Symbiont-dependent sexual reproduction in Rhizopus microsporus involves transcriptional reprogramming and RNAi-associated small-RNA responses.

Symbioses play a central role in shaping host development and physiology, yet how multiple symbiotic partners collectively influence complex biological processes remains poorly understood. In the fungal holobiont Rhizopus microsporus, bacterial (Mycetohabitans) and viral (narnaviruses) endosymbionts contribute to the formation of zygospores between compatible sexual partners, although their influence on early sexual reproduction remains largely unexplored. Here, we used mRNA sequencing data to evaluate the impact of bacterial and viral symbionts on the fungal transcriptional landscape at the onset of sexual development, revealing a substantial transcriptional reprogramming. Through network analysis, we identified candidate hub genes, including transcription factors, protein kinases, and signal transduction components, that may contribute to this developmental transition. By integrating sRNA-sequencing data, we found small-RNA signals correlating with early sexual success. Furthermore, we uncovered distinct processing patterns for the RmNV-20S and RmNV-23S viral symbionts, revealing that RmNV-23S processing shifted during the initiation of fungal sexual reproduction in the absence of the bacterial partner. Target prediction analyses further suggest that viral-derived sRNAs could influence distinct functional signatures in the fungal genome. Moreover, predicted interactions between endogenous or viral sRNAs and fungal transcripts suggest a potential role for sRNAs in fine-tuning the host transcriptional profile. Altogether, our findings provide a novel framework for understanding how multi-kingdom symbioses modulate host development and identify potential molecular factors that may contribute to the onset of sexual reproduction in R. microsporus.