A viral effector blocks the turnover of a plant NLR receptor to trigger a robust immune response.

Plant intracellular nucleotide-binding and leucine-rich repeat immune receptors (NLRs) play a key role in activating a strong pathogen defense response. Plant NLR proteins are tightly regulated and accumulate at very low levels in the absence of pathogen effectors. However, little is known about how this low level of NLR proteins is able to induce robust immune responses upon recognition of pathogen effectors. Here, we report that, in the absence of effector, the inactive form of the tomato NLR Sw-5b is targeted for ubiquitination by the E3 ligase SBP1. Interaction of SBP1 with Sw-5b via only its N-terminal domain leads to slow turnover. In contrast, in its auto-active state, Sw-5b is rapidly turned over as SBP1 is upregulated and interacts with both its N-terminal and NB-LRR domains. During infection with the tomato spotted wilt virus, the viral effector NSm interacts with Sw-5b and disrupts the interaction of Sw-5b with SBP1, thereby stabilizing the active Sw-5b and allowing it to induce a robust immune response.

Comparative analysis on natural variants of fire blight resistance protein FB_MR5 indicates distinct effector recognition mechanisms.

FB_MR5 is a nucleotide-binding domain and leucine-rich repeat protein identified from wild apple species Malus × robusta 5 conferring disease resistance to bacterial fire blight. FB_MR5 (hereafter MrMR5) recognizes the cysteine protease effector EaAvrRpt2 secreted from the causal agent of bacterial fire blight, Erwinia amylovora. We previously reported that MrMR5 is activated by the C-terminal cleavage product (ACP3) of Malus domestica RIN4 (MdRIN4) produced by EaAvrRpt2-directed proteolysis. We show that MbMR5 from a wild apple species Malus baccata shares 99.4% amino acid sequence identity with MrMR5. Surprisingly, transient expression of MbMR5 in Nicotiana benthamiana showed autoactivity in contrast to MrMR5. Domain swap and mutational analyses revealed that 1 amino acid polymorphism in the MbMR5 CC domain is critical in enhancing autoactivity. We further demonstrated that MrMR5 carrying 7 amino acid polymorphisms present in MbMR5 is not activated by MdRIN4 ACP3 but recognizes AvrRpt2 without MdRIN4 in N. benthamiana. Our findings indicate that naturally occurring polymorphisms of MR5 natural variants can confer its cell death-inducing activity and the effector recognition mechanism likely due to altered compatibility with RIN4.

Structural diversification of fungal cell wall in response to the stress signaling and remodeling during fungal pathogenesis.

Fungi are one of the most diverse organisms found in our surroundings. The heterotrophic lifestyle of fungi and the ever-changing external environmental factors pose numerous challenges for their survival. Despite all adversities, fungi continuously develop new survival strategies to secure nutrition and space from their host. During host-pathogen interaction, filamentous phytopathogens in particular, effectively infect their hosts by maintaining polarised growth at the tips of hyphae. The fungal cell wall, being the prime component of host contact, plays a crucial role in fortifying the intracellular environment against the harsh external environment. Structurally, the fungal cell wall is a highly dynamic yet rigid component, responsible for maintaining cellular morphology. Filamentous pathogens actively maintain their dynamic cell wall to compensate rapid growth on the host. Additionally, they secrete effectors to dampen the sophisticated mechanisms of plant defense and initiate various downstream signaling cascades to repair the damage inflicted by the host. Thus, the fungal cell wall serves as a key modulator of fungal pathogenicity. The fungal cell wall with their associated signaling mechanisms emerge as intriguing targets for host immunity. This review comprehensively examines and summarizes the multifaceted findings of various research groups regarding the dynamics of the cell wall in filamentous fungal pathogens during host invasion.

Recent advances in the molecular understanding of immunoglobulin A.

Immunoglobulin A (IgA) plays a crucial role in the human immune system, particularly in mucosal immunity. IgA antibodies that target the mucosal surface are made up of two to five IgA monomers linked together by the joining chain, forming polymeric molecules. These IgA polymers are transported across mucosal epithelial cells by the polymeric immunoglobulin receptor pIgR, resulting in the formation of secretory IgA (SIgA). This review aims to explore recent advancements in our molecular understanding of IgA, with a specific focus on SIgA, and the interaction between IgA and pathogen molecules.

Genetic breakthroughs in the Brassica napus-Sclerotinia sclerotiorum interactions.

Sclerotinia sclerotiorum (Lib.) de Bary is a highly destructive fungal pathogen that seriously damages the yield and quality of Brassica napus worldwide. The complex interaction between the B. napus and S. sclerotiorum system has presented significant challenges in researching rapeseed defense strategies. Here, we focus on the infection process of S. sclerotiorum, the defense mechanisms of rapeseed, and recent research progress in this system. The response of rapeseed to S. sclerotiorum is multifaceted; this review aims to provide a theoretical basis for rapeseed defense strategies.

Plant NLRs: Evolving with pathogen effectors and engineerable to improve resistance.

Pathogens are important threats to many plants throughout their lifetimes. Plants have developed different strategies to overcome them. In the plant immunity system, nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs) are the most common components. And recent studies have greatly expanded our understanding of how NLRs function in plants. In this review, we summarize the studies on the mechanism of NLRs in the processes of effector recognition, resistosome formation, and defense activation. Typical NLRs are divided into three groups according to the different domains at their N termini and function in interrelated ways in immunity. Atypical NLRs contain additional integrated domains (IDs), some of which directly interact with pathogen effectors. Plant NLRs evolve with pathogen effectors and exhibit specific recognition. Meanwhile, some NLRs have been successfully engineered to confer resistance to new pathogens based on accumulated studies. In summary, some pioneering processes have been obtained in NLR researches, though more questions arise as a result of the huge number of NLRs. However, with a broadened understanding of the mechanism, NLRs will be important components for engineering in plant resistance improvement.

Activation and Regulation of NLR Immune Receptor Networks.

Plants have many types of immune receptors that recognize diverse pathogen molecules and activate the innate immune system. The intracellular immune receptor family of nucleotide-binding domain leucine-rich repeat-containing proteins (NLRs) perceives translocated pathogen effector proteins and executes a robust immune response, including programmed cell death. Many plant NLRs have functionally specialized to sense pathogen effectors (sensor NLRs) or to execute immune signaling (helper NLRs). Sub-functionalized NLRs form a network-type receptor system known as the NLR network. In this review, we highlight the concept of NLR networks, discussing how they are formed, activated and regulated. Two main types of NLR networks have been described in plants: the ACTIVATED DISEASE RESISTANCE 1/N REQUIREMENT GENE 1 network and the NLR-REQUIRED FOR CELL DEATH network. In both networks, multiple helper NLRs function as signaling hubs for sensor NLRs and cell-surface-localized immune receptors. Additionally, the networks are regulated at the transcriptional and posttranscriptional levels, and are also modulated by other host proteins to ensure proper network activation and prevent autoimmunity. Plant pathogens in turn have converged on suppressing NLR networks, thereby facilitating infection and disease. Understanding the NLR immune system at the network level could inform future breeding programs by highlighting the appropriate genetic combinations of immunoreceptors to use while avoiding deleterious autoimmunity and suppression by pathogens.

Optimization of immune receptor-related hypersensitive cell death response assay using agrobacterium-mediated transient expression in tobacco plants.

BACKGROUND: The study of the regulatory mechanisms of evolutionarily conserved Nucleotide-binding leucine-rich repeat (NLR) resistance (R) proteins in animals and plants is of increasing importance due to understanding basic immunity and the value of various crop engineering applications of NLR immune receptors. The importance of temperature is also emerging when applying NLR to crops responding to global climate change. In particular, studies of pathogen effector recognition and autoimmune activity of NLRs in plants can quickly and easily determine their function in tobacco using agro-mediated transient assay. However, there are conditions that should not be overlooked in these cell death-related assays in tobacco. RESULTS: Environmental conditions play an important role in the immune response of plants. The system used in this study was to establish conditions for optimal hypertensive response (HR) cell death analysis by using the paired NLR RPS4/RRS1 autoimmune and AvrRps4 effector recognition system. The most suitable greenhouse temperature for growing plants was fixed at 22 °C. In this study, RPS4/RRS1-mediated autoimmune activity, RPS4 TIR domain-dependent cell death, and RPS4/RRS1-mediated HR cell death upon AvrRps4 perception significantly inhibited under conditions of 65% humidity. The HR is strongly activated when the humidity is below 10%. Besides, the leaf position of tobacco is important for HR cell death. Position #4 of the leaf from the top in 4-5 weeks old tobacco plants showed the most effective HR cell death. CONCLUSIONS: As whole genome sequencing (WGS) or resistance gene enrichment sequencing (RenSeq) of various crops continues, different types of NLRs and their functions will be studied. At this time, if we optimize the conditions for evaluating NLR-mediated HR cell death, it will help to more accurately identify the function of NLRs. In addition, it will be possible to contribute to crop development in response to global climate change through NLR engineering.

NLR receptor networks in plants.

To fight off diverse pathogens and pests, the plant immune system must recognize these invaders; however, as plant immune receptors evolve to recognize a pathogen, the pathogen often evolves to escape this recognition. Plant-pathogen co-evolution has led to the vast expansion of a family of intracellular immune receptors-nucleotide-binding domain and leucine-rich repeat proteins (NLRs). When an NLR receptor recognizes a pathogen ligand, it activates immune signaling and thus initiates defense responses. However, in contrast with the model of NLRs acting individually to activate resistance, an emerging paradigm holds that plants have complex receptor networks where the large repertoire of functionally specialized NLRs function together to act against the large repertoire of rapidly evolving pathogen effectors. In this article, we highlight key aspects of immune receptor networks in plant NLR biology and discuss NLR network architecture, the advantages of this receptor network system, and the evolution of the NLR network in asterid plants.

Host-specific activation of a pathogen effector Aave_4606 from Acidovorax citrulli, the causal agent for bacterial fruit blotch.

RipAY, an effector protein from the plant bacterial pathogen Ralstonia solanacearum, exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the host cellular glutathione (GSH) when stimulated by host eukaryotic-type thioredoxins (Trxs). Aave_4606 from Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbit plants, shows significant homology to RipAY. Based on its homology, it was predicted that the GGCT activity of Aave_4606 is also stimulated by host Trxs. The GGCT activity of a recombinant Aave_4606 protein was investigated in the presence of various Trxs, such as yeast (ScTrx1), Arabidopsis thaliana (AtTrx-h1, AtTrx-h2, AtTrx-h3, and AtTrx-h5), or watermelon (Cla022460/ClTrx). Unlike RipAY, the GGCT activity of Aave_4606 is stimulated only by AtTrx-h1, AtTrx-h3, AtTrx-h5 and ClTrx from a watermelon, the primary host of A. citrulli, but not by ScTrx1, AtTrx-h2. Interestingly, GGCT activity of Aave_4606 is more efficiently stimulated by AtTrx-h1 and ClTrx than AtTrx-h5. These results suggested that Aave_4606 recognizes host-specific Trxs, which specifically activates the GGCT activity of Aave_4606 to decrease the host cellular GSH. These findings provide new insights into that effector is one of the host-range determinants for pathogenic bacteria via its host-dependent activation.

Molecular insights into the biochemical functions and signalling mechanisms of plant NLRs.

Plant intracellular immune receptors known as NLR (nucleotide-binding leucine-rich repeat) proteins confer immunity and cause cell death. Plant NLR proteins that directly or indirectly recognize pathogen effector proteins to initiate immune signalling are regarded as sensor NLRs. Some NLR protein families function downstream of sensor NLRs to transduce immune signalling and are known as helper NLRs. Recent breakthrough studies on plant NLR protein structures and biochemical functions greatly advanced our understanding of NLR biology. Comprehensive and detailed knowledge on NLR biology requires future efforts to solve more NLR protein structures and investigate the signalling events between sensor and helper NLRs, and downstream of helper NLRs.

Transcriptional modification of host cells harboring Toxoplasma gondii bradyzoites prevents IFN gamma-mediated cell death.

Toxoplasma gondii develops a latent infection in the muscle and central nervous system that acts as a reservoir for acute-stage reactivation in vulnerable patients. Little is understood about how parasites manipulate host cells during latent infection and the impact this has on survival. We show that bradyzoites impart a unique transcriptional signature on infected host cells. Many of these transcriptional changes rely on protein export and result in the suppression of type I interferon (IFN) and IFNγ signaling more so than in acute stages. Loss of the protein export component, MYR1, abrogates transcriptional remodeling and prevents suppression of IFN signaling. Among the exported proteins, the inhibitor of STAT1 transcription (IST) plays a key role in limiting IFNγ signaling in bradyzoites. Furthermore, bradyzoite protein export protects host cells from IFNγ-mediated cell death, even when export is restricted to latent stages. These findings highlight the functional importance of host manipulation in Toxoplasma's bradyzoite stages.

Novel Effector RHIFs Identified From Acidovorax avenae Strains N1141 and K1 Play Different Roles in Host and Non-host Plants.

Plant pathogenic bacteria inject effectors into plant cells using type III secretion systems (T3SS) to evade plant immune systems and facilitate infection. In contrast, plants have evolved defense systems called effector-triggered immunity (ETI) that can detect such effectors during co-evolution with pathogens. The rice-avirulent strain N1141 of the bacterial pathogen Acidovorax avenae causes rice ETI, including hypersensitive response (HR) cell death in a T3SS-dependent manner, suggesting that strain N1141 expresses an ETI-inducing effector. By screening 6,200 transposon-tagged N1141 mutants based on their ability to induce HR cell death, we identified 17 mutants lacking this ability. Sequence analysis and T3SS-mediated intracellular transport showed that a protein called rice HR cell death inducing factor (RHIF) is a candidate effector protein that causes HR cell death in rice. RHIF-disrupted N1141 lacks the ability to induce HR cell death, whereas RHIF expression in this mutant complemented this ability. In contrast, RHIF from rice-virulent strain K1 functions as an ETI inducer in the non-host plant finger millet. Furthermore, inoculation of rice and finger millet with either RHIF-deficient N1141 or K1 strains showed that a deficiency of RHIF genes in both strains results in decreased infectivity toward each the host plants. Collectively, novel effector RHIFs identified from A. avenae strains N1141 and K1 function in establishing infection in host plants and in ETI induction in non-host plants.

Bioinformatic tools support decision-making in plant disease management.

Food loss due to pathogens is a major concern in agriculture, requiring the need for advanced disease detection and prevention measures to minimize pathogen damage to plants. Novel bioinformatic tools have opened doors for the low-cost rapid identification of pathogens and prevention of disease. The number of these tools is growing fast and a comprehensive and comparative summary of these resources is currently lacking. Here, we review all current bioinformatic tools used to identify the mechanisms of pathogen pathogenicity, plant resistance protein identification, and the detection and treatment of plant disease. We compare functionality, data volume, data sources, performance, and applicability of all tools to provide a comprehensive toolbox for researchers in plant disease management.

Rpivnt1 provides resistance through the mediation of a light responsive guardee in potato.

The disease triangle describes the interrelationship among pathogen, host, and environment towards disease prevalence in the field. The mechanistic role of environment on NLR-mediated resistance was not known until now. Recently, a comprehensive work revealed that light controls late blight disease reaction in potato caused by the Irish famine pathogen. A specific R gene Rpivnt1 in potato showed dichotomous behavior in disease reaction due to the light-responsive alternate promoter selection of another host gene glycerate 3 kinase (GLYK) during its transcription. The full-length GLYK protein traps the pathogen effector AVRvnt1 into a recognition event which is later sensed by Rpivnt1 in the presence of light. In dark, the truncated GLYK protein devoid of its chloroplastic transit peptide could not able to recognize AVRvnt1 and thus resistance get compromised. A possible model for this event is proposed here for ease in understanding.

The Effect of Virulence and Resistance Mechanisms on the Interactions between Parasitic Plants and Their Hosts.

Parasitic plants have a unique heterotrophic lifestyle based on the extraction of water and nutrients from host plants. Some parasitic plant species, particularly those of the family Orobanchaceae, attack crops and cause substantial yield losses. The breeding of resistant crop varieties is an inexpensive way to control parasitic weeds, but often does not provide a long-lasting solution because the parasites rapidly evolve to overcome resistance. Understanding mechanisms underlying naturally occurring parasitic plant resistance is of great interest and could help to develop methods to control parasitic plants. In this review, we describe the virulence mechanisms of parasitic plants and resistance mechanisms in their hosts, focusing on obligate root parasites of the genera Orobanche and Striga. We noticed that the resistance (R) genes in the host genome often encode proteins with nucleotide-binding and leucine-rich repeat domains (NLR proteins), hence we proposed a mechanism by which host plants use NLR proteins to activate downstream resistance gene expression. We speculated how parasitic plants and their hosts co-evolved and discussed what drives the evolution of virulence effectors in parasitic plants by considering concepts from similar studies of plant-microbe interaction. Most previous studies have focused on the host rather than the parasite, so we also provided an updated summary of genomic resources for parasitic plants and parasitic genes for further research to test our hypotheses. Finally, we discussed new approaches such as CRISPR/Cas9-mediated genome editing and RNAi silencing that can provide deeper insight into the intriguing life cycle of parasitic plants and could potentially contribute to the development of novel strategies for controlling parasitic weeds, thereby enhancing crop productivity and food security globally.

Characterization of the mechanism of thioredoxin-dependent activation of γ-glutamylcyclotransferase, RipAY, from Ralstonia solanacearum.

A class II ChaC protein, RipAY, from phytopathogenic bacterium, Ralstonia solanacearum exhibits γ-glutamylcyclotransferase (GGCT) activity to degrade intracellular glutathione in host cells upon its interaction with host thioredoxins (Trxs). To understand the Trx-dependent activation of RipAY, we constructed various deletion mutants of RipAY and found the determinant region for GGCT activation in the N- and C-terminal sequences of RipAY by analyzing their yeast growth inhibition activity and the interaction with Trxs. Mutational analysis of the active site cysteine residues of Arabidopsis thaliana Trx-h5 (AtTrx-h5), one of the most efficiently stimulating Trxs, revealed that each active site cysteine residue of AtTrx-h5 contributes to efficient RipAY-binding and -activation activity. We also estimated that RipAY and AtTrx-h5 form a complex at a 1:2 M ratio. Furthermore, we found that the constitutive GGCT activity of Gcg1, a yeast class I ChaC protein, is also stimulated by yeast Trx1. These results indicate that class I ChaC proteins can sense the intracellular redox state and interact with Trxs to promote more efficient degradation of glutathione and regulate intracellular redox homeostasis. We hypothesize that RipAY acquired a more efficient and specific Trx-dependent activation mechanism to activate its GGCT activity only in the host eukaryotic cells during the evolution.

Sunflower resistance to multiple downy mildew pathotypes revealed by recognition of conserved effectors of the oomycete Plasmopara halstedii.

Over the last 40 years, new sunflower downy mildew isolates (Plasmopara halstedii) have overcome major gene resistances in sunflower, requiring the identification of additional and possibly more durable broad-spectrum resistances. Here, 354 RXLR effectors defined in silico from our new genomic data were classified in a network of 40 connected components sharing conserved protein domains. Among 205 RXLR effector genes encoding conserved proteins in 17 P. halstedii pathotypes of varying virulence, we selected 30 effectors that were expressed during plant infection as potentially essential genes to target broad-spectrum resistance in sunflower. The transient expression of the 30 core effectors in sunflower and in Nicotiana benthamiana leaves revealed a wide diversity of targeted subcellular compartments, including organelles not so far shown to be targeted by oomycete effectors such as chloroplasts and processing bodies. More than half of the 30 core effectors were able to suppress pattern-triggered immunity in N. benthamiana, and five of these induced hypersensitive responses (HR) in sunflower broad-spectrum resistant lines. HR triggered by PhRXLRC01 co-segregated with Pl22 resistance in F3 populations and both traits localized in 1.7 Mb on chromosome 13 of the sunflower genome. Pl22 resistance was physically mapped on the sunflower genome recently sequenced, unlike all the other downy mildew resistances published so far. PhRXLRC01 and Pl22 are proposed as an avirulence/resistance gene couple not previously described in sunflower. Core effector recognition is a successful strategy to accelerate broad-spectrum resistance gene identification in complex crop genomes such as sunflower.

Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL-INDUCED CELL DEATH1.

The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL-INDUCED CELL DEATH1 (RCD1). We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes. We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)-induced defense genes and alters plant growth responses to light. HaRxL106-mediated suppression of immunity is abolished in RCD1 loss-of-function mutants. We report that RCD1-type proteins are phosphorylated, and we identified Mut9-like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1-interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared with wild-type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling. Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants.

Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.