Protein-Protein Interaction Assay in Phytophthora sojae Using Yeast Two-Hybrid System

An oomycete pathogen, Phytophthora sojae is one of the most serious threats to soybean production worldwide. Transcription factors are crucial for the survival of all living organisms including oomycetes, because of their roles in transcription of the genes. This feature in eukaryotes provides a very useful clue for selection of transcription factors as targets for the future control methods. A potential transcription factor in P. sojae, Ps1365 (PHYSODRAFT_342624) was discovered via yeast one-hybrid system by a former member of the Phuntumart lab (Rutter, 2012). In this research, I aimed to find the proteins which interact with Ps1365 in P. sojae with the hypothesis that these interactive proteins function together with Ps1365 to activate expression of other genes and may also be transcription factors. Ps1365 is a protein composed of 156 amino acids, with a molecular weight of 17,424 Da and isoelectric point 8.39. Genomic analysis indicated that Ps1365 is one of 22 paralogous proteins in P. sojae but only four orthologs were noted in P. infestans. Ps1365 was used as a bait in yeast two-hybrid analysis (Y2H) to screen cDNAs library of the mycelia of P. sojae. Toxicity analysis showed that the pGBKT7-Ps1365 construct was not toxic to the yeast strain Saccharomyces cerevisiae Y2HGold and no-autoactivation was observed by the autoactivation assay. Following the Y2H, high-throughput sequencing was performed and revealed two prey sequences that are potential partners of Ps1365. These sequences were identified as PHYSODRAFT_291312 and PHYSODRAFT_356433. Analysis of deduced amino acid sequence of PHYSODRAFT_291312 showed that it is a small globular protein of 8,288 Da and contains one or two α-helices, the simplest form of a transcription factor. SignalP analysis showed that PHYSODRAFT_291312 lacks signal peptide and do not contain nuclear localized signal. These predictions partly support our hypothesis that PHYSODRAFT_291312 can freely pass through nuclear membrane because of its small size (lower than 40 kDa – the upper limit of molecules to freely pass through nucleus without having a nuclear localization signal) to interact with Ps1365. Co-expression analysis using existing data from the Fungal and Oomycete Genomics Resource (FungiDB) as well as from the National Center for Biotechnology Information (NCBI) showed that both Ps1365 and PHYSODRAFT_291312 expressed together during mycelial growth and during infection, further confirmed that the two proteins may function together to drive expression of genes downstream. Analysis of the deduced amino acid sequence of PHYSODRAFT_356433 showed that it is a globular protein of 11,737.68 Da and contains α-helices. SignalP analysis showed that PHYSODRAFT_356433 lacks signal peptide and a nuclear localized signal. Secondary structure prediction by Phyre2 showed that it has a RNA-binding domain, indicating that it has potential to interact with Ps1365 at either DNA or mRNA level. Co-expression analysis of Ps1365 and PHYSODRAFT_356433 showed a similar expression pattern during the mycelium and cyst stages. All together, these results support the hypothesis that the two candidate P. sojae proteins, PHYSODRAFT_291312 and PHYSODRAFT_356433 may function as interacting proteins, that together with Ps1365 to regulate the expression of virulence factors