“Gene discovery” in cowpea, a drought tolerant African plant

Authors: Inge Gazendam (PhD project) and Nanette Coetzer (MSc project)

Drought is a serious problem affecting crop production throughout the world, especially in areas that are dependent on natural rainfall such as large parts of sub-Saharan Africa. Climate change forecasts an even larger impact on food security.

Since plants are stationary, they cannot move away from conditions that do not suit them. They have developed various mechanisms to adapt to adverse conditions such as drought, salt, heat, cold and various other conditions that are termed “abiotic” stresses.

A large amount of research is going into figuring out the mechanisms that enable plants to survive such unfavourable abiotic conditions. These adaptations include “macro” scale changes to plant morphology such as leaf area and stomata behaviour, and “micro scale” changes like switching on the production of specific protective proteins and other molecules, ion channels and changing membrane characteristics inside cells.

Drought tolerance is governed by multiple genes, and if the most important genes can be identified they can be used to engineer crop plants with improved drought tolerance. In a collaborative project between the Agricultural Research Council Vegetable and Ornamental Plant Institute and the Department of Plant Science, FABI at the University of Pretoria, we chose cowpea (Vigna unguiculata) for drought gene discovery. Some varieties of this important African crop are known to be quite drought tolerant.

Gene discovery can be divided into two main steps:

Step 1: Lab experiments with treated/control plants and isolate the genes; in our case drought-treated cowpea,

Step 2: Use bioinformatics to help you identify the genes responding to the treatment; in our case we developed two bioinformatics software packages.

We grew drought hardy cowpea plants under simulated drought conditions in a greenhouse (Figure 1). Plants of a less hardy variety were well-watered and used to “subtract” the background of genes that are normally expressed. A molecular technique called “suppression subtraction hybridisation” (SSH) was employed which picks out the genes that are present in only the drought-stressed plant.

Because so many genes were potentially involved, the high-throughput technique called microarrays was used to “fish-out” drought tolerance genes. Microarrays are glass slides spotted with small droplets containing DNA of your genes of interest. We used the Microarray robot and scanner at the ACGT Microarray Facility at FABI, UP (http://microarray.up.ac.za).

Microarray experiments produce thousands of data points, so this is where we needed to develop the first software package “SSHscreen”, using the statistical programming language called ‘R’. The input to SSHscreen was a large dataset of intensity values from the DNA spots on the microarray slides. The output from SSHscreen - after various data analysis steps - was a list of potential drought tolerance genes.

The SSHscreen software helped us to choose a selection of genes for DNA sequencing. To help interpret and manage the sequence information we developed the second software, a database called SSHdb (Figure 2). SSHdb allowed us to compare the unknown sequences against all known sequences in the international internet database called “Genbank”, and find out which proteins they are most likely to code for. Our results indicated that cowpea plants protect themselves against drought by detoxification of unwanted compounds, stabilization of useful proteins, and down-regulation of photosynthesis. The next step of the PhD project is to test one of the most promising candidate genes in transgenic plants.

Our work has not only shed light on the drought response in plants but the software pipeline can be used for gene discovery in any biological system, not just plants!

To make it easy for scientists to apply this approach, we have written up the experimental protocol in a book chapter (Berger et al., 2007), and the cowpea work and SSHscreen/SSHdb are described in (Coetzer et al., 2010). The software is freely available from http://microarray.up.ac.za/SSHscreen/. Users can start their own sequence database at SSHdb and share the data with collaborators of their choice by registering at http://sshdb.bi.up.ac.za/.

Further reading

Berger DK, Crampton BG, Hein I, Vos W (2007) Screeing cDNA libraries on glass slide microarrays. In J Brampal, ed, Microarrays, Second Edition, Volume II, Applications and Data Analysis, Ed 2nd. Humana press, Totowa, New Jersey, USA, pp 177-203

Coetzer N, Gazendam I, Oelofse D, Berger D (2010) SSHscreen and SSHdb, generic software for microarray based gene discovery: application to the stress response in cowpea. Plant Methods 6: 10