Abstract

Pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. Effector protein delivery is controlled by the T3S chaperone HpaB, which presumably escorts effector proteins to the secretion apparatus. One intensively studied effector is the transcription activator-like (TAL) effector AvrBs3, which binds to promoter sequences of plant target genes and activates plant gene expression. It was previously reported that type III-dependent delivery of AvrBs3 depends on the N-terminal protein region. The signals that control T3S and translocation of AvrBs3, however, have not yet been characterized. In the present study, we show that T3S and translocation of AvrBs3 depend on the N-terminal 10 and 50 amino acids, respectively. Furthermore, we provide experimental evidence that additional signals in the N-terminal 30 amino acids and the region between amino acids 64 and 152 promote translocation of AvrBs3 in the absence of HpaB. Unexpectedly, in vivo translocation assays revealed that AvrBs3 is delivered into plant cells even in the absence of HrpF, which is the predicted channel-forming component of the T3S translocon in the plant plasma membrane. The presence of HpaB- and HrpF-independent transport routes suggests that the delivery of AvrBs3 is initiated during early stages of the infection process, presumably before the activation of HpaB or the insertion of the translocon into the plant plasma membrane.

Highlights

  • Many Gram-negative bacterial pathogens translocate effector proteins into eukaryotic host cells to modulate host cellular pathways such as defense responses to their own benefit (Raymond et al, 2013; Ashida et al, 2015; Santos and Finlay, 2015; Büttner, 2016; Ensminger, 2016; Grabowski et al, 2017)

  • Escherichia coli strains were cultivated at 37◦C in lysogeny broth (LB) medium and X. campestris pv. vesicatoria strains at 30◦C in nutrient-yeast extract-glycerol (NYG) medium (Daniels et al, 1984) or minimal medium A (Ausubel et al, 1996) at pH 7.0 supplemented with 10 mM sucrose and 0.3% casamino acids

  • AvrBs3 2 fusion proteins were analyzed in X. campestris pv. vesicatoria strain 85∗, which is a derivative of the wild-type strain 85-10 and contains HrpG∗, a constitutively active version of the hrp gene regulator HrpG (Rossier et al, 1999; Wengelnik et al, 1999)

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Summary

Introduction

Many Gram-negative bacterial pathogens translocate effector proteins into eukaryotic host cells to modulate host cellular pathways such as defense responses to their own benefit (Raymond et al, 2013; Ashida et al, 2015; Santos and Finlay, 2015; Büttner, 2016; Ensminger, 2016; Grabowski et al, 2017). Sct proteins are mainly involved in the assembly of the membrane-spanning part of the secretion apparatus, which consists of ring structures in the inner and outer bacterial membrane and a predicted periplasmic inner rod (Büttner, 2012). The inner membrane rings associate with the export apparatus, which is assembled by five transmembrane proteins and presumably forms a transport channel for secreted proteins (Diepold et al, 2011; Büttner, 2012; Dietsche et al, 2016; Deng et al, 2017). Components of the export apparatus are connected with the cytoplasmic ATPase complex, which provides the energy for secretion and/or unfolds secreted proteins during transport (Büttner, 2012; Deng et al, 2017). The ATPase presumably interacts with members of the SctQ family, which assemble as cytoplasmic ring or pod-like structures and are potential docking sites for T3S substrates (Büttner, 2012; Deng et al, 2017; Hu et al, 2017)

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