| Erwinia amylovora genes and gene products involved in pathogenicity in host plants such as apple and in elicitation of defense responses in non-host plants such as tobacco were studied. First, harpin, encoded by hrpN, and an elicitor of the hypersensitive reaction (HR) in many non-host plants, was observed not to affect host plants or cultured cells. Enhanced harpin expression did not alter apparent virulence. Exogenous harpin did not rescue a hrpN strain or enable non-pathogens of apple to multiply in apple leaves. Second, five genes in an eleven-gene operon required for harpin secretion and part of a large cluster of hrp genes (required for HR and pathogenicity) were sequenced. Overall, the operon is highly similar to the type III secretion locus IcrB (yscN to yscU) of Yersinia spp. The operon also encodes a homolog of YopN, a putative cell-contact sensor of Yersinia spp. involved in virulence protein transfer to the host cytoplasm, and Hrpl, previously shown to be required for harpin secretion. Another gene in the operon, hrcT, was demonstrated to be required for harpin secretion also. This gene and others were named, and hrpl renamed, according to a unified nomenclature for conserved hrp genes (type III secretion genes). Evolution of type III secretion was explored using cladistic analysis. Third, a 6.6 kb "disease specific" operon (dsp, required for pathogenicity but dispensible for HR) downstream of hrpN and homologous with avrE of Pseudomonas syringae pathovar (pv.) tomato was shown, as avrE, to convert P. syringae pv. glycinea race 4 to avirulence on soybean. The operon, designated dspE, contains genes dspE and dspF. The dspE product, 198 kD, was expressed in Escherichia coli. The deduced dspF product resembles chaperones of virulence factors of animal pathogens. dspE was shown to be required for disease in host plants but not for HR elicitation by E. amylovora in tobacco and soybean plants. Positive regulation of the dspE operon by the hrp regulator HrpL was demonstrated using a reporter gene. The cloned avrE locus (gift of A. O. Charkowski and A. Collmer) restored dspE strains of E. amylovora to pathogenicity. |
