Macropinocytosis in Phagocyte Function and Immunity.

Phagocytes play critical roles in the maintenance of organismal homeostasis and immunity. Central to their role is their ability to take up and process exogenous material via the related processes of phagocytosis and macropinocytosis. The mechanisms and functions underlying macropinocytosis have remained severely understudied relative to phagocytosis. In recent years, however, there has been a renaissance in macropinocytosis research. Phagocytes can engage in various forms of macropinocytosis including an "induced" form and a "constitutive" form. This chapter, however, will focus on constitutive macropinocytosis and its role in the maintenance of immunity. Functions previously attributed to macropinocytosis, including antigen presentation and immune surveillance, will be revisited in light of recent revelations and emerging concepts will be highlighted.

Arabidopsis RNA polymerase II C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity.

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1 (C-terminal domain phosphatase-like 1) as a negative regulator of microbe-associated molecular pattern (MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide flg22. Furthermore, flg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with flg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefly luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.

Nicotiana benthamiana LRR-RLP NbEIX2 mediates the perception of an EIX-like protein from Verticillium dahliae.

Verticillium wilt diseases caused by the soil-borne fungus Verticillium dahliae result in devastating yield losses in many economically important crops annually. Here, we identified a novel ethylene-inducing xylanase (EIX)-like protein, VdEIX3, from V. dahliae, which exhibits immunity-inducing activity in Nicotiana benthamiana. In vitro-purified VdEIX3 can induce strong oxidative burst, activate the expression of defense-related genes, and increase resistance against oomycete and fungal pathogens in N. benthamiana. VdEIX3 orthologs of other Verticillium pathogens also induce cell death in N. benthamiana, which form a new type of EIX protein family that is distinct from the known EIX proteins. A leucine-rich repeat receptor-like protein, NbEIX2, regulates the perception of VdEIX3 in N. benthamiana. Our results demonstrate that VdEIX3 is a novel EIX-like protein that can be recognized by N. benthamiana NbEIX2, and also suggest that NbEIX2 is a promising receptor-like protein that is potentially applicable to transgenic breeding for improving resistance to Verticillium wilt diseases.

Overexpressed BSR1-Mediated Enhancement of Disease Resistance Depends on the MAMP-Recognition System.

Plant plasma membrane-localized receptors recognize microbe-associated molecular patterns (MAMPs) and activate immune responses via various signaling pathways. Receptor-like cytoplasmic kinases (RLCKs) are considered key signaling factors in plant immunity. BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice RLCK, plays a significant role in disease resistance. Overexpression of BSR1 confers strong resistance against fungal and bacterial pathogens. Our recent study revealed that MAMP-triggered immune responses are mediated by BSR1 in wild-type rice and are hyperactivated in BSR1-overexpressing rice. It was suggested that hyperactivated immune responses were responsible for the enhancement of broad-spectrum disease resistance; however, this remained to be experimentally validated. In this study, we verified the above hypothesis by disrupting the MAMP-recognition system in BSR1-overexpressing rice. To this end, we knocked out OsCERK1, which encodes a well-characterized MAMP-receptor-like protein kinase. In the background of BSR1 overaccumulation, the knockout of OsCERK1 nearly abolished the enhancement of blast resistance. This finding indicates that overexpressed BSR1-mediated enhancement of disease resistance depends on the MAMP-triggered immune system, corroborating our previously suggested model.

Broad-Spectrum Disease Resistance Conferred by the Overexpression of Rice RLCK BSR1 Results from an Enhanced Immune Response to Multiple MAMPs.

Plants activate their immune system through intracellular signaling pathways after perceiving microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases mediate the intracellular signaling downstream of pattern-recognition receptors. BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice (Oryza sativa) receptor-like cytoplasmic kinase subfamily-VII protein, contributes to chitin-triggered immune responses. It is valuable for agriculture because its overexpression confers strong disease resistance to fungal and bacterial pathogens. However, it remains unclear how overexpressed BSR1 reinforces plant immunity. Here we analyzed immune responses using rice suspension-cultured cells and sliced leaf blades overexpressing BSR1. BSR1 overexpression enhances MAMP-triggered production of hydrogen peroxide (H2O2) and transcriptional activation of the defense-related gene in cultured cells and leaf strips. Furthermore, the co-cultivation of leaves with conidia of the blast fungus revealed that BSR1 overexpression allowed host plants to produce detectable oxidative bursts against compatible pathogens. BSR1 was also involved in the immune responses triggered by peptidoglycan and lipopolysaccharide. Thus, we concluded that the hyperactivation of MAMP-triggered immune responses confers BSR1-mediated robust resistance to broad-spectrum pathogens.

The Magnaporthe oryzae Alt A 1-like protein MoHrip1 binds to the plant plasma membrane.

MoHrip1, a protein isolated from Magnaporthe oryzae, belongs to the Alt A 1 (AA1) family. mohrip1 mRNA levels showed inducible expression throughout the infection process in rice. To determine the location of MoHrip1 in M. oryzae, a mohrip1-gfp mutant was generated. Fluorescence microscopy observations and western blotting analysis showed that MoHrip1 was both present in the secretome and abundant in the fungal cell wall. To obtain MoHrip1 protein, we carried out high-yield expression of MoHrip1 in Pichia pastoris. Treatment of tobacco plants with MoHrip1 induced the formation of necrosis, accumulation of reactive oxygen species and expression of several defense-related genes, as well as conferred disease resistance. By fusion to green fluorescent protein, we showed that MoHrip1 was able to bind to the tobacco and rice plant plasma membrane, causing rapid morphological changes at the cellular level, such as cell shrinkage and chloroplast disorganization. These findings indicate that MoHrip1 is a microbe-associated molecular pattern that is perceived by the plant immune system. This is the first study on an AA1 family protein that can bind to the plant plasma membrane.

Arabidopsis E3 ubiquitin ligase PLANT U-BOX13 (PUB13) regulates chitin receptor LYSIN MOTIF RECEPTOR KINASE5 (LYK5) protein abundance.

Long-chain chitooligosaccharides are fungal microbe-associated molecular patterns (MAMPs) that are recognized by LYSIN MOTIF RECEPTOR KINASE5 (LYK5), inducing the formation of a complex with CHITIN ELICITOR RECEPTOR KINASE1 (CERK1). Formation of this complex leads to activation of the CERK1 intracellular kinase domain and induction of plant innate immunity in Arabidopsis. We found that addition of chitooctaose induced LYK5 protein accumulation as a result of de novo gene expression and the inhibition of LYK5 protein degradation. Screening the putative E3 ligases for interaction with LYK5 identified PLANT U-BOX13 (PUB13), which complexed with LYK5, but this complex dissociated upon addition of chitooctaose. Consistent with these results, LYK5 protein abundance was higher in pub13 mutants compared with the wild type without chitooctaose treatment, while similar abundance was detected with the addition of chitooctaose. The pub13 mutants showed hypersensitivity to chitooctaose-induced rapid responses, such as the production of reactive oxygen species (ROS) and mitogen-activated protein (MAP) kinase phosphorylation, but exhibited normal responses to subsequent long-term chitooctaose treatment, such as gene expression and callose deposition. In addition, PUB13 could ubiquitinate the LYK5 kinase domain in vitro. Taken together, our results suggest an important regulatory function for the turnover of LYK5 mediated by the E3 ligase PUB13.

The Arabidopsis leaf transcriptome reveals distinct but also overlapping responses to colonization by phyllosphere commensals and pathogen infection with impact on plant health.

Plants are colonized by a variety of bacteria, most of which are not pathogenic. Currently, the plant responses to phyllosphere commensals or to pathogen infection in the presence of commensals are not well understood. Here, we examined the transcriptional response of Arabidopsis thaliana leaves to colonization by common commensal bacteria in a gnotobiotic system using RNA sequencing and conducted plant mutant assays. Arabidopsis responded differently to the model bacteria Sphingomonas melonis Fr1 (S.Fr1) and Methylobacterium extorquens PA1 (M.PA1). Whereas M.PA1 only marginally affected the expression of plant genes (< 10), S.Fr1 colonization changed the expression of almost 400 genes. For the latter, genes related to defense responses were activated and partly overlapped with those elicited by the pathogen Pseudomonas syringae DC3000 (Pst). As S.Fr1 is able to mediate plant protective activity against Pst, we tested plant immunity mutants and found that the pattern-recognition co-receptor mutant bak1/bkk1 showed attenuated S.Fr1-dependent plant protection. The experiments demonstrate that the plant responds differently to members of its natural phyllosphere microbiota. A subset of commensals trigger expression of defense-related genes and thereby may contribute to plant health upon pathogen encounter.

Bacterial RNAs activate innate immunity in Arabidopsis.

The common molecular patterns of microbes play a critical role in the regulation of plant innate immunity. However, little is known about the role of nucleic acids in this process in plants. We pre-infiltrated Arabidopsis leaves with total RNAs from Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) and subsequently inoculated these plants with the same bacterial cells. Total Pto DC3000 RNAs pre-infiltrated into Arabidopsis leaves elicited plant immune responses against Pto DC3000. However, sheared RNAs and RNase A application failed to induce immunity, suggesting that intact bacterial RNAs function in plant innate immunity. This notion was supported by the positive regulation of superoxide anion levels, callose deposition, two mitogen-activated protein kinases and defense-related genes observed in bacterial RNA-pre-treated leaves. Intriguingly, the Pto DC3000 population was not compromised in known pattern recognition receptor mutants for chitin, flagellin and elongation factor-Tu (EF-Tu). Plant defense-related mutant analyses further revealed that bacterial RNA-elicited innate immunity was normally required for salicylic and jasmonic acid signaling. Notably, among total RNAs, the abundant bacterial RNA species 16S and 23S ribosomal RNAs were the major determinants of this response. Our findings provide evidence that bacterial RNA serves as a microbe-associated molecular pattern in plants.

Guard cell SLAC1-type anion channels mediate flagellin-induced stomatal closure.

During infection plants recognize microbe-associated molecular patterns (MAMPs), and this leads to stomatal closure. This study analyzes the molecular mechanisms underlying this MAMP response and its interrelation with ABA signaling. Stomata in intact Arabidopsis thaliana plants were stimulated with the bacterial MAMP flg22, or the stress hormone ABA, by using the noninvasive nanoinfusion technique. Intracellular double-barreled microelectrodes were applied to measure the activity of plasma membrane ion channels. Flg22 induced rapid stomatal closure and stimulated the SLAC1 and SLAH3 anion channels in guard cells. Loss of both channels resulted in cells that lacked flg22-induced anion channel activity and stomata that did not close in response to flg22 or ABA. Rapid flg22-dependent stomatal closure was impaired in plants that were flagellin receptor (FLS2)-deficient, as well as in the ost1-2 (Open Stomata 1) mutant, which lacks a key ABA-signaling protein kinase. By contrast, stomata of the ABA protein phosphatase mutant abi1-1 (ABscisic acid Insensitive 1) remained flg22-responsive. These data suggest that the initial steps in flg22 and ABA signaling are different, but that the pathways merge at the level of OST1 and lead to activation of SLAC1 and SLAH3 anion channels.

The grapevine flagellin receptor VvFLS2 differentially recognizes flagellin-derived epitopes from the endophytic growth-promoting bacterium Burkholderia phytofirmans and plant pathogenic bacteria.

• The role of flagellin perception in the context of plant beneficial bacteria still remains unclear. Here, we characterized the flagellin sensing system flg22-FLAGELLIN SENSING 2 (FLS2) in grapevine, and analyzed the flagellin perception in the interaction with the endophytic plant growth-promoting rhizobacterium (PGPR) Burkholderia phytofirmans. • The functionality of the grapevine FLS2 receptor, VvFLS2, was demonstrated by complementation assays in the Arabidopsis thaliana fls2 mutant, which restored flg22-induced H2O2 production and growth inhibition. Using synthetic flg22 peptides from different bacterial origins, we compared recognition specificities between VvFLS2 and AtFLS2. • In grapevine, flg22-triggered immune responses are conserved and led to partial resistance against Botrytis cinerea. Unlike flg22 peptides derived from Pseudomonas aeruginosa or Xanthomonas campestris, flg22 peptide derived from B. phytofirmans triggered only a small oxidative burst, weak and transient defense gene induction and no growth inhibition in grapevine. Although, in Arabidopsis, all the flg22 epitopes exhibited similar biological activities, the expression of VvFLS2 into the fls2 background conferred differential flg22 responses characteristic for grapevine. • These results demonstrate that VvFLS2 differentially recognizes flg22 from different bacteria, and suggest that flagellin from the beneficial PGPR B. phytofirmans has evolved to evade this grapevine immune recognition system.

Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility.

The bacterial flagellin (FliC) epitopes flg22 and flgII-28 are microbe-associated molecular patterns (MAMPs). Although flg22 is recognized by many plant species via the pattern recognition receptor FLS2, neither the flgII-28 receptor nor the extent of flgII-28 recognition by different plant families is known. Here, we tested the significance of flgII-28 as a MAMP and the importance of allelic diversity in flg22 and flgII-28 in plant-pathogen interactions using purified peptides and a Pseudomonas syringae ∆fliC mutant complemented with different fliC alleles. The plant genotype and allelic diversity in flg22 and flgII-28 were found to significantly affect the plant immune response, but not bacterial motility. The recognition of flgII-28 is restricted to a number of solanaceous species. Although the flgII-28 peptide does not trigger any immune response in Arabidopsis, mutations in both flg22 and flgII-28 have FLS2-dependent effects on virulence. However, the expression of a tomato allele of FLS2 does not confer to Nicotiana benthamiana the ability to detect flgII-28, and tomato plants silenced for FLS2 are not altered in flgII-28 recognition. Therefore, MAMP diversification is an effective pathogen virulence strategy, and flgII-28 appears to be perceived by an as yet unidentified receptor in the Solanaceae, although it has an FLS2-dependent virulence effect in Arabidopsis.

The grapevine LysM receptor-like kinase VvLYK5-1 recognizes chitin oligomers through its association with VvLYK1-1.

The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomer-triggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity.

Two divergent immune receptors of the allopolyploid Nicotiana benthamiana reinforce the recognition of a fungal microbe-associated molecular pattern VdEIX3.

The allotetraploid Solanaceae plant Nicotiana benthamiana contains two closely related receptor-like proteins (RLPs), NbEIX2 and NbRXEG1, which regulate the recognition of VdEIX3 and PsXEG1, respectively. VdEIX3, PsXEG1, and their homologs represent two types of microbe-associated molecular patterns (MAMPs) that are widespread in diverse pathogens. Here, we report that NbRXEG1 also participates in VdEIX3 recognition. Both eix2 and rxeg1 single mutants exhibited significantly impaired but not abolished ability to mediate VdEIX3-triggered immune responses, which are nearly abolished in eix2 rxeg1 double mutants. Moreover, a dominant negative mutant of eix2 that contains a 60 bp deletion failed to respond to VdEIX3 and could suppress VdEIX3-induced cell death in the wild-type N. benthamiana. Further phylogenetic analyses showed that NbEIX2 and NbRXEG1 are obtained from different diploid ancestors by hybridization. These results demonstrate that the allotetraploid N. benthamiana recognizes two types of MAMPs by two homologous but diverged RLPs, which provides a model in which an allopolyploid plant probably exhibits defense hybrid vigor by acquiring divergent immune receptors from different ancestors.

Underwhelming or Misunderstood? Genetic Variability of Pattern Recognition Receptors in Immune Responses and Resistance to Mycobacterium tuberculosis.

Human genetic control is thought to affect a considerable part of the outcome of infection with Mycobacterium tuberculosis (Mtb). Most of us deal with the pathogen by containment (associated with clinical "latency") or sterilization, but tragically millions each year do not. After decades of studies on host genetic susceptibility to Mtb infection, genetic variation has been discovered to play a role in tuberculous immunoreactivity and tuberculosis (TB) disease. Genes encoding pattern recognition receptors (PRRs) enable a consistent, molecularly direct interaction between humans and Mtb which suggests the potential for co-evolution. In this review, we explore the roles ascribed to PRRs during Mtb infection and ask whether such a longstanding and intimate interface between our immune system and this pathogen plays a critical role in determining the outcome of Mtb infection. The scientific evidence to date suggests that PRR variation is clearly implicated in altered immunity to Mtb but has a more subtle role in limiting the pathogen and pathogenesis. In contrast to 'effectors' like IFN-γ, IL-12, Nitric Oxide and TNF that are critical for Mtb control, 'sensors' like PRRs are less critical for the outcome of Mtb infection. This is potentially due to redundancy of the numerous PRRs in the innate arsenal, such that Mtb rarely goes unnoticed. Genetic association studies investigating PRRs during Mtb infection should therefore be designed to investigate endophenotypes of infection - such as immunological or clinical variation - rather than just TB disease, if we hope to understand the molecular interface between innate immunity and Mtb.

Abundance of Plant-Associated Gammaproteobacteria Correlates with Immunostimulatory Activity of Angelica sinensis.

Background: Angelica sinensis is a medicinal plant known for a variety of biological effects, including its ability to stimulate innate immune cells in humans. Recent studies indicate that the immunostimulatory activity of A. sinensis arises from microbe-associated molecular patterns (MAMPs) of plant-associated bacteria. However, it is unknown which bacterial taxa in A. sinensis are responsible for the production of immunostimulatory MAMPs. Methods: Samples of A. sinensis were subjected to a cell-based assay to detect monocyte-stimulation and 16S ribosomal RNA amplicon sequencing, which revealed their immunostimulatory activity and microbial communities. The resulting data were analyzed by Linear discriminant analysis effect size (LEfSe), an online biostatistical tool for metagenomic biomarker discovery, to identify the bacterial taxonomical features correlated with the immunostimulatory activity. Results: A series of bacterial taxa under Gammaproteobacteria correlated positively with the immunostimulatory activity, whereas several Gram-positive taxa and Betaproteobacteria correlated negatively with the activity. Conclusions: The identified bacterial taxa set a new stage to characterize immunostimulatory MAMPs in plants.

Recognition of Elicitors in Grapevine: From MAMP and DAMP Perception to Induced Resistance.

In a context of a sustainable viticulture, the implementation of innovative eco-friendly strategies, such as elicitor-triggered immunity, requires a deep knowledge of the molecular mechanisms underlying grapevine defense activation, from pathogen perception to resistance induction. During plant-pathogen interaction, the first step of plant defense activation is ensured by the recognition of microbe-associated molecular patterns, which are elicitors directly derived from pathogenic or beneficial microbes. Vitis vinifera, like other plants, can perceive elicitors of different nature, including proteins, amphiphilic glycolipid, and lipopeptide molecules as well as polysaccharides, thanks to their cognate pattern recognition receptors, the discovery of which recently began in this plant species. Furthermore, damage-associated molecular patterns are another class of elicitors perceived by V. vinifera as an invader's hallmark. They are mainly polysaccharides derived from the plant cell wall and are generally released through the activity of cell wall-degrading enzymes secreted by microbes. Elicitor perception and subsequent activation of grapevine immunity end in some cases in efficient grapevine resistance against pathogens. Using complementary approaches, several molecular markers have been identified as hallmarks of this induced resistance stage. This review thus focuses on the recognition of elicitors by Vitis vinifera describing the molecular mechanisms triggered from the elicitor perception to the activation of immune responses. Finally, we discuss the fact that the link between elicitation and induced resistance is not so obvious and that the formulation of resistance inducers remains a key step before their application in vineyards.

Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis.

Production of reactive oxygen species (ROS) is a conserved immune response primarily mediated by NADPH oxidases (NOXs), also known in plants as respiratory burst oxidase homologs (RBOHs). Most microbe-associated molecular patterns (MAMPs) trigger a very fast and transient ROS burst in plants. However, recently, we found that lipopolysaccharides (LPS), a typical bacterial MAMP, triggered a biphasic ROS burst. In this study, we isolated mutants defective in LPS-triggered biphasic ROS burst (delt) in Arabidopsis, and cloned the DELT1 gene that was shown to encode RBOHD. In the delt1-2 allele, the antepenultimate residue, glutamic acid (E919), at the C-terminus of RBOHD was mutated to lysine (K). E919 is a highly conserved residue in NADPH oxidases, and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease. Consistently, we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure. It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein's stability and complex assembly. However, we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association, suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs. Taken together, our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.