Alternative transcripts of the RPS4 resistance gene in Arabidopsis: Do they produce truncated proteins?

Abstract

Plant disease due to pathogenic infection causes large economic losses worldwide. The study of plant disease resistance will potentially lead to crop improvements in an effort to feed the growing world population. This study focuses on the RPS4 resistance gene in Arabidopsis thaliana, which confers resistance to the pathogenic bacteria Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 in a gene-for-gene specific interaction. Like many related resistance genes, RPS4 produces multiple alternative transcripts whose functions are not yet fully understood. The dominant RPS4 alternative transcripts are produced via alternative splicing causing the retention of either intron 3 or introns 2 and 3. Previous research revealed the combined presence of both the full and alternative transcripts was required for proper RPS4 function. These alternative transcripts have the potential to be translated into truncated proteins due to the presence of premature in-frame stop codons within the retained introns. The goal of this study was to detect and analyze the function of these putative truncated proteins. To detect truncated proteins, N- and C-terminal epitope tags were added to both the genomic RPS4 sequence and cDNAs of different lengths. These constructs will be transiently expressed in tobacco leaves using Agrobacterium. Protein samples will be extracted for an immunoblot assay to reveal the presence or absence of truncated RPS4 proteins. Subcloning RPS4 transcripts into plasmid vectors for Agrobacterium transformation required the majority of time and effort for this project. To determine the biological function of the putative truncated proteins, an Agrobacterium-mediated transient assay on tobacco will be performed using the same RPS4 constructs. The plants will be observed for the presence of a ubiquitous defense response called the hypersensitive response (HR). If a certain RPS4 construct or combination of constructs causes HR, it will lend evidence toward the functional importance of those constructs in generating defense responses. It has been hypothesized that in resting cells, R-proteins are self-inhibited by intramolecular interactions. When the plant recognizes an invading pathogen, these intramolecular interactions are disrupted, leading to defense responses. To test this hypothesis on the RPS4 gene, another transient assay will be performed using RPS4 constructs with N- and C-terminal serial deletions in various combinations. If a certain combination of constructs causes HR, it will provide evidence that these sequences interact intramolecularly. The serial deletion constructs are in the initial stages of development and will be used in the future to perform this experiment. The sum of this research will improve our understanding of alternative transcripts and their function in plant resistance.