- Open Access
Unraveling regulatory networks in plant defense using microarrays
© BioMed Central Ltd. 2001
- Published: 3 January 2001
DNA microarrays are being used to comprehensively examine gene expression networks during the plant defense response that is triggered when a plant encounters a pathogen or an elicitor molecule. In addition to identifying new genes induced during defense, these studies are providing new insights into the complex pathways governing defense gene regulation.
- Salicylic Acid
- Methyl Jasmonate
- Methyl Jasmonate
- Systemic Acquire Resistance
- WRKY Factor
Plants induce an array of defenses in response to pathogen attack . For example, a rapid defense mechanism known as the hypersensitive response is often induced upon recognition of a pathogen-derived molecule . The hypersensitive response results in rapid death of plant cells at the site of infection, which probably serves to prevent pathogen spread. Subsequent to the hypersensitive response, a long-lasting enhanced resistance develops throughout the plant, termed systemic acquired resistance (SAR) . SAR confers resistance not only to secondary challenge by the initial infecting pathogen, but also to a wide range of normally virulent pathogens. SAR is associated with increased levels of salicylic acid (SA), both at the infection site and systemically. SA is necessary and sufficient for SAR: application of SA onto plants can induce SAR, whereas expression of an SA-oxidizing enzyme, salicylate hydroxylase, suppresses SAR. SAR is also correlated with the induced expression of genes in uninfected secondary tissues. These genes include the pathogenesis-related (PR) genes, some of whose protein products have antimicrobial activity. A limited set of SAR marker genes has been identified using traditional methods of screening [3,4]. Now, Maleck et al.  have applied microarray technology to provide a much more comprehensive description of SAR genes from Arabidopsis thaliana. Additionally, knowledge of the genomic sequence for Arabidopsis has enabled them to identify a common promoter element associated with a particular set of SAR-induced genes.
Maleck et al.  used an Arabidopsis microarray containing 10,000 expressed sequence tags (ESTs). Because of redundancy in the EST set, this represented about 7,000 genes or 25-30% of all Arabidopsis genes. This microarray was used to profile gene expression changes under 14 different conditions related to SAR. Most of these conditions corresponded to fully induced SAR generated by either chemical or biological means. For example, plants were treated with benzothiodiazol, a chemical analog of SA, and RNA was extracted 48 hours later. RNA was also collected from uninfected secondary tissue 44 hours after infection of primary leaves with avirulent bacteria. Three Arabidopsis mutants (cim6, 7, and 11) that have a constitutive SAR phenotype and one mutant (npr1/nim1) that is compromised for SAR were also analyzed. Maleck et al.  then compared the gene expression profiles between the 14 experiments involving SAR and identified 413 ESTs (about 300 genes) that differ in expression during SAR. The criterion for selecting these ESTs was conservative: ESTs had to exhibit differential expression equal to or greater than 2.5-fold in at least two SAR-relevant samples.
The identification of these SAR genes, many of which are novel, is a significant achievement in itself. This study goes much further, however, by analyzing in detail the expression profiles of the 413 SAR-associated ESTs. Two types of clustering algorithms were used to derive groups of SAR genes with highly similar regulation patterns. The cluster group containing the PR-1 gene, termed the PR-1 regulon, was further analyzed; PR-1 is a commonly used marker for SAR. The induction of the Arabidopsis PR-1 gene is dependent on the protein NPR1 (also known as NIM1) [6,7]. NPR1 has been shown to interact with members of the TGA family of basic leucine zipper (bZIP) transcription factors [8,9,10]. The promoter of the Arabidopsis PR-1 gene contains a binding site for TGA-bZIP factors (the sequence TGACG) that serves as a positive cis-acting element for SA induction . Thus, it was expected that all genes of the PR-1 regulon would contain a TGA-bZIP binding site in their promoters. Maleck et al.  inspected the promoters (about 1 kb of upstream sequence) of 26 PR-1 regulon genes for known cis elements. Surprisingly, the minimum TGA-bZIP recognition site (TGACG) is absent from 9 of 26 PR-1 regulon promoters and its overall occurrence in the promoter set is less than that expected at random. This suggests that TGA-bZIP factors are unlikely to act as common regulators of PR-1 regulon genes. In contrast, the core binding site for WRKY transcription factors , known as a W box (the sequence TTGAC), is found in all 26 promoters. It is present on average 4.3 times per promoter and the boxes are often clustered. The clustering of W boxes has been shown to be important for a strong and rapid transcriptional response . Inspection of the promoter sequences of a random set of genes that are not co-regulated with PR-1 gives an average occurrence of less than two WRKY factor sites per promoter. These data suggest that WRKY factors are essential for the co-regulation of PR-1 regulon genes. Although the core binding site for TGA-bZIP and WRKY factors are similar, a distinction can be made between these two sites because no functional W box has been found that has a G after the TTGAC core sequence .
Maleck et al.  have uncovered an interesting biological insight regarding transcriptional reprogramming during SAR in Arabidopsis. This insight was made possible through whole genome analysis using DNA microarrays. Another recent study using Arabidopsis DNA microarrays examined gene expression changes of 2,375 genes after inoculation with the fungal pathogen Altemaria brassicicola or treatment with the defense-related signaling molecules SA, methyl jasmonate (MJ), or ethylene . It was found that 705 ESTs on the microarray showed differential expression in one or more of these treatments. The high proportion of ESTs showing changes was due to preferential representation of putative defense-associated ESTs on the microarray. The interesting finding from this study was the level of coordinated gene expression changes between the four treatments. A comparison of the expression profiles from the four treatments showed that 126 genes were induced by multiple treatments. For example, SA and MJ coinduced 55 genes. Furthermore, half the genes induced by ethylene were also induced by MJ and 50 genes induced by the fungus were also induced by SA, MJ, and/or ethylene. Previous studies have indicated that there is coordination between different plant defense pathways, but these studies have only focused on one or a few genes at a time. The extent of the overlap between the pathways can only be fully appreciated with a more global analysis of gene expression, however.
Future microarray experiments will provide further insights into the signaling networks governing plant defense. Microarrays containing the full complement of Arabidopsis genes will provide a more complete analysis. Microarrays developed for other plant species, including important crop species, will also provide further information. It is anticipated that the genome-scale information garnered from these experiments will in due course provide avenues for enhancing disease resistance in crop plants.
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