Investigating the molecular components that result in the activation of fumonisin B1-induced programmed cell death in Arabidopsis thaliana

Abstract

Programmed cell death (PCD) is a gene expression-dependent cell suicide program invoked by plants for selective elimination of unwanted cells during adaptive responses to stress or during growth and organ development. The signalling processes and protein/gene networks controlling plant PCD are not fully understood. Various experimental systems have been used to study plant PCD. The Arabidopsis-fumonisin b1 (FB1) system was selected in this project for its applicability to both in vitro cell suspension cultures or detached leaf disc experiments and whole plant experiments. FB1 is a cell death-activating mycotoxin produced by the maize fungal pathogen Fusarium verticillioides. FB1-induced PCD in Arabidopsis is dependent on light and is regulated by the plant defence hormone, salicylic acid. FB1 triggers PCD in light-grown Arabidopsis cell cultures, while dark-grown cell cultures are immune. Furthermore, by removing dark-grown cells from their growth medium and incubating them in medium taken from light-grown cells compromised this immunity to FB1, even when these cells were incubated in the dark. This demonstrated that soluble factors secreted into the extracellular matrix control the light dependency of FB1-induced PCD in Arabidopsis. A screen to identify extracellular proteins secreted into the growth medium with a putative regulatory function on FB1-induced cell death was setup using Arabidopsis cell cultures grown either in the light or in the dark. Isobaric tags for relative and absolute quantification (iTRAQ) technology was used to identify proteins differentially expressed in response to light, with quantitative reverse transcription polymerase chain reaction (qRT-PCR) evaluating the effects of light on the transcriptional response of selected candidates to FB1 exposure. Amongst numerous proteins with an expression profile corresponding with what would be expected for a protein with a putative function in regulating PCD, Arabidopsis RIBONUCLEASE 1 (RNS1) was selected for further analysis. RNS1 expression is activated by FB1 under light conditions and darkness suppresses this response. Consistent with a putative function in PCD, RNS1 overexpressing plants were more susceptible to FB1, while loss-of-function transfer-DNA insertional mutants (rns1) gained immunity to FB1. Furthermore, complementation with native, but catalytically inactive RNS1, mimicked the rns1 mutant, and also gained immunity.
In addition to the relationship between FB1 and light regulation, another platform to stimulate plant cells into initiating PCD focused on the relationship between FB1 and extracellular ATP (eATP). During FB1 accumulation, a rapid depletion of eATP occurs, instigating a cascade of defence signalling. Using this antagonism to manipulate cell cultures, whole genome microarray analysis lead to the identification of a zinc-finger protein in the C2H2 transcription factor family, referred to as Extracellular ATP Responsive Protein 1 (EARP1). EARP1 expression is activated by FB1 in cell cultures, and this response is inhibited by additional treatment of exogenous ATP. Loss-of-function transfer-DNA insertional mutant lines (earp1) showed a phenotype of reduced cell death in response to FB1 treatment. In order to determine the down-stream signalling components of the EARP1 transcription factor, another whole genome microarray analysis was devised to compare the genetic profile of earp1 against the wild type, in response to FB1. The microarray identified EARP1 as a potential ‘hub-gene’, responsible for the regulation of over 70% of FB1-responsive genes. EARP1 will be a useful molecular tool to identify crucial regulatory genes of FB1-incduced PCD in Arabidopsis.
A final effort to hone in on key PCD-regulatory components used whole genome microarray analysis to develop a screen focusing on the antagonistic relationship between eATP and SA. SA is an important phytohormone for FB1-induced PCD, so much so that Arabidopsis lines that are unable to accumulate SA are incapable of activating PCD. The screen was successful in identifying SA-responsive genes with either a synergistic or antagonistic response upon the addition of exogenous ATP. Furthermore, the majority of selected candidates from the screen responded to FB1 treatments. In addition to identifying a number of FB1-responsive genes, a number of genes selected from the whole-genome analysis showed altered expression levels in loss-of-function transfer-DNA insertional mutants of ICS1. ICS1 is essential for the synthesis of SA via the ISOCHORISMATE SYNTHASE (ICS) pathway and this project has revealed that FB1-induced SA biosynthesis occurs via this pathway.
This PhD project has made great strides in elucidating the molecular mechanisms involved in FB1 signalling through the development of proteomic- and transcriptomic screens. Each screen has also contributed to an in-house database, which sets a foundation for future projects investigating PCD.