Transcription activator-like effectors from endosymbiotic bacteria control the reproduction of their fungal host

Abstract

ABSTRACT The phytopathogenic fungus Rhizopus microsporus harbors a bacterial endosymbiont ( Mycetohabitans rhizoxinica ) that produces a crucial virulence factor responsible for the characteristic symptoms of rice seedling blight. The persistence of this unique microbial alliance is ensured by evasion of fungal defense mechanisms by the resident endosymbionts and their strict control of fungal reproduction. A functional bacterial Type 3 secretion system (T3SS) is essential for the establishment of the symbiosis. Yet, the nature of the effector(s) secreted through the T3SS, and their effects on fungal physiology, has remained elusive. Using bioinformatic analysis, we identified genes encoding potential T3SS effectors, namely an alanine-tryptophan-arginine triad (AWR) peptide and a set of transcription activator-like (TAL) effectors, in the genomes of eight endofungal Mycetohabitans strains. Co-culture experiments demonstrated that the sporulating phenotype of apo-symbiotic R. microsporus is unaffected upon reinfection with M. rhizoxinica Δ awr compared to with wild-type M. rhizoxinica . In contrast, the ability of apo-symbiotic R. microsporus to produce mature sporangiophores when reinfected with M. rhizoxinica strains deficient in individual MTALs ( M. rhizoxinica Δ mtal1 , M. rhizoxinica Δ mtal2 , and M. rhizoxinica Δ mtal3 ) is significantly reduced. Trans -complementation experiments showed restoration of fungal sporulation, thus confirming that TAL effectors produced by M. rhizoxinica (MTAL1, MTAL2, and MTAL3) are needed for fungal sporulation. Using fluorescence microscopy, we show that AWR- and MTAL-deficient M. rhizoxinica strains successfully colonize apo-symbiotic R. microsporus , revealing the importance of bacterial MTALs in establishing a stable symbiosis after fungal colonization. Our findings attribute a new function to members of the MTAL family of T3SS-associated effectors and provide deeper insights into host control by prokaryotic symbionts. IMPORTANCE Interactions between fungi and bacteria are critically important in ecology, medicine, and biotechnology. In this study, we shed light on factors that promote the persistence of a toxin-producing, phytopathogenic Rhizopus-Mycetohabitans symbiosis that causes severe crop losses in Asia. We present an unprecedented case where bacterially produced transcription activator-like (TAL) effectors are key to maintaining a stable endosymbiosis. In their absence, fungal sporulation is abrogated, leading to collapse of the phytopathogenic alliance. The Mycetohabitans TAL (MTAL)-mediated mechanism of host control illustrates a unique role of bacterial effector molecules that has broader implications, potentially serving as a model to understand how prokaryotic symbionts interact with their eukaryotic hosts.