With the discovery and isolation of genes that are involved in disease resistance mechanisms in plants, attempts have been made to engineer durable resistance in economically important crop plants (Stu-iver and Custers, 2001). Constitutive overexpression of single proteins that are toxic or otherwise interfere with pathogen proliferation, such as viral coat proteins, toxins, enzymes of phytoalexin biosynthesis, chitinases, glucanases, and many other PR proteins, has already been proven successful for enhancing plant resistance (Kombrink and Somssich, 1995, 1997; Stuiver and Custers, 2001) (see Chapter 7). However, a major drawback of modulating resistance by transfer of a single gene is that in many cases broad-spectrum disease control is not provided.
Alternatively, targeted activation of endogenous mechanisms that lead to enhanced resistance might be a useful approach. As outlined earlier, HR cell death is a common and mostly efficient defense system utilized by plants which comprises a complex array of defense responses and signaling mechanisms. Although the HR may stop different kinds of pathogens, including viruses, bacteria, and fungi, it is usually triggered only after specific recognition of pathogen-derived elicitors or Avr proteins. In addition, deregulation of cellular responses located downstream of the initial recognition event, such as formation of ROI, modulation of ion channel activity, or initiation of protein degradation, can also induce HR-like cell death. Based on this knowledge, a number of strategies have been devised to utilize induced HR cell death to engineer resistance.
The "two-component-systems" consist of pathogen-derived avr genes or genes encoding elicitors that are introduced and expressed in a plant containing the corresponding recognition system (i.e., R protein or elicitor receptor) to initiate HR cell death. With this approach, successful generation of an HR has been achieved with genes encoding Avr9 from Cladosporium fulvum, AvrRpt2 from Pseudomonas syrin-
gae, and the elicitor protein cryptogein (elicitin) from Phytophthora cryptogea (Hammond-Kosack et al., 1994; McNellis et al., 1998; Keller et al., 1999). An obvious key to the success of this strategy is the selection of an appropriate, tightly regulated promoter, which should be inducible by a variety of pathogens to extend race-specific resistance to broad-spectrum disease resistance. Leakiness of the promoter would result in spontaneous cell death formation with a detrimental influence on plant vigor and yield.
An alternative and more general approach that circumvents the limitations caused by the need for a defined genetic background is to induce cell death by expression of so-called killer genes, encoding products that directly interfere with essential cellular functions. Candidates for such products are RNases, DNases, specific proteases, toxins, etc., several of which have been experimentally evaluated (Mittler and Rizhsky, 2000). For example, expression of the barnase gene, encoding an RNase, under the control of a PR gene promoter resulted in transgenic potato plants with enhanced resistance to Phy-tophthora infestans, supporting the hypothesis that HR cell death at infection sites plays an important role in preventing pathogen proliferation (Strittmatter et al., 1995). However, growth under greenhouse or field conditions ultimately led to self-destruction of the plants, indicative of an endogenous activation of the transgene in aging plants, which underscores the need for specific, pathogen-responsive promoters. Correspondingly, identification and isolation of such selectively activated and tightly regulated promoters or cis-acting promoter elements is presently an intensively studied area (Keller et al., 1999; Pontier et al., 2001; Rushton et al., 2002).
Other transgenes inducing lesion-mimic phenotypes encode components of downstream signaling pathways involved in HR development or compounds that activate or interfere with their function. Thus compounds that mimic ion fluxes across the plasma membrane, such as the bacterio-opsin, a bacterial proton pump, or cholera toxin, an inhibitor of GTPase and G-protein signaling, both induce HR-like cell death, which is correlated with PR gene expression and elevated disease resistance (Mittler and Rizhsky, 2000). Likewise, expression of metabolic enzymes that either generate peroxides (e.g., glucose oxidase) or antisense suppression of those that catalyze their detoxification (e.g., catalase, ascorbate peroxidase) was also found to induce the formation of lesions, expression of defense-related genes and enhanced resistance (Wuetal., 1995;Chamnongpoletal., 1996;Mittler and Rizhsky, 2000). These results clearly impress upon the tight connection between enhanced ROI production and hR cell death. Manipulation of ubiquitin-regulated protein degradation pathways can also result in lesion-mimic phenotypes (Becker et al., 1993). Transformation of tobacco with a modified ubiquitin that is unable to polymerize an essential step in the ubiquitin-degradation pathway led to spontaneous lesion formation; however, challenging these plants with TMV resulted in fewer lesions and reduced virus replication in comparison to control plants.
Although artificial generation of HR seems a promising approach to control biotrophic and hemibiotrophic pathogens, it may be counterproductive with respect to necrotrophic pathogens, which apparently depend on dead host cells for growth. Accordingly, suppression of HR cell death appears to be an appropriate measure to limit these types of pathogens. Experimental evidence for the validity of this concept has recently been obtained. Transgenic tobacco plants expressing negative regulators of apoptosis, such as the human Bcl-2, human Bcl-XL or nematode Ced-9 genes, exhibited resistance to several necro-trophic fungal pathogens, including Sclerotinia sclerotiorum, Botry-tis cinerea, and Cercospora nicotianae, as well as to tomato spotted wilt virus (Dickman et al., 2001). Plants harboring Bcl-XL with a loss-of-function point mutation did not protect against pathogen challenge, demonstrating that resistance was dependent on a functional transgene and was not due to unspecific stress caused by the heterologous gene product (Dickman et al., 2001). These results further suggest that cellular pathways and mechanisms for cell death control are conserved between animals and plants.
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