Discovery Of Endogenous Plant Small Rnas And Their Role In Trans Species Gene Regulation PDF Download

Endogenous small RNAs (20 - 24 nt) engage in complex regulation of gene expression and thus shape and direct plant development, defense, stress response and the epigenome. Based on their biogenesis and functions, endogenous small RNAs can be divided into many categories and subcategories. MicroRNAs (miRNAs) represent the most well-annotated type of small RNAs that regulate gene expression via transcript cleavage or translational repression. However, MIRNAs only contribute to a minor fraction of all the expressed small RNAs in plants. Small RNA genes other than MIRNAs remain poorly annotated, which limits complete elucidation of their regulatory roles. Furthermore, inconsistent MIRNA discovery methodologies in published studies have resulted in widespread discrepancies among existing annotations. To address these issues, and to improve current understanding of small RNA gene functions, we developed robust methodologies for de novo annotation of plant small RNA genes. Our comprehensive small RNA loci discovery based on deep sequencing data and small RNA biogenesis patterns provided refinement of existing MIRNA annotations and their functions in the basal land plant Physcomitrella patens. We also identified numerous P. patens siRNA loci producing almost equal mixture of 23-24 nt small RNAs, confirming that the heterochromatic siRNA pathway is present in the bryophyte lineage. Our de novo annotation of small RNA genes in Amborella trichopoda, the basal-most lineage of flowering plants, revealed a striking predominance of lineage-specific, intronic 23-24 nt MIRNAs and hairpin RNAs that has not been reported in any plants so far. Most of these non-canonical MIRNAs lacked easily identifiable targets in the transcriptome, suggesting these may have functions other than sequence-dependent targeting. In the monocot rice, 24 nt long intronic miRNAs function in RNA dependent DNA methylation. It is possible that A. trichopoda 23-24 nt MIRNAs function in a similar way, and such non-canonical miRNA pathways may have been retained in specific lineages of flowering plants. At least 19 A. trichopoda miRNA families were broadly conserved across land plants, and most of these also had conserved targets. These findings confirmed the presence of all major small RNA gene classes in the basal lineage of flowering plants, as well as the existence of species-specific diversities in small RNA populations expressed in non-model plants. Finally, we explored the potential exchange of endogenous small RNAs between parasitic plants and their hosts. Parasitic plants intimately connect to their hosts through a specialized feeding organ called haustoria. Bidirectional exchange of thousands of mRNAs between the stem parasite C. campestris and its hosts have been previously reported. Host-induced gene silencing has also been shown in several parasitic species including Cuscuta and Triphysaria versicolor (root parasite). De novo annotation of small RNA genes from C. campestris - A. thaliana associations revealed an unprecedented abundance of 22 nt parasite miRNAs in the haustorial interface. Several of these interface-induced C. campestris miRNAs directed slicing of six host mRNAs and triggered secondary siRNA production specifically in interface. Among these targets, Botrytis Induced Kinase 1 (BIK1) encodes a receptor-like cytoplasmic kinase and functions in in plant immunity. Another target, Sieve-Element-Occlusion-Related 1 (SEOR1) encodes a protein thought to be involved in sealing phloem sieve elements after wounding. Additionally, mRNAs encoding three auxin receptors, TIR1, AFB2, and AFB3 were targeted by a C. campestris miRNA and showed a unique pattern of secondary siRNA production in parasite-host interface. Such secondary siRNA production depended on host machinery for RNA interference. Growth of C. campestris on seor1 mutant significantly increased parasite biomass accumulation compared to wild type. Furthermore, interface-induced parasite miRNA-directed cleavage of host TIR1/AFB was also detected in C. campestris -N. benthamiana. Our findings thus confirm conserved trans-species targeting by C. campestris miRNAs across the haustorial interface, and the potential roles of these miRNAs as virulence factors in plant parasitism.