2470 bp 1891 bp WT 1 2 3 1 2 3 4549 bp 2314 bp 1 2 3 4 A B Fig. S1. Verification with PCR amplification of the deletion mutant of pgα1. For the detailed description, please see the Materials and Methods. WT: wild type, M: marker ladder. The size of the bands was indicated.

Fig. S2. Alignment for the sequence of the 4549-bp PCR product that covered the deleted pgα1 locus against ura3 gene (panel A) and pgα1 (panel B). Solid lines are the identical parts of the compared sequences. This result verified the replacement at the pgα1 locus occurred as anticipated. Alignment was conducted with the Software Omega 2.0 (Oxford University).

WT Δpgα1 pgα-c WT Δpgα1 pgα-c pgα1 probe ura3 4906 bp 622 bp 3282 bp 7221 bp Fig. S3. Southern blotting for confirmation of the disruption of pgα1. Genomic DNA from the wild-type strain, Δpgα1 and complement strains were digested with Hind III. Left panel: the membrane was hybridized with pgα1 ORF sequence. Two bands were expected for the wild type 4906 bp and 622 bp (Lane 1), while 7221 bp and 622 bp were detected for the mutant (Lane 2), and in complementation strains, three bands were detected, 7221 bp, 3282 bp and 622 bp (Lane 3). Right panel: the same membrane was reprobed with ura3 sequence, no hybridization fragment was observed in wild type (Lane 1); only one band (7221 bp) was detected as expected in the transformants, and one band (3282 bp) was detected in the complementation strains (Δpgα1-c) (Lane 2 & 3).

Δpgα1-C M wt 1 2 3 4 5 Δpgα1 2.3 kb 4.5 kb A Fig. S4. PCR confirmatin shows that the wild-type copy of pgα1 has been successfully introduced into the complemented strain Δpgα1-C. A 2.3kb pgα1 fragment covering the whole length pgα1 including its ORF and promoter region and terminator region was transformed into Δpgα1. Gα1PT(s)/Gα1PT(as), gave rise to a 4.5-kb fragment in Δpgα1 due to its 2.2-kb ura3 marker, and a 2.3-kb fragment in the complements and the wild type. WT: wild type, M: marker ladder. The size of the bands was indicated. This PCR result confirmed again the 2.3-bp pgα1 fragment restored the pgα1 locus to the wild-type state. .

Pgα1-C B Fig. S4. PCR confirmation of the wild-type copy of pgα1 in the complemented strain Δpgα1-C .

hyg ura3 pgα1 pt Pm wt spores Pm[Pgα1] Pks-down Pks-up ΔPgα 1(ura3) spores Δpgα1pks1 Agrobacterium-mediated OSCAR method Fig. S5. Plasmid construction for the overexpression and double mutant strains.

Pm[Pgα1] WT M A B 3814 bp 5114 bp 2271 bp Fig. S6 A. PCR confirmation of the wild-type copy of pgα1 in the overexpression strain pm[Pgα1]. Panel A: a 3814-bp fragment was amplified with the pair of primers, Gα1PT(s)/hyg(as), in the four overexpression strains (pm[Pgα1]), while no band was obtained in the wild type strain (WT). Panel B shows a 5114-bp fragment was amplified in pm[Pgα1], with the primers Gα1PT(s)/ura3(as), but a 2271-bp fragment in the wild type. The results indicates that the overexpression strain acquired the wild-type copy of pgα1.

Wt 4d Wt 6d pm[Pgα1]4d 6d actin pgα1 pks1 Fig. S6. B. Transcriptional levels of pks1 and pgα1 in overexpression strain and wt strain were also examined by RT-PCR at 3 d and 6 d. The pgα1 gene was up-regulated in overexpression strain compared with that in wt strain, whereas expression levels of the core pks1 gene in overexpression strain was slightly less than wt strain in all cases, respectively.

Δpgα1pks1 WT Δpgα1pks1 WT Δpgα1pks1 A B 1639 bp 1911 bp Fig. S7. Verification with PCR amplification of the deletion mutant of pks1 in the pgα1 mutant strains with hyg marker. Uppks-hyg/hyg(s) and Downpks-hyg/hyg(as), produced a 1911-bp up band (Panel A) and a 1639-bp down band in Δpgα1pks1 (Panel B), while no band in the wild type. The PCR results indicates that the pks1 gene of pgα1 mutant strain was inserted by hygromycin resistance gene hph in the Δpgα1pks1 double mutant strains.

hyg ura3 hid1 pt ΔPgα1 (ura3) spores Δpgα1[Hid1] Agrobacterium-mediated OSCAR method OSCAR method hid1-up hyg hid1-down Agrobacterium-mediated ΔPgα1 (ura3) spores Δpgα1hid1 Fig. S8. Plasmid construction for the overexpression and double mutant strains.

Δpgα1hid1 WT Δpgα1hid1 Δpgα1hid1 WT 225 bp 1573 bp Δpgα1hid1 WT 2330 bp Fig. S9. Verification with PCR amplification of the deletion mutant of pgα1 in the hid1 mutant strains with hyg marker. Panel A : a 225-bp fragment was amplified in Δpgα1hid1, with the primers Gα-ins/Gα-inas, while no band was obtained in the wild type strain (WT). Panel B : Up-gα1/hyg(as) and Down-gα1/hyg(s), produced a 1573-bp up band (Left) and a 2330-bp down band in Δpgα1hid1 (Right), while no band in the wild type. The PCR results indicates that the pgα1 gene of hid1 mutant strain was inserted by hygromycin resistance gene hph in the Δpgα1hid1 double mutant strains.

Δpgα1[Hid1] WT Δpgα1[hid1] Δpgα1(hid1) WT WT 1 2 3 actin hid1 A B 6906 bp 4629 bp Fig. S10. PCR confirmation of the wild-type copy of hid1 in the overexpression strain Δpgα1[Hid1]. Panel A: Left: a 4629-bp fragment was amplified with the pair of primers, Hid1PT(s)/hyg(as), in the four overexpression strains (Δpgα1[Hid1]), while no band was obtained in the wild type strain (WT). Right: a 6906-bp fragment was amplified in Δpgα1[Hid1], with the primers Hid1PT(s)/ura3(as), while no band was obtained in the wild type strain (WT). Panel B shows Transcriptional levels of hid1 in overexpression strain and wt strain at 4 d. The pgα1 gene was up-regulated in overexpression strain compared with that in wt strain .The results indicates that the overexpression strain acquired the wild-type copy of hid1.

2 d 2.5 d 3 d 4 d 2 d 2.5 d 3 d 4 d pks1 act Δpgα1 WT Fig. S11. Pgα1 regulates pks1 expression. Transcriptional levels of pks1 and pgα1 were determined by RT-PCR at 2 d, 2.5 d, 3 d and 4 d.

mutant WT M mutant WT Δpgα2 a Δpgα3 Δpgα3 Δ pgα2 Δpgα1 A B b Fig. S12 Verifications with PCR amplification of the deletion mutants of pgα2 , pgα3. A.(a) gα2-up/Hyg (as) and gα2-down/Hyg (s), which generated approximately 2478 bp and 1803 bp fragments respectively, for the upstream and downstream of the integration site in the mutant Δpgα2, but no band for the wild type. (b) gα3-up/Hyg (as) and gα3-down/Hyg (s), generating approximately 2020 bp and 2579 bp fragments respectively, for the upstream and downstream of the integration site in the mutant Δpgα3, but no band for the wild type. W: wild type, M: marker ladder. The size of the bands was indicated. B. Disruption of 2 other types of 3 G-alphas genes, pgα2 and pgα3 showed no apparent phenotype, comparing with the wild type.