Prevalence and diversity of TAL effector-like proteins in fungal endosymbioticMycetohabitans spp

Abstract

AbstractEndofungalMycetohabitans(formerlyBurkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus,Rhizopus microsporus.The one known secreted effector family fromMycetohabitansconsists of homologs of transcription activator-like (TAL) effectors, which are used by plant pathogenicXanthomonasandRalstoniaspp. to activate host genes that promote disease. These ‘BurkholderiaTAL-like (Btl)’ proteins bind corresponding specific DNA sequences in a predictable manner, but their impact on transcription and their genomic target(s) in the fungus are not yet known. Recent characterization of two Btl proteins (Btl19-13 and MTAL1/Btl21-1), each from a differentMycetohabitansspecies, revealed different phenotypes inRhizopus, underscoring the need to assess the sequence and functional diversity of Btl proteins. We sequenced and assembled nineMycetohabitansspp. genomes using long-read PacBio technology. All assemblies contained fragments ofbtlgenes, and most had intact copies. We then mined fungal-bacterial metagenomes assembled as part of the ZygoLife project. This analysis showed thatbtlgenes are present across diverseMycetohabitansstrains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested thatMycetohabitansmight contain more species than previously recognized. Within our data set, Btl proteins were more conserved acrossMycetohabitans rhizoxinica strainsthan acrossMycetohabitans endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial-fungal symbioses in myriad ways.ImportanceMany Mucoromycota fungi harbor endosymbiotic bacteria, includingRhizopusspp. that are food fermenters and pathogens of plants and immunocompromised people.Rhizopus microsporushas endofungalMycetohabitans(formerlyBurkholderia) spp. that deploy proteins related to DNA-binding ‘transcription activator-like’ effectors of plant pathogens, which enter plant nuclei and activate disease susceptibility genes. One ‘Burkholderia TAL-like (Btl)’ protein, inM. endofungorum, enhances fungal membrane stress tolerance. Another, fromM. rhizoxinica, promotes bacterial colonization of the fungus. By sequencing isolated bacteria and mining fungal holobiont sequences, we found Btl proteins in diverseMycetohabitansstrains, varying in DNA binding specificity, thus in potential host targets. Btl proteins were more conserved withinM. rhizoxinicasuggesting distinctions among the two named species. The results suggest that Btl proteins contribute to symbiosis in diverse ways, providing insight into effector evolution and arguing for functional characterization of additional Btl proteins to understand establishment and maintenance of these important fungal-bacterial interactions.