The plant immune system: From discovery to deployment.

Plant diseases cause famines, drive human migration, and present challenges to agricultural sustainability as pathogen ranges shift under climate change. Plant breeders discovered Mendelian genetic loci conferring disease resistance to specific pathogen isolates over 100 years ago. Subsequent breeding for disease resistance underpins modern agriculture and, along with the emergence and focus on model plants for genetics and genomics research, has provided rich resources for molecular biological exploration over the last 50 years. These studies led to the identification of extracellular and intracellular receptors that convert recognition of extracellular microbe-encoded molecular patterns or intracellular pathogen-delivered virulence effectors into defense activation. These receptor systems, and downstream responses, define plant immune systems that have evolved since the migration of plants to land ∼500 million years ago. Our current understanding of plant immune systems provides the platform for development of rational resistance enhancement to control the many diseases that continue to plague crop production.

The phosphorylation landscape of infection-related development by the rice blast fungus.

Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.

WeiTsing, a pericycle-expressed ion channel, safeguards the stele to confer clubroot resistance.

Plant roots encounter numerous pathogenic microbes that often cause devastating diseases. One such pathogen, Plasmodiophora brassicae (Pb), causes clubroot disease and severe yield losses on cruciferous crops worldwide. Here, we report the isolation and characterization of WeiTsing (WTS), a broad-spectrum clubroot resistance gene from Arabidopsis. WTS is transcriptionally activated in the pericycle upon Pb infection to prevent pathogen colonization in the stele. Brassica napus carrying the WTS transgene displayed strong resistance to Pb. WTS encodes a small protein localized in the endoplasmic reticulum (ER), and its expression in plants induces immune responses. The cryoelectron microscopy (cryo-EM) structure of WTS revealed a previously unknown pentameric architecture with a central pore. Electrophysiology analyses demonstrated that WTS is a calcium-permeable cation-selective channel. Structure-guided mutagenesis indicated that channel activity is strictly required for triggering defenses. The findings uncover an ion channel analogous to resistosomes that triggers immune signaling in the pericycle.

Mining metatranscriptomes reveals a vast world of viroid-like circular RNAs.

Viroids and viroid-like covalently closed circular (ccc) RNAs are minimal replicators that typically encode no proteins and hijack cellular enzymes for replication. The extent and diversity of viroid-like agents are poorly understood. We developed a computational pipeline to identify viroid-like cccRNAs and applied it to 5,131 metatranscriptomes and 1,344 plant transcriptomes. The search yielded 11,378 viroid-like cccRNAs spanning 4,409 species-level clusters, a 5-fold increase compared to the previously identified viroid-like elements. Within this diverse collection, we discovered numerous putative viroids, satellite RNAs, retrozymes, and ribozy-like viruses. Diverse ribozyme combinations and unusual ribozymes within the cccRNAs were identified. Self-cleaving ribozymes were identified in ambiviruses, some mito-like viruses and capsid-encoding satellite virus-like cccRNAs. The broad presence of viroid-like cccRNAs in diverse transcriptomes and ecosystems implies that their host range is far broader than currently known, and matches to CRISPR spacers suggest that some cccRNAs replicate in prokaryotes.

Pathogen protein modularity enables elaborate mimicry of a host phosphatase.

Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruits the serine/threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.

Diversifying the menu for crop powdery mildew resistance.

Powdery mildew, a potentially severe crop disease, can be controlled by mlo mutations, which suppress fungal proliferation but typically also reduce yield. Li et al. (2022) demonstrate that productivity can be restored by overexpressing a host sugar transporter, thus offering a new option for economically and environmentally benign disease control.

Inactivation of a wheat protein kinase gene confers broad-spectrum resistance to rust fungi.

Wheat crops are frequently devastated by pandemic stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). Here, we identify and characterize a wheat receptor-like cytoplasmic kinase gene, TaPsIPK1, that confers susceptibility to this pathogen. PsSpg1, a secreted fungal effector vital for Pst virulence, can bind TaPsIPK1, enhance its kinase activity, and promote its nuclear localization, where it phosphorylates the transcription factor TaCBF1d for gene regulation. The phosphorylation of TaCBF1d switches its transcriptional activity on the downstream genes. CRISPR-Cas9 inactivation of TaPsIPK1 in wheat confers broad-spectrum resistance against Pst without impacting important agronomic traits in two years of field tests. The disruption of TaPsIPK1 leads to immune priming without constitutive activation of defense responses. Taken together, TaPsIPK1 is a susceptibility gene known to be targeted by rust effectors, and it has great potential for developing durable resistance against rust by genetic modifications.

TIR domains of plant immune receptors are 2',3'-cAMP/cGMP synthetases mediating cell death.

2',3'-cAMP is a positional isomer of the well-established second messenger 3',5'-cAMP, but little is known about the biology of this noncanonical cyclic nucleotide monophosphate (cNMP). Toll/interleukin-1 receptor (TIR) domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors have the NADase function necessary but insufficient to activate plant immune responses. Here, we show that plant TIR proteins, besides being NADases, act as 2',3'-cAMP/cGMP synthetases by hydrolyzing RNA/DNA. Structural data show that a TIR domain adopts distinct oligomers with mutually exclusive NADase and synthetase activity. Mutations specifically disrupting the synthetase activity abrogate TIR-mediated cell death in Nicotiana benthamiana (Nb), supporting an important role for these cNMPs in TIR signaling. Furthermore, the Arabidopsis negative regulator of TIR-NLR signaling, NUDT7, displays 2',3'-cAMP/cGMP but not 3',5'-cAMP/cGMP phosphodiesterase activity and suppresses cell death activity of TIRs in Nb. Our study identifies a family of 2',3'-cAMP/cGMP synthetases and establishes a critical role for them in plant immune responses.

Ca2+ sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector.

Plant immunity is activated upon pathogen perception and often affects growth and yield when it is constitutively active. How plants fine-tune immune homeostasis in their natural habitats remains elusive. Here, we discover a conserved immune suppression network in cereals that orchestrates immune homeostasis, centering on a Ca2+-sensor, RESISTANCE OF RICE TO DISEASES1 (ROD1). ROD1 promotes reactive oxygen species (ROS) scavenging by stimulating catalase activity, and its protein stability is regulated by ubiquitination. ROD1 disruption confers resistance to multiple pathogens, whereas a natural ROD1 allele prevalent in indica rice with agroecology-specific distribution enhances resistance without yield penalty. The fungal effector AvrPiz-t structurally mimics ROD1 and activates the same ROS-scavenging cascade to suppress host immunity and promote virulence. We thus reveal a molecular framework adopted by both host and pathogen that integrates Ca2+ sensing and ROS homeostasis to suppress plant immunity, suggesting a principle for breeding disease-resistant, high-yield crops.

A Defense Pathway Linking Plasma Membrane and Chloroplasts and Co-opted by Pathogens.

Chloroplasts are crucial players in the activation of defensive hormonal responses during plant-pathogen interactions. Here, we show that a plant virus-encoded protein re-localizes from the plasma membrane to chloroplasts upon activation of plant defense, interfering with the chloroplast-dependent anti-viral salicylic acid (SA) biosynthesis. Strikingly, we have found that plant pathogens from different kingdoms seem to have convergently evolved to target chloroplasts and impair SA-dependent defenses following an association with membranes, which relies on the co-existence of two subcellular targeting signals, an N-myristoylation site and a chloroplast transit peptide. This pattern is also present in plant proteins, at least one of which conversely activates SA defenses from the chloroplast. Taken together, our results suggest that a pathway linking plasma membrane to chloroplasts and activating defense exists in plants and that such pathway has been co-opted by plant pathogens during host-pathogen co-evolution to promote virulence through suppression of SA responses.

Plant Immunity: Danger Perception and Signaling.

Plants employ numerous cell-surface and intracellular immune receptors to perceive a variety of immunogenic signals associated with pathogen infection and subsequently activate defenses. Immune signaling is potentiated by the major defense hormone salicylic acid (SA), which reprograms the transcriptome for defense. Here we highlight recent advances in understanding the mechanisms underlying activation of the main classes of immune receptors, summarize the current understanding of their signaling mechanisms, and discuss an updated model for SA perception and signaling. In addition, we discuss how different receptors are organized into networks and the implications of such networks in the integration of complex danger signals for appropriate defense outputs.

A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato.

In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.

Co-incidence of Damage and Microbial Patterns Controls Localized Immune Responses in Roots.

Recognition of microbe-associated molecular patterns (MAMPs) is crucial for the plant's immune response. How this sophisticated perception system can be usefully deployed in roots, continuously exposed to microbes, remains a mystery. By analyzing MAMP receptor expression and response at cellular resolution in Arabidopsis, we observed that differentiated outer cell layers show low expression of pattern-recognition receptors (PRRs) and lack MAMP responsiveness. Yet, these cells can be gated to become responsive by neighbor cell damage. Laser ablation of small cell clusters strongly upregulates PRR expression in their vicinity, and elevated receptor expression is sufficient to induce responsiveness in non-responsive cells. Finally, localized damage also leads to immune responses to otherwise non-immunogenic, beneficial bacteria. Damage-gating is overridden by receptor overexpression, which antagonizes colonization. Our findings that cellular damage can "switch on" local immune responses helps to conceptualize how MAMP perception can be used despite the presence of microbial patterns in the soil.

Roles of RNA silencing in viral and non-viral plant immunity and in the crosstalk between disease resistance systems.

RNA silencing is a well-established antiviral immunity system in plants, in which small RNAs guide Argonaute proteins to targets in viral RNA or DNA, resulting in virus repression. Virus-encoded suppressors of silencing counteract this defence system. In this Review, we discuss recent findings about antiviral RNA silencing, including the movement of RNA through plasmodesmata and the differentiation between plant self and viral RNAs. We also discuss the emerging role of RNA silencing in plant immunity against non-viral pathogens. This immunity is mediated by transkingdom movement of RNA into and out of the infected plant cells in vesicles or as extracellular nucleoproteins and, like antiviral immunity, is influenced by the silencing suppressors encoded in the pathogens' genomes. Another effect of RNA silencing on general immunity involves host-encoded small RNAs, including microRNAs, that regulate NOD-like receptors and defence signalling pathways in the innate immunity system of plants. These RNA silencing pathways form a network of processes with both positive and negative effects on the immune systems of plants.

A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana.

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.

Opposite Roles of Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Transcriptional Regulation of Plant Immunity.

Salicylic acid (SA) is a plant defense hormone required for immunity. Arabidopsis NPR1 and NPR3/NPR4 were previously shown to bind SA and all three proteins were proposed as SA receptors. NPR1 functions as a transcriptional co-activator, whereas NPR3/NPR4 were suggested to function as E3 ligases that promote NPR1 degradation. Here we report that NPR3/NPR4 function as transcriptional co-repressors and SA inhibits their activities to promote the expression of downstream immune regulators. npr4-4D, a gain-of-function npr4 allele that renders NPR4 unable to bind SA, constitutively represses SA-induced immune responses. In contrast, the equivalent mutation in NPR1 abolishes its ability to bind SA and promote SA-induced defense gene expression. Further analysis revealed that NPR3/NPR4 and NPR1 function independently to regulate SA-induced immune responses. Our study indicates that both NPR1 and NPR3/NPR4 are bona fide SA receptors, but play opposite roles in transcriptional regulation of SA-induced defense gene expression.

A Natural Allele of a Transcription Factor in Rice Confers Broad-Spectrum Blast Resistance.

Rice feeds half the world's population, and rice blast is often a destructive disease that results in significant crop loss. Non-race-specific resistance has been more effective in controlling crop diseases than race-specific resistance because of its broad spectrum and durability. Through a genome-wide association study, we report the identification of a natural allele of a C2H2-type transcription factor in rice that confers non-race-specific resistance to blast. A survey of 3,000 sequenced rice genomes reveals that this allele exists in 10% of rice, suggesting that this favorable trait has been selected through breeding. This allele causes a single nucleotide change in the promoter of the bsr-d1 gene, which results in reduced expression of the gene through the binding of the repressive MYB transcription factor and, consequently, an inhibition of H2O2 degradation and enhanced disease resistance. Our discovery highlights this novel allele as a strategy for breeding durable resistance in rice.

A meta-analysis on global change drivers and the risk of infectious disease.

Anthropogenic change is contributing to the rise in emerging infectious diseases, which are significantly correlated with socioeconomic, environmental and ecological factors1. Studies have shown that infectious disease risk is modified by changes to biodiversity2-6, climate change7-11, chemical pollution12-14, landscape transformations15-20 and species introductions21. However, it remains unclear which global change drivers most increase disease and under what contexts. Here we amassed a dataset from the literature that contains 2,938 observations of infectious disease responses to global change drivers across 1,497 host-parasite combinations, including plant, animal and human hosts. We found that biodiversity loss, chemical pollution, climate change and introduced species are associated with increases in disease-related end points or harm, whereas urbanization is associated with decreases in disease end points. Natural biodiversity gradients, deforestation and forest fragmentation are comparatively unimportant or idiosyncratic as drivers of disease. Overall, these results are consistent across human and non-human diseases. Nevertheless, context-dependent effects of the global change drivers on disease were found to be common. The findings uncovered by this meta-analysis should help target disease management and surveillance efforts towards global change drivers that increase disease. Specifically, reducing greenhouse gas emissions, managing ecosystem health, and preventing biological invasions and biodiversity loss could help to reduce the burden of plant, animal and human diseases, especially when coupled with improvements to social and economic determinants of health.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Molecular basis of methyl-salicylate-mediated plant airborne defence.

Aphids transmit viruses and are destructive crop pests1. Plants that have been attacked by aphids release volatile compounds to elicit airborne defence (AD) in neighbouring plants2-5. However, the mechanism underlying AD is unclear. Here we reveal that methyl-salicylate (MeSA), salicylic acid-binding protein-2 (SABP2), the transcription factor NAC2 and salicylic acid-carboxylmethyltransferase-1 (SAMT1) form a signalling circuit to mediate AD against aphids and viruses. Airborne MeSA is perceived and converted into salicylic acid by SABP2 in neighbouring plants. Salicylic acid then causes a signal transduction cascade to activate the NAC2-SAMT1 module for MeSA biosynthesis to induce plant anti-aphid immunity and reduce virus transmission. To counteract this, some aphid-transmitted viruses encode helicase-containing proteins to suppress AD by interacting with NAC2 to subcellularly relocalize and destabilize NAC2. As a consequence, plants become less repellent to aphids, and more suitable for aphid survival, infestation and viral transmission. Our findings uncover the mechanistic basis of AD and an aphid-virus co-evolutionary mutualism, demonstrating AD as a potential bioinspired strategy to control aphids and viruses.