1.6.5S
MOLECULAR ANALYSIS OF THE REGIONS FLANKING THE TYPE III PROTEIN SECRETION OPERONS OF ERWINIA AMYLOVORA: SEARCHING FOR GENES ENCODING HRP-DEPENDENT PATHOGENICITY, VIRULENCE AND AVIRULENCE PROTEINS

JF KIM, AJ BOGDANOVE, Z WEI and SV BEER

Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA

Background and objectives
Erwinia amylovora causes the devastating fireblight disease on many rosaceous hosts. The pathogenicity of E. amylovora in hosts, and induction of the hypersensitive reaction (HR) in non-hosts, depend on clustered hrp/hrc genes, which appear to be situated in a pathogenicity island. The Hrp-essential region encodes regulatory proteins such as HrpL sigma factor, and proteins that constitute a protein-secretion system called the Hrp (type III) pathway. HrpN harpin has been shown to be secreted through this pathway. In contrast, a locus located next to the Hrp-required region, called dsp, is required for pathogenicity but not for HR elicitation. In Pseudomonas syringae, two avirulence genes have been reported to be linked to the hrp gene cluster. We analysed the regions bordering hrp/hrc genes to investigate the nature of the Dsp phenotype, to prospect for avr genes or avr gene homologues, another harpin gene(s), and genes encoding Hrp-delivered effector proteins.

Results and conclusions
The DNA beyond the region that includes regulatory genes, secretory genes, and the hrpN gene, was sequenced. Open reading frames at the hrpN side were tentatively designated orfA-L, and ones at the hrcU side orf12-25. Putative HrpL-dependent promoters were identified in front of several ORFs. Some of them including hrpW (orfD) and dspE (orfE) were further characterized.

hrpW, located between the hrp and dsp regions, encodes an acidic glycine/serine-rich cysteine-lacking protein, the expression and secretion of which is Hrp-controlled. HrpW contains a harpin-like domain and a domain homologous to pectate lyases of a distinct class; however, no pectate lyase activity was detected. Purified HrpW elicits the HR when infiltrated into the plant apoplast; its activity is heat-stable, protease-sensitive, and depends on plant metabolism. In contrast to hrpN, however, hrpW is dispensable for the Hrp phenotype of E. amylovora, although over-expressed hrpW in trans enhances HR elicitation by a hrpN mutant.

orfB, not required for the Hrp phenotype, encodes a homolog of AvrRxv of X. campestris pv. vesicatoria, Y4lO of Rhizobium sp. NGR234, AvrA of Salmonella enterica, and YopJ/YopP of Yersinia spp. The dspE and dspF (orfF) genes encode pathogenicity proteins homologous to proteins encoded by the avrE locus of P. syringae pv. tomato. Expression of dspE is hrpL-dependent, and dspE and dspF together as transgenes cause P. syringae pv. glycinea to elicit the HR in soyabean cultivars; avrE partially complements dspE mutants of E. amylovora for pathogenicity.

ORFA is a member of the SycH family of chaperones and chaperone-like proteins, and is probably the specific chaperone of ORFB. dspF, located immediately downstream of dspE, encodes a protein that has properties of the chaperones of the type III secretion systems, and appears to be the chaperone of DspE.

Additional homologies were detected from database searches. For example, ORF17 has high sequence similarities to HrpK/HrpY of P. syringae pathovars. ORFG, ORFH and ORFs 20-25 have highly conserved homologues in other enteric bacteria, and therefore are less likely to be involved in hrp pathogenesis.

Analysis of the culture supernatant indicated that the E. amylovora type III pathway secretes several proteins in addition to HrpN, HrpW and DspE. Homologies and other features suggest that HrpA, HrpJ, ORFB, ORFJ, ORF16 and ORF17 might be among them.