HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells

Abstract

The type III secretion system (T3SS) is required by plant pathogenic bacteria for the translocation of certain bacterial proteins to the cytoplasm of plant cells or secretion of some proteins to the apoplast. The T3SS of Erwinia amylovora, which causes fire blight of pear, apple and other rosaceous plants, secretes DspA/E, which is an indispensable pathogenicity factor. Several other proteins, including HrpN, a critical virulence factor, are also secreted by the T3SS. Using a CyaA reporter system, we demonstrated that DspA/E is translocated into the cells of Nicotiana tabacum'Xanthi'. To determine if other T3-secreted proteins are needed for translocation of DspA/E, we examined its translocation in several mutants of E. amylovora strain Ea321. DspA/E was translocated by both hrpW and hrpK mutants, although with some delay, indicating that these two proteins are dispensable in the translocation of DspA/E. Remarkably, translocation of DspA/E was essentially abolished in both hrpN and hrpJ mutants; however, secretion of DspA/E into medium was not affected in any of the mentioned mutants. In contrast to the more virulent strain Ea273, secretion of HrpN was abolished in a hrpJ mutant of strain Ea321. In addition, HrpN was weakly translocated into plant cytoplasm. These results suggest that HrpN plays a significant role in the translocation of DspA/E, and HrpJ affects the translocation of DspA/E by affecting secretion or stability of HrpN. Taken together, these results explain the critical importance of HrpN and HrpJ to the development of fire blight.

Figure 1

Translocation of DspA/E_1‐733‐CyaA into plant cells by Ea321 and several mutants. Plants were infiltrated with suspensions of strains of Ea321 harbouring pCPP1553 at OD₆₀₀ = 0.4. Leaf samples were taken 7 h after infiltration and the amount of cAMP was determined. cAMP is expressed as pmol/mg of total protein. The values for each strain are the means of 12 samples from one experiment. The bars represent standard errors. Values denoted by the same capital letters do not differ significantly at the P = 0.05 level. WT, wild‐type.

Figure 2

Time‐course translocation of DspA/E_1‐733‐CyaA. Plants were infiltrated with suspensions of strains of Ea321 harbouring pCPP1553 at OD₆₀₀ = 0.4. Leaf samples were taken 1, 3, 5 and 7 h after infiltration and the amount of cAMP was determined. cAMP is expressed as pmol/mg of total protein. The values for each strain are the means of four samples from one experiment. The bars represent standard errors. WT, wild‐type.

Figure 3

Expression and secretion of DspA/E_1‐733‐CyaA by several strains of E. amylovora under hrp‐inducing conditions. Strains of Ea321 (pCPP1553) indicated in the figure were grown in hrp gene‐inducing medium at 18 °C for 36 h. Total proteins from cell pellet and supernatant were collected separately and loaded onto 8% SDS‐PAGE gels. The DspA/E_1‐733‐CyaA fusion protein was detected with CyaA antibody. WT, wild‐type.

Figure 4

Secretion of HrpJ by several strains of E. amylovora under hrp gene‐inducing conditions. Strains of Ea321 (pCPP1553) indicated in the figure were grown in hrp‐inducing medium at 18 °C for 36 h. Proteins from supernatant were collected and loaded in 8% SDS‐PAGE gels; detection by Western blotting was carried out with HrpJ polyclonal antibody. WT, wild‐type.

Figure 5

Translocation of HrpN_1‐323‐CyaA into tobacco cells. Plants were infiltrated with bacterial suspensions of the wild‐type Ea273 and T3SS‐deficient mutant, Ea273‐hrcN, harbouring pCPP1553 or pCPP1729 at OD₆₀₀ = 0.4. Leaf samples were taken 7 h after infiltration and the amount of cAMP was determined. cAMP is expressed as pmol/mg of total protein. The values for each strain are the means of six samples from one experiment. The bars represent standard errors. WT, wild‐type.