Analysis of protease production in the microbial antagonist Pythium oligandrum and host/prey Pythium myriotylum protease inhibitors

Previous transcriptome analysis suggested that proteases of the microbial antagonist Pythium oligandrum and protease inhibitors of its host/prey Pythium myriotylum were important in their interactions. P. oligandrum protease activity was induced when cultured with inactivated oomycete mycelial powder, and exo-proteomic analysis showed significantly higher abundance of 25 proteases in cultures with mycelial powder. Two interaction-upregulated P. myriotylum Kazal-type serine protease inhibitors (Pm_KSPI-7 and Pm_KSPI-11) were heterologously produced, and Pm_KSPI-11, but not Pm_KSPI-7, inhibited P. oligandrum protease activities. Pm_KSPI-11 was a strong inhibitor of porcine trypsin activity (Ki = ~6.5 nmol/L), while neither trypsin nor chymotrypsin activity was inhibited by Pm_KSPI-7. P1-P2 loop residues and protein modeling analyses suggested that the lysine P1 residue in Pm_KSPI-11 may explain the differences in inhibitory properties. Pm_KSPI-11 supplementation of skim milk agar reduced P. oligandrum growth, but supplementation of cV8 agar did not substantially reduce P. oligandrum antagonism towards P. myriotylum. The role of Pm_KSPI-11 may be in reducing the ability of P. oligandrum to derive nutrition from peptides rather than directly counter-antagonizing P. oligandrum in a confrontation. Understanding potential P. myriotylum counter-antagonism abilities, such as protease inhibitors, may improve the biological control of crop diseases caused by P. myriotylum in more complex environments.