Cell-type-specific responses to fungal infection in plants revealed by single-cell transcriptomics.

Pathogen infection is a dynamic process. Here, we employ single-cell transcriptomics to investigate plant response heterogeneity. By generating an Arabidopsis thaliana leaf atlas encompassing 95,040 cells during infection by a fungal pathogen, Colletotrichum higginsianum, we unveil cell-type-specific gene expression, notably an enrichment of intracellular immune receptors in vasculature cells. Trajectory inference identifies cells that had different interactions with the invading fungus. This analysis divulges transcriptional reprogramming of abscisic acid signaling specifically occurring in guard cells, which is consistent with a stomatal closure dependent on direct contact with the fungus. Furthermore, we investigate the transcriptional plasticity of genes involved in glucosinolate biosynthesis in cells at the fungal infection sites, emphasizing the contribution of the epidermis-expressed MYB122 to disease resistance. This work underscores spatially dynamic, cell-type-specific plant responses to a fungal pathogen and provides a valuable resource that supports in-depth investigations of plant-pathogen interactions.

Plant HEM1 specifies a condensation domain to control immune gene translation.

Translational reprogramming is a fundamental layer of immune regulation, but how such a global regulatory mechanism operates remains largely unknown. Here we perform a genetic screen and identify Arabidopsis HEM1 as a global translational regulator of plant immunity. The loss of HEM1 causes exaggerated cell death to restrict bacterial growth during effector-triggered immunity (ETI). By improving ribosome footprinting, we reveal that the hem1 mutant increases the translation efficiency of pro-death immune genes. We show that HEM1 contains a plant-specific low-complexity domain (LCD) absent from animal homologues. This LCD endows HEM1 with the capability of phase separation in vitro and in vivo. During ETI, HEM1 interacts and condensates with the translation machinery; this activity is promoted by the LCD. CRISPR removal of this LCD causes more ETI cell death. Our results suggest that HEM1 condensation constitutes a brake mechanism of immune activation by controlling the tissue health and disease resistance trade-off during ETI.

Calcium signaling in plant immunity: a spatiotemporally controlled symphony.

Calcium ions (Ca2+) are prominent intracellular messengers in all eukaryotic cells. Recent studies have emphasized the crucial roles of Ca2+ in plant immunity. Here, we review the latest progress on the spatiotemporal control of Ca2+ function in plant immunity. We discuss discoveries of how Ca2+ influx is triggered upon the activation of immune receptors, how Ca2+-permeable channels are activated, how Ca2+ signals are decoded inside plant cells, and how these signals are switched off. Despite recent advances, many open questions remain and we highlight the existing toolkit and the new technologies to address the outstanding questions of Ca2+ signaling in plant immunity.

Transcriptional regulation of plant innate immunity.

Transcriptional reprogramming is an integral part of plant immunity. Tight regulation of the immune transcriptome is essential for a proper response of plants to different types of pathogens. Consequently, transcriptional regulators are proven targets of pathogens to enhance their virulence. The plant immune transcriptome is regulated by many different, interconnected mechanisms that can determine the rate at which genes are transcribed. These include intracellular calcium signaling, modulation of the redox state, post-translational modifications of transcriptional regulators, histone modifications, DNA methylation, modulation of RNA polymerases, alternative transcription inititation, the Mediator complex and regulation by non-coding RNAs. In addition, on their journey from transcription to translation, mRNAs are further modulated through mechanisms such as nuclear RNA retention, storage of mRNA in stress granules and P-bodies, and post-transcriptional gene silencing. In this review, we highlight the latest insights into these mechanisms. Furthermore, we discuss some emerging technologies that promise to greatly enhance our understanding of the regulation of the plant immune transcriptome in the future.

Thirty years of resistance: Zig-zag through the plant immune system.

Understanding the plant immune system is crucial for using genetics to protect crops from diseases. Plants resist pathogens via a two-tiered innate immune detection-and-response system. The first plant Resistance (R) gene was cloned in 1992 . Since then, many cell-surface pattern recognition receptors (PRRs) have been identified, and R genes that encode intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) have been cloned. Here, we provide a list of characterized PRRs and NLRs. In addition to immune receptors, many components of immune signaling networks were discovered over the last 30 years. We review the signaling pathways, physiological responses, and molecular regulation of both PRR- and NLR-mediated immunity. Recent studies have reinforced the importance of interactions between the two immune systems. We provide an overview of interactions between PRR- and NLR-mediated immunity, highlighting challenges and perspectives for future research.

Plant immune networks.

Plants have both cell-surface and intracellular receptors to recognize diverse self- and non-self molecules. Cell-surface pattern recognition receptors (PRRs) recognize extracellular pathogen-/damage-derived molecules or apoplastic pathogen-derived effectors. Intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) recognize pathogen effectors. Activation of both PRRs and NLRs elevates defense gene expression and accumulation of the phytohormone salicylic acid (SA), which results in SA-dependent transcriptional reprogramming. These receptors, together with their coreceptors, form networks to mediate downstream immune responses. In addition, cell-surface and intracellular immune systems are interdependent and function synergistically to provide robust resistance against pathogens. Here, we summarize the interactions between these immune systems and attempt to provide a holistic picture of plant immune networks. We highlight current challenges and discuss potential new research directions.

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor.

Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY Perception of AvrRps4C by RRS1WRKY is mediated by the ß2-ß3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C Structure-based mutations that disrupt AvrRps4C-RRS1WRKY interactions in vitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKY-W-box DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.

Chromatin accessibility landscapes activated by cell-surface and intracellular immune receptors.

Activation of cell-surface and intracellular receptor-mediated immunity results in rapid transcriptional reprogramming that underpins disease resistance. However, the mechanisms by which co-activation of both immune systems lead to transcriptional changes are not clear. Here, we combine RNA-seq and ATAC-seq to define changes in gene expression and chromatin accessibility. Activation of cell-surface or intracellular receptor-mediated immunity, or both, increases chromatin accessibility at induced defence genes. Analysis of ATAC-seq and RNA-seq data combined with publicly available information on transcription factor DNA-binding motifs enabled comparison of individual gene regulatory networks activated by cell-surface or intracellular receptor-mediated immunity, or by both. These results and analyses reveal overlapping and conserved transcriptional regulatory mechanisms between the two immune systems.

Channeling plant immunity.

Plant intracellular NLR proteins detect pathogen effectors and then form multimeric protein complexes ("resistosomes") that activate immune responses and cell death through unknown mechanisms. In this issue of Cell, Bi et al. show that the ZAR1 resistosome exhibits cation channel activity, enabling calcium influx that activates defense mechanisms and culminates in cell death.

Mutual potentiation of plant immunity by cell-surface and intracellular receptors.

The plant immune system involves cell-surface receptors that detect intercellular pathogen-derived molecules, and intracellular receptors that activate immunity upon detection of pathogen-secreted effector proteins that act inside the plant cell. Immunity mediated by surface receptors has been extensively studied1, but that mediated by intracellular receptors has rarely been investigated in the absence of surface-receptor-mediated immunity. Furthermore, interactions between these two immune pathways are poorly understood. Here, by activating intracellular receptors without inducing surface-receptor-mediated immunity, we analyse interactions between these two distinct immune systems in Arabidopsis. Pathogen recognition by surface receptors activates multiple protein kinases and NADPH oxidases, and we find that intracellular receptors primarily potentiate the activation of these proteins by increasing their abundance through several mechanisms. Likewise, the hypersensitive response that depends on intracellular receptors is strongly enhanced by the activation of surface receptors. Activation of either immune system alone is insufficient to provide effective resistance against the bacterial pathogen Pseudomonas syringae. Thus, immune pathways activated by cell-surface and intracellular receptors in plants mutually potentiate to activate strong defences against pathogens. These findings reshape our understanding of plant immunity and have broad implications for crop improvement.

A Comparative Overview of the Intracellular Guardians of Plants and Animals: NLRs in Innate Immunity and Beyond.

Nucleotide-binding domain leucine-rich repeat receptors (NLRs) play important roles in the innate immune systems of both plants and animals. Recent breakthroughs in NLR biochemistry and biophysics have revolutionized our understanding of how NLR proteins function in plant immunity. In this review, we summarize the latest findings in plant NLR biology and draw direct comparisons to NLRs of animals. We discuss different mechanisms by which NLRs recognize their ligands in plants and animals. The discovery of plant NLR resistosomes that assemble in a comparable way to animal inflammasomes reinforces the striking similarities between the formation of plant and animal NLR complexes. Furthermore, we discuss the mechanisms by which plant NLRs mediate immune responses and draw comparisons to similar mechanisms identified in animals. Finally, we summarize the current knowledge of the complex genetic architecture formed by NLRs in plants and animals and the roles of NLRs beyond pathogen detection.

PTI-ETI crosstalk: an integrative view of plant immunity.

Plants resist attacks by pathogens via innate immune responses, which are initiated by cell surface-localized pattern-recognition receptors (PRRs) and intracellular nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) leading to pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively. Although the two classes of immune receptors involve different activation mechanisms and appear to require different early signalling components, PTI and ETI eventually converge into many similar downstream responses, albeit with distinct amplitudes and dynamics. Increasing evidence suggests the existence of intricate interactions between PRR-mediated and NLR-mediated signalling cascades as well as common signalling components shared by both. Future investigation of the mechanisms underlying signal collaboration between PRR-initiated and NLR-initiated immunity will enable a more complete understanding of the plant immune system. This review discusses recent advances in our understanding of the relationship between the two layers of plant innate immunity.

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants.

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.

Stories of Salicylic Acid: A Plant Defense Hormone.

Salicylic acid (SA) is a key plant hormone required for establishing resistance to many pathogens. SA biosynthesis involves two main metabolic pathways with multiple steps: the isochorismate and the phenylalanine ammonia-lyase pathways. Transcriptional regulations of SA biosynthesis are important for fine-tuning SA level in plants. We highlight here recent discoveries on SA biosynthesis and transcriptional regulations of SA biosynthesis. In addition, SA perception by NPR proteins is important to fulfil its function as a defense hormone. We highlight recent work to give a full picture of how NPR proteins support the role of SA in plant immunity. We also discuss challenges and potential opportunities for future research and application related to the functions of SA in plants.

Phosphorylation-Regulated Activation of the Arabidopsis RRS1-R/RPS4 Immune Receptor Complex Reveals Two Distinct Effector Recognition Mechanisms.

The Arabidopsis immune receptors RPS4 and RRS1 interact to co-confer responsiveness to bacterial effectors. The RRS1-R allele, with RPS4, responds to AvrRps4 and PopP2, whereas RRS1-S responds only to AvrRps4. Here, we show that the C terminus of RRS1-R but not RRS1-S is phosphorylated. Phosphorylation at Thr1214 in the WRKY domain maintains RRS1-R in its inactive state and also inhibits acetylation of RRS1-R by PopP2. PopP2 in turn catalyzes O-acetylation at the same site, thereby preventing its phosphorylation. Phosphorylation at other sites is required for PopP2 but not AvrRps4 responsiveness and facilitates the interaction of RRS1's C terminus with its TIR domain. Derepression of RRS1-R or RRS1-S involves effector-triggered proximity between their TIR domain and C termini. This effector-promoted interaction between these domains relieves inhibition of TIRRPS4 by TIRRRS1. Our data reveal effector-triggered and phosphorylation-regulated conformational changes within RRS1 that results in distinct modes of derepression of the complex by PopP2 and AvrRps4.

Estradiol-inducible AvrRps4 expression reveals distinct properties of TIR-NLR-mediated effector-triggered immunity.

Plant nucleotide-binding domain, leucine-rich repeat receptor (NLR) proteins play important roles in recognition of pathogen-derived effectors. However, the mechanism by which plant NLRs activate immunity is still largely unknown. The paired Arabidopsis NLRs RRS1-R and RPS4, that confer recognition of bacterial effectors AvrRps4 and PopP2, are well studied, but how the RRS1/RPS4 complex activates early immediate downstream responses upon effector detection is still poorly understood. To study RRS1/RPS4 responses without the influence of cell surface receptor immune pathways, we generated an Arabidopsis line with inducible expression of the effector AvrRps4. Induction does not lead to hypersensitive cell death response (HR) but can induce electrolyte leakage, which often correlates with plant cell death. Activation of RRS1 and RPS4 without pathogens cannot activate mitogen-associated protein kinase cascades, but still activates up-regulation of defence genes, and therefore resistance against bacteria.

High-resolution expression profiling of selected gene sets during plant immune activation.

The plant immune system involves detection of pathogens via both cell-surface and intracellular receptors. Both receptor classes can induce transcriptional reprogramming that elevates disease resistance. To assess differential gene expression during plant immunity, we developed and deployed quantitative sequence capture (CAP-I). We designed and synthesized biotinylated single-strand RNA bait libraries targeted to a subset of defense genes, and generated sequence capture data from 99 RNA-seq libraries. We built a data processing pipeline to quantify the RNA-CAP-I-seq data, and visualize differential gene expression. Sequence capture in combination with quantitative RNA-seq enabled cost-effective assessment of the expression profile of a specified subset of genes. Quantitative sequence capture is not limited to RNA-seq or any specific organism and can potentially be incorporated into automated platforms for high-throughput sequencing.

Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue.

Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from polyadenylated mRNA for transcriptome analysis. Both high-quality and high-throughput protocol options include steps of mRNA enrichment through magnetic bead-enabled precipitation of the poly-A tail, cDNA synthesis, and then fragmentation and adapter addition simultaneously through Tn5-mediated 'tagmentation'. All steps of the protocols have been validated with Arabidopsis thaliana leaf and seedling tissues and streamlined to work together, with minimal cost in money and time, thus intended to provide a beginner-friendly start-to-finish RNA-seq library preparation for transcriptome analysis.

Diverse NLR immune receptors activate defence via the RPW8-NLR NRG1.

Most land plant genomes carry genes that encode RPW8-NLR Resistance (R) proteins. Angiosperms carry two RPW8-NLR subclasses: ADR1 and NRG1. ADR1s act as 'helper' NLRs for multiple TIR- and CC-NLR R proteins in Arabidopsis. In angiosperm families, NRG1 co-occurs with TIR-NLR Resistance (R) genes. We tested whether NRG1 is required for signalling of multiple TIR-NLRs. Using CRISPR mutagenesis, we obtained an nrg1a-nrg1b double mutant in two Arabidopsis accessions, and an nrg1 mutant in Nicotiana benthamiana. These mutants are compromised in signalling of all TIR-NLRs tested, including WRR4A, WRR4B, RPP1, RPP2, RPP4 and the pairs RRS1/RPS4, RRS1B/RPS4B, CHS1/SOC3 and CHS3/CSA1. In Arabidopsis, NRG1 is required for the hypersensitive cell death response (HR) and full oomycete resistance, but not for salicylic acid induction or bacterial resistance. By contrast, nrg1 loss of function does not compromise the CC-NLR R proteins RPS5 and MLA. RPM1 and RPS2 (CC-NLRs) function is slightly compromised in an nrg1 mutant. Thus, NRG1 is required for full TIR-NLR function and contributes to the signalling of some CC-NLRs. Some NRG1-dependent R proteins also signal partially via the NRG1 sister clade, ADR1. We propose that some NLRs signal via NRG1 only, some via ADR1 only and some via both or neither.

Pathogens Suppress Host Transcription Factors for Rampant Proliferation.

Root pathogen Verticillium dahliae deploys an effector called VdSCP41 into plants to disrupt the functions of SARD1 and CBP60g, two central transcriptional regulators of plant immunity. This provides new tools to dissect transcriptional regulation of tissue-specific immunity in the root and to understand dynamic interactions between plants and root-associated microorganisms.