Large scale prediction and reverse genetics analysis of programmed cell death genes in Chlamydomonas reinhardtii

Abstract

Programmed cell death (PCD) refers to any form of cell death that is coordinated by the genome. PCD consists of complex molecular pathways which directly cause the death of a cell. The most well-known form of PCD is an animal-specific process known as apoptosis, of which the underlying molecular pathways are well-characterized. However, despite the observation that PCD occurs ubiquitously throughout the tree of life, little is known regarding the molecular mechanisms of PCD in non-animal systems. In response to a number of different environmental stressors, Chlamydomonas reinhardtii undergoes a form of PCD which exhibits characteristics of apoptosis, including DNA laddering, accumulation of reactive oxygen species, and externalization of phosphatidylserine. The presence of these shared features between C. reinhardtii PCD and apoptosis suggests that similar molecular pathways may underlie the two processes. Despite this, many of the genes required for apoptosis in animals appear to be absent in C. reinhardtii. In the present study, we first employed a large-scale, homology-based, bioinformatics approach to predict the gene products that contribute to C. reinhardtii PCD. From the list of sequences that were obtained by these methods, we selected several entries to study in further detail using a reverse genetic approach. We obtained C. reinhardtii mutant strains, each with an insertional mutation in one of the selected genes, from the Chlamydomonas Library Project (CLiP) and validated that the insert had been mapped accurately in each of the mutant strains. To determine the effects of losing any of these selected genes, we subjected the mutant and parental background strains to a PCD-inducing heat stress. First, we sought to determine if the loss of a single selected gene would affect the ability of cells to undergo PCD in response to stress. Second, we wanted to determine if the loss of one of the selected genes would alter either the timing or intensity of phenotypes characteristic of C. reinhardtii PCD. Our results suggest a role for several of the selected genes in PCD, and future studies will be aimed at further characterizing these roles in more detail.