Mating-type specific ribosomal proteins control aspects of sexual reproduction inCryptococcus neoformans

Abstract

29The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by 30 an unusually large structure, spanning over 100 kb. MAT genes have been characterized by 31 functional genetics as being involved in sexual reproduction and virulence. However, classical 32 gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, 33 RPL39), indicating that they are likely essential. In the present study, targeted gene replacement 34 was performed in a diploid strain for both the α and a alleles of the ribosomal genes RPL22 and 35 RPL39. Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential 36 for viability. Ectopic integration of the RPL22 paralog of opposite MAT identity in the 37 heterozygous RPL22a/rpl22αΔ or RPL22α/rpl22aΔ mutant strains failed to complement their 38 essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 39 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. 40 Furthermore, via CRISPR/Cas9 technology the RPL22 alleles were exchanged in haploid MATα 41 and MATa strains of C. neoformans. These RPL22 exchange strains displayed morphological and 42 genetic defects during bilateral mating. The C. neoformans MATa strain in which the RPL22a 43 gene was exchanged with RPL22α displayed sensitivity to hydroxyurea and high temperature, 44 and had a morphological phenotype consistent with a defective cell cycle. These results 45 contribute to elucidate functions of C. neoformans essential mating type genes that may 46 constitute a type of imprinting system to promote inheritance of nuclei of both mating types. 49Infectious diseases cause significant morbidity and mortality worldwide in both 50 developed and developing countries. Fungal infections are common in humans and impact the 51 majority of the world's population, but are often underestimated (BROWN et al. 2012). The 52 Cryptococcus species complex includes basidiomycetous fungal pathogens that can cause lung 53 infections and life-threatening meningoencephalitis in both normal and immunocompromised 54 patients, accounting for approximately 1 million annual infections globally and almost 200,000 55 annual mortalities (RAJASINGHAM et al. 2017). The available drugs to treat Cryptococcus 56 infections are amphotericin B, 5-flucytosine, and azoles. These drugs are characterized by 57 limited spectrum, toxicity, unavailability in some countries, and emergence of drug resistance 58 (BROWN et al. 2012). 59In fungi, the mechanisms that govern sexual reproduction are controlled by specialized 60 regions called mating type (MAT) loci. The genomic organization of the MAT loci can differ 61 among fungi. The tetrapolar mating type includes two MAT loci, the P/R locus encoding 62 pheromones and pheromone receptor genes defining sexual identity and mate recognition, and 63 the HD locus encoding homeodomain transcription factors that govern post-mating 64 developmental processes. In the tetrapolar system the P...

Recombination suppression and evolutionary strata around mating‐type loci in fungi: documenting patterns and understanding evolutionary and mechanistic causes

“…Both the PR and HD mating‐type loci thus constitute ‘supergenes’ – loci transmitted as a single unit but composed of several genes (Schwander et al ., 2014). Some additional genes may have been incorporated into the mating‐type locus for proper nonself‐recognition; for example, in Cryptococcus neoformans it was shown that mating‐type specific alleles of ribosomal proteins were not interchangeable, being likely involved in promoting inheritance of nuclei of both mating types (Ianiri & Fang, 2020). Some basidiomycetes have multiple copies of the same pheromone alleles at their mating‐type locus, which need to be linked to each other and to the pheromone receptor gene for proper nonself‐recognition; such a situation, especially where duplication numbers differ between mating types (e.g.…”

Uncontrolled transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of Cryptococcus neoformans

“…Furthermore, the read depth on both DNA strands was analyzed with SAMtools and custom made perl scripts were used to calculate the read size distribution and 5'-nucleotide preference of the small RNA reads as previously described 87,88 .…”

Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen

“…Normalization of the read counts to reads per million (RPM) was performed, allowing the comparison of the libraries. Furthermore, the read depth on both DNA strands was analyzed with SAMtools and custom made perl scripts were used to calculate the read size distribution and 5’-nucleotide preference of the small RNA reads as previously described 101, 102 .…”