title: Structure-function analysis of the HrpB2-HrcU interaction in the Xanthomonas citri type III secretion system.
abstract: Bacterial type III secretion systems deliver protein virulence factors to host cells. Here we characterize the interaction between HrpB2, a small protein secreted by the Xanthomonas citri subsp. citri type III secretion system, and the cytosolic domain of the inner membrane protein HrcU, a paralog of the flagellar protein FlhB. We show that a recombinant fragment corresponding to the C-terminal cytosolic domain of HrcU produced in E. coli suffers cleavage within a conserved Asn264-Pro265-Thr266-His267 (NPTH) sequence. A recombinant HrcU cytosolic domain with N264A, P265A, T266A mutations at the cleavage site (HrcU(AAAH)) was not cleaved and interacted with HrpB2. Furthermore, a polypeptide corresponding to the sequence following the NPTH cleavage site also interacted with HrpB2 indicating that the site for interaction is located after the NPTH site. Non-polar deletion mutants of the hrcU and hrpB2 genes resulted in a total loss of pathogenicity in susceptible citrus plants and disease symptoms could be recovered by expression of HrpB2 and HrcU from extrachromossomal plasmids. Complementation of the ΔhrcU mutant with HrcU(AAAH) produced canker lesions similar to those observed when complemented with wild-type HrcU. HrpB2 secretion however, was significantly reduced in the ΔhrcU mutant complemented with HrcU(AAAH,) suggesting that an intact and cleavable NPTH site in HrcU is necessary for total functionally of T3SS in X. citri subsp. citri. Complementation of the ΔhrpB2 X. citri subsp. citri strain with a series of hrpB2 gene mutants revealed that the highly conserved HrpB2 C-terminus is essential for T3SS-dependent development of citrus canker symptoms in planta.
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Paola A
authors.initials: PA
authors.lastname: Cappelletti
authors.forename: Rafael Freitas
authors.initials: RF
authors.lastname: dos Santos
authors.forename: Alexandre M
authors.initials: AM
authors.lastname: do Amaral
authors.forename: Rafael Augusto
authors.initials: RA
authors.lastname: Homem
authors.forename: Thaís dos Santos
authors.initials: Tdos S
authors.lastname: Souza
authors.forename: Marcos A
authors.initials: MA
authors.lastname: Machado
authors.forename: Chuck S
authors.initials: CS
authors.lastname: Farah
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002957:Citrus / Q000382:microbiology; D015169:Colony Count, Microbial; D017124:Conserved Sequence; D003470:Culture Media; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005453:Fluorescence; D050296:Microbial Viability; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010935:Plant Diseases / Q000382:microbiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014974:Xanthomonas / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D010455:Peptides