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Reactive oxygen species (ROS) are essential for life and are involved in the regulation of almost all biological processes. ROS production is critical for plant development, response to abiotic stresses and immune responses. Here, we focus on recent discoveries in ROS biology emphasizing abiotic and biotic stress responses. Recent advancements have resulted in the identification of one of the first sensors for extracellular ROS and highlighted waves of ROS production during stress signalling in Arabidopsis. Enzymes that produce ROS, including NADPH oxidases, exhibit precise regulation through diverse post-translational modifications. Discoveries highlight the importance of both amino- and carboxy-terminal regulation of NADPH oxidases through protein phosphorylation and cysteine oxidation. Here, we discuss advancements in ROS compartmentalization, systemic ROS waves, ROS sensing and post-translational modification of ROS-producing enzymes and identify areas where foundational gaps remain. This Review covers the recent advancements in our understanding of reactive oxygen species production, regulation and perception in plants. It is primarily focused on stress responses and the role of NADPH oxidases.

A critical component controlling bacterial virulence is the delivery of pathogen effectors into plant cells during infection. Effectors alter host metabolism and immunity for the benefit of pathogens. Multiple effectors are phosphorylated by host kinases, and this posttranslational modification is important for their activity. We sought to identify host kinases involved in effector phosphorylation. Multiple phosphorylated effector residues matched the proposed consensus motif for the plant calcium-dependent protein kinase (CDPK) and Snf1-related kinase (SnRK) superfamily. The conserved Pseudomonas effector AvrPtoB acts as an E3 ubiquitin ligase and promotes bacterial virulence. In this study, we identified a member of the Arabidopsis SnRK family, SnRK2.8, which interacts with AvrPtoB in yeast and in planta. We showed that SnRK2.8 was required for AvrPtoB virulence functions, including facilitating bacterial colonization, suppression of callose deposition, and targeting the plant defense regulator NPR1 and flagellin receptor FLS2. Mass spectrometry analyses revealed that AvrPtoB phosphorylation occurs at multiple serine residues in planta, with S258 phosphorylation significantly reduced in the snrk2.8 knockout. AvrPtoB phospho-null mutants exhibited compromised virulence functions and were unable to suppress NPR1 accumulation, FLS2 accumulation, or inhibit FLS2-BAK1 complex formation upon flagellin perception. Taken together, these data identify a conserved plant kinase utilized by a pathogen effector to promote disease.

After three decades of amazing progress made in molecular studies of plant-microbe interactions (MPMI), we start to ask ourselves “what are the major questions still remaining?” as if the puzzle has only a few pieces missing. Such an exercise ultimately leads to the realization that we still have many more questions than answers. Therefore, it would be an impossible task for us to project a coherent “big picture” of the MPMI field in a single review. Instead, we provide our collection of opinions on where we would like to go in our own research (Figure 1) as an invitation to the community to join us in this exploration of new frontiers of MPMI.

ER bodies are endoplasmic reticulum–derived organelles present in plants belonging to the Brassicales order. In Arabidopsis thaliana, ER bodies are ubiquitous in cotyledons and roots and are present only in certain cell types in rosette leaves. However, both wounding and jasmonic acid treatment induce the formation of ER bodies in leaves. Formation of this structure is dependent on the transcription factor NAI1. The main components of the ER bodies are β-glucosidases (BGLUs), enzymes that hydrolyze specialized compounds. In Arabidopsis, PYK10 (BGLU23) and BGLU18 are the most abundant ER body proteins. In this work, we found that ER bodies are downregulated as a consequence of the immune responses induced by bacterial flagellin perception. Arabidopsis mutants defective in ER body formation show enhanced responses upon flagellin perception and enhanced resistance to bacterial infections. Furthermore, the bacterial toxin coronatine induces the formation of de novo ER bodies in leaves and its virulence function is partially dependent on this structure. Finally, we show that performance of the polyphagous beet armyworm herbivore Spodoptera exigua increases in plants lacking ER bodies. Altogether, we provide new evidence for the role of the ER bodies in plant immune responses. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Plant–pathogen interactions result in disease development in a susceptible host. Plants actively resist pathogens via a complex immune system comprising both surface-localized receptors that sense the extracellular space as well as intracellular receptors recognizing pathogen effectors. To date, the majority of cloned resistance genes encode intracellular nucleotide-binding leucine-rich repeat receptor proteins. Recent discoveries have revealed tandem kinase proteins (TKPs) as another important family of intracellular proteins involved in plant immune responses. Five TKP genes—barley Rpg1 and wheat WTK1 (Yr15), WTK2 (Sr60), WTK3 (Pm24), and WTK4—protect against devastating fungal diseases. Moreover, a large diversity and numerous putative TKPs exist across the plant kingdom. This review explores our current knowledge of TKPs and serves as a basis for future studies that aim to develop and exploit a deeper understanding of innate plant immunity receptor proteins.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Innate immune perception is the first line of inducible defense against invading pathogens. Plants lack specialized circulating immune cells. Therefore, diverse cell types are able to recognize and respond to pathogens. Surface-localized and intracellular plant innate immune receptors are capable of recognizing diverse pathogen components. Intracellular nucleotide-binding leucine-rich repeat (NLR) receptors recognize pathogen effectors delivered inside host cells. Recent advances shed light onto NLR activation, phosphorylation of defense signaling nodes and overlap in transcriptional responses between pathogen perception and abiotic stress.

International audience; Vector-borne plant diseases have significant ecological and economic impacts, affecting farm profitability and forest composition throughout the world. Bacterial vector-borne pathogens have evolved sophisticated strategies to interact with their hemipteran insect vectors and plant hosts. These pathogens reside in plant vascular tissue, and their study represents an excellent opportunity to uncover novel biological mechanisms regulating intracellular pathogenesis and to contribute to the control of some of the world's most invasive emerging diseases. In this perspective, we highlight recent advances and major unanswered questions in the realm of bacterial vector-borne disease, focusing on liberibacters, phytoplasmas, spiroplasmas, and Xylella fastidiosa.

The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all CLas isolates, as a molecular probe to understand CLas virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants., Greening disease threatens the productivity of citrus crops worldwide yet the pathosystem is poorly understood. Here, Clark et al. show that an effector cloned from the associated bacteria can suppress host plant papain-like cysteine proteases' activity, suggesting its probable role in pathogenesis.

Microbial patterns are recognized by cell-surface receptors to initiate pattern-triggered immunity (PTI) in plants. Receptor-like cytoplasmic kinases (RLCKs), such as BIK1, and calcium-dependent protein kinases (CPKs) are engaged during PTI to activate the NADPH oxidase RBOHD for reactive oxygen species (ROS) production. It is unknown whether protein kinases besides CPKs and RLCKs participate in RBOHD regulation. We screened mutants in all ten Arabidopsis MAP4 kinases (MAP4Ks) and identified the conserved MAP4K SIK1 as a positive regulator of PTI. sik1 mutants were compromised in their ability to elicit the ROS burst in response to microbial features and exhibited compromised PTI to bacterial infection. SIK1 directly interacts with, phosphorylates, and stabilizes BIK1 in a kinase activity-dependent manner. Furthermore, SIK1 directly interacts with and phosphorylates RBOHD upon flagellin perception. Thus, SIK1 positively regulates immunity by stabilizing BIK1 and activating RBOHD to promote the extracellular ROS burst.

Genetic control of plant diseases has traditionally included the deployment of single immune receptors with nucleotide-binding leucine-rich repeat (NLR) domain architecture. These NLRs recognize corresponding pathogen effector proteins inside plant cells, resulting in effector-triggered immunity (ETI). Although ETI triggers robust resistance, deployment of single NLRs can be rapidly overcome by pathogen populations within a single or a few growing seasons. In order to generate more durable disease resistance against devastating plant pathogens, a multitiered strategy that incorporates stacked NLRs combined with other sources of disease resistance is necessary. New genetic and genomic technologies have enabled advancements in identifying conserved pathogen effectors, isolating NLR repertoires from diverse plants, and editing plant genomes to enhance resistance. Significant advancements have also been made in understanding plant immune perception at the receptor level, which has promise for engineering new sources of resistance. Here, we discuss how to utilize recent scientific advancements in a multilayered strategy for developing more durable disease resistance.

Plants possess large arsenals of immune receptors capable of recognizing all pathogen classes. To cause disease, pathogenic organisms must be able to overcome physical barriers, suppress or evade immune perception, and derive nutrients from host tissues. Consequently, to facilitate some of these processes, pathogens secrete effector proteins that promote colonization. This review covers recent advances in the field of effector biology, focusing on conserved cellular processes targeted by effectors from diverse pathogens. The ability of effectors to facilitate pathogen entry into the host interior, suppress plant immune perception, and alter host physiology for pathogen benefit is discussed. Pathogens also deploy effectors in a spatial and temporal manner, depending on infection stage. Recent advances have also enhanced our understanding of effectors acting in specific plant organs and tissues. Effectors are excellent cellular probes that facilitate insight into biological processes as well as key points of vulnerability in plant immune signaling networks.

Plant small RNAs play important roles in gene regulation during pathogen infection. Here we show that miR863-3p is induced by the bacterial pathogen Pseudomonas syringae carrying various effectors. Early during infection, miR863-3p silences two negative regulators of plant defence, atypical receptor-like pseudokinase1 (ARLPK1) and ARLPK2, both lacking extracellular domains and kinase activity, through mRNA degradation to promote immunity. ARLPK1 associates with, and may function through another negative immune regulator ARLPK1-interacting receptor-like kinase 1 (AKIK1), an active kinase with an extracellular domain. Later during infection, miR863-3p silences SERRATE, which is essential for miRNA accumulation and positively regulates defence, through translational inhibition. This results in decreased miR863-3p levels, thus forming a negative feedback loop to attenuate immune responses after successful defence. This is an example of a miRNA that sequentially targets both negative and positive regulators of immunity through two modes of action to fine-tune the timing and amplitude of defence responses., Small RNA plays an important role in regulating the plant defence against bacterial pathogens. Here the authors propose that miR863-3p acts to fine-tune the timing of defence responses by sequentially silencing negative and positive regulators of the plant immune response.

Plant disease resistance proteins commonly belong to the nucleotide binding-leucine rich repeat (NB-LRR) protein family. These specialized immune proteins mediate recognition of diverse pathogen-derived effector proteins and initiate potent defense responses. NB-LRRs exhibit a multidomain architecture and each domain appears to have discrete functions depending on the stage of NB-LRR signaling. Novel proteins that were found to interact with the core HSP90 chaperone complex regulate accumulation and activation of NB-LRR immune receptors. Recent studies have also advanced our understanding of how accessory proteins contribute to NB-LRR activation. The dynamic nature of NB-LRR localization to different subcellular compartments before and after activation suggests that NB-LRRs may activate immune responses in multiple parts of the cell. In this review we highlight recent advances in understanding NB-LRR function.

Summary Gram-negative bacteria inject type III secreted effectors (T3SEs) into host cells to manipulate the immune response. The YopJ family effector HopZ1a produced by the plant pathogen Pseudomonas syringae possesses acetyltransferase activity and acetylates plant proteins to facilitate infection. Using mass spectrometry, we identified a threonine residue, T346, as the main autoacetylation site of HopZ1a. Two neighboring serine residues, S349 and S351, are required for the acetyltransferase activity of HopZ1a in vitro and are indispensable for the virulence function of HopZ1a in Arabidopsis thaliana. Using proton nuclear magnetic resonance (NMR), we observed a conformational change of HopZ1a in the presence of inositol hexakisphosphate (IP6), which acts as a eukaryotic co-factor and significantly enhances the acetyltransferase activity of several YopJ family effectors. S349 and S351 are required for IP6-binding-mediated conformational change of HopZ1a. S349 and S351 are located in a conserved region in the C-terminal domain of YopJ family effectors. Mutations of the corresponding serine(s) in two other effectors, HopZ3 of P. syringae and PopP2 of Ralstonia solanacerum, also abolished their acetyltransferase activity. These results suggest that, in addition to the highly conserved catalytic residues, YopJ family effectors also require conserved serine(s) in the C-terminal domain for their enzymatic activity.

SummaryIn the absence of pathogen infection, plant effector-triggered immune (ETI) receptors are maintained in a preactivation state by intermolecular interactions with other host proteins. Pathogen effector-induced alterations activate the receptor. In Arabidopsis, the ETI receptor RPM1 is activated via bacterial effector AvrB-induced phosphorylation of the RPM1-interacting protein RIN4 at Threonine 166. We find that RIN4 also interacts with the prolyl-peptidyl isomerase (PPIase) ROC1, which is reduced upon RIN4 Thr166 phosphorylation. ROC1 suppresses RPM1 immunity in a PPIase-dependent manner. Consistent with this, RIN4 Pro149 undergoes cis/trans isomerization in the presence of ROC1. While the RIN4P149V mutation abolishes RPM1 resistance, the deletion of Pro149 leads to RPM1 activation in the absence of RIN4 phosphorylation. These results support a model in which RPM1 directly senses conformational changes in RIN4 surrounding Pro149 that is controlled by ROC1. RIN4 Thr166 phosphorylation indirectly regulates RPM1 resistance by modulating the ROC1-mediated RIN4 isomerization.

The ability to infect new hosts can drive the evolution and specialization of secreted pathogen proteins. [Also see Report by Dong et al. ]

Bacterial pathogens can cause multiple plant diseases and plants rely on their innate immune system to recognize and actively respond to these microbes. The plant innate immune system is comprised of extracellular pattern recognition receptors that recognize conserved microbial patterns and intracellular nucleotide binding leucine-rich repeat (NLR) proteins that recognize specific bacterial effectors delivered into host cells. Plants lack the adaptive immune branch present in animals, but still afford flexibility to pathogen attack through systemic and transgenerational resistance. Here, we focus on current research in plant immune responses against bacterial pathogens. Recent studies shed light onto the activation and inactivation of pattern recognition receptors and systemic acquired resistance. New research has also uncovered additional layers of complexity surrounding NLR immune receptor activation, cooperation, and sub-cellular localizations. Taken together, these recent advances bring us closer to understanding the web of inter-molecular interactions responsible for coordinating defense responses and ultimately resistance.

Protein complex purification represents a powerful approach to identify novel players in plant innate immunity. However, the identification of interacting protein partners within a natural context has been a challenge for researchers. In this chapter, we describe a method of immunoaffinity chromatography using purified, antibodies to isolate native protein complexes from wild-type tissue. We detail the antibody purification and immobilization steps in addition to the co-immunoprecipitation protocol. In addition, a method to prepare protein samples for mass spectroscopy analysis is described. This straightforward protocol has been used to isolate and identify novel components of Arabidopsis immunity-associated protein complexes.

© 2017 The American Phytopathological Society. This white paper reports the deliberations of a workshop focused on biotic challenges to plant health held in Washington, D.C. in September 2016. Ensuring health of food plants is critical to maintaining the quality and productivity of crops and for sustenance of the rapidly growing human population. There is a close linkage between food security and societal stability; however, global food security is threatened by the vulnerability of our agricultural systems to numerous pests, pathogens, weeds, and environmental stresses. These threats are aggravated by climate change, the globalization of agriculture, and an overreliance on non-sustainable inputs. New analytical and computational technologies are providing unprecedented resolution at a variety of molecular, cellular, organismal, and population scales for crop plants as well as pathogens, pests, beneficial microbes, and weeds. It is now possible to both characterize useful or deleterious variation as well as precisely manipulate it. Data-driven, informed decisions based on knowledge of the variation of biotic challenges and of natural and synthetic variation in crop plants will enable deployment of durable interventions throughout the world. These should be integral, dynamic components of agricultural strategies for sustainable agriculture.