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Validation of predicted miRNAs in Phytophthora sojae and Phytophthora infestans

Manuel Ospina-Giraldo: Lafayette College

<div>The post-transcriptional regulatory environment in the oomycetes <em>Phytophthora infestans </em>and <em>P. sojae </em>is poorly understood and the expression of microRNAs (miRNAs), and their potential roles in infection, is a topic of increasing interest. We have attempted to experimentally verify the existence of several miRNAs from both <em>P. infestans </em>and <em>P. sojae</em>, which had been predicted using <em>in silico</em> approaches. In addition, we investigated the possibility of these miRNAs playing a role in <em>Phytophthora </em>pathogenicity. The presence of two of the five miRNAs, namely psj-miR8788 (predicted based on its stem-loop structure and sequence conservation between <em>P. sojae</em>, <em>P. ramorum</em>, and <em>P. infestans</em>) and PimiRNA3, predicted in <em>P. infestans</em>, was experimentally confirmed <em>in vivo</em> by amplification using specific stem-loop primers and subsequent cloning and sequencing. The predicted targets of psj-miR8788 include members of the amino acid/auxin permease family, which serve amino acid transport functions, while the targets for PimiRNA3 include a diverse group of proteins, some of them with enzymatic activity. We also measured the transcriptional activity of psj-miR8788 during infection of soybean plants and determined that psj-miR8788 is upregulated mostly at the earlier stages of infection. This suggests that psj-miR8788 is important during the biotrophic stage of infection, and perhaps plays a role in suppressing plant defense systems by modulating amino acid transport systems in the plant.</div>