Small molecules have the potential to simultaneously knockout the function of closely-related members of protein families . This may permit the study of biological functions of functionally redundant proteins. Using traditional genetics, this can be difficult or infeasible due to technical challenges and lethal phenotypes . Yet another advantage over traditional genetics is that bioactive small molecules allow for the study of essential gene functions at any stage in development because transiently active molecules can be added at any time point during plant development or applied at sub-lethal concentrations. In contrast, genetic mutations are permanent and the analysis of plant lines homozygous for a lethal mutation is challenging or impossible. Finally, multiple unrelated gene functions can be knocked out concurrently by using combinations of different bioactive molecules . For the primary screen,dutch buckets system both forward chemical and mutational screens must be specific, meaning that the read-out has to be specific and serve as an unambiguous proxy for the biological process of interest. Chemical genetics requires the screening of many thousands of chemicals in search of one with the ability to stimulate a particular response of interest . The need for chemicals that can manipulate a large diversity of biological processes resulted in the development of large structurally diverse chemical libraries . The concept of chemical genetics is based on the theoretical assumption that for every existing protein in the biosphere there are hypothetical organic structures capable of binding to it and interfering with its function .
The identification of bioactive compounds interfering with any given biological process or target protein requires screening of libraries representing a large diversity of chemical structures. Of key importance for the identification of bioactive compounds are their physicochemical properties. To be a biologically active compound the substance has to be “drug-like”, which means they must be capable of crossing biological membranes and to remain in an active state in the biological target tissue for a sufficient period of time . Lipinski’s ‘rule-of-five’ states 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 . A large sample size of structurally distinct chemicals maximizes the probability that compounds will be identified that induce the desired biological effect. The identification of proteins targeted by a given compound is an integral step but also typically the bottleneck for most chemical genetics projects. Several strategies exist for target identification, some of those include: affinity chromatography, yeast three-hybrid , protein arrays, and screens for mutants with altered sensitivity to a compound of interest . In affinity chromatography, a compound is tagged and immobilized so that interacting proteins can be purified and then identified. In Y3H the compound of interest is tagged with dexamethasone or methotrexate and then applied to yeast cells. The compound then binds the DEX or MTX binding protein, which is fused to the DNA-binding domain of a transcription factor. The activation domain of the transcription factor is translationally fused to a cDNA library. When the compound interacts with a plant protein a complex is created and this results in the transcription of a reporter gene . In protein arrays a fluorescent- or isotope-labeled small molecule is used to screen protein chips . Finally, in a screen for mutants with altered sensitivity, mutagenized organisms are treated with compound and plants showing responses to the compound that differ from wild type are selected for further study .
There are several examples of successful applications of chemical genetics in plant systems. Armstrong et al., performed a high-throughput chemical screen using a 10,000 compound library with the intent of identifying inhibitors of auxin transcriptional activation . Their screening strategy involved the use of a line that expressed GUS in the root elongation zone after application of auxin. This screen resulted in the identification of 30 compounds showing strong inhibition of GUS expression. Four structurally distinct compounds were further studied based on low active concentrations . Two of these compounds impart phenotypes indicative of an altered auxin response, including impaired root development. The two strongest of these compounds displayed similar growth phenotypes after treatment. Additionally, microarray studies using the later two compounds indicated that similar transcriptional changes were induced by both inhibitors . Chemical genetics became popular in plant biology in the past 10 years and numerous successful applications of this approach in plants have been published during the past five years . In 2007 a number of successful chemical screens were reported. The compound 7-ethoxy-4-methyl chromen-2-one was discovered in a screen of 20,000-compound library based on its ability to cause a swollen root phenotype in Arabidopsis . Using live cell imaging of fluorescently labeled cellulose synthase and microtubules, DeBolt et al., showed that treatment with morlin interferes with cortical micro-tubules to alter the movement of cellulose synthase. This interference resulted in unique cytoskeletal defects which produced shorter and more bundled micro-tubules. Morlin proved highly useful for the study of mechanisms that regulate micro-tubule cortical array organization and how it interacts with cellulose synthase .
In a small screen of 120 bioactive molecules Arabidopsis seedlings were used to identify compounds that inhibited early immune responses. This screen resulted in four hits. These compounds reduced flg22 -activated gene expression of MAMP-responsive ATL2 gene. Two of these four compounds, triclosan and fluazinam, interfere with the accumulation of ROIs and transport of the FLS2 receptor. Additionally, the compound Triclosan, which blocks early immune responses, was used to determine a potential role for lipid signaling in flg22-triggered immunity . Hypostatin was discovered in screens as a compound that inhibits hypocotyl growth in a Arabidopsis accession dependent manner . 11 other accession-selective hit molecules were also identified alongside hypostatin, which is an inhibitor of cell expansion. Additionally, a screen for compounds able to disturb microfibril-cellulose attachment resulted in the identification of cobtorin . This study demonstrated that different Arabidopsis accessions can be used to study the activity of interesting new compounds. The enhancement of plant immune responses by exogenous application of chemicals can be traced back to the treatment of tobacco with SA . While SA, JA, and ET can induce defense response, their use in the field or greenhouse setting is restricted based on their shortcomings as defense inducers that are broadly effective on many plant species . The use of environmentally safe plant defense-inducing chemicals, which boost a plant’s innate immune responses, offers an attractive alternative to pesticides. Linda wants me to reference the body of literature that explored SAR inducers as methods of control – klessig + sa An alternative procedure to protect plants against disease is to activate their own defense mechanisms by specific biotic or abiotic elicitors . The classical type of induced resistance is often referred to as systemic acquired resistance . Sodium salicylate , 2,6- dichloroisonicotinic acid , and benzothiadiazole-S-methyl ester are well-known elicitors of SAR in various plants against disease . The expression of SAR, triggered by either pathogen infection or treatment with NaSA or its functional analogs INA or BTH, is tightly associated with the transcriptional activation of genes encoding pathogenesisrelated proteins . The nonprotein amino acid DL-3-aminon-butanoic acid also activates an induced resistance response. BABA induced resistance, involves SA-dependent, SA independent,dutch buckets and ABA-dependent defense mechanisms, and the importance of these defenses varies according to the nature of the challenging pathogen . Many molecules exist that cause an induction of defenses in plants when applied.
Plant-derived small molecules other than SA, JA, and ET have been identified as important to controlling or preventing disease in plants. Some of these phytohormones include abscisic acid , brassinosteroids, gibberellin, cytokinin, and auxin . Like the interactions between SA, JA and ET, other molecules are important for the tailoring of defense responses . Activation of ABA signaling processes and its biosynthesis have also been shown to promote plant disease . Alternatively, brassinosteroid treatment has been shown to enhance resistance against some biotrophs, mediate abiotic stress responses through NPR1, and induce PR1 expression . Gibberellic acid can induce increases in ROS accumulation and attenuation of JA signaling . Cytokinins, which affect cell division and morphogenesis, can also enhance the SA response and thus promote resistance against biotrophs . Finally, auxin signaling may suppress SA biosynthesis and signaling, while SA attenuates auxin signaling . These plant hormones are integral to the function of plants. While many of these hormones were not originally associated with defense, new research suggests they all have roles in coordinating plant response to pathogen invasion. Every year billions of dollars are spent on pesticides which leave residues on produce that control pests and pathogens but can be harmful to consumers and the environment. Such off-target effects make the study of interactions between plants and pathogens an integral field for the reduction of conventional pesticide use. Using model pathosystems, such as Arabidopsis thalianaand Hyaloperonospora arabidopsidis , many important questions related to plant disease resistance are being addressed. A complex transcriptional network controls plant immune responses. Of key importance for the regulation of this defense network, are protein kinases that act at various stages during defense activation. Chemical genomics can be used to study these different stages. Plant defense-inducing molecules identified and characterized using chemical genomics will be valuable tools for the dissection of the plant defense network and will serve as leads for the development of novel environmentally safe pesticides. Genes from the Arabidopsis ACIDcluster are coordinately inducible by the synthetic elicitors DCA and INA. This cluster is enriched for genes encoding protein kinases. Using a forward genetics approach it was demonstrated that 10 of 16 ACID members tested are required for full immunity of Arabidopsis against Hpa. Seven of these 10 ACID members have not been implicated in plant immunity before. In addition, eight novel synthetic elicitors identified and characterized via chemical genomics were reported on here, one of which, called CMP442, is a more potent defense inducer than DCA or INA. While effective in crop protection pesticides leave residues on produce and have off target effects . This makes the design of novel green pesticides highly attractive. Also, the elucidation of the finer points of interactions between plant and pathogen is integral for the design of new approaches to more efficiently and safely prevent crop diseases. The model plant Arabidopsis thaliana and the oomycete Hyaloperonospora arabidopsidis are a naturally coevolving pathosystem with a high level of intra-species genetic diversity . Use of this and other model interactions has revealed that plants have a complex inducible immune system that protects wild species and crops from pathogen infections. When plants recognize the presence of an infecting pathogen, a multitude of signaling events are triggered that ultimately lead to efficient defense . Some of the early responses after resistance -gene recognition include changes in ion fluxes, synthesis of reactive oxygen species , alterations in gene transcription, which can be followed by a hypersensitive response , where the plant cells surrounding the point of infection die to restrict pathogen growth . An ancient and fundamental form of plant defense involves conserved microbe-associated molecular patterns . MAMPs are recognized by plant pattern-recognition receptors resulting in the activation of a complex defense response. This form of plant immunity is referred to as pattern-triggered immunity . A second form of immunity is based upon the recognition of pathogen-secreted effector molecules, which are proteins that promote pathogen virulence in the plant. Here the plant is capable of recognizing the presence of pathogen effectors, or their cellular effects, by disease Rproteins. R-proteins constitute a second class of plant immune receptors, besides PRRs, and induce a strong defense response, which often includes HR. This form of immunity is called effector-triggered immunity . In the absence of a cognate R-protein, the secretion of effectors enables pathogens to successfully infect their hosts. During such compatible interactions, plants can still mount a weakened immune response, called basal defense. Basal defense typically limits the spread of pathogens but is not capable of fully preventing disease .