DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB‐dependent way

Abstract

SummaryIn Erwinia amylovora, the dsp region, required for pathogenicity on the host plant but not for hypersensitive elicitation on tobacco, is separated from the hrp region by 4 kb. The genetic analysis reported in this paper showed that this 4 kb region is not required for pathogenicity on pear seedlings. The environmental conditions allowing expression of a dsp ::lacZ fusion were examined: expression was barely detected in rich medium at 30ЊC, and the highest expression was observed in M9 galactose minimal medium at 25ЊC. A dsp ::uidA fusion appeared to be expressed only in a HrpL-proficient strain, indicating that the dsp region, like the hrp region, is positively controlled via the alternative factor HrpL. Sequence analysis revealed that the dsp cluster encodes two genes, dspA (5517 bp) and dspB (420 bp), and that the insertions leading to the dsp ::lacZ and the dsp ::uidA fusions were within dspA. A HrpL-dependent promoter sequence (GGAACC-N 15 -CAACA) was identified upstream of dspA, and primer extension analysis detected four transcriptional starts 7, 8, 9 and 10 bp downstream of this sequence. A 70 promoter sequence (TTGCCC-N 16 -GATAAT) was observed upstream of dspB. The functionality of this second promoter was confirmed by complementation analysis. This promoter allowed constitutive expression of dspB, as measured by the expression of a dspB ::uidA fusion in rich medium. In M9 galactose medium, however, HrpL was shown to activate dspB, as expression of the dspB ::uidA fusion was twofold higher in a HrpL þ background than in a HrpL ¹ background. Transposon insertions in either dspA or dspB led to a non-pathogenic phenotype. Thus, both DspA and DspB were required for E. amylovora pathogenicity, as dspB could be expressed independently of dspA. DspA and DspB were visualized as polypeptides with apparent sizes of 190 kDa and 15.5 kDa, respectively, when encoded in the T7 polymerase/promoter system. DspA, which showed homology with the protein predicted from the partial sequence of Pseudomonas syringae pv. tomato avrE transcriptional unit III, was shown to be secreted into the external medium via the Hrp secretion pathway. DspB was predicted to be acidic, like the Syc chaperone of Yersinia. A chaperone role for DspB was suggested further by the fact that DspA secretion required a functional DspB protein.

Secretion and translocation signals and DspB/F-binding domains in the type III effector DspA/E of Erwinia amylovora

“…Secretion and translocation of DspA/E with and without DspB/F Gaudriault et al (1997Gaudriault et al ( , 2002 have observed that DspB/F is required for the secretion and intracellular stability of DspA/E in its entirety. In this study, we showed that short lengths of the N-terminal portion of DspA/E could be secreted in the absence of DspB/F, because all DspA/ E : : AvrRpt2 80-255 fusion proteins were secreted from E. coli expressing the Er.…”

“…DspA/E is a large (198 kDa) protein that belongs to the AvrE family of effectors and may have multiple functional domains on account of its size (Bogdanove et al, 1998;Gaudriault et al, 1997). Indeed, like many AvrEfamily effectors, DspA/E requires a chaperone, DspB/F, for its secretion and intracellular stability (Gaudriault et al, 1997(Gaudriault et al, , 2002. However, while a dspA/E mutant of Er.…”

“…Among these effectors, DspA/E has emerged as an indispensable T3-secreted protein that suppresses the host basal defences (DebRoy et al, 2004) and causes cell death in both host and non-host plants (Boureau et al, 2006;Oh et al, 2007), thus enabling and promoting disease. DspA/E is a large (198 kDa) protein that belongs to the AvrE family of effectors and may have multiple functional domains on account of its size (Bogdanove et al, 1998;Gaudriault et al, 1997). Indeed, like many AvrEfamily effectors, DspA/E requires a chaperone, DspB/F, for its secretion and intracellular stability (Gaudriault et al, 1997(Gaudriault et al, , 2002.…”

Perturbation of Maize Phenylpropanoid Metabolism by an AvrE Family Type III Effector fromPantoea stewartii

“…WtsE from Pnss is most closely related to AvrE family T3Es and in particular to DspA/E proteins from Erwinia spp. Just as a functional WtsE is required for Pnss pathogenicity (Frederick et al, 2001), DspA/E is required for the ability of Erwinia amylovora to elicit fire blight symptoms in apple (Malus domestica), Cotoneaster spp., and pear (Pyrus communis; Gaudriault et al, 1997;Bogdanove et al, 1998). Additionally, AvrE, along with the sequence-unrelated but functionally redundant T3E, HopM1, and the AvrE superfamily member HopR1 (Badel et al, 2006;Kvitko et al, 2009), is required for the virulence of Pseudomonas syringae pv tomato on tomato (Solanum lycopersicum), Arabidopsis (Arabidopsis thaliana), and Nicotiana benthamiana.…”

In silico identification of potential chaperone genes that belong to type III and type IV secretion systems in Xanthomonas axonopodis pv citri

“…Together, the results presented here are important for understanding the pathogenicity of Xanthomonas axonopodis pv citri and may be useful in studies of the mechanism of disease caused by this pathogen in orange plantations. To date, few reports have described a requirement for chaperones in the type III secretion systems of plant pathogens (see for instance, Gaudriault et al, 1997;van Dijk et al, 2002). In conclusion, the study of Xanthomonas axonopodis will benefit from the identification of secretion system chaperone genes since uncovering the precise molecular events controlling the delivery of effector proteins should eventually allow the design of compounds that specifically interfere with these processes, hopefully without deleterious side effects to the host plant (Staskawicz, 2001).…”