Genomic Instability of Duplicated Disease Resistance genes in Arabidopsis thaliana
SNC1 is a pathogen resistance (R-) gene in Arabidopisis thaliana. SNC1 duplication leads to the dwarfed mutant classified as bal, whose most notable characteristic is its phenotypic instability following mutagenesis. This mutagen-induced phenotypic instability of the bal mutants was first noted after a genetic suppressor screen in which a statically significant number of phenotypically “normal” plants were recovered; these reversion or suppression events were named bal à BAL*, and subsequent molecular analysis indicated that one SNC1 copy is inactivated in BAL* plants.
Two different models were formulated to explain the high frequency recovery of BAL* plants. The first hypothesis posits that hypermutation of SNC1 occurs due to local stress-induced mutagenesis, as a consequence of DNA damage. This model stands on the idea that as DNA damage occurs it leads to localized mutagenesis through induction of low-fidelity DNA polymerases, and implies that the mutation rate of the SNC1 gene in the bal variant is elevated preferentially. The second hypothesis is that selection within the meristem causes the high frequency recovery of BAL* phenotypes in the progeny. It is reasoned that SNC1 overexpression results in a decrease in fitness, establishing competition among bal and BAL* stem cells. Stem cells carrying a BAL* allele would have a growth advantage and as a result be more likely to contribute to the subsequent formation of reproductive tissues.
These models can be distinguished by observing the types of SNC1 mutations that occur in response to different mutagenic treatments. Previous experiments indicated the EMS treatment of bal plants leads to C/G à T/A mutations in SNC1. My project looked at the types of snc1 mutations that were recovered in BAL* plants generated by fast neutron mutagenesis, which causes deletions and DNA rearrangements versus base-pair changes. To characterize the mutation spectrum, end-point and quantitative PCR was used in addition to Southern blots and Sanger sequencing. The results of my study support the meristem selection model since the mutations that I characterized reflect the types of mutations expected from fast neutron mutagenesis.
The Boyce Thompson Institute internship has provided me with invaluable insight into some of the rigorous and exciting work conducted in a professional research environment. Through the excellent guidance of my mentor, Erika Hughes, I gained a complete understanding of numerous lab techniques, as well as confidence in my own abilities conducting research. While working in the Richards lab I was exposed to a wide range of new research topics, specifically in genetics, seeking to answer fundamental questions in biology. Which has aided me in seeing more clearly the true importance of research in the plant sciences. This internship has also been a good preview for my future, as I plan to matriculate into graduate school to continue my study of biology.