About This Project
Lichens, symbioses between fungi and algae, survive levels of desiccation lethal to most organisms. We recently discovered that seemingly unnecessary extra DNA segments ("introns") present in the lichen DNA coding for ribosomes are central to the induction of desiccation resistance. We hypothesize that the slowdown in ribosome assembly caused by the introns triggers the expression of genes protective against desiccation damage. We plan to identify these protective genes through RNA sequencing.
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What is the context of this research?
All cells have ribosomes, molecular machines that build proteins. Ribosome assembly is sensitive to stresses like desiccation. So we asked if the many introns found in ribosomal DNA (rDNA) of lichen fungi affect their remarkable desiccation tolerance. Using CRISPR, we moved introns from a lichen fungus into the rDNA of yeast, a fungus with no rDNA introns. Yeast desiccation tolerance went up 1000-fold with introns. We think that introns are not causing the resistance directly, but their removal during ribosome assembly slows it down, which then causes activation of genes protecting the cell from desiccation damage. In the presence of rDNA introns such genes remain on, maintaining cells on constant alert even in absence of desiccation.
What is the significance of this project?
Drought is a major problem for land-based organisms. Land plants evolved roots and evaporation controls to counteract water loss, but prolonged drought can lead to death. Worldwide, drought affects 64% of the land area, is projected to increase due to climate change, and causes large food losses. Not surprisingly, drought stress in plants is intensely studied, but contributions from remote corners of biology could open novel possibilities. One such understudied corner is lichens and their remarkable ability to withstand complete desiccation. Since biological mechanisms are not compartmentalized, understanding drought defenses in lichens would not just answer a question in basic science, but might lead to new practical applications to protect crop plants from drought damage.
What are the goals of the project?
We hypothesize that lichen rDNA introns, by slowing ribosome assembly, trigger expression of genes protective against desiccation damage. Using yeast as model, our immediate goal is to identify such genes by comparing transcription in four yeast strains we constructed. Three have lichen introns inserted at two locations ("A" and "B") in the yeast rDNA: the first has an intron in A, the second an intron in B, the third has two introns, one in A and one in B. The fourth has no rDNA introns. RNA from each strain will be sequenced. Gene expression in the four strains (each in three replicates) will be analyzed bioinformatically. Subsequent goals are to validate the genes' protective functions in yeast and in lichens and eventually to apply the findings to plants if feasible.
RNA sequencing is part of a core project already in progress, aimed at understanding the basis of lichen desiccation tolerance. The project has been supported intramurally through undergraduate research funds provided by Duke University, and RNA sequencing is the first step towards a more comprehensive and expensive molecular analysis.
The first milestone (1.5 months) requires optimizing growth, harvesting yeast and extracting RNA of the highest quality. The second milestone (1.5 months) depends on the turnaround time of the sequencing facility, which depends on how busy they are when we submit our RNA samples. The third milestone (2 months) depends on the availability of bioinformatics consultation and on the complexity of the sequence data.
Jan 16, 2019
Mar 15, 2019
Growth of strains and RNA extraction
Apr 30, 2019
Obtaining sequencing data
Jun 30, 2019
Initial bioinformatics analysis results
Dec 31, 2019
Notify backers at each milestone, up to publication
Meet the Team
I came to Duke University from Italy as a graduate student and never left Duke, where I am now an Associate Professor of the Practice of Biology in the Biology Department and teach undergraduate lab courses in Molecular and Cell Biology. Although I worked with fungi most of my life, I came to love lichens late in my career, inspired by Chicita Culberson, a world-class lichen chemist working in the Biology Department. Together with many collaborators, we just completed the first comprehensive lichen genome project on both alga and fungus of the lichen Cladonia grayi, and this project on the function of lichen introns is the first test of an idea born of that genome analysis. A large component of my research is done by undergraduates, and I consider sparking their interest in research my main mission as a professor.
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