The levels of different transcriptional expressions during plant responses to pathogen attack are regulated by cis- and trans-acting elements. Cis-acting elements involved in pathogen-induced R gene expression have been analyzed extensively. Two groups of pathogen-inducible cis-acting elements, the GCC-like elements (Ohme-Tagaki et al., 2000) and the W boxes (Rushton et al., 1996; Eulgem et al., 2000) are among the best characterized in the context of pathogen defense mechanisms. The cis-acting elements with pathogen-inducible R gene functions fall into three groups: the W boxes, the GCC-like boxes, and the box D. The binding site for WRKY (W box) or AP2/ERF (GCC-like box) transcription factors can be sufficient to confer pathogen inductibility. Systematic DNA-binding studies have shown that nucleotides flanking the W boxes and the GCC-like box specify the DNA-protein interactions and subsequent gene activation (Rushton et al., 2002). The pathogen-inducible systemic promoter could have major applications, first as molecular markers, and second, in the engineering of crops with increased disease resistance. Systemic promoters could be used to better characterize the function of a gene or a mutation within a pathogen-inducible gene. Moreover, the use of defined regulatory sequences may allow highly restricted expression of the desired product, which could be accumulated exclusively at the sites of attempted pathogen invasion. Promoter analysis using transient expression assays may be a promising way to characterize several distinct cis-acting elements and the cloning of related transcription factors. This approach could be used to assess the regulatory pattern of promoters in transgenic plants.

The hypersensitive response is characterized by localized cell and tissue death at the site of infection (Van Loon, 1997). As a result, the pathogen remains confined to necrotic lesions near the site of infection. A ring of cells surrounding necrotic lesions become fully refractory to subsequent infection, known as localized acquired resistance (Fritig et al., 1998). These local responses often trigger non-specific resistance throughout the plant, which leads to a systemic acquired resistance, providing durable protection against infection by a broad range of pathogens (Sticher et al., 1997). The metabolic alterations in acquired resistance include: cell wall reinforcement, stimulation of secondary metabolic pathways, which yield molecular compounds with antibiotic activity, and defense regulators such as salicylic acid, ethylene and lipid-derived metabolites (Fritig et al., 1998; Hahn, 1996).

Receptor-mediated recognition at the site of infection initiates cellular and systemic signaling processes that activate multicomponent defense responses at local and systemic levels. The local resistance occurs rapidly while the development of systemic acquired resistance is delayed (Scheel, 1998). The earliest reactions of plant cells include changes in plasma membrane permeability leading to calcium and proton influx, and potassium and chloride efflux, which subsequently induce extracellular production of reactive oxygen intermediates such as superoxide, hydrogen peroxide, and hydroxyl free radicals (McDowell and Dangl, 2000). The localized production of reactive oxygen intermediates and nitric oxide act to induce hypersensitive responses and expression of disease defense genes (Piffanelli et al., 1999). Other components of the signal network are the induction of phospholipases (PLPs),  which  act  on  lipid-bound unsaturated fatty acids within the membrane, resulting in the releasing of jasmonate, methyl jasmonate and related molecules. Recent evidence confirmed the potential role of jasmonate, ethylene and salicylic acid in the signal pathways leading to up-regulation of pathogen defense-related genes in plants (Ananieva and Ananiev, 1999; Reymond and Farmer, 1998).

Phytohormones such as ethylene and jasmonate have been found to synergistically induce the expression of plant-defense genes in response to different pathogen attacks (Penninckx et al., 1998). Exogenous application of methyl jasmonate to Arabidopsis plants reduces disease development after infection by several fungi, such as A. brassicicola, B. cinerea, and P. cucumerina (Thomma et al., 2000). Lorenzo et al. (2003) have demonstrated that ERF1 (Ethylene response factor 1), a downstream component of the ethylene-signaling pathway (Solano et al., 1998), is an early ethylene- and jasmonate-responsive gene. They suggest that ERF1 may be a common component of both ethylene and jasmonate signaling pathways. In order to investigate the involvement of ERF1 in the regulation of the expression of pathogenesis-related genes, Lorenzo et al. (2003) performed a transcriptome analysis of Col-0 and 35S-ERF1 transgenic plants using Arabidopsis GeneChip from “Affymetrix”, which allows the simultaneous monitoring of 8000 genes. They reported about 164 genes, which were constitutively expressed, 77 genes inducible and 70 genes repressed after 6 h of treatment with jasmonate and Ethylene (Lorenzo et al., 2003). Most research has focused on understanding how relevant genes are upregulated during pathogen-defense mechanism (Table 3). However, plant responses to pathogen attack also involve the downregulation of several genes (Table 4). In potato for example, the mRNA and protein levels of Rubisco are drastically reduced by pathogen infection or elicitor treatment (Table 4). In persley, the expression of several genes involved in cell proliferation and cell-cycle regulation as well as flavanoid biosynthesis, are repressed to a large extent during defense responses (Somssich and Hahlbrock, 1998). Reports of Lorenzo et al. (2003) confirmed the combination of positive and negative regulatory mechanisms of pathogen-acquired resistance in plants. The high number of defense-related genes whose expression was upregulated by ERF1 is fully consistent with their previous results (Berrocal-Lobo et al., 2002) demonstrating that overexpression of ERF1 in transgenic plants confers resistance to several pathogens. In summary, different types of phytohormonal signaling pathways are involved in plant defense responses during pathogen attack. These results suggest that hormonal transcription factors are key elements in the integration of signal transduction for the regulation of defense-related genes.

Table 3. Defense related genes upregulated by ethylene and jasmonate treatment, or by pathogen attack.

Table 4. Genes constitutively repressed under pathogen attack.

The cloning of R genes has opened doors for producing disease-resistant crop plants. Transgenic plants that resist pathogen attacks can be engineered either by insertional mutagenesis or by map-based cloning methodology.

Using the insertional mutagenesis method of transformation, a fragment of DNA is inserted into the coding region or regulatory region of a gene, which results in disruption of gene expression. After this, one proceeds to the cloning of the plant DNA that flanks the integrated insertional mutagen by plasmid rescue. The cloned plant DNA can then be used as a hybridization probe to isolate the gene by screening a lambda or cosmid library constructed from the wild-type plant. The final test is to introduce the cloned gene by transforming sensitive plants and examining them in order to determine whether they have become disease-resistant. The most commonly used DNA molecules for insertional mutagenesis are transposons (Baker et al., 1986) and T-DNA from the Ti plasmid.

In the map-based cloning which is also known as positional cloning, one needs to determine the chromosomal location of the gene of interest. The restriction fragment length polymorphism (RFLP) is the most frequently used method for linking the chromosomal position to a particular trait. In this case, co-inheritance of the disease resistance trait with one specific RFLP DNA probe defines the approximate location of the R gene on the chromosome. The linkage between several specific DNA probes and the R gene allows one to use the DNA probe as a starting point to seek, or to land on the R gene. The gene will be identified in the next step after screening for coding sequences (e.g., cDNA) within the specific region using either a yeast artificial chromosome (YAC) vector or a bacterial artificial chromosome (BAC) vector. The final test can be done after transferring the candidate cDNA fragments into susceptible plants by looking for the acquisition of disease resistance in the transgenic plants.

Resistance of plants to pathogen attacks entails a concerted action of many gene products, and thus, alteration in the expression of multiple genes is required. In this respect, transcription factors may be excellent targets of genetic engineering for improving disease resistance in plants. Transcription factors generally regulate a group of genes rather than a single gene. Therefore, manipulating a single transcription factor could have the same effect as manipulating a set of specific genes within the plant. As highlighted above, transgenic plants allow the targeted expression of pathogen-related genes in vivo and are therefore an excellent system to assess the function of/and tolerance conferred by the encoded proteins. Another purpose for using transgenic plants is to improve disease resistance in agronomically valuable plants.

The defense response in plants appears to be activated by ligand/receptor interactions, in which Avr gene and pathogen or plant surface-derived elicitors serve as ligands for receptors located in the plasma membrane or in the cytosol.

Cloning an R gene is an intensive and time-consuming task. However, one can expect many more R genes to be cloned if discoveries in this area are accessible worldwide. Knowing the gene structure will lead to a better understanding of the pathogen-host interactions and the availability of cloned R genes will increase the potential for engineering disease resistance in plants.

Unfortunately, up to date, the functions of many proteins and other molecules that interact with R genes during defense mechanisms are largely unknown. Therefore, additional proteins are likely to participate in Avr perception and subsequent signal transduction. The genetic approaches utilized so far to isolate and characterize disease-related genes may need to be complemented by biochemical studies to understand fully their functions (Kotchoni and Shonukan, 2002). Many of the identified genes, which are associated with pathogen-defense mechanisms, are still at descriptive level and only the functions of few have been established. The production of mutants using an antisense-RNA approach could turn out to be a powerful technique in understanding the potential role of a gene product during defense mechanisms (Kotchoni and Bartels, 2001). This approach could elucidate certain aspect of disease resistance in plants. A combination of novel approaches including molecular techniques and genetics will provide insights into pathogen-defense mechanism and subsequent disease resistance in years to come.

ACKNOWLEDGMENT

The authors would like to thank Dr. Maina M. (Department of plant pathology, Uni-Bonn, Germany) for critical reading of the manuscript.

Disease necrosis: A common, slow-developing disease symptom caused by necrotrophic pathogens. The molecular basis of necrosis and its relationship to the rapid hypersensitive response during incompatible interactions are not known.

Ethylene: A molecular plant growth regulator involved in response to abiotic stresses, fruit ripening, leaf senescence, and plant tolerance/resistance to pathogens

Induced systemic resistance: A long-lasting and broad-spectrum resistance induced throughout the plant cells as a result of prior exposure to pathogen and abiotic stresses.

Jasmonic acid: Jasmonic acid and methyl jasmonate are plant growth regulators derived from the octadecanoid-signaling pathway elicited by wounding and insect chewing. They are required for signal transduction leading to resistance to insects and certain fungal pathogens.

Local resistance: A local plant defense observed in response to infection by avirulent or virulent pathogens.

Pathogenesis-related (PR) proteins: Proteins induced throughout in the plant in response to pathogen infection, which are associated to systemic acquired resistance. Some PR proteins such as chitinases and glucanases exhibit antifungal activity in vitro.

Phytoalexins: Anti-microbial compounds produced by plants in response to infection.

Salicylic acid: Is an endogenous messenger for the activation of multiple resistance responses against pathogens and abiotic stresses

Systemic acquired resistance: A long-lasting, broad-spectrum resistance induced throughout the plant cells by prior local infection of necrotic pathogens or pretreatment of plant with certain chemicals such as salicylic acid.