Evidence That the Pseudomonas syringae pv. syringae hrp-Linked hrmA Gene Encodes an Avr-Like Protein That Acts in an hrp-Dependent Manner Within Tobacco Cells

Abstract

A 25-kb DNA region, previously cloned from Pseudomonas syringae pv. syringae 61 in cosmid pHIR11, enables non-pathogenic bacteria such as Pseudomonas fluorescens and Escherichia coli to elicit the hypersensitive response (HR) in tobacco (Nicotiana tabacum). hrmA is located within this region, adjacent to a conserved cluster of hrp genes, and is essential for nonpathogens to elicit the HR. DNA sequence analysis suggested that hrmA was the second of two genes in an operon and was preceded by an open reading frame (ORF), ORF1, which is predicted to encode a 10.9-kDa protein. DNA gel blot analysis revealed that sequences hybridizing with a DNA fragment internal to hrmA were absent from P. syringae pv. syringae B728a, P. syringae pv. tabaci 11528, and P. syringae pv. glycinea race 4 U1, but present in P. syringae pv. tomato DC3000. A 2.4-kb BamHI-AvrII fragment carrying hrmA, ORF1, and native regulatory sequences was subcloned into broad-host-range vector pDSK519 and electroporated into P. syringae pv. syringae B728a and P. syringae pv. tabaci 11528. The presence of the hrmA locus had no apparent effect on the ability of P. syringae pv. syringae B728a to cause brown spot of bean, but it caused P. syringae pv. tabaci 11528 to elicit the defense-associated HR rather than disease in N. tabacum cvs. Xanthi N and Xanthi NC and N. clevelandii. Furthermore, N. debeyii, N. glutinosa, N. rustica, and N. tabacum cvs. Petit Havana and Samsun responded with the HR to P. fluorescens(pHIR11). In contrast, N. benthamiana-P. syringae pv. tabaci interactions were unaffected by the presence of HrmA, and P. fluorescens(pHIR11) did not elicit the HR in N. benthamiana. The hrmA ORF was sub-cloned into pFLAG-CTC, which expressed HrmA with a C-terminal FLAG synthetic epitope fusion. Escherichia coli MC4100 cells carrying the functional hrp cluster and the hrmA-FLAG derivative secreted the HrpZ harpin, but not HrmA-FLAG, to the medium, as indicated by immunoblot analysis with M2 anti-FLAG and polyclonal anti-HrpZ antibodies. The hrmA ORF was also subcloned into plant expression vector pFF19 and then biolistically delivered, along with pFF19G (expressing β-glucuroni-dase), into suspension-cultured tobacco cells. Histochemical staining 24 h later revealed substantial β-glucuroni-dase activity in cells receiving pFF19G and pFF19 but not in those receiving pFF19G and pFF19-HrmA. Thus, internal production of HrmA was deleterious to tobacco cells.