Distinct evolutionary trajectories following loss of RNA interference in Cryptococcus neoformans

Area of Science:

• Microbiology
• Genetics
• Evolutionary Biology

Background:

• Microbial hypermutation can be adaptive, unlike in multicellular organisms.
• Previous work identified hypermutators in *Cryptococcus neoformans* with Cnl1 retrotransposon activity, linked to a Znf3 mutation affecting RNAi.
• The role of RNAi loss in microbial adaptation requires further elucidation.

Purpose of the Study:

• To identify and characterize RNAi loss-of-function mutations in *C. neoformans*.
• To investigate the link between RNAi loss, transposon accumulation, and hypermutation.
• To explore the evolutionary consequences of RNAi deficiency in *C. neoformans*.

Main Methods:

• Development of two bioinformatic pipelines to detect RNAi loss-of-function mutations in 387 *C. neoformans* isolates.
• Introduction of identified RNAi loss-of-function mutations into a non-hypermutator strain.
• *In vitro* passaging of RNAi-loss strains and genetic crosses.

Main Results:

• Several *C. neoformans* isolates with RNAi loss-of-function mutations were identified that were not hypermutators.
• Experimental introduction of these mutations induced hypermutation in a strain with a high transposon burden.
• Passaging experiments showed hypermutation in *C. deneoformans* with increased transposon burden, and F1 progeny from crosses exhibited hypermutation due to Cnl1 and KDZ1 transposon insertions.

Conclusions:

• RNAi loss is relatively common (~1.8%) in *C. neoformans* and provides a foundation for distinct evolutionary trajectories.
• RNAi loss can enable hypermutation through transposon accumulation or allow survival without increased mutation rates.
• This study highlights the adaptive potential of RNAi deficiency in microbial evolution.