Fig. 4.

The PaxG C-terminus tripeptide GRV is a peroxisomal targeting sequence. a Expression analysis of PaxG-EGFP, EGFP-N368PaxG and EGFP-GRV strains. RT-PCR analysis of total RNA isolated from 6 day old mycelia of P. paxilli wild-type derivative strains PN2559, PN2731 and PN2564 containing the fusion constructs pSS27 (PaxG-EGFP), pSS54 (EGFP-N368PaxG) and pSS46 (EGFP-GRV), respectively. These strains were confirmed by Southern to contain the respective chimeric genes. Numbers on the right correspond to the fragment sizes indicated in kb. b EGFP-GRV fusion protein is localized to punctuate organelles. P. paxilli wild-type derivative strain PN2564 containing the fusion construct pSS46 (EGFP-GRV) was grown on PD agar at 22°C for 2 days. Bright Field (BF) and EGFP fluorescence images of the mycelium are shown. c EGFP-GRV fusion co-localizes with DsRed-SKL fusion in punctuate organelles (peroxisomes). P. paxilli wild-type derivative strain PN2570 containing both the fusion constructs pSS41 (DsRed-SKL) and pSS46 (EGFP-GRV) was grown on PD agar at 22°C for 2 days. Bright Field (BF), EGFP and DsRed fluorescence images, and merged images of EGFP and DsRed images of the mycelium are shown. d Oleic acid induces proliferation of the punctuate organelles (peroxisomes) containing the EGFP-GRV or DsRed-SKL fusions. P. paxilli wild-type derivative strains PN2564 and PN2566 containing the fusion constructs pSS46 (EGFP-GRV) and pSS41 (DsRed-SKL), respectively, were grown on PD agar or PD agar + 5 mM oleic acid at 22°C for 2 days. Bright Field (BF), EGFP and DsRed fluorescence images of mycelia 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