Function and maintenance of trans-species small RNAs in the Cuscuta genus of parasitic plants
Open Access
- Author:
- Johnson, Nathan
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 25, 2019
- Committee Members:
- Michael Axtell, Dissertation Advisor/Co-Advisor
Michael Axtell, Committee Chair/Co-Chair
Claude Walker Depamphilis, Committee Member
Gabriele Brigitte Monshausen, Committee Member
Cristina Rosa, Outside Member
Teh-Hui Kao, Program Head/Chair - Keywords:
- Small RNAs
Parasitic plants
Small RNA alignment
Trans-species small RNAs
Cuscuta - Abstract:
- Regulatory small RNAs are important components involved in the regulation of many cellular processes in eukaryotes. Typically 20-24 nucleotides in length, small RNAs can repress gene expression through directed degradation of messenger RNA or through the recruitment/maintenance of repressive chromatin marks in the genome. In plants, large-scale annotations of small RNAs have languished despite the definition of several common classes (microRNAs, secondary siRNAs, heterochromatic siRNAs). ShortStack, a tool developed for the alignment and annotation of small RNAs from high-throughput sequencing reads has made large advancements in this process. The alignment step in small RNA analysis represents a particular challenge. In this step, reads are aligned to the genome based on sequence identity, however large proportions of small RNA sequencing reads (>50%) are known to map ambiguously to multiple places in the genome. This motivated the improvement of algorithms for the placement of these reads. Utilizing the density of unambiguously aligned reads in vicinity as a guiding principle, ShortStack chooses a place for ambiguously mapping reads in the genome. With both simulated and real datasets, this method greatly increases alignment precision for a modest reduction in sensitivity, compared to widely-used alternatives. It also improves the approximation of small RNA locus expression miRNAs. This tool was tested with small-RNA-sequencing libraries from three different plants, showing its superior performance with varied input. Through this improved algorithm, alignments and resulting annotations have minimized false positive rates, improving their quality and enhancing the results obtained from small-RNA-sequencing experiments. Species-species interactions are of a particular interest for small RNA research. Work on fungal and oomycete pathogens of plants show that small RNAs are able to regulate gene expression in trans-species manner. Recently, the parasitic plant genus Cuscuta has been a focus in this context, as it has been shown that bi-directional movement of biomolecules readily occurs in its interaction with hosts. Small-RNA-sequencing of Cuscuta campestris parasitizing the host Arabidopsis thaliana showed that numerous small RNAs are highly expressed in the host-parasite interface. Many of these were shown to be microRNAs and indeed target messenger RNAs originating from the host. These trans-species microRNAs were expressed similarly when parasitizing different hosts, but lack of expression on non-living hosts, hinting that a host-based trigger might be required. Secondary siRNAs resulting from targeting by these microRNAs are produced using protein machinery from the host, shown through their loss on mutant host plants. This implies processing takes place inside the host tissue. However, loss of secondary siRNAs did not measurably affect the growth of the parasite. Growing the parasite on hosts with loss of function mutations in targets of trans-species microRNAs showed enhanced growth for several targets, indicating their importance in host defense. These data broaden the picture of how small RNAs can be used in species interactions, making the case that this process may be used to enhance parasitic success. It is not clear how trans-species microRNAs from C. campestris are able to maintain targeting of host messenger RNAs in the light of adaptive pressures. To investigate this, small RNAs from host-parasite interactions in three more Cuscuta species were sequenced, including several different isolates within species. Many small RNAs were found to be highly expressed in the host-parasite interface. Targeting of host messenger RNAs was confirmed for several small RNAs in all species and isolates, indicating that the mechanism of trans-species small RNAs may be present throughout Cuscuta. Grouping distantly similar small RNAs based on sequence shows superfamilies that are retained between species groups. This analysis shows that several similar small RNAs are produced in a single species or isolate, accounting for variation in the target sites among possible hosts. This may also be used as a way to target numerous homologous messenger RNAs in a single host, regulating entire gene families. This provides a mechanism by which Cuscuta can ensure targeting despite any selection in the host to invalidate targeting. This discovery is a major expansion of the scope of trans-species small RNAs in plants and hints that this process may be widespread in nature.