Endofungal Mycetohabitans (formerly Burkholderia) 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 from Mycetohabitans consists of homologs of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These Burkholderia TAL-like (Btl) proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one M. endofungorum strain enhances fungal membrane stress tolerance, while others in a M. rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across Mycetohabitans rhizoxinica strains than across Mycetohabitans 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.

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19 -13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

Endofungal Mycetohabitans (formerly Burkholderia) 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 from Mycetohabitans consists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These 'Burkholderia TAL-like (Btl)' proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one Mycetohabitans endofungorum strain enhances fungal membrane stress tolerance, while others in a Mycetohabitans rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across M. rhizoxinica strains than across M. 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.