Every strain used in this work derives from the natural isolate M. lusitanicus CBS277.49. The parental strain of all the mutant strains is the leucine and uridine double auxotroph MU402 (Ura-, Leu-). This strain was used for clr4 deletion mutant transformation following a CRISPR-Cas9 procedure as previously described (28 , 82 (link)). Briefly, we designed two guide RNAs (gRNA) to generate breaks upstream and downstream of the reannotated clr4 locus (gRNA_clr4_5′: CTCCTGGTGACTGGTGAAAGTGG, and gRNA_clr4_3′: GGGTTTTCATTGGCCGTGTCTCC), and a linear DNA construct containing the selectable marker pyrG flanked by 1-kb upstream and downstream regions of the gRNA cleavage sites (SI Appendix, Table S4). Ribonucleoprotein complexes with Cas9 and gRNAs were assembled in vitro following the supplier instructions (Alt-R™ CRISPR Custom Guide RNAs and Cas9 enzyme, Integrated DNA Technologies). The DNA cassette and the ribonucleoprotein complexes were electroporated into Mucor protoplasts. After transformation, colonies were plated on selective minimal medium with casamino acids (MMC) at pH of 3.2 to positively select prototrophic colonies. DNA deletion allele integration and heterokaryosis was assessed by PCR using primers that generate discriminatory amplicons (SI Appendix, Table S4), as described previously (28 ). At least 10 vegetative passages were conducted in an attempt to achieve homokaryotic mutants, consisting of collecting spores from a single asexual sporangium and subsequent plating in MMC medium. Lethality of the clr4Δ allele was assessed by dissecting spores from a heterokaryotic mutant onto rich, nonselective yeast-peptone-dextrose (YPD) medium, and analyzing the clr4 locus as described above. RNAi mutants analyzed in this study include single Dicer mutant dcl1Δ (strain MU406), dcl2Δ (MU410), and double-mutant dcl1Δ/dcl2Δ (MU411) (31 (link), 32 (link)), Argonaute mutants ago1Δ, ago2Δ, and ago3Δ (MU413, MU416 and MU414, respectively) (34 (link)), RdRP mutants rdrp1Δ, rdrp2Δ, and rdrp3Δ (MU419, MU420 and MU439, respectively) (29 (link), 57 (link)), and the alternative ribonuclease R3B2 mutant r3b2Δ (MU412) (35 (link)). Media were supplemented with uridine (200 mg/L) or leucine (20 mg/L) when needed to supplement auxotrophic requirements, and cultures were grown at room temperature unless otherwise stated.