Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis ofMycetohabitans(formerlyBurkholderia)rhizoxinicawith the fungusRhizopus 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 sequencedMycetohabitansspp. 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. Abtl19-13gene 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 15R. microsporusgenes 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 revealedbtlgenes in 14 diverseMycetohabitansisolates. However, banding patterns and available sequences suggest variation, and thebtl19-13phenotype could not be rescued by abtlgene 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 theM. rhizoxinica–R. microsporussymbiosis.