Upon pathogen recognition, PRRs activate a comprehensive set of defense reactions called pattern-triggered immunity . Many pathogens independently acquired the ability to attenuate PTI through the release of effector molecules, thus enabling infection . In this case the pathogen is virulent and the host susceptible . During compatible interactions plants can still mount a weakened immune response, called basal defense. Basal defense can limit the spread of pathogens but is not capable of fully preventing disease . As a countermeasure to ETS, plants evolved the ability to recognize the presence of effectors, directly or indirectly, by highly specific plant resistance proteins, which mediate a strong immune response termed effector-triggered immunity [ETI; ]. Numerous studies have shown that ETI, basal defense, and PTI utilize a common set of signaling components including multiple regulatory phytohormones, which include salicylic acid , and jasmonic acid . While basal defense seems to be a weakened form of PTI, ETI has been proposed to result from boosted basal defense- or PTI-associated responses . The plant immune system can be subdivided into various defined interacting sectors . For example, distinct defense signaling sectors dependent on early MAMP-activated MAP kinases or the messenger molecules SA or JA, have been described. Interestingly, some of these sectors were found to largely interact in an additive or synergistic fashion during PTI,grow lights while they are partially antagonistic to each other during ETI . The latter phenomenon seems to allow for compensatory effects if a defined sector is disabled due to interferences with pathogen effectors.

While plants have generally developed highly effective mechanisms to cope with pathogens, contemporary crops often have lost substantial parts of their innate immune system due to unbalanced breeding efforts . Consequently, plant diseases cause dramatic losses in crop production every year. In the United States 500 million kg of various pesticides are applied each year at a cost of $10 billion to farmers. Despite this staggering price tag, more than a third of all potential food crops are still destroyed by diseases . The lingering residues of pesticides have been a health concern often covered by the media in recent years . Many pesticides currently in use are carcinogenic and rely on direct anti-pathogenic activity, which often leads to undesirable ecological side effects that can have far reaching consequences both for humans and the environment . This disquiet over the dangers of pesticides has spawned considerable interest in alternative methods of disease control . The use of environmentally safe plant defense-inducing chemicals , which protect plants from diseases by boosting their natural innate immune responses, instead of being toxic to pathogens, offers an attractive alternative for disease control regimes. Examples of such compounds include 2,6-dichloroisonicotinic acid and acibenzolar-S-methyl benzo thiadiazole-7-carbothioic acid S-methyl ester . While these compounds do induce defense responses in some plants, their effects are not universal across all species and their success in crop protection under field conditions has been modest . In addition, BTH has been shown to bephytotoxic in some situations and INA and BTH proved to negatively affect plant growth . The shortcomings of these established synthetic elicitors make the identification of additional types of plant defense inducing compounds very appealing. A chemical genomics-based approach to identify and utilize new types of synthetic elicitors for the dissection of the plant immune system and the development of novel types of pesticides was initiated .

By high-throughput chemical screening, 114 drug-like organic compounds that induce the pathogen-responsive pCaBP22-333::GUS reporter gene in transgenic Arabidopsis were identified. One of them, 3,5-dicholoroanthranlilic acid triggered fast, strong and transient disease resistance against as the pathogenic oomycete Hyaloperonospora arabidopsidis and the bacterial pathogen Pseudomonas syringae . Experiments of the defense inducing activity of DCA in various Arabidopsis defense mutants showed that this synthetic elicitor activates a signaling route dependent on the WRKY70 transcription factor. In contrast to INA and BTH-mediated immunity, which is fully dependent on the transcriptional co-factor NPR1 , DCA-mediated immunity is only partially NPR1- dependent . Thus, the mode-of action utilized by DCA in defense induction is distinct from that of INA and BTH. In this chapter another novel synthetic elicitors, 2–thiazolidine-4-carboxylic acid is described. Like DCA, CMP442 was also able to quickly and transiently induce disease resistance. However its mode-of-action was distinct from that of DCA. CMP442 was synthesized quickly and easily with a high degree of purity. In addition, low doses of CMP442 enhanced the growth of roots and aerial parts of both Arabidopsis and Solanum lycopersicum , while high concentrations inhibited plant growth. These effects were reminiscent of the general phenomenon of hormesis, which has been described in various biological systems for low doses of a wide range of stimuli that are toxic or otherwise detrimental at higher doses . A useful side effect of DCA is that it inhibits the development of roots in Arabidopsis seedlings grown on artificial growth medium . This effect allowed lab members to screen a large population of 50,000 EMS-generated Arabidopsis mutants for individuals that exhibit reduced sensitivity to DCA and, thus, show normal root growth on DCA containing growth medium.

Mutants with altered sensitivity for a drug-like compound may have a defect in a gene required for compound uptake, metabolization, or may encode an in/direct target protein operating in a pathway critical for the compound-triggered phenotype. In the case of DCA, ideally a mutant would have been identified that is compromised either in the direct cellular target of DCA or in a defense signaling component operating downstream from DCA perception. However, this screen was not successful and no mutants with the desired properties were identified . A possible reason for this is that targets of DCA are encoded by multiple redundant genes. Mutations in any one of these redundant genes should not result in a detectable phenotypic effect. For this reason screening of activation tagging libraries was used as an alternative approach. Any protein that may be involved in the perception of DCA or the transduction of DCA-induced signals should exhibit enhanced activity, if over-expressed. Thus, activation tagging mutants that show elevated expression of a gene encoding a DCA target protein should be more sensitive to DCA and exhibit enhanced DCA-mediated reduction of root growth compared to wild type plants. Arabidopsis activation-tagging lines containing randomly inserted T-DNA vectors with four copies of the Cauliflower Mosaic Virus 35S enhancer,led grow lights which can cause overexpression of nearby genes were used . Arabidopsis lines with this T-DNA near a gene encoding the direct DCA target or a protein with a role in DCA uptake, metabolism, or signaling may show enhanced sensitivity to DCA. Seedlings were plated on solid media with 1 to 100 µM of DCA. The plates were placed upright in a growth chamber and root lengths were measured at 0 d, 3 d, 5 d, 7 d, 11 d, and 14 d. Optimization experiments were performed to identify a time point and concentration of DCA leading to a moderate reduction in root growth which can be further increased by higher DCA doses or enhanced activity of a cellular component critical for DCA activity. For this screen, DCA needed to display enhanceable sensitivity. This meant that between the concentrations tested DCA must show significantly diminished root lengths. However, these experiments revealed that DCA-affected Arabidopsis root growth was inappropriate for a reliable high-throughput screen .

This meant that the difference in root length between tested concentrations was too small for a screen. Similar experiments using CMP442 showed that this compound was better suited for the planned screen, as its root growth reducing effect was more robust . Therefore, a screen was designed for Arabidopsis T-DNA activation tagged lines with enhanced sensitivity to CMP442. Increasing the CMP442 concentration from 25 µM to 50 µM resulted in a highly reproducible 7- to 8-fold reduction of Col-0 roots 14 days post planting. Screening of 25 lots of the Weigel activation tag population on 25 µM CMP442, 38 primary hits were identified.Seeds from each candidate were retested for their response to CMP442. However, the phenotype of enhancedCMP442-mediated root growth inhibition was not reproducible for any of the selected primary candidates. While designing the activation tagging screen, it was observed that at low concentrations both DCA and CMP442 caused an enhancement of root lengths compared to that of the control. These dosages were too low to produce an effect in induced disease resistance assays, and therefore, have been termed “sub-optimal dosages”. To further examine whether the enhancement of Arabidopsis root growth observed with low doses of DCA and CMP442 was observed with other synthetic elicitors, root growth assays were performed with SA, BTH and INA along with DCA and CMP442. Each synthetic elicitor was added to the solid media plates at concentrations ranging from 1 to 100 µM. The plates were placed upright in a growth chamber, and root lengths were measured. To merge different data sets, each root length was expressed as a percentage relative to the mean of the control for that data set. Normalizing each root length to the control within a given experiment allowed all replicates to be combined together to calculate the mean, standard error, and perform statistical tests . At day 14, 1 µM of every synthetic elicitor caused an increase in Col-0 root length, while they caused a reduction at higher concentrations. At a concentration of 1 µM, CMP442 clearly enhanced root length at every time point measured. The maximal increase observed for CMP442 was 183.1% at day 14 compared to mock. No other tested synthetic elicitor more dramatically enhanced Col-0 root growth at any concentration or at any time point. To determine whether this increased root length phenotype translated into an increase in the aerial weight of Arabidopsis, soil-sown-seedlings were drenched with synthetic elicitor . After 14 days the aerial portions of the plants were cut off and their weights measured. Only CMP442 treated plants showed a significant increase in their aerial weight compared to the control, with a maximum increase of 119% at 50 µM. These data clearly show that suboptimal dosages of some synthetic elicitors caused hormetic effects leading to increased root length and that CMP442 caused an increase in aerial weight. To our knowledge compounds of this class have not been reported to act as plant defense inducers. While reports on plant based studies are not available, some studies have been performed on PTCs in other systems and show that these compounds have anticancer, antioxidant, antifungal, and antimicrobial activities . None of these studies examined the effect of PTCs on plant pathogens. The diversity of biological activities of PTCs suggested that these compounds are highly suitable for interactions with a wide range of different cellular targets. Both major moieties of CMP442 are necessary for strong elicitor activity, as neither the 4-carboxy-4-thiazolidinyl portion, nor the 5-bromo-2-hydroxyphenyl portion induced strong immunity in our assays. While changes of the substituents of the phenyl group resulted in reduced elicitor activity, the PTCderivatives, CMP140, CMP254, and CMP492 that carry at least one substituent at the phenyl group suppressed the formation of HpaNoco2 spores in Arabidopsis, though at higher concentrations. Of those PTCs tested in our study, CMP442 was the most potent plant defense inducer of this compound class. All synthetic elicitors identified by our previous chemical screen are expected to induce a common set of defense reactions, which include transcriptional activation of the LURP gene cluster and at least some other responses known to be dependent on SA . Nonetheless, we found DCA and CMP442 employed different modes-of action. Their defense-inducing activities differed sharply in the Arabidopsis nahG line, which is unable to accumulate SA due to expression of a bacterial salicylic acid hydroxylase . Immunity against Hpa and CaBP22 and WRKY70 transcript accumulation triggered by DCA were not affected in nahG plants, while the ability of CMP442 to induce these responses was fully blocked in this line. Tests in additional lines deficient for SA-signaling, such as sid2-2 and pad4-1, showed similar trends. Thus, CMP442 is likely to interfere with a step in defense signaling that acts upstream from the accumulation of SA, while DCA disturbs processes downstream from SA.