Fig. 6.

GgsA is localized to punctuate organelles. a Expression analysis of GgsA-EGFP and EGFP-GgsA strains. RT-PCR analysis of total RNA isolated from 6 day old mycelia of P. paxilli wild-type derivative strains PN2555 and PN2549 containing the fusion constructs pSS29 (GgsA-EGFP) and pSS30 (EGFP-GgsA), respectively. These strains were confirmed by Southern to contain the respective fused genes. Numbers on the right correspond to the fragment sizes indicated in kb. b GgsA-EGFP fusion protein localizes to punctuate organelles. P. paxilli wild-type derivative strain PN2555 containing the fusion construct pSS29 (GgsA-EGFP) was grown on PD agar at 22°C for 2 days. Bright Field (BF) and EGFP fluorescence images of the mycelium are shown. c GgsA-EGFP fusion protein does not co-localize with DsRed-SKL fusion. P. paxilli wild-type derivative strain PN2575 containing both the fusion constructs pSS29 (GgsA-EGFP) and pSS41 (DsRed-SKL) was grown on PD agar at 22°C for 2 days. EGFP and DsRed fluorescence images, and merged images of EGFP and DsRed images of the mycelium are shown. Organelles containing the GgsA-EGFP fusion protein are not stained with MitoTracker (d), Hoechst (e) and FM 4-64 (f). P. paxilli wild-type derivative strain PN2555 containing the fusion construct pSS29 (GgsA-EGFP) was grown in PD broth at 22°C with shaking at 150 rpm for 2 days. Mycelia were than stained with 0.1 mM MitoTracker^® Red CMXRos or 10 mM FM 4-64 for 30 min in the same growth medium. Bright Field (BF), EGFP fluorescence, MitoTracker and FM 4-64 staining images, and merged images of EGFP and MitoTracker staining images and EGFP and FM 4-64 staining images of the mycelium are shown. All the reporter gene constructs are under the control of the A. pullulans TEF promoter and A. nidulans trpC terminator. Bars = 10 μM