Following the activation of initial and local defense responses are a set of delayed and systemic responses that include systemic acquired resistance . SAR is an induced form of defense that is activated remotely from the point of pathogen infection conferring a broad spectrum disease resistance against a variety of pathogens . Like many local defense responses, activation of SAR requires the accumulation of the signaling molecule salicylic acid . A complex regulatory network has been shown to be required for proper regulation of these plant immune responses . Many components of this network are commonly utilized by PTI, basal defense, ETI, and SAR. Major regulators of plant defense responses are protein kinases, which act at various hierarchical levels within the plant defense network . There are more than 1000 protein kinases in Arabidopsis . In particular, receptor protein kinases , Ca2+-dependent protein kinases and mitogen-activated protein kinases have been implicated in the regulation of plant immune responses . RPKs are comprised of a transmembrane domain with amino-terminal extracellular domains implicated in ligand recognition and protein–protein interactions, in addition to a carboxyl-terminal intracellular kinase domain involved in signal transduction . The three major sub-classes of RPKs are differentiated based upon their kinase domain substrate specificities. The sub-classes include: receptor-tyrosine kinases,grow bucket receptor-serine/threonine kinases, and receptor-histidine kinases .
Most plant RPKs are proteins containing an: extracellular signal sequence, extracellular leucine-rich repeats , a transmembrane helix, and cytoplasmic kinase domain with the serine/threonine consensus sequence . One variant in the RPK group is the receptor-like kinases , which belong to a large family known as the RLK/Pelle family. The Arabidopsis RLK family is divided into 45 subfamilies with over 600 members that comprise more than 2% of the Arabidopsis genome . One of the main criteria that distinguishes these subfamilies is the existence and type of extracellular domain . There are 15 classifications for RLK extracellular domains, which include: CRINKLY4-like, C-type lectin-like, CrRLK1-like, DUF26-like, extensin-like, legume -lectin-like, LRK10-like, LRR-like, LysM-like, PERK-like, RKF3-like, Sdomain-like, thaumatin-like, URK1- like, and WAK-like . The LRR domain is the most common and represents the largest RLK group with 216 members subdivided into 13 subfamilies . Most RLKs have a conserved arginine and an aspartate in the activation loop of subdomain VI, which acts as a kinase activator by enhancing phosphotransferase efficiency . Often kinases with arginine and aspartate are important for developmental regulation, while those without these conserved residues are important in innate immunity . Accordingly, plant RLKs can be further subdivided into two major categories based upon their functions: one is involved in cell growth and development and the other in plant–pathogen interactions and defense responses . Examples of this second group are PRRs: Xa21 from rice and FLS2 from Arabidopsis , which interact with certain MAMP-type epitopes. Xa21 is membrane bound serine/threonine protein that is activated by AxYS22, a 17- amino acid peptide conserved in strains of Xanthomonas.
FLS2 is a transmembrane protein that recognizes a number of bacterial MAMPs including peptides derived from the flagellin such as flg22 . Another RLK, CERK1, belongs to a distinct subfamily and is required for immune signaling triggered by fungal chitin. In addition, CERK1 binds and recognizes bacterial peptidoglycans contributing to immunity against bacteria . Another group of kinases important for defense are CDPKs, which are encoded by a 34-member gene family in Arabidopsis and make up one of the largest family of Ca2+ sensors in plants . Host proteins must be able to sense alterations in Ca2+ levels and respond accordingly . CDPKs have N-terminal protein serine/threonine kinase domains attached through an autoinhibitory junction domain to a C-terminal Ca2+-binding calmodulin-like domain . CDPKs bind Ca2+ at their C-terminal domain, which activates their protein kinase activity and facilitates their function as transducers of Ca2+signals. A possible role of Ca2+ in plant defense was proposed when CDPK transcripts were found to be elevated in tobacco, maize, tomato or pepper in response to pathogens or their elicitors . Upon pathogen recognition, cytosolic Ca2+ levels increase . The duration and amplitude of these increases are specific for the respective defense-related stimulus, resulting in the differential activation of downstream components . Two proteins have been suggested as potential substrates for CDPKs in plant defense: PAL and plasma membrane associated NADPH oxidase . PAL appears to be phosphorylated in bean cells challenged with a general elicitor but the significance of this observation remains to be demonstrated. In addition, a CDPK was shown to enhance NADPH oxidase activity stimulating an oxidative burst in tomato protoplasts although the significance of this interaction is also not clear . Romeis et al., demonstrated defense-associated activation of CDPKs in tobacco cell cultures transformed with the Cf-9 gene from tomato. Cf-9 is responsible for providing resistance to Cladosporium fulvum in the presence of its corresponding avirulence gene Avr9.
They established that the presence of Avr9 and Cf-9 a kinase was phosphorylated, causing an increase in kinase activity. They further demonstrated that this kinase is of the CDPK-type, because it required Ca2+ . This was the first direct demonstration of CDPK enzyme activity in plant defense. Meanwhile it has become clear that CDPKs are important not only in plant defense signaling but also serve as key points of convergence of various regulatory pathways due to their ability to respond to different hormonal or environmental cues . To better understand CDPK function in plant defense, additional pathogen-induced CDPK-phosphorylated substrates need to be identified. Mitogen-activated protein kinase cascades transmit and magnify signals through a phosphorelay mechanism involving: MAPK-kinase-kinases , MAPK-kinases , and MAPKs. They link upstream recognition events to downstream targets and their sequential phosphorylation targets substrate proteins in the cytoplasm or nucleus. MAPK activation is one of the earliest conserved signaling events after pathogen recognition . Many signaling cascades are shared between different activating stimuli . Cross-inhibition, feedback control, and the use of defined scaffolding proteins connecting distinct signaling components are utilized to enforce specific relationships between activating stimuli and the respective biological responses . Cross inhibition is manifested in the mutual inhibition between two pathways . Feedback control can be represented by negative feedback loops, where the activation of one component down-regulates the function of another. Scaffold proteins bring components together, which enhances specificity within signaling chains . Little is known about specific scaffolding proteins within plants. Two putative plant scaffolding proteins include alfalfa OMTK1, which interacts in protoplasts with the MAPK MMK3 in response to H2O2 and Arabidopsis MEKK1, which binds to MKK2 and MPK4 . MAPKs pathways are known to be involved in plant development, programmed cell death, responses to some abiotic stressors, and defense signaling. The Arabidopsis genome codes for 110 MAPK cascade components, which includes 20 MAPKs, ten MAPKKs and 80 MAPKKKs . Few MAPKs have been studied due to their lethal mutant phenotypes in plants . The most well understood MAPKs are MPK4, MPK3, and MPK6,dutch bucket for tomatoes with the latter two acting as positive regulators for defense responses and the former being a negative regulator of SAR. The Flg22 peptide is recognized by the receptor FLS2 which complexes with BRI1-ASSOCIATED KINASE and triggers MAPK signaling cascades. This cascade includes the activation of MPK3, MPK4 and MPK6 . MPK4 and MPK6 are also activated by hrp proteins from some bacteria and their activation results in the induction of PR genes which sometimes encode proteins with antimicrobial activities . Further studies must identify and elucidate MAPK cascades and find ways around the widespread mutant lethal phenotypes which inhibit kinase pathway studies. Traditional mutational analyses have been unable to circumvent functional redundancy and lethal mutant phenotypes . Thus, additional types of experimental approaches are necessary for the continued elucidation of the intricate and elaborate circuits within plant immune networks. One novel approach, chemical genetics, offers distinct advantages over traditional techniques.
Chemical genetics allows bioactive small molecules to be used in a reversible manner, since frequently their effects on organisms are not permanent. In addition, it provides more temporal control over experiments, since chemicals typically interfere with their targets immediately after application. In contrast, the timing of pathogen infections is often poorly reproducible, as the germination of spores or pathogen growth and spread in plants is asynchronous and often highly sensitive to subtle changes in environmental conditions. Chemicals also have the ability to simultaneously affect multiple members of highly-related protein families, permitting the study of biological functions of functionally redundant proteins. Using traditional genetics to knock out the function of an entire gene family often proves difficult or infeasible due to technical challenges and lethal phenotypes. Yet another advantage over traditional genetics is that bioactive chemicals allow for the study of essential gene functions at any stage in development because transiently active compounds can be added at any time or any concentration. In contrast, genetic mutations are of permanent nature. If they confer lethal phenotypes, no studies can be performed. Finally, the function of multiple structurally unrelated genes can be knocked out concurrently by using combinations of chemicals while also varying the concentration of each chemical allowing the study of quantitative relationships between defined stimuli and phenotypes . Chemical genomics requires tens of thousands or even hundreds of thousands of chemicals to be screened for their ability to stimulate a particular phenotype of interest . The increase in demand for chemicals that can manipulate a diverse set of biological processes resulted in the need for inexpensive large and structurally diverse chemical libraries for screening. As a result, the concept of combinatorial chemistry was developed . This high-throughput approach is based on simultaneously occurring synthesis steps. During each step a set of distinct chemical building blocks is used, yielding a vast number of structural combinations, referred to as “a combinatorial libraries”. The ease of this novel form of synthesis made these libraries widely available and cost-effective to many fields of academic research . Thus, the large sample size of available structurally distinct chemicals maximizes the probability that compounds will be identified that induces the desired biological effect. The Eulgem lab uses chemical genomics to identify and characterize synthetic elicitors, which are small drug-like molecules that induce plant defense responses . Their ability to induce defense responses provides us with a highly attractive alternative to conventional pesticides, if proven to be less toxic.This toxicity often leads to off-target effects against other organisms and the environment. As a result, the dangers of pesticide poisoning become more of a concern, making the identification of compounds that are not toxic, but instead stimulate plant’s inherent defenses very appealing. In addition to their potential use as pesticide replacements, synthetic elicitors can also be utilized as highly specific stimuli to perform more refined functional analyses of the plant defense network by interference with distinct network nodes. Their use should allow for the selective activation of certain regulatory circuits within this network. The identification of cellular targets of synthetic elicitors can uncover novel components of the plant immune system. Taken together the use of synthetic elicitors is likely to enable us to gain a deeper and more comprehensive understanding of the structure and function of the plant defense network. This report highlights the functional characterization of some members of the ACID cluster, a group of genes identified by micro-array experiments after treatment with two synthetic elicitors, DCA and INA . These 137 genes were found to be enriched for protein kinases, which may play key roles in plant defense signaling. Of the 16 ACID genes examined, ten were required for full basal defense of Arabidopsis against Hpa. Seven of the ten ACID genes have not been implicated as components of the plant immune system yet. While important for basal defense, these genes were not essential for immunity mediated by two distinct R-genes. Although they are transcriptionally activated by DCA, DCA mediated immunity was not compromised in their mutants. In addition, eight novel synthetic elicitors identified in the screen performed by Knoth et al., were further characterized. Notably, a synthetic elicitor was identified with a substantially lower active concentration than DCA. It was noted that DCA-mediated immunity in the acid mutants did not display the same hypersusceptibility seen in basal defense assays .