RNA interference knock-down suggests that PIRL6 functions in the male gametophyte of Arabidopsis thaliana

Signal transduction pathways are critical to the success of multicellular organisms because they direct normal cellular processes and healthy responses to stimuli. Conversely, signaling pathway malfunctions can have large consequences for the survival of an organism and its progeny. Leucine-rich repeat proteins (LRRs) are a superfamily of proteins found in all eukaryotes that are capable of forming specific protein-protein interactions, and thus are well-suited to function in these signal transduction pathways. The Ras-group LRRs are one class of LRRs that interact with Ras or Ras-like small G-proteins. In the plant model system Arabidopsis thaliana, the Plant Intracellular Ras-group Leucine-rich-repeat (PIRL) gene family encodes LRR proteins that share structural homology with important Ras-group proteins found in animals and humans. PIRL6 is an interesting member of the PIRL family, partially because no true T-DNA knock-out mutants have been identified for this locus. Expression of a functional PIRL6 transcript occurs primarily in male gametophyte tissue, and together these facts led me to hypothesize a gametophytic function for the PIRL6 gene product. I utilized the RNA interference (RNAi) mechanism of post-transcriptional gene regulation to knock-down PIRL6 expression in Arabidopsis and then analyzed the male gametophytes for phenotypic defects. After transformation with an RNAi-inducing anti-PIRL6 construct, I identified forty-four transformant plants based on herbicide resistance. Using both light and confocal microscopy, I observed a substantial amount of dead and abnormal pollen in the transformant plants. Of the forty-four first generation transformant plants, twenty-nine showed defective pollen, defined as having percentages of dead or abnormal pollen at least two times wild-type WS Arabidopsis. By examining progression of male gametophyte development from the microspore through the mature, tri-cellular stage in PIRL6 knock-down plants, I found that the defective pollen phenotype appeared after the microspore stage of development. Though there was reduced transmission of the construct to the second generation, potentially due to a gametophyte-lethal effect of PIRL6 knock-down, the second generation transformant plants generally possessed defective pollen phenotypes comparable to their parental plants, demonstrating heritability of the construct. Overall, my reverse genetics strategy using RNAi successfully demonstrated the critical importance of PIRL6 in pollen development.