Identification of Defense-related Genes Through Genomics and Proteomics Studies

Dr. Shauna Somerville:
http://www-ciwdpb.stanford.edu/research/research_ssomerville.php

Dr. Katrina M. Ramonell:
katrina@andrew2.stanford.edu

A number of distinct pathways by which plants protect themselves from both fungal, bacterial, viral and insect attacks are known. These defense mechanisms include gene-for-gene interaction, systemic acquired resistance (SAR) and jasmonic acid (JA) and ethylene signal transduction networks. Two milestones accomplished during recent years have facilitated defense-related studies. One is the sequencing of the Arabidopsis genome, which has made it possible to identify genes that trigger defense responses. The other is the development of DNA microarray technology. Microarrays enable investigators to study the regulation of sets of genes in pathways, such as those involved in defense responses or in stress-related responses. This is a considerable advancement over traditional techniques, which permitted the regulation of only single genes or small groups of genes to be determined. Advances in proteomics techniques are similarly beginning to allow researchers to determine the abundance of sets of proteins in various pathways, as well as track post-translational modifications triggered by various conditions.
In a review article published in the August,2002 issue of Current Opinion in Plant Biology (vol. 5:291-294), Katrina M. Ramonell and Shauna Somerville (Department of Plant Biology, Carnegie Institution of Washington, Stanford) discuss the most recent applications of DNA microarrays, cDNA-AFLP analysis and proteomics in deciphering the mechanisms of plant defense systems. The authors consider future approaches to tackling unsolved questions in plant pathogenesis.
The authors describe the two types of microarrays currently available, two-color microarrays and oligonucleotide microarrays. In the two-color microarray, DNA samples such as collections of ESTs (expressed sequence tags) are deposited by means of robotics onto a glass slide. Various types of samples may be used to make a microarray, such as anonymous clones arising from genomic, differentially displayed or normalized libraries or from commercially synthesized long (50-70 nt) oligonucleotides. Two-color microarrays do not require prior sequence knowledge of the clones used to generate the array. Oligonucleotide microarrays make use of 25-base long oligonucleotides, which are synthesized in situ on a solid substrate. These types of microarrays have proven to yield highly consistent results and allow sequence-specific analysis gene expression, which is critical when studying gene families. However, oligonucleotide arrays require knowledge of exact sequence information and careful bioinformatic design before manufacture of the microarray. They are also more expensive and require purchase of arrays from commercial sources.
The authors describe an experiment in which an Arabidopsis microarray containing 10,000 ESTs representing 7000 genes was used to investigate gene expression in plants under 14 different systemic acquired-resistance (SAR)-related conditions, including infection with an avirulent strain of bacteria. The study indicated that the expression levels of 300 genes changed at least 2.5-fold in response to SAR in two or more samples. The data were clustered using two different algorithms to identify genes with similar patterns of regulation. Upon analysis of a cluster that included the pathogenesis-related-1 (PR-1) gene, a common marker for SAR, the researchers found that the core binding site for the WRKY class of transcription factors, the W-box, was present in the promoters of all 26 genes in the cluster. This suggests that WRKY* transcription factors play a significant role in PR-1 regulation and SAR.
Previous evidence had suggested that the methyl jasmonate (MeJA) and salicylate (SA) pathways are antagonistic. Microarray experiments comparing gene expression profiles resulting from treatment with MeJA or SA indicate that these pathways may overlap, as 55 genes were induced by both treatments. Comparison of the profiles with other arising from treatment with ethylene indicated that there may also be coordination between the ethylene and MeJA pathways. An extension of this study identified five “early systemic” genes that were up-regulated in response to the fungal pathogen Alternaria brassicicola, four of which are transcription factors.Another study to identify genes regulated in response to MeJA found that over 280 ESTs out of 2880 on the microarray responded to MeJA. MeJA signaling genes of both known and unknown function were identified, including genes encoding a myrosinase-binding protein (MBP) and a glutathione-S-transferase. MBP and glutathione-S-transferase had previously been shown to be up-regulated by MeJA treatment in brassica and tobacco, respectively.
The authors describe another technique known cDNA-AFLP (cDNA amplified fragment length polymorphism). The technique has three advantages: it requires no prior sequence information, it allows detection of low levels of transcripts, and it costs far less than that microarray analysis because no special equipment is necessary to carry out this analysis. cDNA-AFLP is performed by restricting double-stranded cDNA with two restriction enzymes, followed by ligation of each fragment with specific adapters and selective amplification by PCR. Samples are analyzed using a polyacrylamide gel, and fragments that are present in one sample but not another may be sequenced. cDNA AFLP analysis has limitations; it does not yield quantitative data on levels of gene expression, and data on differentially expressed genes are limited to those samples that are sequenced. However, it has proven to be a valuable tool for gene discovery, particularly in plant species for which microarray techniques are not yet suitable.
AFLP has been used to study the gene expression profiles of the plant Ageratum conyzoides in response to infection with Agrobacterium compared to mock-infection. Two hundred fifty-one out the 16,000 cDNA fragments were differentially expressed 48 hours after the infection with Agrobacterium tumefaciens. The authors observed that 20 of the differentially-expressed genes had homology to genes known to play a role in signal perception, signal transduction and plant defense. The authors were able to identify sets of gene that were specifically regulated by Agrobacterium tumefaciens, genes that appear to participate in a generalized response to bacterial infection, and an overlapping group common to both sets.
The authors describe microarray studies indicating that the responses of plants to pathogens and to various environmental stresses are quite similar. Durrant et al. used transgenic tobacco plants expressing the tomato Cf-9 resistance gene to identify rapidly-elicited genes (called ACRE genes, for Avr9/Cf-9 Rapidly Elicited) up-regulated upon infiltration of the Avr9 peptide elicitor into leaves. Some of the ACRE genes were also up-regulated during mock-infection, though, suggesting that responses to physical stress and elicitors may involve overlapping pathways. In another microarray study, Reymond et al. analyzed the expression of 150 defense-related genes in Arabidopsis after wounding the plant. Wounding activated three pathogenesis-related (PR) genes (PR-1, PR-2 and PR-3), previously characterized as being involved in defense responses, within 15 minutes. With the help of AFGC-provided (Arabidopsis Functional Genomics Consortium) microarrays, Yu and associates observed that there is a strong parallel between the responses of plant cells subjected to the mitochondrial electron transport inhibitor antimycin A and the responses of Arabidopsis to a variety of stresses such as virus infection and excessive amounts of aluminum, cadmium or hydrogen peroxide.

Proteomics techniques allow the systematic identification of sets of proteins that accumulate differentially in a tissue, cell or subcellular compartment or in response to certain conditions. Proteomics techniques are now being applied to the study of plant pathology. In general, proteomics studies involve separation of protein samples using two-dimensional electrophoresis, comparison of protein profiles arising from different treatments or present in different tissues, cell types, or subcellular compartments, and using mass spectrometry techniques to identify target protein. Proteins that are modified post-translationally (for example, by phosphorylation) may be identified using proteomics techniques.
Proteomics studies have enabled identification of a number of proteins implicated in defense responses. For instance, the AtPhos43 protein was demonstrated to undergo phosphorylation within minutes of exposure to both a microbial elicitor (flagellin) and a fungal elicitor (chitin). The phosphorylation occurred independently of SA and Enhanced Disease Susceptibility1 (EDS1), a putative lipase involved in defense signaling. Other proteins phosphorylated in response to microbial elicitors include a syntaxin implicated in defense signaling. Another proteomic study analyzed the protein profiles of leaves of rice plants infected with the blast fungus pathogen (Magnaporthe grisea) and fertilized with varying levels of nitrogen. The nitrogen treatment was of interest because rice grown on high levels of nitrogen are more susceptible to blast fungus. Twelve proteins were identified whose levels changed with different levels of nitrogen treatment. In another study, rice proteins associated with resistance or susceptibility to sheath blight (caused by Xanthomonas axonopodis pv. Citri) were identified.
The development of powerful tools such as DNA microarrays, cDNA AFLP and proteomics techniques are allowing the identification of novel genes and proteins that function in defense responses. The authors suggest that this knowledge about defense mechanisms may permit the design and fabrication of custom-made microarrays and protein arrays for more extensive studies of defense responses to a specific disease or pest. The authors state that the development of comprehensive public databases of pathogenesis-related data will be a key factor for integration of data. The authors predict that, given the availability of annotated base sequence information for more and more plant species, microarrays will be used extensively to understand mechanisms of plant defense responses.
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*Found exclusively in plants, WRKY proteins are zinc-finger transcription factors. These proteins were shown to bind specifically to W Box-type [(C/T)TGAC(C/T)] DNA sequence elements both in vitro and in vivo.

 

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