|Authors: ||L. Triplett, M. Melotto, S.Y. He, G. Sundin|
|Keywords: ||DspF, hrp secretion, CyaA|
Pathogenesis of Erwinia amylovora, the causal agent of fire blight, requires injection of the effector protein DspE into host cells via the type III secretion system (T3SS). Secretion is facilitated by the chaperone protein DspF, but the portion of DspE required for secretion and chaperone interaction has not been previously determined.
An adenylate cyclase reporter system was used to map the portion of DspE required for translocation into tobacco cells. E. amylovora strains containing 15, 30, 51, 77, 101, 151, 203 or 737 N-terminal amino acids of DspE fused to the CyaA protein of Bordetella pertussis were infiltrated into tobacco, and cAMP accumulation was measured as an indicator of protein translocation.
Results showed that the first 51 amino acids of DspE were sufficient for minimal protein translocation into the host cell and that translocation levels increased with increasing DspE fragment length.
Yeast-two-hybrid experiments were conducted to map the chaperone binding domain of DspE. A putative chaperone binding domain was identified within the first 100 residues of DspE. To further characterize the DspE-DspF interaction, computational methods were used to create a homology-based structural model of DspF. Based upon this model, 12 amino acids of DspF were identified as putative exposed substrate-binding residues.
Site-directed mutagenesis PCR was used to replace codons for candidate residues with alanine, yielding 12 single-site dspF mutant clones.
Mutants were introduced into Ea1189ΔdspF:km, a chaperone knockout strain exhibiting a reduced-virulence phenotype.
Five of the 12 dspF mutants failed to complement the virulence phenotype of the dspF knockout.
These results demonstrated that DspF residues L32, E39, E44, H55 and F78 are important for virulence, and yeast-two-hybrid experiments showed that DspF residues E39, E44 and H55 are required for binding to DspE.
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