Consequences of adaptation of TAL effectors on host susceptibility to Xanthomonas

Abstract

Transcription activator-like effectors (TALEs) are virulence factors of Xanthomonas that induce the expression of host susceptibility (S) genes by specifically binding to effector binding elements (EBEs) in their promoter regions. The DNA binding specificity of TALEs is dictated by their tandem repeat regions, which are highly variable between different TALEs. Mutation of the EBEs of S genes is being utilized as a key strategy to generate resistant crops against TALE-dependent pathogens. However, TALE adaptations through rearrangement of their repeat regions is a potential obstacle for successful implementation of this strategy. We investigated the consequences of TALE adaptations in the citrus pathogen Xanthomonas citri subsp. citri (Xcc), in which PthA4 is the TALE required for pathogenicity, whereas CsLOB1 is the corresponding susceptibility gene, on host resistance. Seven TALEs, containing two-to-nine mismatching-repeats to the EBEPthA4 that were unable to induce CsLOB1 expression, were introduced into Xcc pthA4:Tn5 and adaptation was simulated by repeated inoculations into and isolations from sweet orange for a duration of 30 cycles. While initially all strains failed to promote disease, symptoms started to appear between 9-28 passages in four TALEs, which originally harbored two-to-five mismatches. Sequence analysis of adapted TALEs identified deletions and mutations within the TALE repeat regions which enhanced putative affinity to the CsLOB1 promoter. Sequence analyses suggest that TALEs adaptations result from recombinations between repeats of the TALEs. Reintroduction of these adapted TALEs into Xcc pthA4:Tn5 restored the ability to induce the expression of CsLOB1, promote disease symptoms and colonize host plants. TALEs harboring seven-to-nine mismatches were unable to adapt to overcome the incompatible interaction. Our study experimentally documented TALE adaptations to incompatible EBE and provided strategic guidance for generation of disease resistant crops against TALE-dependent pathogens.

Fig 1. Variability of X. citri LOB1 targeting TALEs and the LOB1 EBE region in Rutaceae species.

A. RVD repeat arrays of LOB1 targeting TALEs from X. citri species (Sources are elaborated in Table 1). B. Sequence alignment of allelic variants (Sources are elaborated in Table 2) of the surrounding region of the TALE effector-binding elements (EBEs) from Rutaceae plants. Sequence alignment was conducted with Clustal Omega Multiple Sequence Alignment feature (https://www.ebi.ac.uk/Tools/msa/clustalo/) using default settings. Conserved residues in EBE region are marked in red. Variations in the EBE compared to allelic variant A are marked in blue. Variations in the area outside of the EBE compared to allelic variant A are marked in green. Thymidine residues proceeding EBEs are underlined. C. Target finding scores (lower scores indicate higher predicted binding affinity) of LOB1 targeting TALEs against allelic variants of Rutaceae LOB1 promoter according to TAL Effector Nucleotide Targeter 2.0 using Target Finder tool (https://tale-nt.cac.cornell.edu/). Scores are depicted in colored heat maps correlating to the ruler placed on the top of the table. D and E. Induced expression of sweet orange and Swingle citrumelo LOB1 by PthA4. Nicotiana benthamiana leaves were inoculated with Agrobacterium to co-express His-PthA4 or an empty vector with GUS reporter under the control of the LOB1 promoter from sweet orange (Citrus sinensis) or Swingle citrumelo (Poncirus trifoliata x Citrus paradisi). Expression of His-PthA4 was driven by an estradiol-inducible system and 17β-estradiol was applied at 24 h after agro-infiltration. D. Histochemical GUS staining of inoculated leave at 72 h after 17β-estradiol treatment. Experiment was repeated three times with similar results. E. GUS activity (arbitrary units [AU]) in inoculated areas was determined at 72 h after 17β-estradiol treatment. Values are means ± SE of nine biological replicates. The experiment was conducted three times and each experiment was composed of three biological replicates. Letters denote significant differences based on analysis of variance (Anova) and comparisons for all pairs using Student’s t-test (P-value < 0.05).

Fig 2. Experimental evolution of TALEs.

A. RVD repeat arrays of PthA4 (XACb0065) and dTALEs used in experimental evolution test. The nucleotide sequence of the effector-binding element of CsLOB1 from sweet orange (Citrus sinensis) is represented at the bottom. “Adapted” column indicates whether the dTALE variant was able to adapt in the duration of the experiment. B. Schematic representation of the experimental evolution workflow. Scheme was created with Biorender (https://biorender.com/).

Fig 3. Functional characterization of adapted dTALEs.

Sweet orange leaves were syringe-infiltrated with suspensions (1 × 10⁸ CFU/mL for A and B, 1 × 10⁶ CFU/mL for C) of Xcc 306 (Xcc WT), Xcc pthA4:Tn5 or Xcc pthA4:Tn5 transformed with the parental and adapted dTALEs depicted in Fig 4A. A. Inoculated leaves were photographed at 7 days post inoculation. The experiments were repeated three times with similar results. B. The gene expression of CsLOB1 was quantified at 36 and 72 h post inoculation (hpi) using quantitative reverse transcription PCR. The GAPDH gene was used as an endogenous control. Values are means ± SE of three biological replicates. C. Bacterial growth in planta. Values represent means ± SE of three biological replicates. The experiments were repeated three times with similar results. B and C. Asterisks indicate a significant difference (Student’s t-test, P-value < 0.05) compared to Xcc pthA4:Tn5.

Fig 4. Repeat rearrangements in adapted dTALEs.

A. RVD repeat arrays of parental and adapted dTALEs. Red-colored RVDs represent original mismatches compared to dTALEWTLOB1. Blue color indicates deleted or altered repeats in the adapted dTALEs compared to parental dTALEs. B. Predicted binding of adapted dTALEs [determined according to TAL Effector Nucleotide Targeter 2.0 using Target Finder tool (https://tale-nt.cac.cornell.edu/] to the CsLOB1 in sweet orange (Chromosome 7, 28358599–28358574, allelic variant A) EBE. The PthA4 effector-binding element (EBE) is labeled in green and thymidine residue proceeding the EBE is underlined.