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IDENTIFICATION AND CHARACTERIZATION OF THE ACTIVATED DEFENSE RESPONSE IN THE COMMERCIALLY IMPORTANT AGAROPYTE, GRACILARIA GRACILIS, AFTER EXPOSURE TO. Thanks to Wiesner Vos for help with the statistical analysis of the microarray data.

Introduction

Additionally, disease epidemics appear to be most common in aquaculture settings (Farley, 1992 and Ganzhorn et al., 1992), but degradation of natural habitats and pollution can also facilitate disease outbreaks (Osterhaus et al., 1995). In addition to serving as habitats for several marine macro- and micro-organisms, seaweeds act as primary producers in the marine food chain (Chan et al., 2006).

The genus Gracilaria

Besides being agarophytes, Gracilaria species have been used for human consumption (Levring et al. 1969), as food for the cultivation of invertebrates (Chiang, 1981), as fertilizers (Zaneveld, 1959), in the pharmaceutical industry (Chapman, 1950), in the treatment tertiary wastewater treatment (Ryther et al., 1979) and for biogas production (Hanisak, 1981).

Figure 1.1  Basic frond morphology in Gracilaria.  (a) G. verrucosa  (Plastino, 1985); (b) G
Figure 1.1 Basic frond morphology in Gracilaria. (a) G. verrucosa (Plastino, 1985); (b) G

Commercial cultivation of Gracilaria in South Africa

The construction was believed to cause changes in water flow characteristics within the bay, favoring the development of strong thermal stratification of the water column (Anderson et al., 1996). The unavailability of nutrients (especially nitrogen) and the elevated temperature led to the collapse of the Gracilaria resource.

Figure 1.3  Gracilaria  gracilis  beach-cast collected for the local industry in Saldanha Bay in the
Figure 1.3 Gracilaria gracilis beach-cast collected for the local industry in Saldanha Bay in the

Important biotic and abiotic factors that impact marine aquaculture of

Adverse environmental conditions such as low nutrients and elevated temperature, as in the case of Saldanha Bay, can alter the complex balance between host and epiphyte and lead to pathogenesis (Jaffray et al., 1997). A similar phenomenon has been observed in the cultivation of Laminaria japonica (Ishikawa and Saga, 1989), Eucheuma (Ask and Azanza, 2002) and Kappaphycus (Hurtado et al., 2006).

Mechanisms of disease resistance in macroalgae

  • Pathogen-induced defence in macroalgae and higher plants

In higher plants, HR regulation involves sensing changes in intracellular ROS homeostasis during the oxidative burst ( Delledonne et al., 2001 ). This knockout exhibited sensitivity to respiratory inhibitors and resulted in decreased PCD (Weinberger et al., 1999).

Figure 1.4  Hypothetical, simplified scheme of the complex sensory and signalling mechanisms  following the oxidative burst induced by recognition of exogenous PAMPs or endogenous elicitors based  on the current knowledge for marine algae
Figure 1.4 Hypothetical, simplified scheme of the complex sensory and signalling mechanisms following the oxidative burst induced by recognition of exogenous PAMPs or endogenous elicitors based on the current knowledge for marine algae

Functional genomics and its role in the identification of macroalgal defence

  • High throughput DNA sequencing
  • Molecular tools for transcriptome analysis
  • Overview of cDNA microarray technology

The ratio of the gene of interest (target) expressed in a sample versus a calibrator (control) sample compared to a reference gene (reference). A protein of the expected size (30 kDa), indicated by the arrow, was observed and quantified with the protein molecular weight marker (lane M).

Figure 1.5  Simplified schematic depiction of the  cDNA microarray procedure used to measure  mRNA transcript changes between test samples (Duggan et al., 1999)
Figure 1.5 Simplified schematic depiction of the cDNA microarray procedure used to measure mRNA transcript changes between test samples (Duggan et al., 1999)

Significance of this study

INTRODUCTION

Recently, cDNA microarrays were successfully used to identify differentially expressed genes in the red macroalga Gracilaria changii (Ho et al., 2009; Teo et al., 2009). For example, fluorescent labeling procedures require large amounts of RNA per cDNA experiment (Zhoa et al., 2002).

MATERIALS AND METHODS

  • Construction of microarray
    • PCR amplification of cDNA
    • Purification of amplified cDNA products
    • Printing of microarray slides
  • Seaweed sample acquisition for exposure to disease elicitors
  • RNA extraction protocol
    • Quantitative and qualitative analysis of RNA
    • RNA amplification and aminoallyl labelling
  • Microarray hybridization of RNA targets to cDNA probes
    • Target hybridization mixture
    • Pre-hybridization of microarray slides
    • Target and probe hybridization reaction
  • Image acquisition
    • Extraction of features from spots on the microarray
  • Data processing, analysis and normalization
  • Identification of differentially expressed genes
  • Plasmid DNA isolation and sequencing of putative defence genes
  • Bioinformatics and functional annotation of cDNA sequences

Ratio measurements at 230, 260 and 280 nm were used to determine the purity of the RNA samples. Genes were considered to be differentially expressed (95% confidence level) if their M-values ​​deviated more than 1.96(σ) from the mean (µ) of the global distribution.

Table 2.1  Constituents of PCR master mix used for colony PCR.
Table 2.1 Constituents of PCR master mix used for colony PCR.

RESULTS AND DISCUSSION

  • Array construction
  • RNA isolation and preparation of RNA targets
  • RNA amplification and aminoallyl labelling
  • Microarray hybridizations and pre-processing of scanned images
  • Normalization of microarray data
  • Statistical analysis and identification of differentially expressed genes
    • Sequencing of cDNA inserts and bioinformatics for functional
  • Possible roles of functionally annotated G. gracilis genes in the context
    • Significantly differentially expressed G. gracilis genes
    • G. gracilis genes that failed to meet the criteria for significant

These include glutathione S-transferae (GST), heat shock protein (HSP), and an oxidoreductase protein (Taki et al., 2005). To date, the specificities and functions of the different isoforms in plants remain largely unknown (Maeda et al., 2003). In the context of an activated defense response to macroalgae, Collén et al. 2007) observed transcriptional repression of genes involved in general metabolism.

In the context of macroalgae activated defenses, Weinberger et al. 1999) showed that after exposure to agar or agar oligosaccharides, G. The protein tRNA-dihydrouridine synthase catalyzes the synthesis of dihydrouridine, a modified base found in the D-loop of most tRNAs (Bishop et al., 2002) . GDP-fucose is synthesized in the cytosol via two pathways, namely the salvage pathway and the de novo pathway (Moriwaki et al., 2007).

This is one of the most common modifications involving oligosaccharides such as glycoproteins and glycolipids (Hirschberg et al., 1998).

Figure 2.4  Denaturing formaldehyde agarose gel electrophoresis of total RNA preparations (1  µg) from G
Figure 2.4 Denaturing formaldehyde agarose gel electrophoresis of total RNA preparations (1 µg) from G

CONCLUSION

Normalization of the data removed these unwarranted influences and allowed comparison of gene expression data across three separate biological repeats. Nevertheless, the gene expression data must be validated independently of the microarray experiment before the differentially expressed genes identified in this study can be accepted with certainty. In addition, other microarray studies in red macroalgae (Collén et al., 2007; Collén et al., 2006) suggest that the most marked changes in gene expression in response to biotic stress occur within the first 6 hours after stress application. .

Therefore, the validity of gene expression data for a specific number of genes will be discussed in chapter 3. In addition, transcriptional regulation between 0 and 24 hours of exposure to disease triggers will be assessed to characterize the transcriptional regulation of genes of selected more comprehensively.

INTRODUCTION

When RNA is a limiting factor, conventional semiquantitative reverse transcription-PCR can overcome this problem, but quantification remains difficult and relies on endpoint analysis of the PCR product (Siebert, 1997; Wittwer et al., 1997; Higuchi et al. al., 1993). On the other hand, real-time PCR (qPCR) is relatively fast, inexpensive, and requires only pico-nanogram amounts of RNA (Chuaqui et al., 2002). Various fluorescence-based chemistries are compatible with qPCR and the technique is sensitive enough to quantify the accumulation of specific mRNAs by analyzing the efficiency and speed of each PCR reaction (Walker, 2002; Wittwer et al., 1997 ; Higuchi et al., 1993).

Real-time PCR is therefore a combination of three separate steps: (i) the reverse transcriptase-dependent conversion of RNA into cDNA, (ii) amplification of the cDNA using PCR and (iii) detection and quantification of amplification products in real time (Gibson et al., 1996). The more target present in the starting cDNA material, the lower the Ct value (Nolan et al., 2006).

MATERIALS AND METHODS

  • Seaweed sample acquisition for exposure to disease elicitors
  • RNA extraction
    • Twenty four time-course experiment
    • Thirty minute time-course experiment
    • Assessment of DNAse efficacy and presence of
  • Quantitative and qualitative analysis of RNA
  • Conversion of total RNA to complementary DNA (cDNA synthesis)
  • Designing of qPCR primers
  • Identification of reference genes for normalization of qPCR data
  • Normalization and relative quantitation of gene expression
  • Statistical analysis and validation of microarray experiment

A positive control consisted of 10 ng of the 18S rRNA gene cloned into a plasmid, while nuclease-free H2O was added to the no template control (NTC). Thus, PCR amplification of the high copy number 18S rRNA gene was assessed using 450 ng of total RNA as template. The positive control consisted of 10 ng of the 18S rRNA gene cloned into a plasmid, while nuclease-free H2O was added to the no template controls (NTC).

Reference gene-specific qPCR primers were designed at the 3' ends of the DNA sequences using FastPCR (Table 3.2). Real-time PCR analyzes of the 24 h and 30 min time course experiments were used to assess the transcriptional regulation of five putative defense genes (GOIs) and four reference genes (RGs).

Table 3.1  Gene specific qPCR primers for five putative defence genes and the expected  amplicon sizes
Table 3.1 Gene specific qPCR primers for five putative defence genes and the expected amplicon sizes

RESULTS

  • Extraction of total RNA
  • Assessment of DNAse efficiency and genomic DNA contamination in
  • Identification of reference gene for normalization of 24 hour qPCR
  • Identification of reference gene for normalization of 30 min qPCR data
  • Relative quantitation of putative defence genes during the 24 hour
  • Relative quantitation of putative defence genes during the 30 min
  • Validation of microarray experiment

Similarly, a t-test was used to analyze PRX2F expression in the experimental and control samples after 8, 12 and 24 hours. Analysis of PSD expression in the 24 hour time course experiment revealed a very clear repression in transcription in the samples exposed to elicitors (Figure 3.15). Parametric t-tests revealed statistically significant differences in PSD gene expression in the experimental and control samples (indicated by

As expected, RG 329 expression did not fluctuate significantly in the control and experimental samples over the 30 minute time course (Figure 3.18). The expression of thioredoxin in the experimental samples at 15 and 30 minutes pdi was suppressed approximately 3-fold and induced 3-fold relative to the respective time point controls (Figure 3.24).

Figure 3.2  Assessment of RNA quality and integrity by electrophoresis of 1.2 µg total RNA  through a 1.2 % formaldehyde-agarose gel
Figure 3.2 Assessment of RNA quality and integrity by electrophoresis of 1.2 µg total RNA through a 1.2 % formaldehyde-agarose gel

DISCUSSION

  • Validation of microarray experiment
  • Analysis of transcriptional regulation of five genes involved in defence

On the other hand, the results of the 24 h time-course qPCR experiments (although subject to large variability in the gene expression data and consequently low power of t-tests) clearly demonstrated that significant changes in gene expression occurred in G. Although point bleaching was not assessed in this study, the significant transcriptional upregulation of the G. In this context (i.e. three distinct phases of a .. activated defense response), the pattern of transcriptional upregulation of the G.

Before accepting this proposed hypothesis (activation of all three phases in activated defense in G. gracilis), the translational regulation of the gene product (protein) in response to disease triggers over a 24-hour course must be evaluated. Throughout the 24 h time course, a significant suppression of PSD expression in G.

CONCLUSION

The significant transcriptional downregulation of PSD, a gene responsible for the bioconversion of PtdSer, was an interesting observation. Furthermore, PtdSer is directly involved in the initiation of PCD via its deposition in plasma membranes. Thus, suppression of the gene responsible for the bioconversion of PtdSer would theoretically have resulted in a higher concentration of PtdSer in the cell, thus implicating HR and PCD in the activated defense response of G.

The putative serine protease-like gene also showed transcriptional regulation in response to pathogens, but its physiological role in the defense response of G. Characterization of the transcriptional regulation of these five putative defense genes was only the first step in understanding the presumably highly complex activated defense response of G.

INTRODUCTION

Thus, the aim of this chapter was to determine this relationship for two putative defense genes. The mRNA transcript for the peroxiredoxin-like gene (PRX2F) has been shown to be significantly increased over a 24-hour period in response to pathogens. The mRNA transcript for a putative serine protease-like gene appeared to be repressed after pathogen exposure (noting that statistical analysis did not detect statistically significant changes in gene expression due to low power of the assays and possible type II error). ).

In such cases, proteomic studies involving western hybridization analyzes are often compromised because commercially available antibodies provide low cross-reactivity with G. An alternative to this problem was to use a protein expression system in which the coding region of G.

Figure 4.1  Central pathway of molecular biology depicting the biological parameters  that  influence the mRNA-protein correlation (Maier et al., 2009)
Figure 4.1 Central pathway of molecular biology depicting the biological parameters that influence the mRNA-protein correlation (Maier et al., 2009)

MATERIALS AND METHODS

  • Determination of full-length genes
  • PCR amplification of full-length genes
  • Cloning of open reading frames into protein expression vector
  • Recombinant protein expression
  • Affinity purification of recombinant proteins
  • Antibody production and determination of antibody titre
  • Purification of antibodies by polyethylene glycol precipitation
  • Seaweed sample acquisition for exposure to disease elicitors
  • Isolation of seaweed total protein
  • Determination of antibody specificity and optimal amount of G
  • SDS-PAGE and western hybridization analysis of the 24 hour time-
  • Densitometry analysis of western hybridization immunoblots
  • Statistical analyses of western hybridization analysis
  • Determination of full-length genes
  • PCR amplification of G. gracilis ORFs and cloning into pET29a
  • Recombinant protein expression
  • Affinity purification of recombinant proteins
  • Polyclonal antibody production and determination of the titre and
  • Western hybridization analysis of 24 hour time-course experiment

The expected sizes of the PRX2F-like and serine protease coding regions were 675 and 999 bp, respectively. Lane (1) represented PRX2F gene amplification, lane (2) represented serine protease-like gene amplification, and λ Pst represented DNA molecular weight scale. The predicted 94% solubility of the recombinant serine protease-like protein was contradicted by its observed insolubility in E.

In contrast, expression of the recombinant serine protease-like protein could not be achieved in the auto-inducing media of the small-scale experiment. Aliquots of the broth were collected before induction (lane 1) and 22 hours after induction (lane 2). Prior to western hybridization analyzes of PRX2F and serine protease-like proteins, extracting total protein from all G.

This proved to be highly effective as only a single positive signal, corresponding to the expected size of the G. gracilis serine protease-like protein, was observed in all three biological replicate immunoblots (Figure 4.23; panels A – C).

Figure 4.2   Prediction of open reading frames present in the cDNA clone harbouring the G
Figure 4.2 Prediction of open reading frames present in the cDNA clone harbouring the G

DISCUSSION

CONCLUSION

General Discussion

Future Work

Predefined filter for use in image processing

Script used for microarray data analysis

Multiple sequence alignments with PSP genes

Sequence alignment with G. gracilis and C. crispus peroxiredoxin-like genes

Transit peptide prediction using G. gracilis thioredoxin gene sequence

Creator TM SMART TM cDNA library construction kit protocol

Figure

Figure 1.1  Basic frond morphology in Gracilaria.  (a) G. verrucosa  (Plastino, 1985); (b) G
Figure 1.2  Map of Saldanha Bay / Langebaan Lagoon system on the west coast of South Africa
Figure 1.3  Gracilaria  gracilis  beach-cast collected for the local industry in Saldanha Bay in the
Figure 1.4  Hypothetical, simplified scheme of the complex sensory and signalling mechanisms  following the oxidative burst induced by recognition of exogenous PAMPs or endogenous elicitors based  on the current knowledge for marine algae
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References

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