Host specificity of plant-associated microeukayotes in roots and leaves of aquatic and terrestrial plants based on a blocking peptide-nucleic acid (PNA) amplification approach

The study of plantmicrobe interactions, whether mutualistic, antagonistic, parasitic, or commensal, has given a clearer view of how ecological circumstances affect the structure of vegetation. In addition to speeding up these studies, new sequencing technologies and other molecular approaches reveal the relationships between microorganisms and plants in a far higher resolution. Here we incorporated common methods in similar studies with currently developing tools such as long-read PacBio single-molecule sequencing technology and using a novel amplification-blocking protein-nucleic acid (PNA) approach. Assuming that fungi are relatively poorly adjusted to tissues of aquatic plants compared to more specialized unicellular organisms, and that plant tissues in both water and anoxic sediment are more subjected to random colonization, this study aimed to effectively document plantassociated eukaryote composition in different plant organs and different habitats and to reveal particular patterns in host specificity. We selected four terrestrial and four aquatic plant species common in a split sampling area between water and the shore of lake Kaariku, South Estonia. We developed blocking PNAs for both forward and reverse primers by using SILVA alignments for rRNA 18S and 28S genes supplemented by sequence data from fungal lineages, traced in the regions between primers and the ITS region. Our blocking agents cover the ITS4 primer and a combination of four positions conserved in plants but variable in other organisms. Analyses of the PacBio sequence data obtained from regular and PNA-enriched amplicons demonstrated that: 1- eukaryotic microbiome associated with leaves of aquatic plants has a much larger proportion of non-fungal microbes; 2- leaves include a more similar eukaryotic microbiome than in roots; and 3- partner specificity of terrestrial plants is relatively higher than aquatic plants.