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Supplementary Table 1 Average colony growth rate of the wild type, ∆ncs-1, ncs-1G2A, ncs-1E120Q, and ncs-1R175A mutant strains at various concentrations of CaCl2
Strains / Average colony growth rate (cm/h) at various concentrations of CaCl2 (M)0 / 0.2 / 0.3 / 0.4
Wild type / 0.363±0.007 / 0.337±0.018 / 0.295±0.018 / 0.251±0.016
∆ncs-1 / 0.260±0.006 / 0.156±0.004 / 0.080±0.010 / 0.020±0.017
ncs-1G2A hop / 0.500±0.012 / 0.395±0.008 / 0.339±0.005 / 0.279±0.008
ncs-1E120Q hop / 0.274±0.018 / 0.163±0.049 / 0.102±0.020 / 0.047±0.005
ncs-1R175A hop / 0.285±0.011 / 0.156±0.012 / 0.080±0.005 / 0.020±0.005
Supplementary Table2 Average colony growth rate of the wild type, ∆ncs-1,ncs-1Rat,ncs-1Rat(E120Q)homokaryotic transformant, and Rat ncs-1E120Qmutant strains at various concentrations of CaCl2
Strains / Average colonygrowth rate (cm/h) at various concentrations of CaCl2 (M)0 / 0.2 / 0.3 / 0.4
Wild type / 0.339±0.006 / 0.320±0.002 / 0. 239±0.002 / 0. 123±0.002
∆ncs-1 / 0.280±0.006 / 0.160±0.002 / 0.090±0.001 / 0.034±0.002
ncs-1Rat hop / 0.314±0.001 / 0.268±0.002 / 0.204±0.002 / 0.110±0.008
ncs-1Rat (E120Q) hop / 0.278±0.002 / 0.161±0.002 / 0.095±0.001 / 0.035±0.002
Supplementary Fig.1Cloning of the ncs-1 from the N. crassausing double joint PCR for site-directed mutational analysis. (A)Amplification of individual fragments. PCR products were resolved in 0.8% agarose gel; lane 1, 1 kb DNA ladder (NEB);lane 2, PCR product of size ~3.992 kb that contains the ncs-1 gene (using primers RD-NCS-1-5F and NCU04379-3R);lane 3, PCR product of the 5′ flank of size ~1.587 kb of the ncs-1 gene (using primers 7NCS-1F and 8NCS-1R); lane 4, PCR product of the selectable marker bar cassette of ~1.2 kb in size (using primers BAR-FW and BAR-RV). (B)Double joint PCR for joining the individual fragments. PCR products were resolved in 0.8% agarose gel; lane 1, 1 kb DNA ladder (NEB); lanes 2 and 3, PCR using primers 7NCS-1F and NCU04379-3Rfor the full length construct of ~6.779 kb generated after joining all the three individual fragments; lane 4, PCR amplicon (control)generated by joining the bar cassette and the 5′ flank (~2.7 kb); lane 5, PCR amplicon (control) of the functional ncs-1 gene (~3.992 kb); lane 6, pRS426 vector (~5.7 kb)digested with the SmaIrestriction enzyme.
Supplementary Fig. 2PCR verification of the ∆mus-53∆ncs-1 a (15) strain transformed with the plasmid construct pRD-2. PCR products were resolved in 0.8% agarose gel; lane 1, 1 kb DNA ladder (NEB); lanes 2 and 3, PCR product (~2.8 kb) obtained using the template DNA from a heterokaryotic transformant HT-1-∆mus-53 ∆ncs-1a (15)and a homokaryotic transformant HoP-3-a (generated from a cross of HT-1-∆mus-53 ∆ncs-1a (15)with ncs-1 A), respectively, and primers HI-ncs-1-F and BAR-RV. The BAR-RV reverse primer is specific for the bar cassette. HI-ncs-1-F forward primer, which is ~100 bp upstream of the 5’ flank of the ncs-1 gene, was used with the BAR-RV primer to verify the homologous integration of the final assembled ncs-1::bar construct(6.779kb) cloned in the pRD-2 vector.
Supplementary Fig. 3PCR amplification of the N. crassahomokaryotic strainstransformed with the plasmid construct pDG-5. (A) ncs-1RatcDNA fragment of length 570 bp was amplified using the primers Rat ncs-1-Fand Rat ncs-1-R,and resolved in 1.2% agarose gel;lane 1, 100 bpDNA ladder (NEB); lanes 2, 4,and 5,PCR amplification of 570 bp of ncs-1RatcDNA in pDG-5 construct (control amplicon), and two homokaryotic strains RB10-7 and RB10-14 (Table 1), respectively; and lane 3,absence of amplification (control) for the wild type strain.(B) The hphfragment of length 2.89 kb was amplified using the primers HI-ncs-1-F and 5hph-Rv,and resolved in 0.8% agarose gel.Lane 1, 1 kb DNA ladder (NEB);lanes 2, 4,and 5,PCR amplification of 2.89 kb of the hph cassette in ∆ncs-1 strain (control amplicon), RB10-7 and RB10-14 strains (Table 1), respectively; and lane 3,absence of amplicon (control) for the wildtype.
SupplementaryFig. 4 PCR amplification of the N. crassa homokaryotic strains transformed with the plasmid construct pDG-5E120Q. (A) ncs-1RatcDNA fragment of length 570 bp was amplified using primers Rat ncs-1-F and Rat ncs-1-R, and resolved in 1.2% agarose gel;lane 1, 100 bp DNA ladder (NEB); lanes 2, 4, and 5,PCR amplification of 570 bp of ncs-1RatcDNA in pDG5 construct (control amplicon), and in the E5-28 and E5-3 strains (Table 1),respectively; and lane 3, absence of amplicon (control) for the wild type strain. (B) The hphfragment of length 2.89 kb was amplified using primers HI-ncs-1-F and 5hph-Rv, and resolved in 0.8% agarose gel. Lane 1, 1 kb DNA ladder (NEB); lanes 2, 4 and 5, PCR amplification of 2.89 kb of hph cassette in the ∆ncs-1 (control amplification), E5-28, and E5-3 strains, respectively; and lane 3,absence of amplicon(control) for the wildtype.