A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host

Abstract

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.

Keywords: Btl proteins; Rhizopus microsporus; TAL effector; symbiosis; type III secretion.

Fig. 1.

Structure, expression, T3 secretion, and nuclear localization of Btl19-13. (A) Domain structure of a Xanthomonas TAL effector, a Ralstonia TAL effector, and a Btl protein. T3, type III secretion signal; T3?, putative type III secretion signal; NLS, nuclear localization signal; “RIRK,” aa sequence of a putative NLS in Btl19-13; AD, activation domain. (B) Confocal microscopy of Mycetohabitans sp. B13 cells constitutively expressing EYFP and expressing Btl19-13:mCherry under the native btl19-13 promoter, within a R. microsporus hypha. DAPI staining of the nuclei is included for reference. (C) Quantification of cAMP by ELISA in Nicotiana benthamiana leaf punches 6 h after infiltration with Pseudomonas syringae pv. tomato DC3000 (Pst) expressing the following: AvrPto:Cya (61 kDa), Btl19-13:Cya (118.6 kDa), Btl19-13:Cya missing the first 45 aa (Btl19-13Δ1-45, 110 kDa), and the first 45 aa of Btl19-13 fused to CyaA (Btl19-13[1-45], 51 kDa). T3SS designates an hrpQ-U mutant incapable of type III secretion (28). Each bar shows the results from three biological replicates with two technical replicates each. Error bars denote SD. The experiment was repeated twice with similar results. Below, a Western blot of leaf homogenates probed with anti-Cya, shown with a Stain-Free loading control (Bio-Rad). Asterisks (*) indicate bands corresponding to the expected size. (D) Confocal microscopy of Saccharomyces cerevisiae cells expressing the indicated proteins and stained with NucBlue to locate nuclei. Protein expression was induced with galactose 24 h prior to imaging. “AAAA” indicates an alanine substitution of the “RIRK” motif in Btl19-13 (residues 692–695).

Fig. 2.

Effect of Btl19-13 on reporter gene activity in a transient expression assay in N. benthamiana leaves. (A) Schematic of the two reporter constructs used. GUS is driven by a pepper Bs3 minimal promoter harboring a binding element for the Xanthomonas TAL effector AvrBs3 and either a binding element for Btl19-13 (BE) or a scrambled binding element (sBE). (B and C) Fluorometric assays of GUS activity in planta. N. benthamiana leaves were coinfiltrated with Agrobacterium tumefaciens strains delivering one of the two reporter constructs, as indicated, and one (B) or more (C) expression constructs for the following effector proteins: AvrBs3; dT19-13, a designer TAL effector with similar RVD sequence to Btl19-13; Btl19-13; Tal1c, a Xanthomonas TAL effector which has no predicted binding element in the Bs3 promoter. GUS activity was assayed 48 hpi and is shown relative to the corresponding reporter construct with only Tal1c. Each value is an average of three replicates, and the experiment was repeated twice with similar results. An asterisk (*) over two values indicates a significant difference (paired Student’s t test, P < 0.05). Error bars denote SD.

Fig. 3.

Effect of Btl19-13 on R. microsporus cell membrane stress tolerance and global gene expression. (A and B) Colony diameter of R. microsporus infected with wild-type Mycetohabitans sp. B13 (WT), B13∆btl19-13 (mutant), or the mutant strain carrying btl19-13 on a plasmid (pBtl19-13). Fungi were grown at 28 °C on half-strength potato dextrose agar without (A) or with (B) 0.005% SDS for 3 and 6 d, respectively. Data shown represent 10 biological replicates for each bacterial genotype and were analyzed by ANOVA with a post hoc Tukey’s test to determine significance as indicated by lowercase letters (P < 0.001). The experiment was repeated twice and yielded similar results. (C) Venn diagram of the genes that were differentially expressed between R. microsporus infected with wild-type B13 or the complement, B13∆btl19-13(pBtl19-13), and the mutant B13∆btl19-13. Differential expression was determined by RNA sequencing. Data were analyzed with DESeq2 (37) using an adjusted P value < 0.05 as the threshold for significance. Three biological replicates were sequenced, each containing tissue from three plates.

Fig. 4.

Presence of btl genes in geographically diverse isolates of Mycetohabitans spp. and the effect of substituting btl18-14 for btl19-13. (A) World map showing the location and substrate from which the R. microsporus hosts of the Mycetohabitans spp. assessed were isolated, generated using rworldmap (39). More information on the strains is available in SI Appendix, Table S3. (B) Southern blots of genomic DNA from each strain, digested with AatII and probed with btl19-13 amplified from B13. Strains are identified by the culture collection accession number of their fungal hosts. ATCC 52813 represents B13, ATCC 52814 B14, and ATCC 62417 B1. The arrow points to a faint high-molecular-weight band in an otherwise empty lane, which can be more clearly seen in SI Appendix, Fig. S5. (C and D) Growth of R. microsporus infected with wild-type B13 (WT), B13∆btl19-13 (mutant), the mutant strain with pBtl19-13, or the mutant strain with pBtl18-14, on half-strength potato dextrose agar without (C) or with (D) 0.005% SDS after 3 or 6 d, respectively, at 28 °C. Data shown represent values from 10 replicate plates each and were analyzed by ANOVA with a post hoc Tukey’s test. The experiment was repeated twice and yielded the same result. In each plot, different lowercase letters above any two groups indicate a significant difference between the means (P < 0.001).