Therefore each set of R genes incorporated into a cultivar must be evaluated systematically requiring a significant investment of time. However, it is clear that gene pyramiding offers an attractive mechanism for combining the individual specificities of R genes as well as taking advantage of their synergistic effects to generate broad-spectrum resistance.An alternate strategy to breeding is to directly introduce a cloned resistance gene into a plant via transgenic technology. Introduction of a gene by transgenic means can overcome the limitations of traditional breeding, namely inter species sterility. Additionally, transgenic technologies allows multiple genes to be inserted simultaneously. However, validation of the function of the transgene and its stability and heritability after transformation requires a significant investment of time and resources. Further, the transgenic lines must also undergo subsequent analysis for agronomic traits before release. While the creation of transgenic plants may be relatively straightforward for a number of species, the strategy has its own substantial time requirements. The greatest advantage of transgenic technology is its ability to overcome fertility barriers for the dissemination of genes originating from a different species; two examples from the Solanaceae family highlight this advance. Bs2, as mentioned above, was identified originally in pepper and its resistance has been durable in the field against isolates of X. campestris . Due to the fitness requirement associated with avrBs2 locus,ebb flow tray the incorporation of the resistance locus Bs2 via transgenic technology may offer durable resistance in a number of plant systems affected by X. campetris.

To assess this hypothesis, tomato was transformed with the Bs2 gene from pepper. Inoculations of X. c. pv vesicatoria isolates onto Bs2– containing transgenic tomato plants failed to cause disease therefore Bs2 function was conserved in tomato . Tomato and pepper when crossed cannot form a fertile hybrid and this resistance could not have been utilized with standard breeding protocols. In another example the N gene from tobacco, conferring resistance to the tobacco mosaic virus , was transferred into tomato . The resulting transgenic tomato plants, expressing the N resistance gene, were inoculated with TMV and complete resistance was observed. While TMV is not as devastating economically to tomato as is X. campestris, the conceptual notion that resistance loci can be transferred among species while retaining their function points illustrates a key advance for engineering resistance using transgenic technology. These examples demonstrate conservation in disease signaling pathways that can be exploited for cultivar improvement.Disease resistance research has largely focused on understanding the specific pathogen–host interactions mediated by R and avr loci. Recently, studies have revealed signaling components that function downstream of R genes or other pathogen sensors. Studies on broad-spectrum resistance pathways, such as the rhizobacteria-mediated, induced systemic resistance pathway and the insect-responsive pathway involving jasmonic acid are rapidly gaining momentum . However, research on the pathway transducing a broad-spectrum defense response termed the systemic acquired resistance response has progressed most rapidly. Chemical and abiotic inducers of SAR, along with inherent signaling components of this pathway identified by basic research in model plant systems, are among the initial targets being used to engineer multi-pathogen disease resistance in important crop plants.

The SAR defense response is manifested when a plant host is inoculated with a pathogen that results in a localized infection. This primary infection subsequently primes the host to resist secondary infections by viral, oomycete and bacterial pathogens . In the model plant Arabidopsis, SAR is associated with a rise of internal levels of the plant hormone salicylic acid , and is correlated with the increased expression of a set of genes termed pathogenesis related genes . Several PR genes encode proteins with antimicrobial activity and thus contribute to an overall defense response directly . Research aimed at modulating this pathway and generating broad-spectrum resistance has largely targeted three parts of this response for further study: the ability of SA to trigger the response, the increased expression of PR genes and the identification and modulation of other signaling components.Therefore, investigations into the costs of induced resistance have started by assaying the effects of using chemical inducers. Heil et al. have studied the fitness of wheat plants treated with BTH in the absence of pathogens. When plants were grown either hydroponically or in the field, water-treated control plants were able to achieve greater biomass than their BTH-treated cohorts. In field experiments, however, significant growth differences were not seen until approximately 6 weeks after treatment. The authors suggest that many of the potential fitness costs associated with induced resistance responses may be masked in laboratory experiments where growth conditions are kept optimal, and support this hypothesis with experiments performed growing plants under differing nitrogen concentrations. In addition, when the age of the plants induced for SAR was considered it was found that the growth-costs of BTH treatment could be reduced if the BTH was applied after the lateral shoot formation was complete .

These data also underscore the importance of factoring plant developmental programs into any efficient strategy to enhance plant resistance by chemical treatment or genetic engineering.Another unwanted effect that may arise from transgenic manipulation of genes involved in defense signaling pathways is spontaneous cell death. Spontaneous cell death has been uncovered in many genetic screens for enhanced disease resistance and recently, has been seen in transgenic plants. These mutants and transgenic plants are often collectively referred to as lesion-mimic mutants since they display lesions similar to those observed in a defense response even in the absence of pathogens. This form of cell death in plants is sometimes influenced by alterations in environmental conditions such as light, temperature and humidity . Therefore, both in basic research and in applied experiments, it will be important to understand the parameters controlling cell death. This research is critical not only for optimizing the situations where transgenes and chemicals will be most useful to generate disease resistance, but also to minimize negative effects on important agronomic factors such as development, fertility and yield.Many of the examples listed above,flood and drain tray may appear as substantial challenges to engineering disease resistance, however, these challenges provide opportunities to create plants that are even more resistant than plants engineered based on our current knowledge. For instance if the already identified components of a signaling pathway are not the best candidates for durable resistance in the field, technologies such as micro-arrays will help to pinpoint novel targets of interest . When mutations involved in disease resistance have already been identified, but are recessive in nature such as the mlo, edr1 and mpk4 mutants, classical breeding strategies can be employed. These mutants cannot be placed into heterologous systems using transgenic technology but, as with gene-pyramiding, they are still useful in breeding. Or, as technology continues to improve, gene knockouts and silencing of homologs may be employed to generate mutants in diverse species. If research continues to suggest crosstalk between ISR, SAR and insect defense signaling pathways, there may be great potential for additive defense effects by manipulating overlapping components. So, while limitations and cost of engineering broad-spectrum defenses warrant much attention, it is useful to look at such challenges as means for streamlining and improving upon current engineering strategies.Another promising strategy for enhancing resistance in plants is the use of RNA homology-dependent silencing to combat viral and bacterial disease . The nature of this silencing has been evaluated in a number of systems where similar phenomena are called by different names; RNAi in animals and quelling in fungi . One conserved step leading to RNA homology dependent silencing is the formation of a double stranded RNA intermediate. This dsRNA intermediate is recognized by an enzymatic complex which targets degradation of all corresponding homologous RNA transcripts . Several cases detailed below illustrate the possibilities for generating disease resistant plants by taking advantage of this inherent biological process.Viral resistance using RNA homology-dependent silencing has been successfully engineered into many plant systems. Single or multiple viral-derived transgenes can be expressed in plants leading to RNA homology-dependent silencing and subsequent viral resistance.

The use of this transgenic technology may be particularly effective in thwarting viral diseases where little or no genetic resistance has been identified. Resistance to rice yellow mottle virus is one example where traditional breeding cannot be used for improvement due to fertility barriers and genetic resistance being a poorly defined polygenic trait . The RYMV open reading frame 2 was highly expressed in transgenic rice. The resultant RYMV resistant lines carried very low or non-detectable amounts of the ORF2 RNA transcript. Conversely, transgenic lines that were susceptible had abundant amounts of the ORF2 transcript. Therefore the resistance phenotype was correlated with the loss of the viral transgene expression. This indicates that the mechanism of resistance was due to silencing of the ORF2 present as the transgene and in the RYMV RNA genome. The ORF2 sequence variation among different RYMV field isolates was found to be less than 10% at the nucleotide level suggesting that an RNA homology-dependent silencing approach may be effective in the field . Viral resistance utilizing endogenous silencing mechanisms is not restricted to using a single open reading frame from one virus. Two ORF fragments from different viruses can be fused into a chimeric expression cassette to confer resistance to both viruses. One clear example was generated from using tomato spotted wilt virus and turnip mosaic virus . The open reading frame for the N gene encoding the nucleocapsid from TSWV was fused to the coat protein of TuMV and the resulting chimeric construct was used to transform tobacco. As with the example using RYMV,resistance of the transgenic plants to both viruses corresponded with the loss of transcript accumulation from both viruses as detected by northern analysis. Transgenic plants susceptible to both viruses showed accumulation of the gene fragment transcript for both viruses. These two examples have been evaluated in greenhouse experiments; however, a well-described example of RNA homology-dependent silencing for viral resistance is presently being utilized successfully in the field.One clear commercial success of generating enhanced resistance by stable expression of a viral gene is against the papaya ring spot virus . Papaya is grown throughout the tropics and subtropics and no natural resistance has been described for PRSV. A PRSV control strategy for the Hawaiian islands was developed using RNA homology-dependent silencing by expressing a mutated open reading frame for the coat protein from PRSV . Resistant transgenic plants were generated and were found to be devoid of the CP RNA indicating the RNA homology-dependent silencing of the plant-derived transgene and PRSV gene . All PRSV strains present in Hawaii have been effectively controlled using silencing constructs derived from this mutant CP ORF. Sequence analysis demonstrated that these Hawaiian isolates had 97% or greater sequence homology to the mutant CP transgene. However, isolates of PRSV from outside of Hawaii can cause disease on the transgenic papaya lines. These geographically distinct isolates were found to have a lower sequence homology to the CP than the isolates from Hawaii. Thus, silencing of PRSV was contingent upon levels of sequence homology above 97% . Interestingly, PRSV and RYMV require different levels of homology between transgene and the endogenous gene to induce silencing. The silencing in RYMV was successful for all variations tested as compared with less than 3% divergence allowed for successful silencing in PRSV. Silencing is not only dependent upon the degree of homology but also the target sequence that is chosen. Much like the transgenic approach with R genes, each silencing construct must be carefully validated. Overall, RNA homology-dependant silencing has proven its utility in both the greenhouse and the field, and appears to be among the most versatile mechanisms currently available to engineer resistance to viruses.Crown-gall is a perennial problem in nurseries of fruit trees, nut trees and some bushy ornamental plants. Prevention of gall formation is a target for engineering resistance in these trees since breeding programs for resistance are not practical due to temporal considerations . When replanted, the trunks suffer cuts that are an entry point for the bacterium Agrobacterium tumefaciens, the causal agent of the disease, and infection becomes apparent with the formation of galls.