The C-Terminal Half of the HrpN Virulence Protein of the Fire Blight PathogenErwinia amylovoraIs Essential for Its Secretion and for Its Virulence and Avirulence Activities

Abstract

The HrpN (harpin) protein of the fire blight pathogen Erwinia amylovora is an essential virulence factor secreted via the bacterial type III secretion system. HrpN also has avirulence activity when delivered to tobacco by E. amylovora and has defense elicitor activity when applied to plants as a cell-free protein extract. Here, we characterize a series of random mutations in hrpN that altered the predicted amino acid sequence of the protein. Amino acid substitutions and deletions in the highly conserved, C-terminal portion of HrpN disrupted the virulence and avirulence activities of the protein. Several of these mutations produced a dominant-negative effect on E. amylovora avirulence on tobacco. None of the mutations clearly separated the virulence and avirulence activities of HrpN. Some C-terminal mutations abolished secretion of HrpN by E. amylovora. The results indicate that the C-terminal half of HrpN is essential for its secretion by E. amylovora, for its virulence activity on apple and pear, and for its avirulence activity on tobacco. In contrast, the C-terminal half of HrpN was not required for cell-free elicitor activity. This suggests that the N-terminal and C-terminal halves of HrpN mediate cell-free elicitor activity and avirulence activity, respectively.

Mutation of the Erwinia amylovora argD Gene Causes Arginine Auxotrophy, Nonpathogenicity in Apples, and Reduced Virulence in Pears

“…Cultures were grown at 28°C with rotary shaking at 150 rpm. Each strain population was quantified by serial dilution plating at 0 and 48 h after starting the culture (45,46).…”

“…Immature apple and pear fruits were collected at the Pennsylvania State University Fruit Research and Extension Center orchards in Biglerville, PA. Bacteria were suspended in 10 mM MgCl 2 solution to an absorbance of 0.1 ( ϭ 600). Then, 20 l of this cell suspension was introduced onto wounds on the skin side of halved immature 'Gala' apple and 'Bosc' pear fruits (46). Bacterial populations in the immature fruits were quantified at 0 and 7 dpi by macerating the apples in 10 mM MgCl 2 solution, followed by serial dilution plating, and expressed as CFU per gram of plant tissue (36).…”

Pathogenicity and infection strategies of the fire blight pathogen Erwinia amylovora in Rosaceae: State of the art

“…These proteins are glycine-rich, lack cysteine and are involved in inducing the hypersensitive response in non-host plants. Unlike HrpW, HrpN is required for full virulence in plants (Kim & Beer, 1998;Reboutier et al, 2007;Sinn et al, 2008;Wei et al, 1992) and plays an important role in the translocation of DspA/E (Bocsanczy et al, 2008). HrpJ has been postulated to act as an essential extracellular chaperone to prevent aggregation of harpins in the apoplast, and thus facilitate translocation of effector proteins into the host cells (Nissinen et al, 2007).…”

Membrane-Targeted HrpNEaCan Modulate Apple Defense Gene Expression

“…Like HpaG and HrpZ, HrpN Ea was more recently found to undergo amyloidogenesis in plant apoplast-like conditions , and three LZ-like and three HR-inducing motifs were found in its sequence (Table 1; Fig. 1, blocks 6 and 7) (Hao and Wei 2001;Sinn et al 2008;Tarafdar et al 2009). Among these motifs, the C-terminal one seems to be essential for virulence as well as for the HR-inducing activity (Sinn et al 2008).…”

“…1, blocks 6 and 7) (Hao and Wei 2001;Sinn et al 2008;Tarafdar et al 2009). Among these motifs, the C-terminal one seems to be essential for virulence as well as for the HR-inducing activity (Sinn et al 2008). In accordance with its shared characteristics with amyloid proteins, namely its pore-forming activity, HrpN Ea was also found to be required for translocation of DspA/E, the essential pathogenicity effector of E. amylovora, into tobacco cells (Bocsanczy et al 2008).…”