Generation and Screening of T-DNA Insertion Mutants that alter Localization of PDLP5

Abstract

Plasmodesmata (PD) are structures unique to plants, which serve as a signaling pathway between distinctly separate neighboring cells and facilitate cell-to-cell communication. PD has been shown to play a role in plant growth, developmental processes, and plant defense. Recently a family of proteins was found to localize to the plasmodemata, indicating a potential role in these multiple functions. The fifth isoform of the family has been implicated in a plant defense role via the regulation of components associated with PD, facilitation of cellular signaling, and alteration of PD size exclusion. Else wise, little is known about PDLP5 including the molecular players that interact with the protein to aid in function or the specific localization to PD. In order to elucidate these players, a genetic screen was designed utilizing the molecular mechanisms of a certain bacteria, Agrobacterium tumefaciens. Mutants were generated using A. tumefaciens to insert transfer DNA into transgenic Arabidopsis thaliana lines with fluorescent proteins fused to PDLP5. The resulting plants were screened for any alteration of normal fluorescent signal. Based on the probability of an insertion into the DNA sequence of molecular player that interacts with PDLP5 and subsequent disruption or altered expression of such a player, few mutants were found to alter the localization of our protein of interest. Albeit, the study provided proof of concept indicating the methodology is sound for future investigations of this nature, as continued study would result in the isolation of additional mutants.