Distribution and Expression of Elicitin-like Protein Genes of the Biocontrol Agent Pythium oligandrum

Pythium oligandrum has the ability to induce plant defence reactions, and four elicitin-like proteins (POD-1, POD-2, POS-1 and oligandrin) that are produced by this oomycete have been identified as elicitor proteins. The first three are cell wall protein elicitors (CWPs), and the latter is an extracellular protein. Pythium oligandrum isolates have been previously divided into two groups based on the CWPs: the D-type isolate containing POD-1 and POD-2, and the S-type isolate containing POS-1. We identified the genes encoding these elicitin-like proteins and analyzed the distribution of these genes among 10 P. oligandrum isolates. A genomic fosmid library of the D-type isolate MMR2 was constructed and genomic regions containing the elicitin-like protein genes were identified. Southern blot analyses with probes derived from pod-1 and an oligandrin gene indicated that the 10 P. oligandrum isolates could be divided into the same groups as those based on the CWPs. The D-type isolates carried pod-1, pod-2 and two oligandrin genes, termed oli-d1 and oli-d2, while the S-type isolates carried pos-1 and one oligandrin gene termed oli-s1. Phylogenetic analysis of POD-1, POD-2, POS-1, Oli-D1, Oli-D2 and Oli-S1 with the previously defined elicitins and elicitin-like proteins of Phytophthora and Pythium species showed the specific clade. These genes occurred as single copies and were present in the P. oligandrum genomes but not in the other nine Pythium species (Pythium iwayamai, Pythium volutum, Pythium vanterpoolii, Pythium spinosum, Pythium torulosum, Pythium irregulare, Pythium ultimum, Pythium aphanidermutum and Pythium butleri). Furthermore, RT-PCR analysis demonstrated that all of these genes were expressed during the colonization of tomato roots by P. oligandrum, supporting the idea that they encode potential elicitor proteins. To investigate the genetic relationships between the D-type and the S-type isolates, physical maps of the flanking regions around pod-1, pod-2, pos-1 and the oligandrin genes were constructed. The maps suggest that the D-type isolates may be derived from the S-type isolates due to gene duplication and deletion events.