A cloned Erwinia chrysanthemi Hrp (type III protein secretion) system functions in Escherichia coli to deliver Pseudomonas syringae Avr signals to plant cells and to secrete Avr proteins in culture

The Hrp (type III protein secretion) system is essential for the plant parasitic ability of Pseudomonas syringae and most Gram-negative bacterial plant pathogens. AvrB and AvrPto are two P. syringae proteins that have biological activity when produced via heterologous gene expression inside plant cells or when produced by Hrp(+) bacteria. Avr-like proteins, presumably injected by the Hrp system on bacterial contact with plant cells, appear to underlie pathogenic interactions, but none has been observed outside of the bacterial cytoplasm, and identifying novel genes encoding them is tedious and uncertain without a phenotype in culture. Here we describe a cloned Hrp secretion system that functions heterologously in Escherichia coli to secret AvrB and AvrPto in culture and to promote AvrB and AvrPto biological activity in inoculated plants. The hrp gene cluster, carried on cosmid pCPP2156, was cloned from Erwinia chrysanthemi, a pathogen that differs from P. syringae in being host promiscuous. E. coli DH5 alpha carrying pCPP2156, but not related Hrp-deficient cosmids, elicited a hypersensitive response in Nicotiana clevelandii only when also expressing avrB in trans. The use of pAVRB-FLAG2 and pAVRPTO-FLAG, which produce Avr proteins with a C-terminal FLAG-epitope fusion, enabled immunoblot detection of the secretion of these proteins to E. coli(pCPP2156) culture media. Secretion was Hrp dependent, occurred without leakage of a cytoplasmic marker, and did not occur with E. coli(pHIR11), which encodes a functional P. syringae Hrp system. E. coli(pCPP2156) will promote investigation of Avr protein secretion and systematic prospecting for the effector proteins underlying bacterial plant pathogenicity.