Distinct evolutionary trajectories following loss of RNA interference in <i>Cryptococcus neoformans</i>

While increased mutation rates typically have negative consequences in multicellular organisms, hypermutation can be advantageous for microbes adapting to the environment. Previously, we identified two hypermutator <i>Cryptococcus neoformans</i> clinical isolates that rapidly develop drug resistance due to transposition of a retrotransposon, Cnl1. Cnl1-mediated hypermutation is caused by a nonsense mutation in a gene encoding an RNA interference (RNAi) component, <i>ZNF3</i>, combined with a tremendous transposon burden. To elucidate adaptive mechanisms following RNAi loss, two bioinformatic pipelines were developed to identify RNAi loss-of-function (LOF) mutations in a collection of 387 sequenced <i>C. neoformans</i> isolates. Remarkably, several RNAi-loss isolates were identified that are not hypermutators and have not accumulated transposons. To test whether these RNAi LOF mutations can cause hypermutation, the mutations were introduced into a nonhypermutator strain with a high transposon burden, which resulted in a hypermutator phenotype. To further investigate whether RNAi-loss isolates can become hypermutators, in vitro passaging was performed. Although no hypermutators were found in two <i>C. neoformans</i> RNAi-loss strains after short-term passage, hypermutation was observed in a passaged <i>Cryptococcus deneoformans</i> strain with an increased transposon burden. Consistent with a two-step evolution, when an RNAi-loss isolate was crossed with an isolate containing a high Cnl1 burden, F1 hypermutator progeny inheriting a high transposon burden were identified. In addition to Cnl1 transpositions, insertions of a gigantic DNA transposon KDZ1 (~11 kb) contributed to hypermutation in the progeny. Our results suggest that RNAi loss is relatively common (7/387, ~1.8%) and enables distinct evolutionary trajectories: hypermutation following transposon accumulation or survival without hypermutation.