## 2 Materials and Methods
### Strain Construction  
This work used the TRACE (Transient CRISPR-Cas9 Coupled with Electroporation) method to create a \(cnb1\Delta::\)_NeoR_ mutation in the wild-type strain H99, as previously published [23; 24]. Briefly, pYF24 was used as a template to amplify the Cas9 endonuclease gene under the control of the \(GPD1\) promoter by PCR. A 9 Kb band was purified by QiaQuick (Qiagen, Hilden, Germany). EuPaGDT (Eukaryotic Pathogen CRISPR guide RNA/DNA Design Tool) embedded in the FungiDB site was used to design the gRNA target site ([[http://gna.ctegd.uga.edu/](http://gna.ctegd.uga.edu/)]([http://gna.ctegd.uga.edu/](http://gna.ctegd.uga.edu/)) accessed on 25 January 2021). The target site built was 20 nucleotides long, excluding the NGG sequence, to avoid self-cleavage of the gRNA. The nucleotide sequence selected as the target site was fused at its 3\({}^{\prime}\) end to the sequences of the guide RNA scaffold to create the forward primer. A reverse primer for gRNA was also created, and the primer pair was used to amplify part of the guide RNA using the pDD162 (AddGene, Watertown, MA, USA) plasmid as the template [25]. The complementary sequence of the target site was incorporated into the reverse primer containing a sequence at its 3\({}^{\prime}\) end complementary to sequences of the U6 promoter, and the forward primer to amplify the U6 promoter was also designed. The primer pair was used to PCR amplify the U6 promoter using _C. neoformans_ strain JEC21 genomic DNA. A second PCR reaction, using the two PCR-amplified fragments as the template, was carried out to join the promoter and the gRNA sequence together using nested primers. This reaction generated a DNA fragment containing a 20-nucleotide target site fused to the guide DNA scaffold driven by the U6 promoter. This 300 bp fusion product was purified with QiaQuick. The \(cnb1\Delta::\)_NeoR_ deletion construct containing 500 bp of the promoter and terminator regions of the \(CNB1\) gene flanking the _NeoR_ resistance gene driven by the actin promoter was made by overlapping PCR, as previously described [26]. All three DNA fragments encoding the gRNA, Cas9, and deletion construct were purified, eluted in water, and introduced into the wild-type strain H99 by electroporation according to the protocol previously published [24]. Transformants resistant to G418 were subjected to 5 cycles of subculture in YEPD without antibiotics to eliminate transient transformants. Finally, the transformants were inoculated in YEPD supplemented with G418. They were also subjected to diagnostic PCR to identify homologous recombination at the \(CNB1\) locus. Among forty transformants, six were positive in the diagnostic PCR (15% of homologous integration) and were designated CNU160 to CNU165. All of them were temperature-sensitive at 37 \({}^{\circ}\)C, a marked phenotype of the calcineurin mutants. CNU160 was chosen for further analysis.  
Strains containing the _GFP-GPP2_ allele were constructed by PCR amplification of the _GPP2_ gene from the Start to Stop codon plus 300 bp of the terminator region. gDNA of the H99 wild-type strain was used as a template with PRCP472 and PRCP473 primers (Supplementary Information, Table S3). The band was excised from agarose gel, and the DNA was cloned into the pCN19 plasmid and digested with _Bam_HI and _Spe_I restriction enzymes. Gibson assembly (NEB) was used to reconstitute the circular plasmid containing the _GPP2_ gene. The vector contains the _GFP_ sequence driven by the Histone H3 promoter and the nonweothricin resistance gene as a selectable marker. The plasmid was sequenced to confirm translational fusion between _GFP_ and _GPP2_ sequences. After confirmation, the plasmid (pRCP115) was introduced into the wild-type strain H99 (CNU151) and into the mutant strains CNU160 (\(cnb1\Delta\)) and CNU166 (\(cna1\Delta\)), generating the strains CNU189 and CNU193, respectively.