Abstract
M.Sc.
LPS, as a pathogen associated molecular pattern (PAMP) molecule can interact with
eukaryotic host cells. Interaction occurs by either direct contact or due to the release of
micelles containing LPS from bacterial cell surfaces. LPS activates innate host defence
systems in both invertebrate and vertebrate animal/insect cells via analogous pathways,
where the lipid A component,is responsible for the activities. LPS from several plant
pathogens have been shown to activate a number of defence-related responses in plants.
Initial concentration studies and cell viability assays were conducted to assess
isonitrosoacetophenone (INAP) and LPS as elicitors of defensive responses in tobacco
(Nicotiana tabacum cv. Samsun) cell suspensions. The effective concentrations were
found to be 100vM INAP and 100μg/ml LPS. RNA was isolated, quantified and analysed
to confirm the quality of the starting material for differential display analysis.
The DDRT-PCR technique was successfully applied in order to obtain comparative
"displays" of PCR amplicons derived from three sub-divided mRNA pools (i.e. each of
the three different anchor primers, per treatment). Significant differences in the profiles
of control, INAP and LPS treated cells were observed, indicating that the eliciting agents
had prominent effects on cellular homeostasis, resulting in an altered gene expression
profile.
DDRT-PCR can be technically challenging at a number of steps. Modifications were
incorporated to initially obtain differentially expressed transcripts (DETs), as well as reamplify
the DETs. 223 Putative DETs were isolated from denaturing polyacrylamide
sequencing gels. 172 Putative DETs were re-amplified, of which 126 appeared as good
candidates for further analysis. Finally, 96 putative DETs were chosen for reverse
Northern analysis. DDRT-PCR has been reported to be plagued by false positives.
Reverse Northern analysis confirms the presence of the putative DET from the subdivided
RNA pool, as well as affirming the differential expression, compared between the
control and inducer blots. 26 DETs were selected for cloning, of which 16 were
sequenced. Homologies between the DETs and known sequences were determined using
BLASTN and BLASTX alignments, DNAssist software, as well as MIPS alignments to
the Arabidopsis genome.
Five of the DETs were assigned putative functions in plant signal perception,
transduction and the defence response, based on their respective sequence homologies to
sequences involved in innate immunity.
It is proposed that the DET, HAP3-15, represents the plant equivalent of a component of
the innate immunity pathway in mammals and Drosophila. It is further proposed that
HAP3-15 represents a S-Receptor kinase protein (SRK), with a defensive role in
distinguishing self from potential pathogens. Therefore, as a SRK, HAP3-15 would
function as a transmembrane receptor able to conduct an external signal through the
membrane to the cytoplasm as a form of signal perception. Subsequently HAP3-15 could
ii
play a role in phosphorylation cascades through the kinase domain and, consequently, be
responsible for signal transduction. In addition, LPS would then represent the ligand
creating the signal perceived by the SRK, HAP3-15, with oligosaccharide binding ability.
HAP3-15 was also identified as a true positive by the INAP probe in reverse Northerns,
implying that both the biological and chemical inducers used, activated the same receptor
kinase. Whether the same signalling pathway was followed during the phosphorylation
cascades has not been determined.
Further analysis will require Northern blots in a time study to investigate the kinetics of
induction. In addition, longer sequence information for each of the five DETs needs to be
obtained to identify the corresponding genes in order to investigate their roles in innate
immunity in plants.