Journal: bioRxiv

Article Title: Expansion and optimization of the auxin-inducible degron 2 (AID2) system in Candida pathogens

doi: 10.64898/2026.03.27.714890

Figure Lengend Snippet: Plasmid constructs for ( A ) integration of OsTIR1 F74A -3Myc expression cassette, ( B ) integration of the AID* degron and epitope tags at the 3’ end of target genes, ( C ) 3’ integration of AID* with mNeonGreen fluorescent protein tag, ( D ) integration of 3V5-AID* at the 5’ end of genes for N-terminal tagging with natural promoter control, and ( E ) integration of composite tagging cassettes at the 3’ end of target genes. In (D), pHLP907 contains the promoter region for PPH21 ; users will replace this feature with the equivalent promoter regions from other target genes. ( F ) The structure of the final genomic loci resulting from use of the new N-terminal tagging and composite cassettes shown in (D) and (E). Variable features in each series are indicated outside the circular map (see for complete list of plasmids). In each map, red arrows indicate the position of PCR primers for integration cassette amplification (see Table S1 for PCR primer sequences). bla, β-lactamase (encoding ampicillin resistance in E. coli ); cat, chloramphenicol acetyltransferase (encoding chloramphenicol resistance in E. coli ); ‘ori’ - E. coli origin of replication; ‘t’ - transcriptional terminator; green arrows with labels ‘p xxx ’ are promoters; CDS – coding sequence; SAT1, CaKan, CaHyB – Codon-optimized Candida selection markers encoding nourseothricin, G418, and hygromycin B resistance, respectively; FLP – coding sequence for Flp recombinase that recognizes FRT direct repeats.

Article Snippet: HGLA100 was generated using an OsTIR1 F74A -3Myc base strain kindly provided by Dr. Scott Briggs (Purdue University) and gene tagging reagents described previously ( ).

Techniques: Plasmid Preparation, Construct, Expressing, Control, Amplification, Sequencing, Selection

Journal: bioRxiv

Article Title: Expansion and optimization of the auxin-inducible degron 2 (AID2) system in Candida pathogens

doi: 10.64898/2026.03.27.714890

Figure Lengend Snippet: (A) Cdc14-AID*-3HA (HCAL196), Glc7-AID*-3HA (HCAL184), and 3V5-AID*-Pph21 (HCAL232) degradation in cells expressing OsTir1 F74A -3Myc was measured by anti-HA or anti-V5 immunoblotting after treatment of log phase liquid YPD cultures with the indicated 5-Ad-IAA concentrations for 60 minutes. The load control was PSTAIR (Cdc28). (B) . Dose–response curves were generated from experiments represented in (A) by quantifying percent protein remaining relative to the untreated culture using a digital imager. EC₅₀ values for Cdc14 (2 nM), Glc7 (0.5 nM), and Pph21 (2 nM) were calculated by fitting data with a dose response function. (C) Kinetics of Cdc14-AID*-3HA (HCAL196), Glc7-AID*-3HA (HCAL184), and 3V5-AID*-Pph21 (HCAL232) degradation after treatment with 50 nM 5-Ad-IAA were measured by anti-HA or anti-V5 immunoblotting as in (A). (D) The kinetic assays represented in (C) were quantified as described in (B) and half-life values for Cdc14 (3 minutes), Glc7 (4 minutes), and Pph21 (5 minutes) determined by fitting data with a single phase exponential decay function. In (B) and (D) data points represent means of 2 independent experiments with bars indicating range (min-max).

Article Snippet: HGLA100 was generated using an OsTIR1 F74A -3Myc base strain kindly provided by Dr. Scott Briggs (Purdue University) and gene tagging reagents described previously ( ).

Techniques: Expressing, Western Blot, Control, Generated

Journal: bioRxiv

Article Title: Expansion and optimization of the auxin-inducible degron 2 (AID2) system in Candida pathogens

doi: 10.64898/2026.03.27.714890

Figure Lengend Snippet: (A) Growth kinetics of wild-type (SC5314) and GLC7-AID*-3HA strains with (HCAL 184) or without (HCAL185) integrated OsTIR1 F74A . Saturated YPD cultures were diluted to equal starting densities in the presence or absence of 100 nM 5-Ad-IAA, and optical density at 600 nm (OD 600 ) measured over time. Graphs were generated from mean values of 3 independent cultures. (B) Patch assay of C. albicans SC5314 and GLC7-AID*-3HA with and without OsTIR1 F74A -3Myc (HCAL184 and HCAL185, respectively) was performed on YPD agar with or without 100 nM 5-Ad-IAA. Plates were imaged after incubation at 30 °C for 1 day. (C) Growth kinetics of C. albicans SC5314 and 3V5-AID*-PPH21 (HCAL 232) as in (A). (D) Bright field images of SC5314 and HCAL232 log phase YPD cultures with either 100 nM 5-Ad-IAA or mock treatment for 3.5 hours at 30 °C. (E) SC5314 and HCAL232 were grown on YPD agar with or without 100 nM 5-Ad-IAA and colonies imaged after 2 days at 30 °C. Scale bar = 1 mm. (F) SC5314 and HCAL232 were grown in embedded YPS agar at 30°C for 2 days in the presence or absence of 100 nM 5-Ad-IAA prior to imaging. Scale bars (0.5 mm) in (E) and (F) indicate identical fields of view in all panels.

Article Snippet: HGLA100 was generated using an OsTIR1 F74A -3Myc base strain kindly provided by Dr. Scott Briggs (Purdue University) and gene tagging reagents described previously ( ).

Techniques: Generated, Incubation, Imaging

Journal: bioRxiv

Article Title: Expansion and optimization of the auxin-inducible degron 2 (AID2) system in Candida pathogens

doi: 10.64898/2026.03.27.714890

Figure Lengend Snippet: (A) Cdc14-AID*-9Myc degradation in C. glabrata HGLA100 was measured by anti-Myc immunoblotting after treatment of log phase 30 °C YPD cultures with the indicated 5-Ad-IAA or 5-Ph-IAA concentrations for 60 minutes. (B) Percent protein remaining relative to the untreated culture from experiments represented in (A), using anti-PSTAIR as a loading control, was plotted and fit with a dose response function in Graphpad Prism to calculate EC 50 (5-Ad-IAA = 11 nM; 5-Ph-IAA = 521 nM). Data points are means of 2 independent measurements and bars denote the range (min-max). (C) Liquid cultures of HGLA100 and the parent strain CBS138 were diluted to equal starting densities in YPD in a 96-well microplate, supplemented with 500 nM 5-Ad-IAA or 5-Ph-IAA or solvent alone and grown at 30°C with shaking for 24 hours in a microplate spectrophotometer measuring OD 600 . (D) Agar patch assay with independent HGLA100 isolates 1 and 2 and the CBS138 parent strain on YPD and YPD supplemented with 500 nM 5-Ad-IAA or 5-Ph-IAA. Plates were incubated at 30°C for 72 hours prior to imaging. (E-F) Cdc14-AID*-3HA (E) or Glc7-AID*-3HA (F) degradation was measured by immunoblotting after treatment of log phase C. albicans HCAL196 or HCAL184 YPD cultures with the indicated 5-Ph-IAA concentrations for 60 minutes. Data were quantified and plotted as in panels (A-B) to determine EC 50 (Cdc14 = 25 nM; Glc7 = 2 nM). The experiment is identical to that in , and the values for 5-Ad-IAA-treated cultures are replicated from for comparison. (G) Growth assays with HCAL184 cultures supplemented with 100 nM 5-Ad-IAA or 5-Ph-IAA or solvent alone as described for panel (C). (H-I) Glc7-AID*-3HA degradation in C. albicans HCAL203 expressing the OsTir1 F74G -3Myc variant was measured by anti-HA immunoblotting after treatment with the indicated concentrations of 5-Ad-IAA (H) or 5-Ph-IAA (I). Data were processed and quantified as described in panels (A-B) and compared to identical data for HCAL184 expressing OsTir1 F74A -3Myc duplicated from panel (F). (J) OsTir1 F74A -3Myc or OsTir1 F74G -3Myc expression in samples from experiments in panels (H-I) was compared by anti-Myc immunoblotting, with anti-PSTAIR (Cdc28) as a load control.

Article Snippet: HGLA100 was generated using an OsTIR1 F74A -3Myc base strain kindly provided by Dr. Scott Briggs (Purdue University) and gene tagging reagents described previously ( ).

Techniques: Western Blot, Control, Solvent, Spectrophotometry, Incubation, Imaging, Comparison, Expressing, Variant Assay