Ergosterol, an orphan fungal microbe-associated molecular pattern (MAMP).

Plants are endowed with innate immune system to resist pathogen attack. Pathogen-associated molecular patterns (PAMPs) and host-associated molecular patterns (HAMPs) are danger/alarm signals to switch on the plant immune systems. Plants possess receptor systems called plant pattern recognition receptors (PRRs) for sensing pathogen- or host-derived patterns to trigger inducible immune defenses. Most of the PRRs identified are receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Plants use the PRRs to defend themselves from microbial pathogens. PRRs do not act alone but rather function as part of multi-protein complexes at the plasma membrane. The PRRs interact with several transmembrane LRR receptor-like kinase proteins that act as signaling adapters or amplifiers to achieve full functionality. The PRRs are localized at the plasma membrane and the PAMPs activate expression of the genes encoding various PRRs and PRR interacting regulators to trigger defense responses against pathogens. PRRs play important role in triggering defense responses, PAMP perception by PRR is required for full immunity and plants deficient in the specific PRR become more susceptible to pathogens. However, pathogens secrete effectors, which directly target the PRRs and suppress the PRR-activated downstream immune responses. Early and robust activation of PRRs before the pathogens invade and secrete virulence effectors seems to be necessary for triggering strong defense responses and for effective management of crop diseases. Transgenic plants constitutively expressing various PRRs including FLS2, EF-Tu Receptor, XA21, and WAK1 show enhanced resistance against various fungal, oomycete, and bacterial diseases. Pathogens that are adapted to a particular host plant may be adept at suppressing the PRRs of that host by their effectors. The effectors of the pathogens might not recognize PRRs from other host plants and development of transgenic plants expressing PRRs from other plant species may provide good resistance against various pathogens. Transfer of Arabidopsis and rice PRRs to various economically important crop plants has revealed high potential of the PRRs for crop disease management. Engineering PRR-interacting protein complexes has also shown to be useful technology for crop disease management.

The research problem: The detection of pathogen-associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs) is a key mechanism by which plants activate an effective immune response against pathogen attack. MAPK cascades are important signaling components downstream of PRRs that transduce the PAMP signal to activate various defense responses. Preliminary experiments suggested that the receptor-like cytoplasmickinase (RLCK) Mai5 plays a positive role in pattern-triggered immunity (PTI) and interacts with the MAPKKK M3Kε. We thus hypothesized that Mai5, as other RLCKs, functions as a component PRR complexes and acts as a molecular link between PAMP perception and activation of MAPK cascades. Original goals: The central goal of this research was to investigate the molecular mechanisms by which Mai5 and M3Kε regulate plant immunity. Specific objectives were to: 1. Determine the spectrum of PAMPs whose perception is transmitted by M3Kε; 2. Identify plant proteins that act downstream of M3Kε to mediate PTI; 3. Investigate how and where Mai5 interacts with M3Kε in the plant cell; 4. Examine the mechanism by which Mai5 contributes to PTI. Changes in research directions: We did not find convincing evidence for the involvement of M3Kε in PTI signaling and substituted objectives 1 and 3 with research activities aimed at the analysis of transcriptomic profiles of tomato plants during the onset of plant immunity, isolation of the novel tomato PRR FLS3, and investigation of the involvement of the RLCKBSKs in PTI. Main achievements during this research program are in the following major areas: 1. Functional characterization of Mai5. The function of Mai5 in PTI signaling was demonstrated by testing the effect of silencing the Mai5 gene by virus-induced gene silencing (VIGS) experiments and in cell death assays. Domains of Mai5 that interact with MAPKKKs and subcellular localization of Mai5 were analyzed in detail. 2. Analysis of transcriptional profiles during the tomato immune responses to Pseudomonas syringae (Pombo et al., 2014). We identified tomato genes whose expression is induced specifically in PTI or in effector-triggered immunity (ETI). Thirty ETI-specific genes were examined by VIGS for their involvement in immunity and the MAPKKK EPK1, was found to be required for ETI. 3. Dissection of MAP kinase cascades downstream of M3Kε (Oh et al., 2013; Teper et al., 2015). We identified genes that encode positive (SGT and EDS1) and negative (WRKY1 and WRKY2) regulators of the ETI-associated cell death mediated by M3Kε. In addition, the MKK2 MAPKK, which acts downstream of M3Kε, was found to interact with the MPK3 MAPK and specific MPK3 amino acids involved interaction were identified and found to be required for induction of cell death. We also identified 5 type III effectors of the bacterial pathogen Xanthomonaseuvesicatoria that inhibited cell death induced by components of ETI-associated MAP kinase cascades. 4. Isolation of the tomato PRR FLS3 (Hind et al., submitted). FLS3, a novel PRR of the LRR-RLK family that specifically recognizes the flagellinepitope flgII-28 was isolated. FLS3 was shown to bind flgII-28, to require kinase activity for function, to act in concert with BAK1, and to enhance disease resistance to Pseudomonas syringae. 5. Functional analysis of RLCKs of the brassinosteroid signaling kinase (BSK) family.Arabidopsis and tomato BSKs were found to interact with PRRs. In addition, certain ArabidospsisBSK mutants were found to be impaired in PAMP-induced resistance to Pseudomonas syringae. Scientific and agricultural significance: Our research activities discovered and characterized new molecular components of signaling pathways mediating recognition of invading pathogens and activation of immune responses against them. Increased understanding of molecular mechanisms of immunity will allow them to be manipulated by both molecular breeding and genetic engineering to produce plants with enhanced natural defense against disease.

Pulmonary infection is the most common risk factor for acute lung injury (ALI). Innate immune responses induced by Microbe-Associated Molecular Pattern (MAMP) molecules are essential for lung defense but can lead to tissue injury. Little is known about how MAMP molecules are degraded in the lung or how MAMP degradation/inactivation helps prevent or ameliorate the harmful inflammation that produces ALI. Acyloxyacyl hydrolase (AOAH) is a host lipase that inactivates Gram-negative bacterial endotoxin (lipopolysaccharide, or LPS). We report here that alveolar macrophages increase AOAH expression upon exposure to LPS and that Aoah+/+ mice recover more rapidly than do Aoah-/- mice from ALI induced by nasally instilled LPS or Klebsiella pneumoniae. Aoah-/- mouse lungs had more prolonged leukocyte infiltration, greater pro- and anti-inflammatory cytokine expression, and longer-lasting alveolar barrier damage. We also describe evidence that the persistently bioactive LPS in Aoah-/- alveoli can stimulate alveolar macrophages directly and epithelial cells indirectly to produce chemoattractants that recruit neutrophils to the lung and may prevent their clearance. Distinct from the prolonged tolerance observed in LPS-exposed Aoah-/- peritoneal macrophages, alveolar macrophages that lacked AOAH maintained or increased their responses to bioactive LPS and sustained inflammation. Inactivation of LPS by AOAH is a previously unappreciated mechanism for promoting resolution of pulmonary inflammation/injury induced by Gram-negative bacterial infection.

Pathogen-associated molecular patterns (PAMPs) are recognized by plant pattern recognition receptors, activating PAMP-triggered immunity to restrict pathogen infection. The fungal pathogen Lasiodiplodia theobromae poses a major threat to global agriculture, yet its PAMPs and their interaction with plant pattern recognition receptors remain poorly understood. In this study, we identify LtSCRE11 as a key virulence factor of L. theobromae that also acts as a PAMP, inducing cell death and immune responses in Nicotiana benthamiana and peach (Prunus persica). In peach, LtSCRE11 is directly recognized by the L-type lectin receptor kinase PpLecRK-IX.1, which is essential for LtSCRE11-triggered cell death and immune activation. Remarkably, different plant lineages appear to have independently evolved distinct receptors to recognize LtSCRE11. PpLecRK-IX.1 binds LtSCRE11 through its extracellular lectin domain, forming a complex with the leucine-rich repeat receptor-like kinase PpBAK1 to mediate defense signaling. Our findings highlight the dual role of LtSCRE11 as both a virulence factor and a PAMP that activates plant immunity through PpLecRK-IX.1 in peach. This work provides insights into plant-fungal interactions and presents promising strategies for developing broad-spectrum disease resistance in peach.

Plants possess innate immune system to resist pathogen attack. Innate immunity is the first line of defense against invading microorganisms. Pathogens possess pathogen-associated molecular patterns (PAMPs). The PAMPs are primary danger/alarm signal molecules to switch on the plant immune systems. PAMPs are evolutionarily conserved building blocks of microbial surfaces that directly bind to plant pattern recognition receptors (PRRs). Plants use the PRRs to defend themselves from microbial pathogens. The PRRs are localized at the plasma membrane and the PAMPs activate expression of the genes encoding various PRRs. When activated by the PAMP, the PRR is translocated to endocytic compartments and endocytosis of the PRR is important for activation of several downstream signaling events. The plant immune system uses several second messengers to encode information generated by the PAMPs and deliver the information downstream of PRRs to proteins which decode and interpret the signals and initiate defense gene expression. G-proteins act as molecular switches in signal transduction system. Calcium ion is an important intracellular second messenger and carries the PAMP signal downstream to initiate immune responses. Reactive oxygen species (ROS) serve as second messengers transmitting the message. ROS appears to interact with various defense signaling systems. It plays a central role in launching the defense response. Nitric oxide (NO) is a diffusible molecular messenger that plays an important role in plant immune response signal transduction. Mitogen-activated protein kinase (MAPK) cascades are major pathways downstream of PAMP/PRR signaling complex that transduce extracellular stimuli into intracellular responses in plants. The plant hormones salicylic acid (SA), jasmonates (JA), ethylene (ET), abscisic acid (ABA), auxin (AUX), cytokinin (CK), gibberellin (GA), and brassinosteroid (BR) play important role in intercellular and systemic signaling systems triggering expression of various defense-responsive genes. SA signaling is involved in triggering systemic acquired resistance (SAR). SAR is associated with priming of defense responses and the priming results in a faster and stronger induction of defense responses after pathogen attack. The priming can be inherited epigenetically and descendants of primed plants exhibit next-generation systemic acquired resistance. Thus when pathogens land on the plant surface, the PAMPs trigger highly complex defense responses against the pathogens and suppress disease development. However virulent pathogen may modify its PAMP structure during its pathogenesis to reduce its elicitor activity. Virulent pathogens may also contain inefficient PAMPs and trigger subdued defense responses favoring disease development. The reduced activity of PAMPs might facilitate the virulent pathogens to cause disease. Besides PAMP molecules, pathogens produce effectors, which play an important role in pathogenesis. Effectors specifically contribute to virulence of pathogens by targeting host plant innate immunity. The effectors secreted by various pathogens have been shown to suppress the PAMP-triggered immunity. Effectors disrupt binding of PAMP with PRR in the PAMP-PRR signaling complex. Effectors may promote ubiquitin-proteasome-mediated degradation of PRRs to impede PAMP-triggered plant immunity. Effectors have been shown to target the receptor kinase activity of the PRRs and inhibit the kinase activity to block PAMP-triggered immunity. Autophosphorylation of PRRs results in activation of PRRs and the effectors may inhibit the autophosphorylation of PRRs to suppress the PAMP-triggered immune system. Some effectors have been shown to block the action of the PRR signal amplifier BAK1. Several receptor-like cytoplasmic kinases (RLCK) including BIK1, PBS1, and PBS1-like (PBL) proteins play important role in regulation of the signaling pathways downstream of PAMP-PRR-BAK1 signaling complex and the effectors have been shown to block the action of these RLCKs. Effectors may also suppress the MAPK signaling cascade triggered by PAMPs. Effectors have been shown to suppress SA signaling system, which is involved in triggering defense responses against a broad range of plant pathogens. Pathogens may induce specific signaling systems, which may favor disease development. Pathogen hijacks ABA signaling system to suppress SA-mediated defense responses promoting disease development. JA signaling system has been reported to confer susceptibility against some pathogens and pathogens may hijack JA signaling system to cause disease. Necrotrophic pathogens use SA signaling pathway to promote disease development by suppressing JA signaling pathway. Auxin signaling system has been shown to be involved in promoting susceptibility to pathogens and inducing disease development. Pathogens hijack the host auxin metabolism leading to the accumulation of a conjugated form of the hormone, indole-3-acetic acid (IAA) – Asp, to promote disease development. Pathogens may hijack BR signaling machinery to interfere with effectual SA- and GA-controlled defenses. These studies suggest that various signals and signaling systems in plants modulate the pathogenesis inducing susceptibility and disease resistance and precise manipulation of these signaling systems will be an ideal tool to manage crop diseases.

Plant PRRs and the Activation of Innate Immune Signaling

Plant pattern recognition receptors (PRRs) are essential for immune responses and establishing symbiosis. Plants detect invaders via the recognition of pathogen-associated molecular patterns (PAMPs) by PRRs. This phenomenon is termed PAMP-triggered immunity (PTI). We investigated disease resistance in Vitis amurensis to identify PRRs that are important for resistance against downy mildew, analyzed the PRRs that were upregulated by incompatible Plasmopara viticola infection, and cloned the full-length cDNA of the VaHAESA gene. We then analyzed the structure, subcellular localization, and relative disease resistance of VaHAESA. VaHAESA and PRR-receptor-like kinase 5 (RLK5) are highly similar, belonging to the leucine-rich repeat (LRR)-RLK family and localizing to the plasma membrane. The expression of PRR genes changed after the inoculation of V. amurensis with compatible and incompatible P. viticola; during early disease development, transiently transformed V. vinifera plants expressing VaHAESA were more resistant to pathogens than those transformed with the empty vector and untransformed controls, potentially due to increased H2O2, NO, and callose levels in the transformants. Furthermore, transgenic Arabidopsis thaliana showed upregulated expression of genes related to the PTI pathway and improved disease resistance. These results show that VaHAESA is a positive regulator of resistance against downy mildew in grapevines.

Chitin is a microbe-associated molecular pattern (MAMP) molecule in fungal cell wall that functions as elicitor which induces immune response in plants. Plants have its own defence system to protect itself from any invasion of pathogens. Chitin Elicitor Binding Protein (CEBiP) is one of the plant pattern recognition receptors (PRRs) that will detect the presence of the pathogen-associated molecular patterns (PAMPs) and activate plant defence system. In this study, we used Oryza sativa variety UKMRC9 to study the function of Chitin Elicitor Binding Protein (CEBiP) gene in plant immune system. The sample was extracted and amplified using the primer that were specifically designed for CEBiP based on the full gene sequence obtained from MSU Rice Genome Annotation Project database. The sample then was transformed into pGEM-T cloning vector. The plasmid construct of vector pCAMBIA 1305.2 was completed using PmII and BstEII as the restriction enzyme. The sequencing results of pGEM-T-CEBiP-RC9 obtained are subjected to multiple sequence alignment and the generation of a phylogenetic tree. Analysis obtained from BLAST shows that CEBiP UKMRC9 has 100% identity with four sequences from NCBI database with high query coverage. Multiple sequence alignment also shows that there are some nucleotides that have been inserted and deleted (indels) in the amplified sequence. Pairwise alignment indicates high similarity between CEBiP UKMRC9 and Chitin elicitor binding protein [Oryza sativa Japonica Group], PREDICTED: chitin elicitor-binding protein [Oryza sativa Japonica Group], Chain A, Crystal Structure of Oscebip, Chain A, and Crystal Structure of Oscebip Complex in BLAST. Results from phylogeny analysis places CEBiP UKMRC9 under the same clade with Oryza sativa cv Nipponbare CEBiP MSU. Even within the graminaceae, there is cladal separation of organisms based on genus and species.

A highly evolved surveillance system in plants is able to detect a broad range of signals originating from pathogens, damaged tissues, or altered developmental processes, initiating sophisticated molecular mechanisms that result in defence, wound healing, and development. Microbe-associated molecular pattern molecules (MAMPs), damage-associated molecular pattern molecules (DAMPs), virulence factors, secreted proteins, and processed peptides can be recognized directly or indirectly by this surveillance system. Nucleotide binding-leucine rich repeat proteins (NB-LRR) are intracellular receptors and have been targeted by breeders for decades to elicit resistance to crop pathogens in the field. Receptor-like kinases (RLKs) or receptor like proteins (RLPs) are membrane bound signalling molecules with an extracellular receptor domain. They provide an early warning system for the presence of potential pathogens and activate protective immune signalling in plants. In addition, they act as a signal amplifier in the case of tissue damage, establishing symbiotic relationships and effecting developmental processes. The identification of several important ligands for the RLK-type receptors provided an opportunity to understand how plants differentiate, how they distinguish beneficial and detrimental stimuli, and how they co-ordinate the role of various types of receptors under varying environmental conditions. The diverse roles of extra-and intracellular plant receptors are examined here and the recent findings on how they promote defence and development is reviewed.

Plant Immunity: AvrPto Targets the Frontline

In order to recognize a vast variety of attackers, plants possess a plethora of sophisticated detection systems. Perception of microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs) by the plant pattern recognition receptors (PRRs) leads to subsequent initiation of defense responses, a process collectively referred to as pattern-triggered immunity (PTI). PTI has been extensively studied in plant leaves, especially of the model organism Arabidopsis thaliana, whereas the mechanisms underlying PTI in roots so far attracted less attention. However, since a vast number of plant pests are soil-borne and attack roots in order to propagate and colonize whole plants, understanding the mechanisms underlying basic defense at the root level is of high interest for the development of new tools to combat root pathogens of crop plants. It has been demonstrated that recognition of flg22, the conserved epitope of the bacterial flagellin protein, leads to tissue-specific defense responses in roots. In order to investigate the cause for this tissue-specific induction of downstream responses, several approaches were employed during the course of this work. By studying the cellular localization of the PRR recognizing flg22, FLAGELLIN-SENSING 2 (FLS2), we were able to depict an expression map of FLS2 in wild-type Arabidopsis plants. Our study revealed that FLS2 was expressed in a highly tissue-specific manner in roots and shoots and that the FLS2 promoter activity was inducible upon environmental stimuli as well as during developmental processes, changing not only in intensity in expressing tissues but also in tissue-specificity. These results indicate an important role of the tissue-specific PRR localization in immunity mechanisms. In a parallel study, we expressed FLS2 under the control of several root tissue-specific promoters, which allowed us to analyze the competence of these tissues to detect flg22. Unexpectedly, all investigated root tissues were able to perceive externally applied flg22. In fact, PTI responses could be activated in intact roots as well as in dissected roots, suggesting that the peptide is able to penetrate through the different tissue layers. Remarkably, the expression level of the receptor was not the major parameter determining the magnitude of the immune response output. Thus, we postulated that perception of flg22 by certain tissues leads to stronger PTI responses potentially indicating why plants restrict immune receptor accumulation to tissue-specific locations possibly in order to balance the outcome of the defense activation. Due to the fact that many developmental or immunity processes in plants depend on systemic communication between different plant organs and that beneficial root microbes are known to prime and enhance resistance in aerial plant tissues, we hypothesized that MAMP perception by roots might induce a signaling event from roots to shoots. In order to address the potential existence of such systemic alarm signals, various methods were implemented. However, we encountered several technical limitations mainly concerning elicitor diffusion. Therefore, we focused on the development of an improved application method for studying systemic root-to-shoot signaling in Arabidopsis plants. Our system proved suitable to perform systemic signaling analysis and revealed that at the transcriptional level no systemically activated defense gene modifications were detectable in distal shoots of root-treated plants in our conditions. Like root pathogens, also viruses constitute a major threat in agro-economy and are responsible for immense crop losses. The basal defense response against viruses is thought to be mediated by RNA silencing, a process by which viral replication intermediates are cleaved and degraded by the plant silencing machinery through the recognition of virus-derived small RNAs. Intriguingly, a recent study conducted in our lab demonstrated a role of BRASSINOSTEROID INSENSITIVE1 (BRI1)-ASSOCIATED RECEPTOR KINASE 1 (BAK1), a coreceptor of several PRRs involved in immunity and development, in antiviral defense. These results indicated that PTI may also contribute to antiviral resistance but the exact recognition process remained elusive. Because dsRNA produced during viral replication has been shown to act as a PAMP in animals, we decided to test whether dsRNA is perceived as a viral PAMP in planta as well. We found that natural as well as synthetic dsRNA is indeed perceived as a PAMP by Arabidopsis, leading to the activation of typical PTI responses. Remarkably, dsRNA application also promoted protection of Arabidopsis plants against viral infection. Taken together, this study provides new insights into the recognition mechanisms of bacteria- and virus-associated molecular patterns by different plant organs and contributes to elucidate the molecular defense strategy of plants against agriculturally important diseases.

Salicylic Acid Signaling in Plant Innate Immunity

Switching on Plant Immune Signaling Systems Using Microbe-Associated Molecular Patterns

Loci associated with variation in maize responses to two microbe-associated molecular patterns (MAMPs) were identified. MAMP responses were correlated. No relationship between MAMP responses and quantitative disease resistance was identified. Microbe-associated molecular patterns (MAMPs) are highly conserved molecules commonly found in microbes which can be recognized by plant pattern recognition receptors. Recognition triggers a suite of responses including production of reactive oxygen species (ROS) and nitric oxide (NO) and expression changes of defense-related genes. In this study, we used two well-studied MAMPs (flg22 and chitooctaose) to challenge different maize lines to determine whether there was variation in the level of responses to these MAMPs, to dissect the genetic basis underlying that variation and to understand the relationship between MAMP response and quantitative disease resistance (QDR). Naturally occurring quantitative variation in ROS, NO production, and defense genes expression levels triggered by MAMPs was observed. A major quantitative traits locus (QTL) associated with variation in the ROS production response to both flg22 and chitooctaose was identified on chromosome 2 in a recombinant inbred line (RIL) population derived from the maize inbred lines B73 and CML228. Minor QTL associated with variation in the flg22 ROS response was identified on chromosomes 1 and 4. Comparison of these results with data previously obtained for variation in QDR and the defense response in the same RIL population did not provide any evidence for a common genetic basis controlling variation in these traits.

Plants never had it easy. When their ancestors colonized the land about 460 M years ago during the Ordovician period, they were immediately followed by crustaceans—which eventually developed into insects—that found plants a ready source of food. Ever since plants have been at the bottom of the food chain for pathogens and predators from viruses to mammalian herbivores. Unlike animals, plants cannot run away, having given up motility to maximize their surface exposed to sunlight. In lieu, they became masters in chemical warfare and communication to recognize and fend off pathogens and herbivores. Their “claws and teeth” are a dazzling arsenal of chemicals, metabolic and regulatory pathways, producing sensing and communication molecules that attract increasing interest from chemists and biologists for their potential use in medicine and agriculture. > Humans have also learned to use toxic compounds such as digitalis, atropine, opium or nicotine for medicinal, cosmetic or recreational use. Among these molecules are many spices, including the pungent piperine and capsaicin from peppers, or the glucosinolates from mustard and horseradish, and the bitter polyphenols, flavonoids or terpenes from red grapes, coffee beans, tea or cabbage. Humans have long come to appreciate these tastes—that originally evolved as a warning signal to herbivores—for centuries, spices were the most valuable commodity in global trade. Humans have also learned to use toxic compounds such as digitalis, atropine, opium or nicotine for medicinal, cosmetic or recreational use. The alkaloid taxol from the bark of the Pacific yew is a potent chemotherapeutic drug for cancer treatment; artemisinin, isolated from the plant Artemisia annua, or sweet wormwood, has long been employed in Chinese herbal medicine and been refined into a treatment for malaria. The metabolic pathways of plant compounds with medicinal value are therefore an active field of research with the objective of engineering these into E. coli or …