A TAL-Based Reporter Assay for Monitoring Type III-Dependent Protein Translocation in Xanthomonas

Gram-negative plant- and animal-pathogenic bacteria use type III secretion (T3S) systems to translocate effector proteins into eukaryotic host cells. Type III-dependent delivery of effector proteins depends on a secretion and translocation signal, which is often located in the N-terminal protein region and is not conserved on the amino acid level. Translocation signals in effector proteins have been experimentally confirmed by employing reporter proteins, which are specifically activated inside eukaryotic cells. Here, we describe a method to monitor effector protein translocation using a deletion derivative of the transcription activator-like (TAL) effector protein AvrBs3 as reporter. AvrBs3 is a type III effector of the tomato and pepper pathogen X. campestris pv. vesicatoria and is imported into the plant cell nucleus where it binds to specific promoter elements of target genes and activates their transcription. The N-terminal deletion derivative AvrBs3∆2 lacks a functional T3S and translocation signal but contains the effector domain and induces plant gene expression when fused to a functional translocation signal. In resistant pepper plants, AvrBs3 and translocated AvrBs3∆2 fusion proteins induce the expression of the Bs3-resistance gene, which triggers a strong, macroscopically visible defense response. The protocol for translocation assays with AvrBs3∆2 fusion proteins includes (1) the generation of expression constructs by Golden Gate cloning, (2) the transfer of expression constructs into bacterial recipient strains, (3) in vitro secretion assays with reporter fusion proteins and (4) infection of AvrBs3-responsive pepper plants.