Citrus Huanglongbing (HLB) associated with 'Candidatus Liberibacter asiaticus' (CLas), a biotrophic pathogen, is a devastating citrus diseases worldwide. Enhancing host plant resistance through elicitor treatments has been exploited for crop disease management. HLB was suggested to be a pathogen-triggered immune disease in HLB susceptible varieties. Here, we hypothesized that boosting citrus immunity to CLas with biotic elicitors including microbe-associated molecular patterns (MAMPs) can increase citrus resistance to HLB. In greenhouse assays, foliar spray of chitosan, harpins, or lipopolysaccharides (LPS) activated sweet orange plant immunity in a dosage-dependent manner. Peak expression levels of citrus defense genes including FRK1, MKK4, PAL1, WRKY22, and WRKY29 were recorded at 12 to 72 hours post treatment, and chitosan and harpins induced stronger gene expression than LPS. Additionally, all the three MAMPs elicited reactive oxygen species (ROS) burst in treated leaves, and both chitosan and harpins induced a higher level of ROS than LPS. Moreover, chitosan and harpins, sprayed weekly at 1.0 and 0.25 mg/mL respectively, significantly reduced CLas growth in sweet orange trees and suppressed plant growth. In a 2-year field trial under natural CLas infection conditions, foliar spray of chitosan, harpins, or salicylic acid monthly delayed CLas infection and slowed down the increase of CLas titer, without suppressing plant growth. This study demonstrated that promoting immunity can be used as a useful strategy in controlling a pathogen-triggered immune disease caused by a biotrophic pathogen.

Plants lack specialized immune cells and instead rely on coordinated cellular responses to restrict pathogen invasion while preserving tissue integrity. How plant immunity spatially organizes these responses remains unclear. Using live-cell reporters in Arabidopsis infected with Pseudomonas syringae, we show that effector-triggered immunity, superimposed on pattern-triggered immunity, establishes a sustained yet spatially confined immune architecture at the infection front. Defense activation persists for days but remains restricted to a narrow ring of cells surrounding viable bacterial microcolonies. Over time, immune activity spreads to adjacent layers, forming a coordinated, multilayered defense zone. This zonation extends beyond transcriptional activation to polarized callose deposition at pathogen-facing cell walls, reinforcing a localized containment boundary that limits pathogen spread. Consistent with previous single-reporter studies, simultaneous visualization of salicylic acid (SA) and jasmonic acid (JA) biosynthesis and response markers reveals a radial hormone gradient, with SA-enriched cells proximal to bacterial colonies and JA-enriched cells in surrounding regions. Individual cells predominantly activate one pathway, indicating that SA-JA antagonism is resolved through spatial compartmentalization across neighboring cells. Together, these findings establish immune zonation as a strategy for robust pathogen containment while minimizing collateral tissue damage.

Wheat is a critical global food source, yet its production is threatened by stripe rust caused by Puccinia striiformis f. sp. tritici (Pst), which causes substantial yield losses worldwide. The resistance gene Yr15, derived from wild emmer wheat, encodes the tandem kinase protein WTK1 and confers wide-spectrum resistance to more than 2,000 Pst isolates. Here, we examined how WTK1-mediated resistance is shaped by pathogen load, isolate identity, and host genetic background, with emphasis on the histopathological dynamics of infection. Susceptible genotypes Kronos S (tetraploid) and Avocet S (hexaploid) showed stable levels of susceptibility across inoculum gradients, whereas their near-isogenic lines carrying WTK1 (Kronos R and Avocet R) exhibited dose-dependent hypersensitive responses, with Avocet R showing slightly stronger reactions. An inoculum of 10 mg/ml consistently distinguished susceptible from resistant responses, providing a reliable threshold for phenotyping. Importantly, WTK1-carrying lines resisted all tested isolates, including the highly virulent Pst#5006. A 336-h postinoculation (hpi) time course revealed that fungal growth diverged between resistant and susceptible plants beginning at 144 hpi. In Kronos R, fungal colonies were detectable up to 96 hpi but were subsequently curtailed by localized programmed cell death. Biomass quantification confirmed no significant increase in fungal load in WTK1 lines from 12 to 336 hpi. Microscopic analysis further showed that defense activation occurred after haustorium formation, indicating a post-haustorial mechanism of resistance. Together, these findings provide the first detailed temporal map of tandem kinase protein-mediated defense in wheat and underscore the robustness of WTK1 across pathogen pressures.

Genomic clusters of immune genes, including those encoding nucleotide-binding leucine-rich repeat (NLR) proteins, are a model for exploring the dynamics of genomic regions in flux. Rapid sequence evolution of immune genes, including NLRs, and variation in their gene content, may enable long-lived plants, which lack adaptive immune systems, to keep pace with the fast evolution of pathogens. To explore the patterns and processes shaping the evolution of NLR gene content in a genus of long-lived tree species, we unified the annotation of NLR genes across 11 accessions (or 15 haplotypes) from the genus Citrus and its relatives, including three new diploid genome assemblies. A majority of NLRs were arranged in genomic clusters composed of paralogous genes, typically from a single gene family. Even larger clusters, with 10 or more NLRs, were limited to genes derived from one or few gene families. These patterns suggested that genomic clustering of NLRs arose through local expansion of phylogenetically related NLRs, but the mechanistic processes driving these patterns are not clear. Local gene duplication can be mediated by multiple processes, including transposon-mediated gene capture and subsequent proliferation, and non-allelic repair of double stranded breaks, including unequal recombination. Examples of retrotransposon-mediated duplication of NLRs were identified, but these were not sufficient to explain massive regional expansions. Signatures of unequal recombination are challenging to identify. Focusing on recent lineage-specific sequence duplications, at least one case of unequal recombination was identified, supporting a role for unequal recombination in shaping genomic variation in these regions. ### Competing Interest Statement The authors have declared no competing interest. National Institute of Food and Agriculture, https://ror.org/05qx3fv49, 2020-70029-33202, 2023-70029-41305, 2019-70016-29796 Citrus Research Board, https://ror.org/04q4bdj41, 5200-171 U.S. National Science Foundation, https://ror.org/021nxhr62, DBI-1922642

Abstract Pattern recognition receptors (PRRs) mediate plant immune responses by detecting extracellular immunogenic patterns, including microbe-associated molecular patterns (MAMPs). PRR signaling is commonly assessed using assays such as reactive oxygen species (ROS) bursts, cytosolic calcium influx, mitogen-activated protein kinase (MAPK) activation, and seedling growth inhibition (SGI), which are performed in distinct experimental systems, including seedlings grown on artificial media and soil-grown rosettes. Here, we systematically compare receptor kinase immune outputs triggered by the bacterial MAMPs elf18 and flg22 in Arabidopsis thaliana seedlings and rosettes across a range of concentrations. Rosettes exhibited greater sensitivity than seedlings in ROS assays, whereas cytosolic calcium responses measured using the Aeq cyt /pMAQ2 reporter were stronger in seedlings, correlating with reduced reporter transcript accumulation in rosette tissue. MAPK activation was consistently stronger in rosettes, whereas SGI assays revealed higher sensitivity to elf18 than flg22 in seedlings despite flg22 inducing stronger early signaling outputs. Together, these results demonstrate that canonical PRR-mediated immune outputs are differentially sensitive to experimental context and should not be interpreted as interchangeable measures of immune activation. These findings highlight the importance of considering experimental conditions when comparing immune responses across assays and developmental stages.

To combat soilborne pathogens, roots activate pattern-triggered immunity (PTI) through pattern-recognition receptors (PRRs) that recognise microbe-associated molecular patterns (MAMPs). Root PTI pathways can differ from their above-ground counterparts and have been well-characterised in the model plant Arabidopsis thaliana but are not well-defined in crops. Gene repurposing coupled with differences in root tissues and root architecture in tomato species (Solanum lycopersicum and S. pimpinellifolium) led us to hypothesise that signalling pathways of Solanaceous-specific PRRs diverge from canonical pathways. The objective of this study was to characterise PTI signalling pathways and responses (ROS, MAPK, gene expression, and growth inhibition) in roots of wild and domesticated tomatoes downstream of three immune receptors: the well-conserved SlFLS2 and the Solanaeceous-specific FLS3 and CORE. We find that Solanum root PTI responses are concentrated in early differentiating root regions compared to late differentiating regions or whole roots, and that FLS3 and CORE signalling pathways are overlapping but distinct from each other and from FLS2. Although the early differentiating root region had strong PTI responses across Solanum cultivars and species, different genetic backgrounds varied in their response dynamics. Our results underscore the complexity of PTI signalling across species and highlight the developmental-stage specificity of tomato root immunity.

Plant pathogens pose a continuous threat to global food production. Recent discoveries in plant immunity research unveiled a unique protein family characterized by an unusual resistance protein structure that combines two kinase domains. This study demonstrates the widespread occurrence of tandem kinase proteins (TKPs) across the plant kingdom. An examination of 104 plant species' genomes uncovered 2,682 TKPs. The majority (95.6%) of these kinase domains are part of the receptor-like kinase-Pelle family, which is crucial for cell surface responses in plant immunity. Notably, 90% of TKPs comprise dual kinase domains, with over 50% being pseudokinases. Over 56% of these proteins harbor 127 different integrated domains, and over 47% include a transmembrane domain. TKP pseudokinases and/or integrated domains probably serve as decoys, engaging with pathogen effectors to trigger plant immunity. The TKP Atlas we created sheds light on the mechanisms of TKP convergent molecular evolution and potential function.

The type III secretion system in Pseudomonas syringae complex pathogens delivers type III effectors (T3Es) into plant cells to manipulate host processes, enhance survival, and promote disease. While substantial research has focused on herbaceous pathogens, T3Es in strains infecting woody hosts are less understood. This study investigates the HopBL family of effectors in Pseudomonas savastanoi, a pathogen of woody plants. HopBL1 and HopBL2, core effectors in P. savastanoi, are restricted to phylogroup 3 strains of the P. syringae complex, all isolated from woody hosts. Phylogenetic analysis suggests recent horizontal acquisition of these effectors across multiple P. syringae pathovars, integrated into genomic islands flanked by mobile genetic elements. Structural analysis shows that both HopBL effectors contain SUMO protease and DNA-binding domains, with HopBL1 also possessing an ethylene-responsive motif, all characteristic of XopD from Xanthomonas spp. Despite low sequence identity, HopBL effectors exhibit structural similarity to XopD, with HopBL1 showing greater resemblance, particularly in the arrangement of these domains. Functional assays in olive and oleander revealed strain-specific contributions of HopBL1 and HopBL2 to virulence. In oleander, the natural host of P. savastanoi pv. nerii, mutation of either effector gene resulted in reduced symptom development. We show that HopBL2 localised predominantly to subnuclear foci and associated with plasmodesmata, with partial overlap observed along microtubules, suggesting a potential role in cytoskeleton manipulation. These findings underscore the importance of T3Es unique to P. syringae strains infecting woody hosts and their adaptation to modulate host cellular structures to promote disease.

The surface-localized receptor kinase FLS2 detects the flg22 epitope from bacterial flagella. FLS2 is conserved across land plants, but bacterial pathogens exhibit polymorphic flg22 epitopes. Most FLS2 homologues possess narrow perception ranges, but four with expanded perception have been identified. Using diversity analyses, AlphaFold modelling and amino acid properties, key residues enabling expanded recognition were mapped to FLS2’s concave surface, interacting with the co-receptor and polymorphic flg22 residues. Synthetic biology enabled engineering of expanded recognition from QvFLS2 (Quercus variabilis) into a homologue with canonical perception. A similar approach enabled transfer of Agrobacterium perception from FLS2XL (Vitis riparia) into VrFLS2. Evolutionary analyses across three plant orders showed residues under positive selection aligning with those binding the co-receptor and flg22’s C terminus, suggesting more alleles with expanded perception exist. Our experimental data enabled the identification of specific receptor amino acid properties and AlphaFold3 metrics that facilitate predicting FLS2–flg22 recognition. This study provides a framework for rational receptor engineering to enhance pathogen restriction. Receptor kinase FLS2 detects the flg22 epitope of bacterial flagellin. Here the authors identify key residues on FLS2’s concave surface that enable expanded perception of flg22 variants, allowing the engineering of synthetic receptors with enhanced pathogen recognition.

Plants have intricate innate immune receptors that detect pathogens. Research has intensely focused on two receptor classes recognizing external and internal threats. Recent research has identified a class of disease-resistance proteins called tandem kinase proteins (TKPs). We investigated RWT4, a wheat TKP that confers resistance to the devastating fungal pathogen Magnaporthe oryzae. We established a rice protoplast system, revealing RWT4 specifically recognizes the AvrPWT4 effector, leading to the transcription of defense genes and inducing cell death. RWT4 possesses both kinase and pseudokinase domains, with its kinase activity essential for defense. RWT4 directly interacts with and transphosphorylates AvrPWT4. Biolayer interferometry revealed both RWT4 kinase and pseudokinase regions bind the effector. Sequence similarity and structural modeling revealed a partial kinase duplication in RWT4's kinase region as critical for effector interaction and defense activation. Collectively, these findings demonstrate that TKPs can directly bind a recognized effector, leading to downstream defense activation.

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis (Arabidopsis thaliana) NADPH oxidase respiratory burst oxidase homolog D (RBOHD) is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase Pbl13-interacting RING domain E3 ligase (PIRE). Arabidopsis PIRE knockouts exhibit enhanced ROS production and resistance to the foliar pathogen Pseudomonas syringae. Here, we identified 170 PIRE homologs, which emerged in tracheophytes and expanded in angiosperms. We investigated the role of tomato (Solanum lycopersicum) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a PIRE-dependent manner. Using genome editing, we generated mutants in 2 S. lycopersicum PIRE (SlPIRE) homologs. SlPIRE1 edited lines (Slpire1) in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, Slpire1 exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens P. syringae and Xanthomonas campestris. However, Slpire1 exhibited similar levels of colonization as wild type upon inoculation with diverse soil-borne pathogens. These results indicate that PIRE regulates RBOHs in multiple plant species and is a promising target for foliar disease control. This study also highlights the pathogen-specific role of PIRE, indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated pathogenic interactions.

The Triticeae tribe comprises species representing some of the world's largest food and forage crops, including common wheat, durum, barley, rye, and oat. Crop yields are continuously threatened by various plant diseases and deploying disease resistance (R) genes is a key strategy for protection. More than 70 different Triticeae R gene loci have been cloned, with approximately 60% derived from wild relatives. These R genes belong to diverse protein families, such as receptor kinases (RKs), nucleotide-binding leucine-rich repeat (NLR) immune receptors, tandem kinase proteins, and kinase fusion proteins as well as noncanonical R genes related to membrane, transcription, and detoxification. RKs and NLRs often confer race-specific resistance by recognizing pathogen effectors, whereas noncanonical R genes can provide broad-spectrum resistance. This review provides an overview of the diverse R genes cloned from Triticeae and their evolutionary origins, modes of action, and application in resistance breeding.

Plant pathogens pose a continuous threat to global food production. Recent discoveries in plant immunity research unveiled a unique protein family characterized by an unusual resistance protein structure that combines two kinase domains (KD). This study demonstrates the widespread occurrence of tandem-kinase proteins (TKPs) across the plant kingdom. An examination of 104 plant species' genomes uncovered 2,682 TKPs. The majority (95.6%) of these KDs are part of the RLK-Pelle receptor-like kinase family, which is crucial for cell surface responses in plant immunity. Notably, 90% of TKPs comprise dual KDs, with over 50% being pseudokinases. Over 56% of these proteins harbor 127 different integrated domains, and over 47% include a transmembrane domain. TKP pseudokinases and/or integrated domains probably serve as decoys, engaging with pathogen effectors to trigger plant immunity. The TKP Atlas we created sheds light on the mechanisms of TKP convergent molecular evolution and potential function.

Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

Original images for figures and quantifications.

"Candidatus Liberibacter spp." are insect-vectored, fastidious, and vascular-limited phytopathogens. They are the presumptive causal agents of potato zebra chip, tomato vein clearing, and the devastating citrus greening disease worldwide. There is an urgent need to develop new strategies to control them. In this study, we characterized a dual-specificity serine/tyrosine phosphatase (STP) that is well conserved among thirty-three geographically diverse "Candidatus Liberibacter spp." and strains that infect multiple Solanaceaea and citrus spp. The STP is expressed in infected plant tissues, localized at the plant cytosol and plasma membrane, and interferes with plant cell death responses. We employed an in silico target-based molecular modeling and ligand screen to identify two small molecules with high binding affinity to STP. Efficacy studies demonstrated that the two molecules can inhibit "Candidatus Liberibacter spp." but not unrelated pathogens and confer plant disease tolerance. The inhibitors and strategies are promising means to control "Candidatus Liberibacter spp."