Structure-function analysis of Phytophthora parasitica elicitins

Elicitins are elicitor proteins produced by oomycete phytopathogens, primarily Phytophthora species. To further characterize the biological properties of elicitins, a structure-function study was undertaken. Systematic mutants, site-directed mutants and non-canonical elicitins were all evaluated for the ability to induce a hypersensitive response (HR) in tobacco. Some of the mutant proteins were evaluated for the ability to induce acquired resistance (AR) and for the ability to bind sterols.; Three alanine-scanning mutants, AS-4, AS-8 and BW-3, were 50 to 100-fold impaired in the ability to induce an HR. In addition, mutant AS-1 was unaffected in the ability to induce an HR, yet was unable to induce AR. Several site-directed mutants with significant phenotypes were identified. The mutant L15E was 10 to 100-fold reduced in the ability to induce an HR and was unable to induce AR to Phytophthora parasitica var. nicotianae. Furthermore, the mutant proteins L41D and L41K were unable to induce an HR in young tobacco plants and were also unable to induce AR. The proteins L41G and L41Q were not impaired, suggesting that a charged residue at position 41 disrupts the parasiticein-tobacco, interaction. The non-canonical elicitins were unaltered relative to the canonical parasiticein in the ability to induce an HR. However, the ELC2 proteins were not able to induce AR. This, and the result from mutant AS-1 would suggest that the ability of elicitins to induce an HR can be separated from the ability to induce acquired resistance.; The sterol-binding parameters were determined for parasiticein and seven of the mutant or non-canonical elicitins. Two proteins were unaltered in HR but altered in sterol binding. Y12R was better able to bind sterols, while the elicitin domain of ELC3 was significantly reduced in the ability to bind sterols. Two additional proteins (L15E and L41D) were dramatically reduced in the ability to induce an HR, but only slightly altered in the ability to bind sterols. These results suggest that the ability to induce an HR in tobacco and the ability to bind sterols in vitro are not correlated.