Plant cell surface pattern recognition receptors (PRRs) perceive non- or altered-self elicitors to induce immune responses. PRRs relay information across the plasma membrane and trigger downstream signalling via receptor-like cytoplasmic kinases such as BOTRYTIS-INDUCED KINASE 1 (BIK1). BIK1 associates with several PRRs and acts as a key executor of immune responses through the phosphorylation of substrate proteins. However, a comprehensive understanding of how BIK1 targets specific substrates and a full repertoire of these substrates are lacking. Here we defined the substrate specificity of BIK1 and used these data to predict candidate substrates in Arabidopsis. Using high-throughput biochemical and genetic screening of these candidates, we confirmed many as direct BIK1 substrates in vitro and novel regulators of plant immunity. Among the BIK1 substrates identified are MULTIPLE C2 DOMAIN AND TRANSMEMBRANE REGION PROTEIN 3, which we reveal regulates flagellin 22 (flg22)-induced plasmodesmata closure and immunity, and members of the largely uncharacterized CYCLIN-DEPENDENT KINASE-LIKE family, which we uncover as novel negative regulators of immunity. In parallel, we interrogated intracellular NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT (NLR) immune receptors for potential BIK1 phosphorylation motifs and identified multiple NLRs as direct BIK1 substrates. We reveal that BIK1 phosphorylation regulates NLR oligomerization, thus controlling a key activation step for these immune receptors. Together, our unbiased biochemical screens shed light on the central role of BIK1 as a key kinase shaping multiple layers of plant immune signalling. Cell surface receptors perceive immunogenic elicitors, triggering downstream signalling via receptor-like cytoplasmic kinases such as BIK1. Here the authors define and use the phosphorylation motif of BIK1 to find novel substrate candidates.

Plant V-ATPase serves as a primary active proton pump of the endomembrane system and is crucial for the stress response. However, the role of the C subunit of V-ATPase (VHA-C) in effector-triggered immunity remains poorly understood. Here, we reveal that Phytophthora infestans evolved a pair of RxLR effectors, AL3 and Avr2, which are expressed sequentially and both target the host VHA-C (StATP6V1C1) and StBSL1. In the early stage of P. infestans infection, AL3 promotes the assembly of StATP6V1C1 with subunits G and E, leading to increased V-ATPase activity and cytoplasmic acidification. Subsequently, Avr2 inhibits the StWNK10-catalyzed Ser-261 phosphorylation of StATP6V1C1, thereby retarding V-ATPase activity and causing intracellular alkalinization. In cultivars absence of two immune receptors, this pH shift facilitates the interactions of the two effectors with downstream susceptibility factors of StBSL1 at various stages of infection, which may promote the onset and development of the disease. As coping strategy, plants independently evolve two NLRs, R2 and Rpi-mcq1, guard both StATP6V1C1 and StBSLs to perceive effectors thereby mitigating the risk of late blight. Our findings establish a new arms race battlefield between plants and oomycetes, highlighting the role of intracellular pH homeostasis in both effector-triggered susceptibility (ETS) and effector-triggered immunity (ETI).

The advent of single-cell genomic technologies has revolutionized plant cell biology by revealing cellular heterogeneity in plant tissues and organs with unprecedented resolution. Methods like single-cell transcriptomics, epigenomics, and multi-omics integration have deepend insights into molecular mechanisms governing plant development, function, and evolutionary adaptations (Xu and Jackson, 2025). Notably, these approaches have facilitated comprehensive cell atlases mapping gene expression, chromatin accessibility, and regulatory networks across species (Wang et al., 2025; Guo et al., 2025), identifying marker genes, differentiation trajectories, and spatiotemporal regulatory in processes like root development, and seed germination (Zhang et al., 2021; Yao et al., 2024). However, proliferating single-cell studies face a major bottleneck from inconsistent cell type annotations, which impede data integration and crossspecies comparison. A unified, phylogenetically informed atlas of plant cellular diversity is essential for elucidating molecular developmental and evolutionary principles (Sebé-Pedrós et al., 2025).

• The morphology of starch granules is a major determinant of the functional and nutritional properties of starch and is highly variable among cereal species. Much of this morphological variation stems from differences in the spatial and temporal patterns of starch granule initiation in amyloplasts during grain development. Simple granules are thought to arise from a single initiation per amyloplast (e.g. in Brachypodium distachyon), whereas compound granules develop from multiple initiations per amyloplast (e.g. in rice).
• We used live-cell imaging to visualise amyloplasts in the developing endosperm of Brachypodium, using transgenic lines expressing a fluorescent amyloplast reporter.
• We discovered that the simple-type starch granules in Brachypodium can arise from multiple initiations per amyloplast. The amyloplasts showed dynamic changes in their structure and formed two types of stromules: stable stromules that formed a stromal continuum between amyloplasts, and short-lived stromules that were more dynamic. We also observed actin-dependent movement of amyloplasts within endosperm cells, and movement of starch granules within the amyloplasts.
• Our results suggest complex and pleiomorphic amyloplast organisation and mobility that could influence granule formation. This goes beyond the existing ‘one granule, one amyloplast’ model for simple-type granules and advances our understanding of both amyloplast biogenesis and granule formation.

Following the perception of pathogen virulence proteins in plants, nucleotide-binding and leucine-rich repeat immune receptors (NLRs) are activated via a wide range of mechanisms. Singleton NLRs can both perceive effectors and trigger an immune response, whereas other NLRs specialise in either pathogen recognition (sensor NLRs) or activation of the immune response (helper NLRs). Sensor and helper NLRs can function as genetically linked pairs or in unlinked receptor networks. Although growing evidence suggests that NLRs conditionally oligomerise upon activation, our understanding of the resting state of NLRs prior to effector perception remains limited. Here, we investigated the oligomeric state of the genetically linked rice (Oryza sativa) sensor Pik-1 and helper Pik-2 NLR pair prior to effector activation when transiently expressed in Nicotiana benthamiana leaves. We show that both wild-type Pikm-1 and engineered Pikm-1Enhancer sensors associate with Pikm-2 and form ∼1 MDa hetero-complexes in the resting state that accumulate at the plasma membrane. Our findings contribute to the growing evidence that pre-activation mechanisms vary widely across NLRs. This knowledge could be leveraged for disease resistance engineering strategies complementary to approaches focussing solely on effector binding.

Transposon silencing via RNA-directed DNA methylation (RdDM) is mediated primarily through 24-nucleotide (nt) small interfering RNAs (siRNAs) and two plant-specific RNA polymerases, Pol IV and Pol V. We generated and characterized RNA-interference (RNAi) lines targeting the largest subunit of Pol IV and Pol V and the second-largest subunit shared by Pol IV and Pol V in soybean. Our analyses showed that the canonical roles of Pol IV and Pol V in RdDM as described in Arabidopsis, whereby Pol IV produces 24-nt siRNAs and Pol V recruits siRNAs to homologous loci to trigger DNA methylation, are conserved in soybean. Our analyses also uncovered functions of Pol IV and Pol V in that they repress defense response genes en masse. These genes normally undergo RdDM and are silenced, but they are de-repressed when Pol IV and Pol V are knocked down. Furthermore, the de-repression of a set of defense-related genes channeled their RNAs into the RNAi pathway to produce 21-22-nt siRNAs. Knocking down Pol IV and Pol V either singly or together led to increased resistance against the oomycete pathogen Phytophthora sojae, suggesting that Pol IV- and Pol V-mediated gene silencing regulates plant immunity.

Herbivorous insects can shape the epidemiology of disease in plants by vectoring numerous phytopathogens. While the consequences of infection are often well-characterized in the host plant, the extent to which phytopathogens alter the physiology and development of their insect vectors remains poorly understood. In this review, we highlight how insect-borne phytopathogens can promote vector fitness, consistent with theoretical predictions that selection should favor a mutualistic or commensal phenotype. In doing so, we define the metabolic features predisposing plant pathogens to engage in beneficial partnerships with herbivorous insects and how these mutualisms promote the microbe's propagation to uninfected plants. For the vector, the benefits of co-opting microbial pathways and metabolites can be immense: from balancing a nutritionally deficient diet and unlocking a novel ecological niche to upgrading its defensive biochemistry against natural enemies. Given the independent origins of these tripartite interactions and a number of convergent features, we also discuss the evolutionary and genomic signatures underlying microbial adaptation to its dual lifestyle as both a plant pathogen and an insect mutualist. Finally, as host association can constrain the metabolic potential of microbes over evolutionary time, we outline the stability of these interactions and how they impact the virulence and transmission of plant pathogens.

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. Thorough genetic analyses by four laboratories reinforce the central role of the protein kinase BIK1 in plant immunity. Other unrelated functions proposed for BIK1, however, are associated with genome rearrangement in a single transfer DNA mutant.

Nucleotide-binding leucine-rich repeat (NLR) immune receptors sense pathogen molecules and oligomerize, initiating defense signaling. Some NLRs function poorly at elevated temperatures for unknown reasons. We show that temperature-sensitive NLRs retain ligand binding at elevated temperatures but are impaired in oligomerization. We identify key residues involved in temperature resilience. Structural modeling reveals stabilizing intramolecular interactions of the NB-ARC domain with surface residues of the adjacent leucine-rich repeat (LRR) that preserve receptor integrity and functionality under heat stress. These insights enable in silico classification of NLRs as temperature-sensitive or -tolerant and underpin design of temperature tolerant variants of temperature sensitive NLRs.

These findings provide a mechanistic basis for temperature sensitivity in plant immune receptors and enable engineering of temperature-tolerant disease resistance in crops.

Subjects Bacterial techniques and applicationsFungal biologySymbiosis Eukaryotes often host prokaryotic cells inside their own cells. Hosts of such ‘endosymbionts’ can rely on these organisms for metabolism or the production of other necessary molecules.