Telomeres safeguard the genome, acting as sentinels of oxidative stress and preventing chromosome ends from eliciting a DNA damage response. PROTECTION OF TELOMERES 1 (POT1) is a highly conserved telomere protein, essential for chromosome integrity and telomeric DNA replication. Arabidopsis thaliana encodes two divergent POT1 paralogs: AtPOT1a stimulates telomerase activity, but AtPOT1b function is unknown. Here we show that AtPOT1b modulates reactive oxygen species (ROS) homeostasis. Oxidative stress induces AtPOT1b expression and telomeric accumulation, while AtPOT1b inactivation elevates ROS, increases telomeric and genome-wide oxidation, and causes stochastic telomere length changes. To address how AtPOT1b controls ROS, we report its localization in nuclei and peroxisomes, and association with catalases and peroxidases that enhance ROS scavenging. Impairing AtPOT1b-CAT2 interaction increases ROS accumulation and telomeric oxidation. Moss or human POT1 rescues ROS overaccumulation in Arabidopsis pot1b mutants, but not telomere deficiency in pot1a pot1b mutants, supporting a conserved role for POT1 in modulating ROS homeostasis and genome stability, distinct from canonical telomeric functions.

Plants thrive in complex and often hostile environments by continuously sensing and responding to a wide array of biotic and abiotic challenges. Central to this surveillance system are cell surface receptors, including receptor kinases and receptor proteins. Recent advances have revealed how these receptors not only recognize pathogen- and damage-associated molecular patterns but also perceive endogenous peptides and environmental signals. Importantly, emerging evidence highlights extensive crosstalk between immune and abiotic stress signaling, with shared components and co-regulatory mechanisms shaping an integrated response. In this review, we synthesize recent discoveries on the diverse roles of cell surface receptors in detecting external and internal signals, coordinating defense and stress tolerance, and balancing these processes with nutrient acquisition.

Virus-induced gene silencing (VIGS) has been applied as a functional genomics tool across diverse plant species. Integrated with the Arabidopsis sequence-tagged T-DNA homozygous mutant library, VIGS enables an efficient screening approach that combines features of both forward and reverse genetics, facilitating the identification of novel regulators in plant immunity. Plant defense against pathogens relies on a two-layered immune system, classified as pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Dysregulation of key PTI or ETI components can lead to excessive or uncontrolled cell death. The cell death phenotype offers a unique avenue for genetic screens aimed at identifying suppressors of immune-related cell death. However, conventional genetic approaches face limitations due to seedling lethality and the consequent lack of viable seeds, restricting their efficiency. Here, we describe an Agrobacterium-mediated transient VIGS assay optimized for systematic gene silencing at seedling stages, leading to cell death phenotypes. This method enables high-throughput screening for cell death suppressors using T-DNA homozygous mutant collections. The platform provides a rapid, cost-efficient strategy for uncovering key regulators of plant immune signaling, offering new insights into mechanisms governing immune homeostasis and cell death suppression.

Obligate biotrophic powdery mildew (PM) fungi strictly require living hosts to survive. To search for host factors or processes essential for PM pathogenesis, we conducted a tailored forward genetic screen with the immunocompromised eds1-2/pad4-1/sid2-2 (eps) triple Arabidopsis mutant. This led to the identification of 5 allelic disruptive mutations in Mildew Locus O 2 (MLO2) that are responsible for the compromised immunity yet poor infection (cipi) mutant phenotype upon challenge with an adapted PM isolate. Moreover, the eds1/pad4/sid2/mlo2/mlo6/mlo12 (eps3m) sextuple and the eds1/pad4/sid2/pen1/pen2/pen3/mlo2/mlo6/mlo12 (eps3p3m) nonuple mutants displayed near-complete immunity to adapted and nonadapted PM fungi without signs of defense activation, further strengthening the inference that these 3 clade V MLOs in Arabidopsis may be bona fide host susceptibility factors of PM fungi. Confocal imaging revealed focal accumulation of MLO2-GFP in the peri-penetration peg membranous space, which occurs before and may be required for haustorium differentiation. Ectopic leaf expression analyses of 8 other MLOs belonging to different clades showed that only MLO7 can complement the loss of MLO2, MLO6, and MLO12. Results from domain-swapping analyses between MLO1 and MLO2 suggest a bipartite functional configuration for MLO2: its cytoplasmic C-terminus determines where and when MLO2 functions, while its N-terminal 7 transmembrane domain-containing region executes the cellular function that is critical for PM pathogenesis. Genetic studies further demonstrated that, unlike MLO7 in synergids, focal accumulation of MLO2 does not depend on FERONIA (FER) and its 5 paralogs. Together, these findings define clade V MLOs as host factors co-opted by obligate biotrophic PM fungi for successful host colonization.

Protein phosphatase 5 (PP5) is a conserved serine/threonine phosphatase regulating growth, stress responses, programmed cell death and immunity across eukaryotes. However, the mechanisms underlying its activation remain poorly understood. Here we demonstrate that the disruption of the plant MEKK1-MKK1/2-MPK4 cascade activates LET7, a plant homologue of PP5, triggering nucleotide-binding leucine-rich repeat (NLR) SUMM2-mediated autoimmunity in Arabidopsis. The binding of LET7 to the co-chaperone protein HOP1 disrupts the autoinhibitory interaction between the tetratricopeptide repeat and phosphatase domains of LET7 in releasing its phosphatase activity. Activated LET7 subsequently dephosphorylates CRCK3, a crucial kinase regulating SUMM2 autoimmunity. Furthermore, HOP1 and LET7 stabilize SUMM2 via their tetratricopeptide repeat domains, highlighting the dual role of the HOP1-LET7 module in dephosphorylating CRCK3 and stabilizing SUMM2 in NLR-mediated immunity. Our studies reveal a conserved mechanism of PP5 activation across plants and animals and elucidate a unique dephosphorylation cascade governing NLR activation.

Roots, composed of diverse cell types across longitudinal developmental zones, are vital for plant survival against microbial challenges. Leveraging single-cell transcriptomics and live-cell imaging of Arabidopsis roots, we reveal here that plant-derived phytocytokines elicit more potent immune responses than microbe-derived patterns across root cell types and zones. The differential expression of receptors and key signaling modules in distinct cell types and zones contributes to the response intensity to specific elicitors. Phytocytokines sustain growth-defense trade-offs by suppressing the expression of receptor-like kinase genes associated with root cell division and elongation. The intensity of immune responses in different root zones is associated with fungal and bacterial pathogen invasion sites. Furthermore, motif-informed network inference highlighted key transcriptional regulators driving cell identity-specific transcriptomic immune responses. Our study provides a comprehensive landscape of transcriptional responses in plant roots in response to diverse immune elicitors, highlighting how distinct phytocytokines orchestrate stage- and cell type-dependent transcriptional reprogramming.

The receptor-like cytoplasmic kinase BIK1 and its close homologue PBL1 have been widely recognized as central components of plant immunity. However, most genetic studies of BIK1 and PBL1 functions were carried out with single transfer DNA (T-DNA) insertional mutant alleles. Some phenotypes observed in these mutants, for example autoimmunity, have been difficult to reconcile with the proposed role of BIK1 and PBL1 in pattern-triggered immunity. In this study, we generated several new alleles of bik1 and pbl1 by CRISPR-Cas9-based gene editing and systematically analysed these mutants alongside existing T-DNA insertional lines. These analyses reinforced the central role of BIK1 and PBL1 in pattern-triggered immunity mediated by both receptor kinases and receptor-like proteins. At the same time, however, we revealed several pleiotropic phenotypes associated with T-DNA insertions that are not necessarily linked to loss of BIK1 or PBL1 function. Further analyses of newly generated bik1 pbl1 double mutants uncovered an even greater contribution of these kinases to immune signalling and disease resistance than previously appreciated. These findings clarify longstanding ambiguities surrounding BIK1 and PBL1 functions.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors detect pathogen effectors and activate immunity1. Coiled-coil NLRs (CNLs) form resistosomes as Ca2+-permeable channels in the plasma membrane (PM)2-4. However, the mechanism by which resistosomes activate cell death remains unclear. Here we report that the CNL SUPPRESSOR OF mkk1 mkk2 2 (SUMM2), unlike canonical CNLs that use a MADA motif to penetrate the PM5, tethers to the PM through N-myristoylation, a common feature among many CNLs. PM targeting via N-myristoylation is essential for SUMM2-induced cell death. Upon activation, SUMM2 promotes the association of the lipase-like proteins ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4) with the helper NLR-ACTIVATED DISEASE RESISTANCE 1-LIKE 1 (ADR1-L1). Furthermore, active SUMM2 induces the clustering of multiple ADR1-L1 resistosomes into a ring-like assembly colocalized with the EDS1-PAD4 complex, and the EDS1-PAD4-ADR1 module is essential for SUMM2-activated cell death. Together, these findings reveal that N-myristoylation-mediated PM targeting of SUMM2 promotes the assembly of higher-order EDS1-PAD4-ADR1-L1 resistosome clusters for cell death initiation.

The plant immune system senses infections primarily through two branches of immune receptors: cell surface-resident pattern-recognition receptors (PRRs) and intracellular NOD-like receptors (NLRs). Although distinct in perception and activation, PRR and NLR signaling are interconnected and mutually regulated. A major class of PRRs, receptor kinases (RKs), often activate intracellular kinases, including receptor-like cytoplasmic kinases (RLCKs) and mitogen-activated protein kinases (MAPKs). Some RLCKs act as decoys, activating NLRs upon pathogen effector recognition. Recent advances expand the repertoire of kinases, including RKs, tandem kinase proteins, and calcium-dependent protein kinases, by directly activating or suppressing NLRs through phosphorylation. Furthermore, PRR-regulated RKs and MAPKs play critical roles in restraining NLR activity to maintain immune homeostasis. In response to pathogen perturbations, plants mobilize backup surveillance mechanisms involving RKs and RLCKs to derepress NLR immunity. This review highlights recent advances in the dynamic interplay between PRR and NLR signaling, focusing on protein kinases.

Bacterial transcription activator-like effectors (TALEs) promote pathogenicity by activating host susceptibility (S) genes. To understand the pathogenicity and host adaptation of Xanthomonas citri pv. malvacearum (Xcm), we assemble the genome and the TALE repertoire of three recent Xcm Texas isolates. A newly evolved TALE, Tal7b, activates GhSWEET14a and GhSWEET14b, different from GhSWEET10 targeted by a TALE in an early Xcm isolate. Activation of GhSWEET14a and GhSWEET14b results in water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identify a pectin lyase gene as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9 gene editing supports the function of GhSWEETs in cotton bacterial blight and the promise of disrupting the TALE-binding site in S genes for disease management. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes to promote pathogenicity. Newly evolved Xanthomonas citri pv. malvacearum isolates triggers recent bacterial blight outbreaks in cotton. Here, the authors show that a recently evolved TALE, Tal7b, activates host susceptibility genes GhSWEET14a and GhSWEET14b rather than GhSWEET10 to confer pathogenicity in these new isolates.

Obligate biotrophic powdery mildew (PM) fungi strictly require living host to survive. To search for host factors or processes essential for PM pathogenesis, a tailored genetic screen was conducted with the immuno-compromised eds1-2/pad4-1/sid2-2 ( eps ) triple Arabidopsis mutant. This led to the identification of five allelic disruptive mutations in Mildew Locus O 2 ( MLO2 ) to be responsible for the compromised-immunity-yet-poor infection (cipi) mutant phenotype upon challenge from an adapted PM isolate. Moreover, the eds1/pad4/sid2/mlo2/mlo6/mlo12 ( eps3m ) sextuple mutant display near complete immunity to the adapted PM fungus without sign of defense activation, demonstrating that these three clade V MLOs in Arabidopsis are bona fide host susceptibility factors of PM fungi. Confocal imaging revealed focal accumulation of MLO2- GFP in the peri-penetration peg membranous space, implicating MLO2 in repairing and stabilizing the damaged host plasma membrane, which may be co-opted by PM fungi for haustorium differentiation. Results from domain-swapping analysis between MLO1 and MLO2 suggest a bipartite functional configuration for MLO2: its C-terminus determines where and when MLO2 functions, while its N-terminal seven transmembrane domain region executes the cellular function that is critical for PM pathogenesis. Genetic studies further demonstrate that, unlike MLO7 in synergids, focal accumulation of MLO2 does not depend on FERONIA (FER) and its five other family members, nor does it require phosphatidylinositol 4,5-bisphosphate produced from phosphatidylinositol 4-phosphate 5-kinase 1 (PIP5K1) and PIP5K2. Together, these findings define clade V MLOs as host factors co-opted by obligate biotrophic PM fungi for successful host colonization. ### Competing Interest Statement The authors have declared no competing interest. National Science Foundation, https://ror.org/021nxhr62, IOS-1901566, IOS-2224203

Alternative splicing of precursor mRNAs serves as a crucial mechanism to enhance gene expression plasticity for organismal adaptation. However, the precise regulation and function of alternative splicing in plant immune gene regulation remain elusive. Here, by deploying in-depth transcriptome profiling with deep genome coverage coupled with differential expression, differential alternative splicing, and differential transcript usage analysis, we reveal profound and dynamic changes in alternative splicing following treatment with microbial pattern flg22 peptides in Arabidopsis. Our findings highlight RNA polymerase II C-terminal domain phosphatase-like 3 (CPL3) as a key regulator of alternative splicing, preferentially influencing the splicing patterns of defense genes rather than their expression levels. CPL3 mediates the production of a flg22-induced alternative splicing variant, diacylglycerol kinase 5α (DGK5α), which differs from the canonical DGK5β in its interaction with the upstream kinase BIK1 and subsequent phosphorylation, resulting in reduced flg22-triggered production of phosphatidic acid and reactive oxygen species. Furthermore, our functional analysis suggests that DGK5β, but not DGK5α, contributes to plant resistance against virulent and avirulent bacterial infections. These findings underscore the role of CPL3 in modulating alternative splicing dynamics of defense genes and DGK5 isoform-mediated phosphatidic acid homeostasis, shedding light on the intricate mechanisms underlying plant immune gene regulation.

After cellular damage caused by wounding or pathogens, Arabidopsis thaliana endogenous elicitor peptides (Peps) are released into the apoplast, enhancing innate immunity by directly binding to the membrane-localized leucine-rich repeat receptor kinase PEP RECEPTOR1 (PEPR1). Ligand binding induces PEPR1 heterodimerization with the co-receptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1), followed by PEPR1 internalization, both essential for a subset of Pep1-induced responses. However, the role of BAK1 in Pep1-triggered PEPR1 endocytosis remains unclear. Here, we show that the ligand-induced PEPR1 endocytosis depends on its kinase activity and requires BAK1 C-terminal tail phosphorylation, which is equally indispensable for immune signaling and BAK1 internalization. Using a GFP insertional mutagenesis approach, we generated a partially functional GFP-tagged BAK1 to demonstrate that, following Pep1 elicitation, BAK1 and PEPR1 are endocytosed together with similar dynamics. Our findings identify the BAK1 function as a prerequisite for PEPR1 internalization.

"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."

Plasma membrane-resident receptor kinases (RKs) are crucial for plants to sense endogenous and exogenous signals in regulating growth, development, and stress response. Upon perception of ligands by the extracellular domain, RKs are usually activated by auto- and/or trans-phosphorylation of the cytoplasmic kinase domain, which in turn phosphorylates downstream substrates to relay the signaling. Therefore, monitoring ligand-induced in vivo phosphorylation dynamics of RKs and their associated proteins provides mechanistic insight into RK activation and downstream signal transduction. Phos-tag specifically binds phosphomonoester dianions of phosphorylated serine, threonine, and tyrosine residues, which enables Phos-tag-containing SDS-PAGE gels to separate phosphorylated proteins from non-phosphorylated form. Here, we describe a detailed method of Mn2+-Phos-tag SDS-PAGE analysis to detect the ligand-induced in vivo phosphorylation of RKs and associated proteins.

Plant immune homeostasis is achieved through a balanced immune activation and suppression, enabling effective defense while averting autoimmunity. In Arabidopsis, disrupting a mitogen-activated protein (MAP) kinase cascade triggers nucleotide-binding leucine-rich-repeat (NLR) SUPPRESSOR OF mkk1/2 2 (SUMM2)-mediated autoimmunity. Through an RNAi screen, we identify PUB5, a putative plant U-box E3 ligase, as a critical regulator of SUMM2-mediated autoimmunity. In contrast to typical E3 ligases, PUB5 stabilizes CRCK3, a calmodulin-binding receptor-like cytoplasmic kinase involved in SUMM2 activation. A closely related E3 ligase, PUB44, functions oppositely with PUB5 to degrade CRCK3 through monoubiquitylation and internalization. Furthermore, CRCK3, highly expressed in roots and conserved across plant species, confers resistance to Fusarium oxysporum, a devastating soil-borne fungal pathogen, in both Arabidopsis and cotton. These findings demonstrate the antagonistic role of an E3 ligase pair in fine-tuning kinase proteostasis for the regulation of NLR-mediated autoimmunity and highlight the function of autoimmune activators in governing plant root immunity against fungal pathogens.

In plant immunity, a well-orchestrated cascade is initiated by the dimerization of receptor-like kinases (RLKs), followed by the phosphorylation of receptor-like cytoplasmic kinases (RLCKs) and subsequent activation of NADPH oxidases for ROS generation. Recent findings by Zhong et al. illustrated that a maize signaling module comprising ZmWAKL-ZmWIK-ZmBLK1-ZmRBOH4 governs quantitative disease resistance to grey leaf spot, a pervasive fungal disease in maize worldwide, unveiling the conservation of this signaling quartet in plant immunity.

Foliar pathogens exploit natural openings, such as stomata and hydathodes, to invade plants, multiply in the apoplast, and potentially spread through the vasculature. To counteract these threats, plants dynamically regulate stomatal movement and apoplastic water potential, influencing hydathode guttation and water transport. This review highlights recent advances in understanding how phytocytokines, plant small peptides with immunomodulatory functions, regulate these processes to limit pathogen entry and proliferation. Additionally, we discuss the coordinated actions of stomatal movement, hydathode guttation, and the vascular system in restricting pathogen entry, multiplication, and dissemination. We also explore future perspectives and key questions arising from these findings, aiming to advance our knowledge of plant immunity and improve disease resistance strategies.