Plant Innate Immune Response Research Articles

Three Main Genes in the MAPK Cascade Involved in the Chinese Jujube-Phytoplasma Interaction

Chinese jujube (Ziziphus jujuba Mill.) is an important economic forest species and multipurpose fruit tree in the family of Rhamnaceae. Phytoplasmas are significant prokaryotic pathogens, associated with more than 1000 plant diseases. Jujube witches’ broom disease (JWB) is a typical phytoplasma disease, caused by ‘Candidatus Phytoplasma ziziphi’. Mitogen-activated protein kinase (MAPK) cascades are highly universal signal transduction modules and play crucial roles in regulating innate immune responses in plants. Thus, in the current study, systematical expression profiles of 10 ZjMPK and 4 ZjMPKK genes were conducted in plantlets with JWB disease, plantlets recovered from JWB disease, the tissues showing different disease symptoms, and resistant/susceptible cultivars infected by JWB phytoplasma. We found that most ZjMPK and ZjMKK genes exhibited significant up- or down-regulation expression under phytoplasma infection, but the top three differentially expressed genes (DEGs) were ZjMPK2, ZjMKK2 and ZjMKK4, which showed the biggest times of gene’s significant difference expression in all materials. Based on STRING database analysis, ZjMKK2 and ZjMPK2 were involved in the same plant-pathogen interaction pathway, and Yeast two-hybrid screening showed that ZjMKK2 could interact with ZjMPK2. Finally, we deduced a pathway of jujube MAPK cascades which response to ‘Candidatus Phytoplasma ziziphi’ infection. Our study presents the first gene-family-wide investigation on the systematical expression analysis of MAPK and MAPKK genes in Chinese jujube under phytoplasma infection. These results provide valuable information for the further research on the signaling pathway of phytoplasma infection in Chinese jujube.

The dark side of organic vegetables: interactions of human enteropathogenic bacteria with plants

Many recent studies reported that several pathogenic bacteria rely on multiple hosts during their life cycle. Specifically, Gram-negative enteropathogenic bacteria, such as Salmonella or Escherichia coli O157:H7, infect both human and plant hosts. These multi-kingdom pathogenic bacteria cause food-associated outbreaks in human by active invasion of the host. In the current review, we cover the interactions between human enteropathogenic bacteria and plants. In particular, we describe the current state of knowledge on the mechanisms of adhesion, invasion, and colonization of the plant hosts by human enteropathogenic bacteria, and describe plant innate immune responses to virulence factors produced by these bacteria.

Nematode ascaroside enhances resistance in a broad spectrum of plant–pathogen systems

AbstractRecognition of specific molecule signatures of microbes, including pathogens, induces innate immune responses in plants, as well as in animals. Analogously, a nematode pheromone, the ascaroside ascr#18, induces hallmark plant defences including activation of (a) mitogen‐activated protein kinases, (b) salicylic acid‐ and jasmonic acid‐mediated defence signalling pathways and (c) defence gene expression and provides protection to a broad spectrum of pathogens. Ascr#18 is a member of an evolutionarily conserved family of nematode signalling molecules and is the major ascaroside secreted by plant–parasitic nematodes. Here, we report the effects of ascr#18 on resistance in four of the major economically important crops: maize, rice, wheat and soybean to some of their associated pathogens. Treatment with low nanomolar to low micromolar concentrations of ascr#18 provided from partial to strong protection in seven of eight plant–pathogen systems tested with viruses, bacteria, fungi, oomycetes and nematodes. This research may have potential to improve agricultural sustainability by reducing use of potentially harmful agrochemicals and enhance food security worldwide.

AtNPR4 from Arabidopsis thaliana: expression, purification, crystallization and crystallographic analysis.

Salicylic acid (SA) is an important phytohormone that is involved in the regulation of plant defence, growth and development. A large number of proteins have been shown to have the ability to interact with SA, and NPR4 has been demonstrated to be a receptor of SA that plays significant roles in the innate immune response of plants. In this study, Spodoptera frugiperda (Sf9) cells were used to express full-length AtNPR4 from Arabidopsis thaliana. To facilitate crystallization, T4 lysozyme (T4L) was added to the N-terminus of the AtNPR4 protein. The recombinant T4L-AtNPR4 protein was expressed, purified and crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. The T4L-AtNPR4 crystals have symmetry consistent with space group C2, with unit-cell parameters a = 93.7, b = 85.8, c = 88.2 Å, β = 90° and one molecule per asymmetric unit. The best crystal diffracted to a resolution of 2.75 Å. Structure determination is in progress.

EFR-Mediated Innate Immune Response in Arabidopsis thaliana is a Useful Tool for Identification of Novel ERQC Modulators.

Plants offer a simpler and cheaper alternative to mammalian animal models for the study of endoplasmic reticulum glycoprotein folding quality control (ERQC). In particular, the Arabidopsis thaliana (At) innate immune response to bacterial peptides provides an easy means of assaying ERQC function in vivo. A number of mutants that are useful to study ERQC in planta have been described in the literature, but only for a subset of these mutants the innate immune response to bacterial elicitors has been measured beyond monitoring plant weight and some physio-pathological parameters related to the plant immune response. In order to probe deeper into the role of ERQC in the plant immune response, we monitored expression levels of the Phosphate-induced 1 (PHI-1) and reticulin-oxidase homologue (RET-OX) genes in the At ER α-Glu II rsw3 and the At UGGT uggt1-1 mutant plants, in response to bacterial peptides elf18 and flg22. The elf18 response was impaired in the rsw3 but not completely abrogated in the uggt1-1 mutant plants, raising the possibility that the latter enzyme is partly dispensable for EF-Tu receptor (EFR) signaling. In the rsw3 mutant, seedling growth was impaired only by concomitant application of the At ER α-Glu II NB-DNJ inhibitor at concentrations above 500 nM, compatibly with residual activity in this mutant. The study highlights the need for extending plant innate immune response studies to assays sampling EFR signaling at the molecular level.

The cotton laccase gene GhLAC15 enhances Verticillium wilt resistance via an increase in defence-induced lignification and lignin components in the cell walls of plants.

Summary Verticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The lignification of cell wall appositions is a conserved basal defence mechanism in the plant innate immune response. However, the function of laccase in defence‐induced lignification has not been described. Screening of an SSH library of a resistant cotton cultivar, Jimian20, inoculated with V. dahliae revealed a laccase gene that was strongly induced by the pathogen. This gene was phylogenetically related to AtLAC15 and contained domains conserved by laccases; therefore, we named it GhLAC15. Quantitative reverse transcription‐polymerase chain reaction indicated that GhLAC15 maintained higher expression levels in tolerant than in susceptible cultivars. Overexpression of GhLAC15 enhanced cell wall lignification, resulting in increased total lignin, G monolignol and G/S ratio, which significantly improved the Verticillium wilt resistance of transgenic Arabidopsis. In addition, the levels of arabinose and xylose were higher in transgenic plants than in wild‐type plants, which resulted in transgenic Arabidopsis plants being less easily hydrolysed. Furthermore, suppression of the transcriptional level of GhLAC15 resulted in an increase in susceptibility in cotton. The content of monolignol and the G/S ratio were lower in silenced cotton plants, which led to resistant cotton cv. Jimian20 becoming susceptible. These results demonstrate that GhLAC15 enhances Verticillium wilt resistance via an increase in defence‐induced lignification and arabinose and xylose accumulation in the cell wall of Gossypium hirsutum. This study broadens our knowledge of defence‐induced lignification and cell wall modifications as defence mechanisms against V. dahliae.

Cloning and characterization of the PtVIP1 gene in Populus

The VirE2-interaction protein 1 (VIP1) serves as a regulator of mitogen-activated protein kinase 3 (MPK3)-mediated stress gene modulation under biotic stress, which in turn activates the MPK3 pathway in Arabidopsis. The mode of action of the VIP1 protein in Populus in response to biotic stress remains unknown. In this study, we cloned the full-length cDNA of the PtVIP1 gene from Populus trichocarpa (accession number of GenBank: KY793105). The VIP1 protein harboured a conserved bZIP (basic leucine zipper) domain located in the C-terminus. The VIP1 subcellular localization assay indicated that the VIP1 protein was present in the cytoplasm and nucleus under normal conditions, and that an increase in the amount of the protein in the nucleus occurred after treatment with flg22, the elicitor-active epitope of flagellin which triggers the innate immune response in plants. Transgenic Populus plants overexpressing VIP1 genes (PtVIP1 of Populus; or AtVIP1 of Arabidopsis, as positive control) were generated to investigate the role of VIP1 in vivo. The expression of poplar pathogenesis-related protein 1 (PR1) genes was up-regulated in transgenic- PtVIP1 or AtVIP1 poplar plants. The transgenic poplar plants overexpressing PtVIP1 or AtVIP1 also showed enhanced resistance to Brenneria salicis infection. These results suggest that the VIP1 protein accumulates in the nucleus in response to biotic stress, and that the pathogen resistance of transgenic VIP1 poplar may be associated with the induced expression of PR1 genes in response to pathogen challenge.

Stomatal Closure and SA-, JA/ET-Signaling Pathways Are Essential for Bacillus amyloliquefaciens FZB42 to Restrict Leaf Disease Caused by Phytophthora nicotianae in Nicotiana benthamiana.

Bacillus amyloliquefaciens FZB42 is a plant growth-promoting rhizobacterium that induces resistance to a broad spectrum of pathogens. This study analyzed the mechanism by which FZB42 restricts leaf disease caused by Phytophthora nicotianae in Nicotiana benthamiana. The oomycete foliar pathogen P. nicotianae is able to reopen stomata which had been closed by the plant innate immune response to initiate penetration and infection. Here, we showed that root colonization by B. amyloliquefaciens FZB42 restricted pathogen-mediated stomatal reopening in N. benthamiana. Abscisic acid (ABA) and salicylic acid (SA)-regulated pathways mediated FZB42-induced stomatal closure after pathogen infection. Moreover, the defense-related genes PR-1a, LOX, and ERF1, involved in the SA and jasmonic acid (JA)/ethylene (ET) signaling pathways, respectively, were overexpressed, and levels of the hormones SA, JA, and ET increased in the leaves of B. amyloliquefaciens FZB42-treated wild type plants. Disruption of one of these three pathways in N. benthamiana plants increased susceptibility to the pathogen. These suggest that SA- and JA/ET-dependent signaling pathways were important in plant defenses against the pathogen. Our data thus explain a biocontrol mechanism of soil rhizobacteria in a plant.

Transgenic expression of plant-specific insert of potato aspartic proteases (StAP-PSI) confers enhanced resistance to Botrytis cinerea in Arabidopsis thaliana

The plant-specific insert of Solanum tuberosum aspartic proteases (StAP-PSI) has high structural similarity with NK-lysin and granulysin, two saposin-like proteins (SAPLIPs) with antimicrobial activity. Recombinant StAP-PSI and some SAPLIPs show antimicrobial activity against pathogens that affect human and plants. In this work, we transformed Arabidopsis thaliana plants with StAP-PSI encoding sequence with its corresponding signal peptide under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Results obtained show that StAP-PSI significantly enhances Arabidopsis resistance against Botrytis cinerea infection. StAP-PSI is secreted into the leaf apoplast and acts directly against pathogens; thereby complementing plant innate immune responses. Data obtained from real-time PCR assays show that the constitutive expression of StAP-PSI induces the expression of genes that regulate jasmonic acid signalling pathway, such as PDF1.2, in response to infection due to necrotrophic pathogens. On the other hand, according to the data described for other antimicrobial peptides, the presence of the StAP-PSI protein in the apoplast of A. thaliana leaves is responsible for the expression of salicylic acid-associated genes, such as PR-1, irrespective of infection with B. cinerea. These results indicate that the increased resistance demonstrated by A. thaliana plants that constitutively express StAP-PSI owing to B. cinerea infection compared to the wild-type plants is a consequence of two factors, i.e., the antifungal activity of StAP-PSI and the overexpression of A. thaliana defense genes induced by the constitutive expression of StAP-PSI. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the use of pesticides.We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the spreading of resistance of agriculturally important pathogens.

Diluted Fungal Phytotoxin: An Immerging tool for Boosting Innate Immunity in Plants

Fungal pathogens produce an array of toxin with various chemical structures, specificity, mechanism of action and functions. In recent years fungal toxins have been intensively studied, mainly for enhancing innate immunity in plants. Innate immunity is the only line of defense in plants, whose elicitation by toxin at optimum dilution may trigger the accumulation of various bioactive molecules such as defense enzymes, pathogenesis related proteins and secondary metabolite. Production of these defense related molecules may pave the way for the use of toxic metabolite to counter disease in plants, constituting a potential and cost effective alternative for toxic fungicides. Furthermore, this simple methods might help in enhancement of secondary metabolite production in medicinal plants which eventually be applied in the pharmaceutical industry. Henceforth the present review focus on the role of fungal phytotoxin as an elicitor triggering innate immune responses in plants.

Real-time PCR Analysis of PAMP-induced Marker Gene Expression in Nicotiana benthamiana.

Perception of pathogen-associated molecular patterns (PAMPs) often triggers various innate immune responses in plants. The transcriptional changes of defense-related genes are often used as a marker to assay PAMP-triggered plant immune response. Here we described a protocol to monitor the relative expression level of marker genes in Nicotiana benthamiana upon treatment with PAMPs. The procedure includes leaf treatment using PAMPs, total RNA isolation, cDNA synthesis, quantitative real-time PCR and data analysis. This protocol is applicable to monitor marker gene expression triggered by different PAMPs in N. benthamiana.

A High-Sensitivity, Microtiter-Based Plate Assay for Plant Pattern-Triggered Immunity.

The first step in the plant immune response to pathogen challenge involves the perception of conserved epitopes, called microbe-associated molecular patterns (MAMPs), by cell-surface pattern recognition receptors (PRRs). Given the key roles that MAMPs and PRRs play in plant innate immunity, great effort has been expended to identify these molecules. Current methods for assaying these immune responses are often limited in their resolution and throughput, and consequently, there is a need for medium- to high-throughput methodologies. Here, we describe the development of a 96-well microtiter plate-based assay for plant pattern-triggered immunity that measures the activity of plant peroxidase (POX) enzymes produced in response to treatment with bacterial MAMPs. The system has been optimized to minimize both the amount of plant tissue and MAMPs required and displays up to three orders of magnitude greater sensitivity than the traditional luminol-based reactive oxygen species assay when measuring the plant response to treatment with the bacterial MAMP flg22, reaching detection limits in the picomolar range. This high sensitivity opens the possibility of evaluating the immune-eliciting effects of weaker elicitors. The throughput and material requirements of the assay make it ideal for screens involving quantitative measurement of the plant innate immune response to MAMPs.

A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.

SG2-Type R2R3-MYB Transcription Factor MYB15 Controls Defense-Induced Lignification and Basal Immunity in Arabidopsis.

Lignification of cell wall appositions is a conserved basal defense mechanism in the plant innate immune response. However, the genetic pathway controlling defense-induced lignification remains unknown. Here, we demonstrate the Arabidopsis thaliana SG2-type R2R3-MYB transcription factor MYB15 as a regulator of defense-induced lignification and basal immunity. Loss of MYB15 reduces the content but not the composition of defense-induced lignin, whereas constitutive expression of MYB15 increases lignin content independently of immune activation. Comparative transcriptional and metabolomics analyses implicate MYB15 as necessary for the defense-induced synthesis of guaiacyl lignin and the basal synthesis of the coumarin metabolite scopoletin. MYB15 directly binds to the secondary wall MYB-responsive element consensus sequence, which encompasses the AC elements, to drive lignification. The myb15 and lignin biosynthetic mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with defense-induced lignin having a major role in basal immunity. A scopoletin biosynthetic mutant also shows increased susceptibility independently of immune activation, consistent with a role in preformed defense. Our results support a role for phenylalanine-derived small molecules in preformed and inducible Arabidopsis defense, a role previously dominated by tryptophan-derived small molecules. Understanding the regulatory network linking lignin biosynthesis to plant growth and defense will help lignin engineering efforts to improve the production of biofuels and aromatic industrial products as well as increase disease resistance in energy and agricultural crops.

Overexpression of GhPFN2 enhances protection against Verticillium dahliae invasion in cotton.

Growing evidence indicates that actin cytoskeleton is involved in plant innate immune responses, but the functional mechanism remains largely unknown. Here, we investigated the behavior of a cotton profilin gene (GhPFN2) in response to Verticillium dahliae invasion, and evaluated its contribution to plant defense against this soil-borne fungal pathogen. GhPFN2 expression was up-regulated when cotton root was inoculated with V. dahliae, and the actin architecture was reorganized in the infected root cells, with a clear increase in the density of filamentous actin and the extent of actin bundling. Compared to the wild type, GhPFN2-overexpressing cotton plants showed enhanced protection against V. dahliae infection and the actin cytoskeleton organization in root epidermal cells was clearly altered, which phenocopied that of the wild-type (WT) root cells challenged with V. dahliae. These results provide a solid line of evidence showing that actin cytoskeleton reorganization involving GhPFN2 is important for defense against V. dahliae infection.

CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis.

Plant phosphatidylinositol‐specific phospholipase C at the center of plant innate immunity

Understanding plant resistance to pathogenic microbes requires detailed information on the molecular mechanisms controlling the execution of plant innate immune responses. A growing body of evidence places phosphoinositide-specific phospholipase C (PI-PLC) enzymes immediately downstream of activated immune receptors, well upstream of the initiation of early defense responses. An increase of the cytoplasmic levels of free Ca2+ , lowering of the intercellular pH and the oxidative burst are a few examples of such responses and these are regulated by PI-PLCs. Consequently, PI-PLC activation represents an early primary signaling switch between elicitation and response involving the controlled hydrolysis of essential signaling phospholipids, thereby simultaneously generating lipid and non-lipid second messenger molecules required for a swift cellular defense response. Here, we elaborate on the signals generated by PI-PLCs and their respective downstream effects, while providing an inventory of different types of evidence describing the involvement of PI-PLCs in various aspects of plant immunity. We project the discussed information into a model describing the cellular events occurring after the activation of plant immune receptors. With this review we aim to provide new insights supporting future research on plant PI-PLCs and the development of plants with improved resistance.

Synthesis of Bradyrhizose Oligosaccharides Relevant to the Bradyrhizobium O-Antigen.

The unique α-(1→7)-bradyrhizoside linkages are constructed for the first time via judicious choice of the glycosylation partners and conditions, thus tetra- and penta-bradyrhizosides relevant to the peculiar O-antigen of Bradyrhizobium are synthesized, which are shown to adopt the defined right-handed helical conformations and to be unable to induce innate immune responses in plants.

Synthesis of Bradyrhizose Oligosaccharides Relevant to the Bradyrhizobium O‐Antigen

AbstractThe unique α‐(1→7)‐bradyrhizoside linkages are constructed for the first time via judicious choice of the glycosylation partners and conditions, thus tetra‐ and penta‐bradyrhizosides relevant to the peculiar O‐antigen of Bradyrhizobium are synthesized, which are shown to adopt the defined right‐handed helical conformations and to be unable to induce innate immune responses in plants.

BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation.

Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses.