Construction and characterisation of a pearl millet defence response cDNA library 

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Chapter 2 Construction and characterisation of a pearl millet defence response cDNA library


Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. In order to construct a pearl millet cDNA library enriched for defence response genes, suppression subtractive hybridisation (SSH) was employed following wounding and treatment of pearl millet plants with the pathogen elicitors chitin and flagellin. A forward and reverse library was constructed to identify genes that are up and down regulated during the defence response, respectively. Furthermore, a quantitative procedure for screening cDNA libraries constructed by SSH is presented. Following two colour Cy dye labelling and hybridisation of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). A third enrichment ratio 3 (ER3), was also calculated following hybridisation of unsubtracted tester and unsubtracted driver cDNAs. Graphical representation of ER1 and ER2, or ER3 plotted against inverse ER2 enabled identification of clones that were likely to represent up regulated transcripts. Normalisation of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet clones identified by this quantitative approach was verified by inverse Northern blots. Sequence analysis was performed on clones shown to be up regulated during the defence response identified from plots of ER1 versus ER2, and ER3 versus inverse ER2. This pearl millet cDNA library serves as a basis for further microarray studies to examine the effect of defence signalling molecules and pathogen infection on pearl millet gene expression.


Pearl millet [Pennisetum glaucum (L.) R. Br] is a member of the Gramineae family that includes many major monocotyledonous agricultural crop species such as maize, rice, wheat, sorghum, barley and oats. Many of these species have large, complex genomes that present a substantial challenge to molecular studies (Carson et al., 2002). Much of what is known at the genetic level for crop plants has been obtained through genetic mapping and synteny comparisons with species with relatively small genomes such as sorghum and rice (Devos and Gale, 2000; Gale and Devos, 1998; Keller and Feuillet, 2000). In recent years, completion of the rice genome sequence has added to our knowledge of cereal genome structure and complexity (Goff et al., 2002; Yu et al., 2002). However, information at the level of gene sequence and function is still very limited for most non-model crop species. As a consequence, research groups have employed Expressed Sequence Tags (ESTs) as a method for gene discovery in crop species with complex genomes (Akimoto-Tomiyama et al., 2003; Carson et al., 2002; Hein et al., 2004).
ESTs provide a rapid method to establish an inventory of expressed genes through determination of single pass sequences of 200 to 900 bp from one or both ends of randomly isolated gene transcripts that have been converted to cDNA. The sequences are sloppy and have a relatively high error rate, but, in most cases, they are sufficiently accurate to unambiguously identify the corresponding gene through homology comparisons with known genes. In addition, high throughput technology and EST sequencing projects can result in identification of significant portions of an organism’s gene content and thus can serve as a foundation for initiating genome sequencing projects (Alba et al., 2004). Most importantly, thousands of sequences can be determined with limited investment.
In order to add value to EST data, several techniques exist to isolate and characterise cDNA fragments that are differentially expressed under specific conditions. These include differential display reverse transcriptase PCR (DD RT-PCR), cDNA amplified fragment length polymorphism (cDNA-AFLP), serial analysis of gene expression (SAGE) and suppression subtractive hybridisation (SSH) (Bachem et al., 1996; Diatchenko et al., 1996; Liang and Pardee, 1992; Velculescu et al., 1995). In DD RT-PCR cDNA is synthesised from RNA using reverse transcriptase and an oligo dT primer that anneals to the 3’ polyA tail of mRNA. Thereafter, subsets of cDNA populations for comparison are amplified with short, non-specific oligonucleotide primers, in combination with oligo dT primers, and visualised on polyacrylamide gels. Differentially expressed cDNA fragments are isolated from the gel and sequenced. Like DD RT-PCR, cDNA-AFLP is derived from a DNA fingerprinting method and also involves the selective PCR amplification of subsets of cDNA populations for comparison on polyacrylamide gels. However, cDNA-AFLP is an improvement on DD RT-PCR in that amplification is specific, using primers with higher annealing temperatures that bind to adaptors ligated to the ends of double stranded cDNA molecules following restriction digestion. SAGE is an elegant technique that combines differential display and cDNA sequencing approaches, and it has the advantage of being quantitative. Unfortunately, SAGE is laborious and requires an extensive foundation of sequence information. All three techniques described above are often limited by their ability to capture low abundance transcripts (Alba et al., 2004).
Suppression subtractive hybridisation (SSH) is a powerful technique to enrich libraries with differentially expressed cDNAs, and can be combined with large scale sequencing approaches (Birch and Kamoun, 2000). The SSH technique utilises subtractive hybridisation to selectively remove cDNA from genes that are expressed in both control and experimental samples, and a post hybridisation PCR step to preferentially amplify cDNA unique to the experimental sample (Figure 2.1). One of its main advantages is that it includes a normalisation step that enables the detection of low abundance differentially expressed transcripts such as many of those likely to be involved in signalling and signal transduction, and might thus identify essential regulatory components in several biological processes (Birch and Kamoun, 2000). A further advantage of SSH is that it yields cDNA fragments that can be used directly for the construction of DNA microarrays.
In this study, we aimed to isolated pearl millet genes that are involved in defence response. In order to achieve this, we treated pearl millet seedlings with the pathogen elicitors chitin and flagellin and mechanically wounded the leaves. Differential gene isolation was accomplished through application of SSH to enrich cDNA libraries for genes up- or down regulated in response to elictor treatment. The SSH procedure was chosen for several reasons: it includes a normalisation step, it enriches for differentially expressed transcripts, and it yields cDNA fragments that can be used directly for the construction of DNA microarrays. The normalisation step is particularly important because a few defence genes, such as those encoding the pathogenesis related (PR) proteins, are abundantly induced during defence response, potentially obscuring important defence specific transcripts expressed at much lower levels (Mahalingam et al., 2003).
In previous studies, SSH libraries were screened to identify cloned differentially expressed genes by colony blot hybridisation, inverse Northern analysis or cDNA AFLP (Birch et al., 1999; Hein et al., 2004; Mahalingam et al., 2003). However, these methods are time consuming, and do not allow the level of enrichment of a transcript to be quantified. SSH has also been used as a method to generate a cDNA library to use in subsequent cDNA microarray expression profiling (Yang et al., 1999). In this study, cDNA microarrays were used to screen PCR amplified clones from forward and reverse subtracted SSH libraries to identify genes from pearl millet that are up- or down regulated during defence responses, respectively. This quantitative approach of determining the extent to which transcripts were enriched by the SSH process allowed us to identify and exclude clones that were not derived from differentially expressed transcripts and to determine whether transcripts were rare or abundant. Based on cDNA microarray analysis of forward and reverse subtracted pearl millet SSH libraries, SSH clones were selected for sequence analysis. A number of genes exhibited significant similarities to genes associated with plant defence and stress responses.

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All reagents were purchased from Sigma (Aston Manor, South Africa) unless otherwise stated. Sequences of adaptors and primers used in PCR, SSH and sequencing reactions are shown in Table 2.1.

Plant material and growth

Pearl millet breeding lines ICML12=P7 and 842B were obtained from ICRISAT India and ICRISAT Zimbabwe respectively. ICML12=P7 is resistant to downy mildew caused by the oomycetous fungus Sclerospora graminicola, and rust (causal agent: Puccinia substriata var. indica) (Singh et al., 1990), whereas 842B is moderately susceptible to S. graminicola infections (M. O’Kennedy, personal communication).
Pearl millet seed was sterilised by briefly rinsing with 70% ethanol, followed by 20 min incubation in 0.7% sodium hypochlorite. Following three washes with sterile distilled water, seeds were plated on half strength MS medium (Murashige and Skoog, 1962), and incubated at 25ºC with a 16 hour light/8 hour dark photoperiod.

Elicitor preparation

The fungal elicitor chitin was purchased from Sigma Aldrich (Sigma catalogue number C-3641). The bacterial elicitor flagellin (Felix et al., 1999) was prepared from Bacillus sp. alk 36 (E. Berger, personal communication). Briefly, Luria broth pH 8.5 (Sambrook et al., 1989), was inoculated with a colony of Bacillus sp. alk 36 and grown with shaking at 42ºC for 48 hours. An equal volume of 0.1 N NaOH was added to the culture, which was then left at room temperature for 30 min to strip the flagellin from the cell wall (cell bound fraction). Samples were centrifuged at 6000 rpm for 10 min to pellet bacteria, and the supernatant containing the flagellin was transferred to a new tube. Alternatively, ten millilitres of bacterial culture was pelleted at 6000 rpm for 10 min, and proteins remaining in the supernatant were precipitated (extracellular fraction). An equal volume of 10% trichloroacetic acid was added to the cell bound and extracellular supernatants, which were incubated at -20ºC for one hour to precipitate the proteins. Extracellular and cell bound proteins  were collected by centrifugation at 11 000 rpm for 20 minutes, the protein pellet was dried in a laminar flow bench, and resuspended in a total volume of 1 ml phosphate buffered saline (PBS) (Sambrook et al., 1989). Five micrograms of protein was analysed on a 10% SDS polyacrylamide gel (Ausubel et al., 2005) to assess the presence and quality of flagellin in the crude extracellular protein extract. Proteins were detected by staining the polyacrylamide gel in 0.1% Coomassie Brilliant R, 50% methanol, 10% acetic acid.
Presence of the flagellin in the protein extract was confirmed by Western blot analysis according to the method of (Ausubel et al., 2005). Briefly, 2.5 μg protein was run on a 10% SDS polyacrylamide gel, and transferred to polyvinyldifluoride (PVDF) membrane at 15V overnight in CAPS (3-[cyclohexylamino]-1-propane-suphonic acid) buffer (10 mM CAPS 3% methanol, pH 10.5). Following protein transfer, the membrane was incubated in blocking solution (10 mM Tris, pH 7.5, 150 mM NaCl, 3% milk powder and 0.1% Tween 20) for 2 hours at room temperature. Rabbit antibodies raised against the Bacillus sp. alk36 flagellin protein were diluted 1:2000 in blocking solution and added to the membrane for 2 hour at 37ºC. The membrane was subjected to three washes of five minutes each with washing buffer (10 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) to remove unbound antibodies. Following an hour incubation at 37ºC with Anti-Rabbit IgG (whole molecule) alkaline phosphatase conjugate (Sigma) (diluted 1:1000 in blocking buffer), the membrane was once again subjected to three washes of five minutes each in washing buffer. The membrane was equilibrated in detection buffer (100 mM Tris, pH 9.5, 100 mM NaCl and 50 mM MgCl2) subsequent to antibody detection with NBT/BCIP solution (Roche Diagnostics, Mannheim, Germany) (200 μl in 10 ml detection buffer). The membrane was incubated in the dark until bands appeared. The reaction was stopped by the addition of TE, pH 8.0 (10 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0).

Treatment of pearl millet seedlings with elicitors

Leaves of ten day old ICML12=P7 and 842B pearl millet seedlings were wounded by pricking leaves at one centimetre intervals with a sterile needle. The abaxial surface of ICML12=P7 leaves was inoculated with a total of 100 µl of either 100 mg/ml chitin, or a crude boiled extract of flagellin. Control 842B plants were treated with sterile deionised water and not wounded. Each elicitor and control inoculation was repeated in triplicate. Plates containing pearl millet seedlings were sealed with Micropore™ tape (3M, Isando, South Africa), and were incubated at 25ºC with a 16 hour light/8 hour dark photoperiod. Necrotic lesion formation was observed under a dissecting microscope at 24, 48 and 96 hours post inoculation.

RNA isolation

Pearl millet leaves were harvested 5, 14 and 24 h post elicitor treatment (hpe), and immediately placed in liquid nitrogen. Total RNA was prepared from ten day old chitin or flagellin inoculated ICML12=P7 leaves, or untreated 842B leaves using a Plant RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA yield was determined by measuring absorbency at 260 nm, and RNA integrity was determined by electrophoresing two micrograms total RNA through a 1.2% agarose gel. Poly (A)+ RNA was purified from total RNA using an Oligotex® mRNA Mini Kit (Qiagen).

RT-PCR amplification of the actin gene

A two step reverse transcriptase PCR (RT-PCR) was performed to amplify the actin gene from pearl millet mRNA samples. First strand cDNA synthesis reactions were performed using a C. therm RT-PCR kit (Roche Diagnostics) according to the manufacturer’s instructions. Each 20 μl reaction contained 1 μM actin forward primer (Table 2.1), 200 μM dNTPs, 3% (v/v) DMSO, DTT, 1 X RT buffer (Roche Diagnostics), 3 units C. therm polymerase (Roche Diagnostics) and 50 ng mRNA. The reaction was incubated at 60ºC for 30 min, followed by 94ºC for 2 min to inactivate the reaction.

CHAPTER ONE Literature review Overview of plant defence response mechanisms
1.1 Introduction
1.2 Pearl millet
1.3 Pearl millet diseases
1.4 Pathogen recognition by plant cells
1.5 Plant defence signalling networks
1.6 Biochemistry of plant defence responses.
1.7 DNA microarrays: tools for studying global gene expression changes during plant defence response.
1.8 Aims of the project
1.9 Literature cited
CHAPTER TWO Construction and characterisation of a pearl millet defence response cDNA library 
2.1 Abstract
2.2 Introduction
2.3 Materials and methods.
2.4 Results and discussion .
2.5 Literature cited .
CHAPTER THREE Nitric oxide mediated transcriptional changes in pearl millet 
3.1 Abstract .
3.2 Introduction .
3.3 Materials and methods.
3.4 Results and discussion .
3.5 Literature cited
CHAPTER FOUR Evaluation of pearl millet defence signalling pathways involved in leaf rust (Puccinia substriata) resistance and perception
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.4 Results and discussion
4.5 Literature cited


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