A. Elevation of Endogenous Levels of Salicylic Acid
In the first section, strategies were applied that are based on the expression of a single gene (or a few genes) and a single target mechanism. From epidemiological studies it is evident that such strategies have a high chance of being overcome by pathogens, similar to resistance against chemical pesticides. From this point of view, more complex defense strategies should be beneficial for enhanced long-term pathogen tolerance. One obvious strategy is to make use of the plant's own defense system, for instance, by lowering the threshold level above which a plant mounts an efficient set of defense reactions to combat microbial pathogens. Verbene et al. (47) described the expression of two bacterial genes in plant chloroplasts that lead to salicylic acid biosynthesis from chorismate. Transgenic tobacco plants have high levels (—100 /¿M) of salicylic acid glucoside (the plant's vacuolar storage form) and are resistant against the fungus Oidium lycopersicon. Similarly, lesions after infection with tobacco mosaic virus were much smaller than those in wild-type plants, resembling the establishment of systemic acquired resistance (SAR). The constant production of salicylic acid in the transgenic plants turns on the immune system of plants. Although this strategy was so far tested only in tobacco, it seems to be very promising for many other plants.
Many laboratories have developed mutagenesis-based screens to look for plants with a defect in the plant immune system, simplified set equivalent to SAR. A single mutant was isolated independently by three groups. The gene is synonymously called npr (48) (nonexpresser of PR-genes), nim (49) (non-immunity), or sai (50) (salicylic acid insensitive). Although the biochemical function of the NPR protein is not well understood, it seems to interact with transcription factors required for PR-gene expression in a salicylic acid-dependent manner (51). One obvious experiment after the isolation of the NPR gene was to increase the expression level of this gene in transgenic plants. Luckily, the transgenic Arabidopsis lines showed strongly enhanced tolerance against infection by the biotrophic fungus Peronospora parasitica. This was achieved by a threefold increase in the protein level of NPR, which turned out to be sufficient to activate the complex plant defense system con-stitutively, resulting in SAR and resistance against P. parasitica (52).
A surprisingly small and reactive molecule plays a key role in plant defense: hydrogen peroxide (H202). Previously, H202 was regarded as an unavoidable by-product of respiration and photosynthesis for which the plant cell has no use, and thus it is rapidly detoxified by means of, for instance, catalase or ascorbate peroxidase. Over the past decade a totally different picture of the function of H202 and other reactive oxygen species has emerged from many studies (53,54). For instance, H202 drives the cross-linking of plant cell wall structural proteins. The local toughening of the cell wall is a barrier for penetrating fungi and is beneficial for the plant to mount a local defense to stop the ingression of the fungus. Cross-linked cell walls were shown to be much more resistant towards microbial cell wall cleaving hydrolyses (55,56). Along the same line, the expression of a wheat germin protein in wheat leaves results in enhanced resistance against Blumeria graminis f.sp. tritici, the causal agent of wheat powdery mildew (57). It was shown that germin is oxidatively cross-linked in wheat cell walls at sites of attempted fungal penetration. From a biochemical point of view, it is also clear that other compounds can be polymerized in H202-dependent processes. Many other phenolic secondary compounds are candidates for mixed polyphenols beside the lignin precursors (58). Thus, it is conceivable that part of the enhanced pathogen tolerance of stilbene-producing plants is mediated by the supply of the phenolic compound resveratrol for polyphenol formation rather than the direct antimicrobial activity of this phytoalexin alone.
The plant-pathogen interaction that results in programmed cell death of the hypersensitive reaction (HR) exhibits an extra oxidative burst several hours after contact between plant and microbe. This phenomenon is widespread and therefore seems to be important in plant resistance. The H202 from the oxidative burst was shown to be a signal that diffuses locally around the site of the infection and thereby transcriptionally induces genes in neighboring cells (59). Furthermore, H202 pulses (<10 minutes) induce cell death in soybean cell cultures several hours later. Although the concentrations used for the H202 pulses were relatively high (2-5 mM), a similar result can be obtained by supplying a constant H202 concentration of 1050 /u,M for several hours using the enzyme glucose oxidase (R. Tenhaken and C. Riibel, unpublished).
Following these ideas, transgenic plants were generated in which a catalase gene was largely suppressed by an antisense strategy (60,61). As a consequence, the steady-state levels of H202 are higher and these plants are more sensitive to microbial pathogen attack, resulting in enhanced tolerance. These observations resemble findings in transgenic potato plants that express a glucose oxidase gene from Aspergillus in their cell wall (62,63). The slightly elevated levels of H202 in the potato plants have a strong impact on pathogen resistance.
Why and how a small increase in H202 causes the greatly increased pathogen tolerance remain a mystery waiting to be solved in future studies.
As a final remark, the catalase antisense plants are more resistant to pathogens but they are also more sensitive to oxidative stress caused by photosynthesis under high light conditions. One should be cautious about the likely reduction in cold tolerance and drought stress, two unfavorable environmental conditions that put oxidative stress on the plants. It seems that the concentration of H202 has to remain within a certain margin in order to avoid detrimental side effects on the plant's tolerance to unusual environmental conditions.
D. Cell Death as a Trigger of Plant Resistance
The most potent plant defense against microbial pathogens is the hypersensitive reaction, a form of programmed cell death in plants. The principle of the HR is based on the early recognition of a pathogen, which then actively triggers a particular cell death program in the attacked cell (64). Although it is easily conceivable that an attack of a biotrophic fungus can be stopped efficiently by killing the plant cell in contact with the pathogen, the molecular basis for the general success of the HR to stop pathogen ingression remains to be solved in detail (65). The scientific problem comes down to the question of what else is turned on by the programmed cell death of the HR that is not activated by cell death per se (for instance, wounding).
In general, an HR in one leaf will establish an immune response (SAR) in the whole plant that allows a more efficient HR in the next infection event, as mentioned before for the tobacco mosaic virus-inoculated tobacco plants (2). A lesson from these and other studies is that the plant's endogenous cell death program is a valuable tool for plant resistance.
Plant breeders have used programmed cell death in plants for decades to achieve resistance against pathogens. The barley Mlo gene is probably the best understood example (66). In rare cases, a mutation in the Mlo locus causes programmed cell death of single cells in leaves, a phenomenon termed lesion mimic. The important thing about lesion mimic is the discovery that this cell death causes plant responses very similar to those that HR of a few cells would cause after a primary infection. Thus, lesion mimic is a physiological equivalent of an HR. The Mlo gene was identified by positional cloning and predicted to be a membrane protein that is a negative regulator (repressor) of cell death (67). About 10 years ago, several groups started to look for lesion mimic mutants in Arabidopsis (68,69). Many mutants have been identified, and at least for some of them the molecular mechanism is known. In contrast to the hidden cell death in Mlo barley plants, the Arabidopsis lesion mimics often show drastic phenotypes including dwarfism (70).
2. Transgenic Plants with Induced Limited Cell Death
Meanwhile, several different approaches have been taken to induce cell death in plants. Often the cell death behaves like the cell death in the hypersensitive reaction after pathogen contact and subsequently triggers a whole battery of plant defenses, usually including elevated levels of salicylic acid (SA), induction of PR-genes, and immunity by the SAR process. One example is the bacterial ribonuclease barnase (71), which is a very potent enzyme that kills eukaryotic cells when present at a few molecules per cell. This enzyme is effectively inhibited by the small protein barstar. Using a pathogen-inducible promoter of a glutathione-S-transferase gene from potato, Strittmatter et al. (72) expressed the barnase gene in potato plants. To avoid killing of the whole plant by the leaky promoter, it was necessary to coexpress the barstar inhibitor. Potato lines in which the strength of the pathogen-inducible promoter led to a surplus of free active barnase showed pathogen-dependent cell death after Phytophthora infestans inoculation. Thus, a totally artificial cell death process was able to enhance plant tolerance toward fungal infection. The limited success of the study is most likely due to a nonoptimal choice of the promoters used, but nevertheless it demonstrates impressively the power of artificial cell death-inducing systems.
Another cell death—inducing molecule is the fungal protein cryptogein, secreted by Phytophthora cryptogea. It is one of many similar proteins of Phytophthora species, which are collectively termed elicitins. Expression of the gene encoding cryptogein under the control of the pathogen-inducible hsr203J promoter in tobacco resulted in enhanced tolerance of transgenic lines against Phytophthora parasitica, Thielaviopsis basicola, and Erysiphe cichoracearum infection (73). The mode of action seems to be the induction of a cell death program by the cryptogein protein, which then activates the general plant defense machinery. It is, however, currently anticipated that elicitins cause cell death only in tobacco species, thus limiting the use of this system in crop plants.
A third example of induced cell death was published by Tang et al. (74). The researchers constitutively expressed the /»to-kinase gene in tomato, which was identified some years ago and shown to be required for resistance against the bacterial pathogen Pseudomonas syringae pv. tomato. The constitutive expression of pio-kinase is already sufficient to induce programmed cell death in a limited number of cells in tomato leaves, subsequently activating the SAR response in tomato. These transgenic plants exhibit broad-spectrum pathogen tolerance as expected from the activated SAR response.
Although the latter examples of engineered cell death are specific for particular plant species, a novel more generally applicable system for the induction of artificial cell death emerges from many studies of plant resis tance and microbial avirulence genes. As predicted from the early studies of Flor (1947), the interaction of a plant resistance gene product with a microbial avirulence gene product is the basis for the programmed cell death in the HR. Whereas the first avr genes were cloned from phytopathogenic bacteria in the early 1980s, the first plant resistance genes (R genes) were identified in 1994. The rigorous test of whether the avr gene product by itself is sufficient to induce an HR was impressively answered and confirmed by studies in which the avr gene was transiently expressed in plant cells.
Plant R genes seem to work functionally at least in closely related species. For instance, the tomato Cf-9 resistance gene was transformed into tobacco plants and the resulting transgenic lines still responded to the corresponding avr9 peptide from the tomato pathogenic fungus Cladosporium fulvum (75). When this tobacco line was crossed with a transgenic plant expressing the avr9 avirulence gene, the siblings showed a whole-plant HR and died at the seedling stage, thus confirming the concept. The expression of both avr and R genes in a strictly controlled pathogen-dependent manner is one of the most promising strategies for engineered cell death and subsequent activation of the plant's immune system. A great variety of suitable promoter elements for such experiments is currently under development. The use of two different pathogen-inducible promoters for the resistance and the avirulence gene will help to minimize the detrimental effects of unintentional cell death.
Thus, the regulation of cell death is an important issue and understanding it is necessary for further exploitation of programmed cell death as a novel mechanism for enhanced pathogen tolerance.
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