBD123A dominant-negative and alcA(p)::cflBG18V dominant-activated transformants displayed a decrease in conidiation under inducing conditions that was more severe in the transformants with dominant alleles. (B) Strains were induced during conidiation by growth for 1 day at 25°C and induction for an additional day. The arrowheads indicate conidiophore structures (c, conidia; m, metulae; p, phialides; v, vesicle). The alcA(p)::cflB+, alcA(p)::cflBD123A and alcA(p)::cflBG18V transformants showed conidiophores with inappropriate polarized growth where metulae and phialides could be greatly extended. Scale bars, 20 µm. Images were captured using DIC or DAPI (to observe nuclei).

View larger version (58K): [in a new window]   Fig. 6. Loss of cflB leads to an increase in apical branching in vegetative hyphal cells. Strains were grown for 2 days at 25°C (cflB+ and cflB::pyrG+) or for 1 day and induced for an additional day [alcA(p)::cflBD123A and alcA(p)::cflBG18V]. Apical hyphal cells in the wild type are not branched. The cflB and alcA(p)::cflBD123A dominant-negative strains showed hyperbranched apical hyphal cells. The alcA(p)::cflBG18V dominant-activated transformants possessed unbranched apical cells that displayed slightly irregular morphology. Scale bars, 20 µm. Images were captured using DIC or DAPI (to observe nuclei).

View larger version (44K): [in a new window]   Fig. 7. cflB is necessary for polarized growth of vegetative hyphal cells. Strains were grown for 4 days at 25°C (cflB+ and cflB::pyrG+) or for 2 days and induced for an additional day [alcA(p)::cflBD123A and alcA(p)::cflBG18V]. The cflB strain displays extremely swollen hyphal cells that can be anucleate, uninucleate or multinucleate. The alcA(p)::cflBD123A dominant-negative and alcA(p)::cflBG18V dominant-activated transformants also possess swollen hyphal cells but depolarized to a lesser extent than cflB. Hyphal cells can also be anucleate, uninucleate or multinucleate. Single arrowheads indicate anucleate hyphal cells and double arrowheads indicate multinucleate hyphal cells. Scale bars, 20 µm. Images were captured using DIC or DAPI (to observe nuclei).

View larger version (47K): [in a new window]   Fig. 8. The cflB gene is not required for correct actin localization in arthroconidiating hyphae and in yeast cells. Wild-type and cflB strains were grown for 4 days at 37°C. Actin localization was examined using immunocytochemistry. (A) Colonial morphology at 37°C. Colonies of the cflB strain are reduced in size and possess irregular edges. (B) Arthroconidiating hyphae are shown at 37°C prior to cell separation. The cflB strain possesses swollen arthroconidiating hyphae that are highly septate. The white arrows indicate concentrated actin in both wild-type and cflB strains. (C) Yeast cells at 37°C. The single white arrow indicates actin concentrated in a yeast cell of both the wildtype and the cflB strain. (D) Nascent septation sites in vegetative hyphae at 37°C. Two regions of concentrated actin are indicated by single white arrows in the FITC panel. The corresponding cell wall is indicated by a single arrow in the CAL panel. Double arrows indicate completed double septation sites. (E) Magnification of the actin staining regions marked in D, where the arrows indicate the two regions of concentrated actin. Scale bars, 20 µm.

View larger version (68K): [in a new window]   Fig. 9. The cflB gene is necessary for correct actin localization in vegetative hyphal cells. Strains were grown for 4 days at 25°C. Actin localization was examined using immunocytochemistry. Actin is localized as cortical spots and is also concentrated at sites of polarized growth. (A) Vegetative hyphal growth at 25°C. The white arrow indicates concentrated actin at the hyphal apex, which is absent in the cflB strain. (B) Asexual development at 25°C. The single white arrow indicates actin concentrated in the newly formed conidium, the double white arrow indicates actin concentrated at the distal tip of a phialide. Conidiophore cell types are indicated (c, conidia; m, metulae; p, phialides). The cflB strain lacks actin concentrated in conidiophores. (C) Nascent septation sites in vegetative hyphae at 25°C. Concentrated actin is indicated by single white arrows (FITC panel) and double arrows indicate corresponding cell walls (CAL panel). Scale bars, 20 µm.