Abstract
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.
Impact Statement Many Mucoromycota fungi harbor endosymbiotic bacteria, including Rhizopus spp. that are food fermenters and pathogens of plants and immunocompromised people. Rhizopus microsporus has endofungal Mycetohabitans (formerly Burkholderia) spp. that deploy proteins related to DNA-binding ‘transcription activator-like’ effectors of plant pathogens, which enter plant nuclei and activate disease susceptibility genes. By sequencing isolated bacteria and mining fungal holobiont sequences, we found Btl proteins in diverse Mycetohabitans strains, varying in predicted DNA binding specificity, thus in potential host targets. Btl proteins were more conserved within M. rhizoxinica, suggesting distinctions among the two named species. The results in the context of phenotypic differences observed in other studies 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.
Competing Interest Statement
JES was a paid consultant for Zymergen, Sincarne, and Michroma.
Footnotes
Repositories: NCBI accession numbers are available in Table 2 and the Data Availability summary as individual data types were deposited in different ways.
Updated Figure 1, results, and methods to include pangenome analysis conducted by new author Bhuwan Abbot; introduction and discussion revised for clarity and impact