One possible explanation was that the experimental conditions varied between the assays for basal defense and DCA-mediated immunity. In contrast to the analysis of DCA-mediated immunity , there was no pre-treatment with compound or mock solution before being sprayed with pathogen in the basal defense assays . It was observed in other experiments that this type of pretreatment affected the experimental outcome and indicated that pre-treatment of Arabidopsis plants with mock solution reduced the extent of susceptibility to Hpa. To examine if any of these synthetic elicitors induced phytotoxicity, trypan blue staining of seedlings after saturation treatment , where seedlings were grown and treated in liquid medium, was used. Dark blue staining, indicating cell death, was prevalent in 100% of the seedlings treated for 24 h with 300 µM of DCA, CMP144, CMP994, CMP202, CMP199, and CMP384 . Cell death was evident starting at treatments of 100 µM for CMP199. Therefore, the absence of GUS expression at 100 µM CMP199 was likely due to compound-induced phytotoxicity. For all synthetic elicitors cell death was not observed at concentrations showing effective GUS reporter activation ,blueberry grow pot indicating that cell death was not responsible for the GUS reporter activation.
Based on these preliminary assays, these synthetic elicitors were clearly potent inducers of GUS expression in the pCaBP22::GUS lines. Additional study was warranted to scrutinize their unique properties and ascertain their potential for future research use. To accurately quantify the defense-inducing activity of these eight novel synthetic elicitors, a dose-response analysis measuring the inhibition of HpaNoco2 spore development in three-week-old Col-0 plants was performed . For this experiment Col-0 seedlings were sprayed with the respective compound or mock solution and challenged with HpaNoco2 24 h later. Hpa spore formation was evaluated 7 dpi. Each synthetic elicitor behaved similarly during either Hpa defense or the reporter gene assays. CMP199 showed nearly 70% inhibition of spore development at 1 µM and 100% at 10 µM. CMP199 which showed a high activity in GUS saturation treatments, proved to be a more efficient defenseinducer compared to DCA at every concentration tested . This finding was important since this was the first time that a synthetic elicitor more potent than DCA had been discovered by our program. CMP199 showed great potential for future use. In the defense assays CMP199 and DCA provided most efficient protection against HpaNoco2, in contrast CMP384 was only able to induce significant levels of immunity at the highest tested concentration . The weakest of the examined new synthetic elicitors, CMP384 and CMP608 mediated only a 30% inhibition of spore development at 100 µM. The remaining five compounds exhibited intermediate levels of defense-inducing activity, with 55-75% inhibition of spore development at 100 µM. Different inducers of the same biological response may act antagonistically, additively, or synergistically. Synergistic activities produce a greater effect than the sum of their independent effects on the system.
A suite of synthetic elicitors with distinct modes of action, acting synergistically, may have great potential as environmentally safe pesticides, as they may trigger strong defense responses at extremely low concentrations when applied in combination. In addition, synergistically acting synthetic elicitors may be useful for plant defense network studies by facilitating the discovery of cooperative crosstalk mechanisms linking distinct compound-triggered pathways. Two redundantly acting compounds should not induce pCaBP22-333::GUS expression when each is applied at concentrations below their minimally active concentration. Additively acting compounds should activate the reporter when combined at concentrations equaling the sum of their minimally active ones. However, these compounds should not activate the reporter when applied at lower combined concentrations. If two compounds activate reporter expression at combined concentrations lower than the sum of their minimally active concentrations, their effects are synergistic. Finally, if two compounds act antagonistically the reporter gene response triggered by one compound should be reduced by application of the second compound. Antagonistically acting compounds would be tested at concentrations guaranteed to induce pCaBP22- 333::GUS expression. Here, the synergy of DCA, CMP442 , or CMP199 under saturation treatment conditions was tested. Consistent with previous observations, CMP199 proved to be the most potent of these three synthetic elicitors . CaBP22-333::GUS expression was scored visually based upon intensity of color. The minimal concentration of CMP199 for inducing the pCaBP22-333::GUS reporter was 100 nM, whereas that of DCA was 250 nM, and that of CMP442 was 1 µM. When any two of the synthetic elicitors DCA, CMP442 or CMP199 were combined the resulting responses were neither synergistic nor antagonistic .
GUS expression was weakly evident after the mixture of 50 nM each of DCA and CMP199 . This combination does not indicate synergism because if it was, GUS expression should have been stronger resulting in a more intense blue color. Instead, it appeared that this interaction was additive given that they activated the reporter at concentrations equaling the sum of their minimally active ones. Further experimentation will be necessary to determine more precisely the minimally active concentration of CMP199 to confirm that the tested compounds do not act synergistically. A concentration below 250 nM of DCA must be used in conjunction with CMP442 to distinguish between these two options. However, it is certain that they did not act strongly synergistic, as 50 nM of each, which is below their minimally active concentration, did not induce GUS expression. Finally, CMP199 and CMP442 appeared to act additively when used together. If they acted synergistically, a mix of 50 nM of each compound would have induced GUS expression. Instead 50 nM CMP199 and 250 nM CMP442 induced GUS in an additive manner. Saturation treatments were revealing about the role each synthetic elicitor plays in the activation of defense. Nonetheless, a more comprehensive defense assay may be necessary to confirm these observations while testing a wider range of concentrations. The complex responses associated with the activation of the plant immune system can be dissected and studied with the aid of synthetic elicitors activating the system at distinct points. These synthetic elicitors may serve as powerful tools to complement traditional genetics used to study plant defense networks. The versatility and reversibility of many synthetic elicitors makes the identification of a wider range and spectrum of these compounds a priority. Building on the success experienced by Knoth et al. in their identification of DCA, eight more synthetic elicitors were reported on here. Experiments demonstrated that most of these elicitors, excluding CMP608 and CMP384, have the ability to induce pCaBP22-333::GUS expression at concentrations of at least 10 µM. These low concentrations increase the probability of high target specificity,hydroponic bucket decreasing the likelihood of off-target or unwanted side effects . At synthetic elicitor concentrations that induced defense, no herbicidal effects were observed, while all compounds proved to be phytotoxic at higher concentrations. Each new synthetic elicitor discussed here adheres to Lipinski’s rule of five suggesting they may all be readily absorbed by plant cells. These rules state that properties that favor bioactive compounds include a molecular weight of less than 500 g/mol, a lipophilicity value of more than five, less than five hydrogen-bond donors, and less than 10 hydrogen-bond acceptors . CMP60, CMP994, and CMP202 share a phenyl-1H-pyrazol-1-ylmethanone skeleton as a common substructure.However, several closely related analogs act as potent inhibitors of human neutrophil elastase, antiinflammatory, analgesic, anticonvulsant, anticholinergic, antihistaminic, antibacterial, antiepileptic, and antifungal agents . CMP384 analogs, but not CMP384 itself, were reported to have antimicrobial activities . Of the remaining compounds, the exact structures or closely related analogs of CMP144, CMP174, and CMP199 are not referenced to in any biological studies. The wide range of structures and their apparent uniqueness offers many promising candidates for potential synthetic elicitors. In HpaNoco2 defense assays, the novel synthetic elicitor CMP199 outperformed DCA with a lower active concentration. A lower active concentration may reflect a higher degree of target specificity. Also, a major goal of this project was the identification of compounds with different modes-of-action. A suite of functionally distinct synthetic elicitors is likely to allow for more control and flexibility when studying the plant defense network and is likely to facilitate the creation of novel environmentally friendly pesticides. CMP199 will be further examined by other lab members to determine if its mode of action is distinct from that of DCA.
To determine if the available synthetic elicitors acted synergistically, dual combinations of DCA, CMP199, and CMP442 were tested at a range of different concentrations for each compound. This experiment demonstrated that these synthetic elicitors are likely to act in an additive, but not synergistic, manner. As a confirmation of this data, additional assays should be performed testing disease resistance inducibility with these combinations of synthetic elicitors. These experiments are necessary since it is possible that the GUS assay is not of sufficient sensitivity to display synergistic effects since. Pathogen assays would provide quantitative data instead of the qualitative data provided by the visually read GUS assays. The plant immune system is a highly complex web of interactions that allow plants to modulate their responses to the type of infecting pathogen. Currently, only a small number of defense network components have been identified and characterized. The identification of additional elements that compose this web will lead to a better understanding of plant defense and new strategies to combat diseases. In microarray experiments performed by Knoth et al. , the ACID cluster was identified as a set of genes upregulated at time points corresponding to synthetic elicitor-mediated disease resistance. These 137 ACID genes were hypothesized to be important for plant defense. More than one quarter of the 137 ACID members encode kinases . Of the 16 ACID genes examined in this study, 12 encoded protein kinases. Protein kinases often act as regulatory elements controlling plant defense responses. To increase the probability of success in the functional analysis of ACID members, genes encoding protein kinases were preferentially chosen for study. ACID3 encoded a TIR-NBS-LRR class disease resistance protein and ACID8 a LRR-receptor like protein kinase. ACID9 and ACID13 encoded LRR transmembrane protein kinases and ACID12 was a receptor like protein kinase. Additionally, ACID1, ACID7, ACID11, ACID10, ACID14, ACID15, and ACID16 were genes that encoded for proteins with potential kinase activity. The remaining four ACID genes did not encode protein kinases. Of the remaining genes: ACID2 is an armadillo/beta-catenin repeat family protein, ACID4 a remorin family protein, ACID6 a member of EXO70 gene family, and ACID5 is an ankyrin repeat family protein. These four genes were included based on the availability of two independent homozygous T-DNA insertion lines. For ACID1, ACID4, ACID6, ACID7, ACID8, ACID9, ACID10, ACID12, ACID13, and ACID15 both tested T-DNA mutant lines exhibited enhanced Hpa susceptibility. This indicated that the mutations in/near the genes themselves were likely responsible for the respective defense phenotypes, as opposed to unknown mutations. Only one of the two tested T-DNA insertion lines for ACID5, ACID11, and ACID16 exhibited reduced basal defense to Hpa. It is likely that in each case the second line contained its T-DNA insertion in a position not capable of reducing gene function. For example, the insertion in acid11-1 is located in an exon and caused enhanced susceptibility to HpaNoco2. The insertion in acid11-2 did not affect HpaNoco2 resistance; this T-DNA located in the promoter of ACID11 did not to significantly reduce ACID11 expression. Neither the acid16-2 nor acid16-3 line displayed a defense phenotype. The insertions in these two mutants were intronic and might be spliced out of the ACID16 pre-mRNA . In contrast, the insertion in acid16-1, which is located in an exon, caused enhanced susceptibility to HpaNoco2. The insertion in the promoter in acid5-2, but not the exonic insertion in acid5-1, resulted in a defense-related phenotype. One possible explanation for this is that the insertion in the promoter abolished proper expression of this gene, while the exonic insertion results in a truncated, but functional, protein. Alternately, the insertion loci predicted by TAIR may not be accurately annotated . Among the acid mutants lines that exhibited reduced basal defense phenotype, only acid4-1, acid4-2, acid6-1, acid6-2, acid7-1, acid7-2, acid8-1, acid13-1, acid13-2, acid15-1, and acid15-2 displayed reduced levels of the respective transcripts. The observation that some T-DNA insertions did not affect endogenous ACID levels was not entirely unexpected.