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 EBE _PthA4 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.

Author summary

Mutation of the EBEs of susceptibility (S) genes via genome editing and utilization of naturally occurring EBE variants have been used to generate disease resistant plants. However, TALE adaptations may lead to resistance loss, limiting the long-term efficacy of the strategy.

We utilized an experimental evolution approach to test TALEs adaptations in the Xanthomonas citri-citrus pathosystem using designer TALEs that cannot recognize the EBE of host targets. We identified adaptive TALE mutations and deletions that occurred during less than 30 cycles of repeated infections, which reconstituted the virulence on the host. Adaptive variants originated from TALEs that harbored a small number of mismatches (≤5) to the EBE, whereas designer TALEs that harbored larger number of mismatches (≥7) to the EBE failed to adapt in the duration of this study. Our study experimentally demonstrates adaptive rearrangements of TALEs during host adaptation and suggests that the potential durability in the resistance of modified crops should be a significant factor to be considered prior to their introduction into the field.