75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

317 Using Synthetic RPP8 Gene Clusters To Model R Gene Evolution By Meiotic Unequal
Crossing-Over
Stacey Simon, Bonnie Woffenden, Crystal Gilbert, John Jelesko, John McDowell
Virginia Tech
Disease resistance genes (R genes) are frequently organized as gene clusters. Unequal crossing-over between different
linked genes of a cluster can create new combinations of R genes, as well as chimeric genes, thereby facilitating the
evolution of new R genes. The resulting chimeric R genes could have an altered pathogen recognition specificity. The
Arabidopsis RPP8 gene, for downy mildew resistance, belongs to a two-gene cluster, and sequence comparisons suggest
that unequal crossing-over has significantly affected the evolution of allelic diversity at RPP8. An allelic series of three
different pathogen recognition specificities has been defined at the RPP8 locus. We are utilizing a genetic screen to model
both the frequency and character of unequal crossing-over within a synthetic RPP8 transgenic cluster. We will identify
rare meiotic unequal cross-over events by coupling chimeric gene formation to the activation of the Firefly Luciferase
gene. The recombination breakpoints will be mapped and the pathogen resistance specificities of the chimeric RPP8
genes will be tested. We will also address whether the frequency of meiotic recombination is affected by abiotic and
biotic stress. This study will provide general insights into the frequency and character of meiotic unequal crossing-over
and its impact on the evolution of functional diversity within R gene clusters.
318 PAG1, the α7 Subunit of the 20S Proteasome, is Essential in Pollen Development
Gulsum Soyler-Ogretim, Jed Doelling
Division of Plant and Soil Sciences, West Virginia University, Morgantown, 26506
The 26S proteasome is responsible for the degradation of ubiquitin-tagged proteins in eukaryotic organisms. 20S
core particle of the 26S proteasome consists of 4 stacked rings of 7 proteins each: the two inner rings are each composed
of 7 different β subunits and two outer rings are each composed of 7α subunits. Protein degradation by the proteasome
is tightly regulated and occurs inside the cylinder. Whereas two different genes encode many of the α and β subunits in
Arabidopsis, there is only one gene that encodes the α subunit PAG1. In this study, we use reverse genetics to study the
role of PAG1 and the 26S proteasome during Arabidopsis growth and development.
We acquired a potential PAG1 T-DNA insertion mutant from the Arabidopsis Biological Resource Center based
on search of the database www.arabidopsis.org. The presence of T-DNA insertion within PAG1 was confirmed by
PCR genotyping. Because homozygous mutant plants were not found among the progeny of heterozygous plants,
reciprocal crosses between a heterozygous mutant and a wild type plant were conducted to determine the cause. When
the heterozygous plant was used as the pollen donor, no heterozygous individuals were identified among 100 random
offspring. This suggests that pollen transmission of the mutant allele is hindered: if mutant pollen and wild type pollen
are equal, one would expect half of the offspring to contain a mutant allele. Ovule transmission of the mutant allele was
found to occur at the expected proportion.
We are continuing to characterize PAG1 mutant individuals by conducting complementation tests using endogenous
and inducible promoters and to characterize the development and the function of mutant pollen. The hope is to determine
the consequences of defective ubiquitin-dependent protein degradation at different stages of plant development. Pollen
development will be monitored using microscopy to count nuclei following DAPI staining and analyzing pollen
morphology. Pollen function will be investigated using in vitro pollen germination assays and vital stains.