Pathogen Perception Research Articles

Proteomics of Rice-Magnaporthe oryzae Interaction: What Have We Learned So Far?

Rice blast disease, caused by Magnaporthe oryzae, is one of the major constraints to rice production, which feeds half of the world’s population. Proteomic technologies have been used as effective tools in plant−pathogen interactions to study the biological pathways involved in pathogen infection, plant response, and disease progression. Advancements in mass spectrometry (MS) and apoplastic and plasma membrane protein isolation methods facilitated the identification and quantification of subcellular proteomes during plant-pathogen interaction. Proteomic studies conducted during rice−M. oryzae interaction have led to the identification of several proteins eminently involved in pathogen perception, signal transduction, and the adjustment of metabolism to prevent plant disease. Some of these proteins include receptor-like kinases (RLKs), mitogen-activated protein kinases (MAPKs), and proteins related to reactive oxygen species (ROS) signaling and scavenging, hormone signaling, photosynthesis, secondary metabolism, protein degradation, and other defense responses. Moreover, post−translational modifications (PTMs), such as phosphoproteomics and ubiquitin proteomics, during rice−M. oryzae interaction are also summarized in this review. In essence, proteomic studies carried out to date delineated the molecular mechanisms underlying rice-M. oryzae interactions and provided candidate proteins for the breeding of rice blast resistant cultivars.

Phosphoregulation of Ca2+ Influx in Plant Immunity.

A rapid influx of calcium into the cytosol represents a hallmark of plant immune responses. Recent studies in arabidopsis (Tian etal.) and rice (Wang etal.) reveal that pathogen-responsive receptor-like cytoplasmic kinases phosphorylate and activate calcium-permeable cyclic nucleotide-gated channels to trigger calcium influx, filling a missing link between pathogen perception and calcium signaling.

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.

Time to Fight: Molecular Mechanisms of Age-Related Resistance.

Plant age is a crucial factor in determining the outcome of a host-pathogen interaction. In successive developmental stages throughout their life cycles, plants face dynamic changes in biotic and abiotic conditions that create distinct ecological niches for host-pathogen interactions. As an adaptive strategy, plants have evolved intrinsic regulatory networks that integrate developmental signals with those from pathogen perception and defense activation. As a result, amplitude and timing of defense responses are optimized, so as to balance the cost and benefit of immunity activation. A general term "age-related resistance" refers to a gain of disease resistance against a certain pathogen when plants reach a relatively mature stage. Age-related resistance is a common observation on fruits, vegetables, and row crops for their resistance against viruses, bacteria, fungi, oomycetes pathogens, and insects. This review focuses on the recent advances in understanding the molecular mechanisms of how plants coordinate developmental timing and immune response.

Perspectives on intracellular perception of plant viruses.

SummaryThe intracellular nucleotide‐binding domain leucine‐rich repeat (NLR) class of immune receptors plays an important role in plant viral defence. Plant NLRs recognize viruses through direct or indirect association of viral proteins, triggering a downstream defence response to prevent viral proliferation and movement within the plant. This review focuses on current knowledge of intracellular perception of viral pathogens, activation of NLRs and the downstream signalling components involved in plant viral defence.

Chloroplast clustering around the nucleus is a general response to pathogen perception in Nicotiana benthamiana.

SummaryIt is increasingly clear that chloroplasts play a central role in plant stress responses. Upon activation of immune responses, chloroplasts are the source of multiple defensive signals, including reactive oxygen species (ROS). Intriguingly, it has been described that chloroplasts establish physical contact with the nucleus, through clustering around it and extending stromules, following activation of effector‐triggered immunity (ETI). However, how prevalent this phenomenon is in plant–pathogen interactions, how its induction occurs, and what the underlying biological significance is are important questions that remain unanswered. Here, we describe that the chloroplast perinuclear clustering seems to be a general plant response upon perception of an invasion threat. Indeed, activation of pattern‐triggered immunity, ETI, transient expression of the Rep protein from geminiviruses, or infection with viruses or bacteria all are capable of triggering this response in Nicotiana benthamiana. Interestingly, this response seems non‐cell‐autonomous, and exogenous treatment with H2O2 is sufficient to elicit this relocalization of chloroplasts, which appears to require accumulation of ROS. Taken together, our results indicate that chloroplasts cluster around the nucleus during plant–pathogen interactions, suggesting a fundamental role of this positioning in plant defence, and identify ROS as sufficient and possibly required for the onset of this response.

The cell wall regulates dynamics and size of plasma-membrane nanodomains in Arabidopsis

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

A resistosome-activated 'death switch'.

Pathogen perception triggers a monomeric nucleotide-binding leucine-rich plant immune receptor to form a pentameric wheel-like complex termed a resistosome, with the N-terminal α helices forming a funnel-shaped structure that may perturb plasma membrane integrity to cause hypersensitive cell death.

RRM Transcription Factors Interact with NLRs and Regulate Broad-Spectrum Blast Resistance in Rice.

Nucleotide-binding site leucine-rich repeat (NLR) receptors perceive pathogen effectors and trigger plant immunity. However, the mechanisms underlying NLR-triggered defense responses remain obscure. The recently discovered Pigm locus in rice encodes a cluster of NLRs, including PigmR, which confers broad-spectrum resistance to blast fungus. Here, we identify PIBP1 (PigmR-INTERACTING and BLAST RESISTANCE PROTEIN 1), an RRM (RNA-recognition motif) protein that specifically interacts with PigmR and other similar NLRs to trigger blast resistance. PigmR-promoted nuclear accumulation of PIBP1 ensures full blast resistance. We find that PIBP1 and a homolog, Os06 g02240, bind DNA and function as unconventional transcription factors at the promoters of the defense genes OsWAK14 and OsPAL1, activating their expression. Knockout of PIBP1 and Os06 g02240 greatly attenuated blast resistance. Collectively, our study discovers previously unappreciated RRM transcription factors that directly interact with NLRs to activate plant defense, establishing a direct link between transcriptional activation of immune responses with NLR-mediated pathogen perception.

Differential gene regulatory pathways and co-expression networks associated with fire blight infection in apple (Malus \xd7 domestica)

Apple cultivars with durable resistance are needed for sustainable management of fire blight, the most destructive bacterial disease of apples. Although studies have identified genetic resistance to fire blight in both wild species and cultivated apples, more research is needed to understand the molecular mechanisms underlying host–pathogen interaction and differential genotypic responses to fire blight infection. We have analyzed phenotypic and transcriptional responses of ‘Empire’ and ‘Gala’ apple cultivars to fire blight by infecting them with a highly aggressive E. amylovora strain. Disease progress, based on the percentage of visual shoot necrosis, started showing significant (p < 0.001) differences between ‘Empire’ and ‘Gala’ 4 days after infection (dai). ‘Empire’ seems to slow down bacterial progress more rapidly after this point. We further compared transcriptome profiles of ‘Empire’ and ‘Gala’ at three different time points after fire blight infection. More genes showed differential expression in ‘Gala’ at earlier stages, but the number of differentially expressed genes increased in ‘Empire’ at 3 dai. Functional classes related to defense, cell cycle, response to stress, and biotic stress were identified and a few co-expression gene networks showed particular enrichment for plant defense and abiotic stress response genes. Several of these genes also co-localized in previously identified quantitative trait locus regions for fire blight resistance on linkage groups 7 and 12, and can serve as functional candidates for future research. These results highlight different molecular mechanisms for pathogen perception and control in two apple cultivars and will contribute toward better understanding of E. amylovora-Malus pathosystem.

Genetic buffering of cyclic AMP in Arabidopsis thaliana compromises the plant immune response triggered by an avirulent strain of Pseudomonas syringae pv. tomato.

Cyclic AMP plays important roles in different physiological processes, including plant defence responses. However, as little information is known on plant enzymes responsible for cAMP production/degradation, studies of cAMP functions have relied, to date, on non-specific pharmacological approaches. We therefore developed a more reliable approach, producing transgenic Arabidopsis thaliana lines overexpressing the 'cAMP-sponge' (cAS), a genetic tool that specifically buffers cAMP levels. In response to an avirulent strain of Pseudomonas syringae pv. tomato (PstAvrB), cAS plants showed a higher bacterial growth and a reduced hypersensitive cell death in comparison with wild-type (WT) plants. The low cAMP availability after pathogen infection delayed cytosolic calcium elevation, as well as hydrogen peroxide increase and induction of redox systems. The proteomic analysis, performed 24h post-infection, indicated that a core of 49 proteins was modulated in both genotypes, while 16 and 42 proteins were uniquely modulated in WT and cAS lines, respectively. The involvement of these proteins in the impairment of defence response in cAS plants is discussed in this paper. Moreover, in silico analysis revealed that the promoter regions of the genes coding for proteins uniquely accumulating in WT plants shared the CGCG motif, a target of the calcium-calmodulin-binding transcription factor AtSR1 (Arabidopsis thaliana signal responsive1). Therefore, following pathogen perception, the low free cAMP content, altering timing and levels of defence signals, and likely acting in part through the mis-regulation of AtSR1 activity, affected the speed and strength of the immune response.

Pattern recognition receptors and their interactions with bacterial type III effectors in plants.

Innate immune signaling of plants is initiated by pattern recognition receptors (PRRs) at the plasma membrane. Upon pathogen attack, PRRs recognize pathogen-associated molecular patterns (PAMPs) via ectodomain and lead to signaling cascade via cytoplasmic kinase domain. PAMP-triggered immunity (PTI) activates basal defense responses sufficient to confer broad-spectrum disease resistance by inhibiting pathogen entry and growth. On the other hand, one of the major virulence factors in plant-pathogenic bacteria is type III secretion system, which can deliver effector proteins into the host cell and modulate host cellular processes. Most type III effectors are implicated in PTI suppression, and PRRs have been identified as targets of multiple type III effectors. Mutants defective in T3SS lack pathogenicity in many bacterial species, revealing that T3SS-mediated PTI suppression is critical for host colonization and subsequent disease development. This review summarizes molecular basis of bacterial pathogen perception by plant PRRs and also interaction between PRRs and type III effectors during early stages of plant-pathogen interaction.

Genome-wide functional analyses of plant coiled-coil NLR-type pathogen receptors reveal essential roles of their N-terminal domain in oligomerization, networking, and immunity.

The ability to induce a defense response after pathogen attack is a critical feature of the immune system of any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. However, the mechanism by which defense signaling is triggered by these NLRs in plants is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, the complexes are highly heterometric. Also, in contrast to metazoan NLRs, the N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions in plants is proposed to provide resilience to perturbation by pathogens.

Lipo-chitooligosaccharide signalling blocks a rapid pathogen-induced ROS burst without impeding immunity.

Molecular signals released by microbes at the surface of plant roots and leaves largely determine host responses, notably by triggering either immunity or symbiosis. How these signalling pathways cross-talk upon coincident perception of pathogens and symbionts is poorly described in plants forming symbiosis. Nitrogen fixing symbiotic Rhizobia spp. and arbuscular mycorrhizal fungi produce lipo-chitooligosaccharides (LCOs) to initiate host symbiotic programmes. In Medicago truncatula roots, the perception of LCOs leads to reduced efflux of reactive oxygen species (ROS). By contrast, pathogen perception generally triggers a strong ROS burst and activates defence gene expression. Here we show that incubation of M.truncatula seedlings with culture filtrate (CF) of the legume pathogen Aphanomyces euteiches alone or simultaneously with Sinorhizobium meliloti LCOs, resulted in a strong ROS release. However, this response was completely inhibited if CF was added after pre-incubation of seedlings with LCOs. By contrast, expression of immunity-associated genes in response to CF and disease resistance to A.euteiches remained unaffected by LCO treatment of M.truncatula roots. Our findings suggest that symbiotic plants evolved ROS inhibition response to LCOs to facilitate early steps of symbiosis whilst maintaining a parallel defence mechanisms toward pathogens.

Genome-wide analysis of the soybean CRK-family and transcriptional regulation by biotic stress signals triggering plant immunity.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins that exhibit ectodomains containing the domain of unknown function 26 (DUF26). The CRKs form a large subfamily of receptor-like kinases in plants, and their possible functions remain to be elucidated. Several lines of evidence suggest that CRKs play important roles in plant defense responses to environmental stress, including plant immunity. We performed a genome-wide analysis of CRK encoding genes in soybean (Glycine max). We found 91 GmCRKs distributed in 16 chromosomes, and identified several tandem and segmental duplications, which influenced the expansion of this gene family. According to our phylogenetic analysis, GmCRKs are grouped in four clades. Furthermore, 12% of the members exhibited GmCRKs with a duplicated bi-modular organization of the ectodomains, containing four DUF26 domains. Expression analysis of GmCRKs was performed by exploring publicly available databases, and by RT-qPCR analysis of selected genes in soybean leaves responding to biotic stress signals. GmCRKs exhibited diverse expression patterns in leaves, stems, roots, and other tissues. Some of them were highly expressed in only one type of tissue, suggesting predominant roles in specific tissues. Furthermore, several GmCRKs were induced with PAMPs, DAMPs and the pathogens Phakopsora pachyrhizi and Phytophthora sojae. Expression profiles of several GmCRKs encoding highly similar proteins exhibited antagonist modes of regulation. The results suggest a fine-tuning control of GmCRKs transcriptional regulation in response to external stimuli, including PAMPs and DAMPs. This study offers a comprehensive view of the GmCRKs family in soybean, and provides a foundation for evolutionary and functional analysis of this family of plant proteins involved in the perception of pathogens and activation of plant immunity.

Lipid Profile of Xylella fastidiosa Subsp. pauca Associated With the Olive Quick Decline Syndrome.

Lipids, components of the plasma and intracellular membranes as well as of droplets, provide different biological functions related to energy, carbon storage, and stress responses. Bacterial species display diverse membrane composition that changes in response to the different environmental conditions. During plant–pathogen interactions, lipids might have roles in several aspects such as recognition, signal transduction, and downstream responses. Among lipid entities, free fatty acids (FFAs) and their oxidized form, the oxylipins, represent an important class of signaling molecules in host–pathogen perception, especially related to virulence and defense. In bacteria, FFAs (e.g., diffusible signaling factors) and oxylipins have a crucial role in modulating motility, biofilm formation, and virulence. In this study, we explore by LC-TOF and LC-MS/MS the lipid composition of Xylella fastidiosa subsp. pauca strain De Donno in pure culture; some specific lipids (e.g., ornithine lipids and the oxylipin 7,10-diHOME), characteristic of other pathogenic bacteria, were revealed. Nicotiana tabacum was used for testing the ability of this pathogen in producing such lipids in the host. Different lipid compounds present a clear distribution pattern within the infected plant tissues compared to the uninfected ones.

The calcium sensor TaCBL4 and its interacting protein TaCIPK5 are required for wheat resistance to stripe rust fungus.

Calcineurin B-like proteins (CBLs) act as Ca2+ sensors to activate specific protein kinases, namely CBL-interacting protein kinases (CIPKs). Recent research has demonstrated that the CBL-CIPK complex is not only required for abiotic stress signaling, but is also probably involved in biotic stress perception. However, the role of this complex in immune signaling, including pathogen perception, is unknown. In this study, we isolated one signaling component of the TaCBL-TaCIPK complex (TaCBL4-TaCIPK5) and characterized its role in the interaction between wheat (Triticum aestivum) and Puccinia striiformis f. sp. tritici (Pst, stripe rust fungus). Among all TaCBLs in wheat, TaCBL4 mRNA accumulation markedly increased after infection by Pst. Silencing of TaCBL4 resulted in enhanced susceptibility to avirulent Pst infection. In addition, screening determined that TaCIPK5 physically interacted with TaCBL4 in planta and positively contributed to wheat resistance to Pst. Moreover, the disease resistance phenotype of TaCBL4 and TaCIPK5 co-silenced plants was consistent with that of single-knockdown plants. The accumulation of reactive oxygen species (ROS) was significantly altered in all silenced plants during Pst infection. Together these findings demonstrate that the TaCBL4-TaCIPK5 complex positively modulates wheat resistance in a ROS-dependent manner, and provide new insights into the roles of CBL-CIPK in wheat.

Integrative network analyses of wilt transcriptome in chickpea reveal genotype dependent regulatory hubs in immunity and susceptibility

Host specific resistance and non-host resistance are two plant immune responses to counter pathogen invasion. Gene network organizing principles leading to quantitative differences in resistant and susceptible host during host specific resistance are poorly understood. Vascular wilt caused by root pathogen Fusarium species is complex and governed by host specific resistance in crop plants, including chickpea. Here, we temporally profiled two contrasting chickpea genotypes in disease and immune state to better understand gene expression switches in host specific resistance. Integrative gene-regulatory network elucidated tangible insight into interaction coordinators leading to pathway determination governing distinct (disease or immune) phenotypes. Global network analysis identified five major hubs with 389 co-regulated genes. Functional enrichment revealed immunome containing three subnetworks involving CTI, PTI and ETI and wilt diseasome encompassing four subnetworks highlighting pathogen perception, penetration, colonization and disease establishment. These subnetworks likely represent key components that coordinate various biological processes favouring defence or disease. Furthermore, we identified core 76 disease/immunity related genes through subcellular analysis. Our regularized network with robust statistical assessment captured known and unexpected gene interaction, candidate novel regulators as future biomarkers and first time showed system-wide quantitative architecture corresponding to genotypic characteristics in wilt landscape.

Transfer of tomato immune receptor Ve1 confers Ave1-dependent Verticillium resistance in tobacco and cotton.

SummaryVerticillium wilts caused by soilborne fungal species of the Verticillium genus are economically important plant diseases that affect a wide range of host plants and are notoriously difficult to combat. Perception of pathogen(‐induced) ligands by plant immune receptors is a key component of plant innate immunity. In tomato, race‐specific resistance to Verticillium wilt is governed by the cell surface‐localized immune receptor Ve1 through recognition of the effector protein Ave1 that is secreted by race 1 strains of Verticillium spp. It was previously demonstrated that transgenic expression of tomato Ve1 in the model plant Arabidopsis thaliana leads to Verticillium wilt resistance. Here, we investigated whether tomato Ve1 can confer Verticillium resistance when expressed in the crop species tobacco (Nicotiana tabcum) and cotton (Gossypium hirsutum). We show that transgenic tobacco and cotton plants constitutively expressing tomato Ve1 exhibit enhanced resistance against Verticillium wilt in an Ave1‐dependent manner. Thus, we demonstrate that the functionality of tomato Ve1 in Verticillium wilt resistance through recognition of the Verticillium effector Ave1 is retained after transfer to tobacco and cotton, implying that the Ve1‐mediated immune signalling pathway is evolutionary conserved across these plant species. Moreover, our results suggest that transfer of tomato Ve1 across sexually incompatible plant species can be exploited in breeding programmes to engineer Verticillium wilt resistance.

Multiple strategies for pathogen perception by plant immune receptors.

Contents Summary 17 I. Introduction 17 II. Pathogen perception by NLRs: from direct recognition to integrated decoys 18 III. Multiple activation and signaling pathways for NLRs 18 IV. How to engineer NLR-mediated disease resistance? 21 V. Conclusion 23 Acknowledgements 23 References 23 SUMMARY: Plants have evolved a complex immune system to protect themselves against phytopathogens. A major class of plant immune receptors called nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) is ubiquitous in plants and is widely used for crop disease protection, making these proteins critical contributors to global food security. Until recently, NLRs were thought to be conserved in their modular architecture and functional features. Investigation of their biochemical, functional and structural properties has revealed fascinating mechanisms that enable these proteins to perceive a wide range of pathogens. Here, I review recent insights demonstrating that NLRs are more mechanistically and structurally diverse than previously thought. I also discuss how these findings provide exciting future prospects to improve plant disease resistance.