Structure-Function Analysis of the HrpB2-HrcU Interaction in the Xanthomonas citri Type III Secretion System

Author(s)

Cappelletti, Paola A. ^[1] ; dos Santos, Rafael Freitas ^[1] ; do Amaral, Alexandre M. ^{[2, 3]} ; Homem, Rafael Augusto ^[2] ; Souza, Thais dos Santos ^[1] ; Machado, Marcos A. ^[2] ; Farah, Chuck S. ^[1]

Affiliation

^[1] Univ Sao Paulo, Inst Quim, Dept Bioquim, BR-01498 Sao Paulo - Brazil

^[2] Ctr APTA Citros Sylvio Moreira IAC, Sao Paulo - Brazil

^[3] EMBRAPA Recursos Genet & Biotecnol, Brasilia, DF - Brazil

• Document type: Journal article
• Source: PLoS One; v. 6, n. 3, p. e17614, 2011.
• Web of Science Citations: 6

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 Delta hrcU 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 Delta hrcU 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 Delta hrpB2 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. (AU)

• FAPESP's process: 05/59243-3 - Structural and functional analysis of multi-prosthetic systems for the pathogenicity of Xanthomonas axonopodis pv. citri
• Grantee: Shaker Chuck Farah
• Support Opportunities: Research Projects - Thematic Grants