Identification Of Interactive Proteins Of Pi-PIPKD5in Phytophthora Infestans

Phytophthora is a kind of destructive plant pathogens. Totally, there are over100species of Phytophthora, of which Phytophthora infestans is the most notorious one. It is shown that there are two modes of reproduction in Phytophthora, sexual and asexual reproduction, both of which are regulated by hormone. In eukaryotes, G protein-coupled receptors (GPCRs) are an important group of receptors in signal transduction. Recently, a special class of GPCRs has been found in Phytophthora sojae, whose seven transmembrane region characteristic for GPCRs is followed by a phosphatidylinositol-4-phosphate5-kinas domain (PIPK). This kind of GPCRs is called GPCR-PIPK (GK). It is reported that two GK genes, PsPIPK4and PsPIPK10, regulate asexual and sexual reproduction respectively in Phytophthora sojae.In Phytophthora infestans, the Pi-PIPKD5gene has a high homology with PsPIPK10gene of Phytophthora sojae. Therefore, the Pi-PIPKD5gene could be involved in the sexual reproduction in Phytophthora infestans. In this study, the interactive proteins of Pi-PIPKD5were identified by yeast two-hybrid technology, which was important to discover the function of the Pi-PIPKD5gene. Firstly, two bait plasmids expressed N-terminus and C-terminus of Pi-PIPKD5protein respectively were constructed. Toxicity and self-activation test showed that the C-terminal bait protein was toxic to yeast strains, while the N-terminal bait protein was non-toxic and did not have self-activation activity. Then, a cDNA library of Phytophthora infestans was constructed and used to screen the interactive proteins of Pi-PIPKD5with the N-terminus of Pi-PIPKD5as a bait. The results showed that31interactive proteins were identified and4of which were validated to be the positive interactive proteins through backcross. The positive prey plasmids were sequenced and the protein database of Phytophthora infestans was searched. A plasmid possibly code for a conservative mycelium protein. Protein sequence analysis showed that it had a Ca2+-binding site (EF-Hand), an undetermined domain (DUF59) and an analogous Fe-S cluster assembly domain. According to the study on DUF59family, this domain is associated with the synthesis of Fe-S cluster, suggesting that it could be involved in the signal transduction regulated by Ca2+and synthesis of Fe-S cluster.