Given that JEDI–PDF genes are JA-inducible, this signaling molecule may also be important for the induction of basal defense against Hpa. No other studies on silencing or disruption of the JEDI–PDF genes have been reported on in the literature. Future studies will have to address this possibility. A set of RNAi transgenic lines was developed which can be used to elucidate roles the JEDI-PDF genes play in plant defense. Pathogen assays indicated that these genes may, in fact, play a role in basal defense against HpaNoco2. Since a defense phenotype was observed in some PDF-RNAi lines indicated that this technique may have overcome the problem of redundancy previously limiting the study of these genes. These observations, together with this experimental data suggested a potential role of the JEDI-PDF genes in plant defense against Hpa. The PDF-RNAi lines could prove valuable tools for the further study of the JA/ET signaling network and plant defense responses.Even within different Arabidopsis accessions variation exists in the level of cross-talk between SA and JA signaling, indicating that variation in downstream signaling of plant hormones contributes to natural variation in basal resistance .
SA and JA are also known to contribute differently to defense based upon the invading pathogen . While a large body of research indicates that SA and JA pathways are mutually antagonistic ,rolling benches more recent studies have found indications of synergism between these pathways . Current methods providing resistance against viral infections in plants often involve transgenic plants, which are often not widely accepted by consumers . Additionally, pesticides are being used but are often toxic to humans and the environment or are only useful for a small range of plant-species or virus-species . One recent study performed by Shang et al., 2011 observed that SA accumulates one day after JA during immune responses of Nicotiana benthamiana against viruses. This group also found that the stimulation of JA accumulation followed by external application of SA reduced viral replication by 80–90% . This regime of phytohormone application produced broad-spectrum and effective resistance in Arabidopsis, N. benthamiana, Nicotiana glutinosa, Nicotiana tabacum, Capsicum frutescens , and Solanum lycopersicumagainst Cucumber mosaic virus, Turnip crinkle virus, Tobacco mosaic virus and Tobacco necrosis virus, respectively . PDF1.2 is known as a JA-inducible and SA-repressible gene ; however, its expression level was higher in SA-pretreated virus-infected seedlings than in the virus-infected seedlings without any treatment. This may be due to the complex cross-talk between JA and SA.
Other studies also found that the timing and/or order of pathway induction matters when trying to induce plant defense . During a long-term plant pathogen interaction , PDF1.2a may become SA-inducible, although it is negatively regulated by SA at early time points during pathogenic interactions . To have a complete picture of what is taking place during these interactions, longer time points should be evaluated. This is especially important since much work on pathway crosstalk has been performed at early time points. This data highlights the fact that the use of synthetic elicitors targeting SA and/or JA-dependent defense signaling mechanisms to induce broad spectrum resistance is environmentally friendly, safe, easy to perform, and did not affect agronomical traits in a negative manner. Unfortunately, the cost of JA for the use in agriculture is prohibitively high, while that of MeJA is lower but not low enough for widespread use. However, the cost of SA or BTH is low enough for these compounds to be used in crop protection. Cheaper synthetic elicitors would be beneficial for the agricultural industry. The identification of synthetic elicitors targeting both the SA and JA branch of the defense network may lead to novel agrochemicals that induce effective broad-spectrum diseases resistance but are not toxic to the surrounding biosphere. Plant innate immunity depends on a network of genes that regulate and execute defense reactions.
Using chemical genetics, small molecules can be identified that induce plant immune responses, but are structurally distinct from natural defense elicitors. I have initiated a chemical genomics-based approach to identify, characterize and utilize new types of synthetic elicitors for the dissection of the plant immune network as well as the development of novel types of pesticides. A cluster of genes were identified that are coordinately up-regulated after treatment with the synthetic elicitors, DCA or INA. The ACID cluster is composed of 137 genes, which are upregulated during time-points when defense is also on. The ACID genes were found to be enriched for protein kinases, which I hypothesized play key roles in plant defense signaling. Based on this knowledge, ACID genes that are kinases were selected first for study. Each ACID gene was also selected based upon the availability of two independent insertion lines. I showed that of the 16 ACID genes examined, 10 are required for Arabidopsis basal defense against HpaNoco2. Seven of the 10 ACID genes are unknown components of the plant immune system. While important for basal defense, I found that these genes are not essential for immunity mediated by the two R-genes RPP7 or RPP4 . I next determined if DCA-mediated immunity was compromised in the ACID mutant lines. Although they are transcriptionally activated by DCA, they are not required for DCA-mediated resistance. Additionally, RT-PCRs showed that the up-regulation PDF1.2 transcripts are abolished in many of these mutants. Based on these results I hypothesize that these ACID genes may play a role in later Arabidopsis defense responses and/or have roles in the cross talk between defense pathways. By high-throughput chemical screening we previously identified 114 synthetic elicitors that activate expression of a pathogen-responsive reporter gene in transgenic Arabidopsis. Here I described the characterization of nine novel synthetic elicitors identified in the screen performed by Knoth et al., 2009.
Notably, I identified a synthetic elicitor that has a substantially lower active concentration than DCA. I report in depth on the characterization of one of these compounds, 2–thiazolidine-4-carboxylic acid . CMP442 is able to quickly and transiently induce disease resistance against Hpa in Arabidopsis, has a distinct mode-of-action from DCA, and is structurally unique from previously identified synthetic elicitors. CMP442 can be synthesized quickly, easily, and inexpensively with a high degree of purity. During my efforts to develop a screen for mutants expressing altered sensitivity to CMP442 a surprising root phenotype was identified. I found that at low doses, CMP442 and other synthetic elicitors enhanced growth of roots and aerial parts of Arabidopsis thaliana and tomato , while high concentrations were toxic or inhibited plant growth. The effect of these synthetic elicitors on root growth is suggestive of a hormetic response. Hormesis is characterized by low dose stimulatory or beneficial effects, as evidenced by the increase in root length and plant weight,ebb and flow bench and a high dose inhibitory effect. The ability of CMP442 and other elicitors to beneficially affect both plant immunity and development points to crosstalk between both types of biological processes and may allow for the design of novel types of multi-functional agrochemicals. These agrochemicals would not only induce defense but increase crop yield at the same time. CMP442 may also allow us to uncover fundamental causes of the general phenomenon of hormesis. The ease and inexpensiveness of CMP442 synthesis show great promise for the use of this synthetic elicitor on larger studies. As previously mentioned synthetic elicitors make it possible to dissect and study the plant defense network, which advancing our goal of developing novel “green” pesticides. Here I report on the development of a screen to identify synthetic elicitors that activate the JA/ET branch of the defense network.
Towards this end a set of genes was identified showing a SA-independent upregulation in response to infection of Hpa. Four out of the five genes are PDFs, and one was the JA pathway molecular marker, PDF1.2a. While the resulting transgenic lines may not be appropriate for a high-throughput chemical screen they still are an additional asset for the study of plant-pathogen interactions. Additionally, I describe the creation of transgenic plants with a RNA silencing transgene able to knock down the transcripts of this highly related family of PDFs. Using the PDF-RNAi silenced plants I performed HpaNoco2 defense assays. These pathogen assays indicate that the RNA silencing was a success and that these genes may, in fact, play a role in basal defense against HpaNoco2. The defense phenotype indicates that the JEDI-RNAi lines overcame the problem of redundancy previously limiting the study of these genes. These observations, together with my experimental data suggest a definitive role of the JEDIs in plant defense against Hpa. The previous observations stating that the JA/ET branches of the defense network have no role in resistance against Hpa are challenged by these results. The JEDI-RNAi lines could prove invaluable tools for the further study of the JA/ET signaling network and plant defense responses. Through the use of a synthetic elicitor such as DCA, we have discovered novel components of the plant defense network. My work has firmly established the ACID genes as significant aspects of the defense signaling network. While their roles in plant defense are still yet to be defined it is clear that our synthetic elicitors are powerful tools to dissect plant defense responses. I have identified nine additional synthetic elicitors which show great potential, two of which were studied further here. One of these CMP442 shows a distinct mode of action from DCA and thus proves that a single screen can be used to identify functionally distinct synthetic elicitors. We have not yet been able to identify any targets of our synthetic elicitors. Although, a screen has been developed that could be used to identify mutants that show altered responses to our synthetic elicitors. Any mutants discovered in these screens with altered sensitivity to these compounds could identify novel features of the defense network or the target of our synthetic elicitor. In addition, these synthetic elicitors show great potential for their use in other plant systems which may facilitate the study of homologous processes across species barriers. These elicitors have already proven to be invaluable tools for the study of plant defense and may prove useful in systems not readily accessible by current molecular techniques. There is also great potential for some of these compounds to pave the way for the development of novel pest control regimes. Application of blends of non-toxic defense activators could be a viable alternative to environmentally hazardous toxic pesticides. These elicitors will be exceedingly valuable tools for further dissection of the plant immune system.Plant innate immunity depends on a network of genes that regulate and execute defense reactions. Two major branches of this network have previously been characterized and are known to involve either SA or JA and ET. Using chemical genetics, small molecules can be identified and characterized that induce plant immune responses, but are structurally distinct from natural defense elicitors. These synthetic elicitors can be used to dissect the different branches of the defense network. I have initiated a chemical genomics based approach to identify, characterize and utilize new types of synthetic elicitors for the dissection of the plant immune network as well as the development of novel types of pesticides. Previous work by our lab identified a cluster of Arabidopsis thalianagenes that are coordinately up-regulated after treatment with the synthetic elicitors, DCA or INA . The ACID cluster is composed of 137 genes, which are up-regulated during time-points after DCA or INA treatment when each of these synthetic elicitors triggers disease resistance against Hyaloperonospora arabidopsidis. The ACID genes were found to be enriched for protein kinases, which I hypothesized are likely to play key roles in plant defense signaling. Based on the knowledge that kinases are important for plant defense, a selection of ACID genes predominantly encoding protein kinases were subject to reverse genetics-based functional studies. I showed that of the 16 ACID genes examined, 10 are required for Arabidopsis basal defense against HpaNoco2 . Seven of these 10 ACID genes are so far unknown components of the plant immune system. While important for basal defense, I found that these genes are not required for immunity mediated by the two R-genes, RPP7 or RPP4. I further determined if DCA-mediated immunity is compromised in the ACID mutant lines.