EXPRESSION OF AVRPPHB, AND AVIRULENCE GENE FROM PSEUDOMONAS-SYRINGAE PV PHASEOLICOLA, AND THE DELIVERY OF SIGNALS CAUSING THE HYPERSENSITIVE REACTION IN BEAN

Protein production encoded by the avirulence gene avrPphB from Pseudom onas syringae pv, phaseolicola was examined, Incorporation of [S-35]-l abeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa produc t. The 28-kDa native peptide was isolated from E. coli following over- expression of avrPphB and found not to elicit the hypersensitive respo nse (HR) after infiltration into bean leaves, Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P. syringae pv. phaseolicola; immunoreactiv e peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucro se. Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPph B protein, revealed the accumulation of peptides corresponding to the smaller cleavage products,in both E. coli and P. syringae pv, phaseoli cola. Biochemical localization experiments showed that all AvrPphB pep tides were cytoplasmic in P. syringae pv. phaseolicola. No AvrPphB pep tides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to w ild type were observed in a hrpY(-) strain, although it also failed to cause a confluent HR. Growth of P. syringae pv. phaseolicola under in ducing conditions removed the requirement for rifampicin-sensitive mRN A synthesis by bacteria to allow HR development (the induction time) i n bean and lettuce leaves. Constitutive expression of hrpL reduced but did not remove the induction time, Expression of the hrp gene cluster of P. syringae pv, phaseolicola from plasmid pPPY430 in E. coli enabl ed phenotypic expression of avrPphE (also carried by pPPY430) and avrP phB (if overexpressed from pPPY3031). Despite constitutive expression of the hip and avr genes in E. coli, a protein synthesis dependent ind uction time was still required for development of the HR in bean genot ypes with matching resistance genes. The significance of processing fo r the function of AvrPphB peptides and the delivery of elicitors of th e HR are discussed.