Liquid–liquid phase separation at the frontline of plant–pathogen battles

Abstract

Liquid–liquid phase separation (LLPS) underlies the formation of biomolecular condensates that organize proteins and RNAs without membrane boundaries. In the ongoing arms race between plants and pathogens, LLPS has emerged as a central regulatory mechanism. Pathogens exploit biomolecular condensates to build replication factories or redirect host factors, while plants leverage LLPS to coordinate immune signaling, RNA silencing, and stress adaptation. Recent studies depict a “biomolecular condensate battleground” where the outcome of infection hinges on the dynamic balance between pathogen-induced and host-driven assemblies. In this review, we integrate recent advances in understanding LLPS mediated by viral, fungal, bacterial, and oomycete effectors, along with key host immune regulators. We also discuss the potential of biomolecular condensate engineering as a novel strategy for crop protection and outline the major conceptual and technical challenges that remain. By connecting molecular biophysics with plant pathology, we propose that targeting LLPS offers a timely and promising route for developing durable disease resistance and improving sustainable agricultural productivity.

This review highlights liquid–liquid phase separation (LLPS) as a unifying mechanism that shapes pathogen effector functions, host immune signaling, and antiviral RNA processes. It underscores the dual roles of biomolecular condensates in defense and disease tolerance, offering new perspectives on how LLPS could be leveraged for sustainable crop improvement.

Introduction

Phase separation (PS) is a physicochemical process in which solute molecules demix into two distinct phases with different concentrations. In biological systems, PS can occur in several forms, including liquid–liquid phase separation (LLPS), liquid–gel transition, and liquid–solid transitions (Brangwynne et al., 2009; Wu and Fuxreiter, 2016). LLPS typically generates dynamic, fluid-like condensates that allow rapid molecular exchange with the surrounding environment, whereas gel-like or solid-like assemblies display reduced mobility and often represent aged or transitional states of LLPS (Molliex et al., 2015). Growing evidence suggests that LLPS underlies the formation of diverse membraneless organelles, such as the nucleolus, processing bodies (PBs), stress granules (SGs), and Cajal bodies (CBs), which are collectively known as biomolecular condensates (Banani et al., 2017; Alberti et al., 2019). These condensates assemble through multivalent interactions among proteins and RNAs, driven by structured domains and intrinsically disordered regions (IDRs) or low-complexity domains (LCDs), which together generate weak, dynamic interactions that determine their physical properties and biological functions (Li et al., 2012; Nott et al., 2015). Similar to their functions in animal systems, biomolecular condensates in plants participate in diverse biological processes, including signal transduction, protein trafficking, and immune responses (Ouyang et al., 2020; Wang et al., 2021; Zhou et al., 2023). Unlike membrane-bound organelles that rely on physical barriers for compartmentalization, sessile plants that continuously face environmental fluctuations are more likely to exploit LLPS for rapid adaptation (Wang et al., 2022; Liu et al., 2024; Ma et al., 2025). By enabling fast assembly of dynamic condensates in response to external cues, LLPS allows local enrichment of specific proteins or RNAs and thereby triggering timely stress responses (Hyman et al., 2014; Banani et al., 2017; Ouyang et al., 2020).

Plants encounter a wide range of environmental challenges, including biotic and abiotic stresses, which together impose substantial yield losses and threaten global food security (Raza et al., 2019). Biotic stresses, in particular, can overstimulate immune responses, leading to excessive defense signaling, tissue damage, and growth inhibition (Huot et al., 2014). Recent perspectives have refined the classical growth–defense trade-off model by emphasizing that growth inhibition is not an automatic consequence of immunity but instead reflects context-dependent decisions balancing metabolic costs, hormonal crosstalk, and developmental priorities (Gao et al., 2024; Hou and Xu, 2025). Maintaining immune homeostasis is therefore essential for plant health and productivity. Biomolecular condensates have recently emerged as important regulators in this process, providing a dynamic platform for the spatial and temporal organization of immune signaling components (Tang et al., 2025a; Huang and Dong, 2025). Understanding how these condensates contribute to the balance between defense and growth is thus crucial for developing resilient crop systems.

Beyond classical disease resistance, disease tolerance has recently gained recognition as a complementary defense strategy that helps maintain plant health by limiting infection-associated damage rather than preventing pathogen entry. Recent studies show that plants achieve tolerance by mitigating stress-induced damage such as protein condensation and endoplasmic reticulum stress, thereby preserving cellular homeostasis and productivity during infection (Tang et al., 2024, 2025a, 2025b). These findings position disease tolerance as a major determinant of plant survival under viral attack and suggest that LLPS may serve as a central mechanism linking immune signaling to cellular homeostasis during tolerance responses.

A recent review by Huang and Dong (2025) provided a comprehensive overview of biomolecular condensates in plant immunity, with a particular focus on how condensate dynamics shape the strength and specificity of immune signaling. Building on this foundation, our review offers an updated synthesis that examines the molecular interplay between pathogen effectors and host biomolecular condensates and explores how LLPS contributes to the balance between resistance and disease tolerance. To guide readers through this topic, we first outline current knowledge of how pathogen effectors from viruses, fungi, and bacteria exploit LLPS to promote infection. We then discuss how host-derived condensates modulate immune signaling pathways to coordinate defense activation while maintaining cellular homeostasis. Finally, we highlight emerging technologies for probing condensate dynamics, consider potential applications of LLPS modulation in crop protection, and discuss key remaining challenges. This review focuses on the biological relevance and regulatory functions of LLPS in plant immunity rather than providing an exhaustive account of its molecular and biophysical underpinnings, to maintain clarity and thematic focus.

Phase separation of effector proteins in plant pathogens

Upon entering host cells, pathogens rapidly accumulate nucleic acids and proteins, creating a microenvironment conducive to LLPS. Many plant RNA viruses, for example, induce the formation of viral replication factories (VRFs) or inclusion bodies (IBs)—membraneless structures with liquid-like properties. Viral RNAs, viral replicase proteins (e.g., RdRp), and host factors—including RNA-binding proteins, molecular chaperones, and lipids—coalesce into highly concentrated compartments through LLPS. These condensates markedly promote viral replication and assembly by concentrating essential components, protecting viral RNA from host nucleases, and shielding it from immune surveillance. Well-documented examples include barley yellow striate mosaic virus (BYSMV; a negative-sense RNA virus), tomato bushy stunt virus (TBSV), and carnation Italian ringspot virus (CIRV), whose proteins form condensates indispensable for viral replication and pathogenicity (Fang et al., 2022; Lin and Nagy, 2024). Extending this paradigm to plant negative-strand RNA viruses, the phosphoprotein P of rice stripe mosaic virus (RSMV) forms LLPS condensates that hijack the host ARF1C–PI4KB pathway to generate phosphatidylinositol 4-phosphate (PI4P), thereby expanding PI4P-enriched viral factories and boosting replication (Wang et al., 2025). In some cases, viral proteins further exploit LLPS to recruit or reorganize host factors to enhance viral replication. A striking example is the N protein of tomato yellow mottle-associated virus (TYMaV), which co-assembles with the viral phosphoprotein into liquid-like IB condensates. The dynamic fusion of these condensates depends on the actin–myosin cytoskeleton, and inhibition of this motility effectively blocks viral replication (Liang et al., 2023).

Beyond viruses, a subset of fungal, oomycete, and bacterial pathogens also secrete effectors that undergo LLPS within host cells. This ability is often mediated by IDRs or oligomerization motifs that enable multivalent interactions. For instance, the bacterial type III effector XopR from Xanthomonas campestris forms LLPS-driven condensates through multivalent IDR interactions to remodel the host actin cytoskeleton and suppress immunity (Sun et al., 2021). Similarly, the fungal effector FolSvp2 from Fusarium oxysporum undergoes K205 acetylation-dependent stabilization and forms condensates that sequester the tomato iron-sulfur protein SlISP, thereby reducing reactive oxygen species (ROS) production and weakening host immunity (Li et al., 2024). Structural prediction analyses further suggest that Tin2-like effectors from Ustilago maydis contain glycine- and proline-rich IDRs that may facilitate LLPS (Seong and Krasileva, 2023).

Although the biochemical activities of many effectors are well characterized, their spatiotemporal dynamics and condensation behavior have received far less attention than those of viral proteins. This gap is partly due to the biological properties of these effectors and the technical challenges associated with studying them. Viral genomes are generally smaller and easier to manipulate, allowing rapid dissection of LLPS-related mechanisms. In contrast, the complex secretion systems of bacteria and the extensive effector diversity in fungi make it considerably more difficult to analyze and validate the roles of intrinsically disordered regions (IDRs) in their phase behavior.

Recent advances in in silico structure prediction have begun to mitigate these challenges. Large-scale AlphaFold2-based modeling of 26,653 secreted proteins from diverse fungi and an oomycete revealed that the Ustilago maydis Tin2-like effector cluster is enriched in glycine- and proline-rich IDRs that are intrinsically disordered and lack stable secondary structures (Seong and Krasileva, 2023). Such LLPS-prone effectors may increase their stability or activity while sequestering host immune proteins—including nucleotide-binding leucine-rich repeat (NLR) receptors and RNA surveillance factors—to suppress defense signaling (Li et al., 2025; Ma et al., 2025). Table 1 provides a summary of known plant pathogen effectors exhibiting LLPS characteristics. Growing evidence indicates that the ability of effectors to oligomerize or form higher-order condensates is tightly associated with virulence, positioning LLPS as a critical determinant of pathogenic function across diverse pathogen taxa.

Table 1.

LLPS proteins in plant pathogens and their functions.

Plant pathogens	Key protein	Condensate components and molecular function	Infection consequences	References
PVA	HC-Pro	forms PG condensates containing P0, AGO1, UBP1, VCS, and eIF(iso)4E; involved in RNA granule formation and translation control.	VPg suppresses PG abundance; HC-Pro/VCS promote, while UBP1 inhibits, VPg-driven translation.	(Hafrén et al., 2015)
BYSMV	P	P protein forms granules recruiting N and L (RdRp), tethered to the ER/actin network.	CK1 blocks P condensation; dephosphorylated P promotes viroplasm replication.	(Fang et al., 2022)
PEMV2	p26	forms nucleolar condensates via fibrillarin (Fib2) interaction; partitions into G3BP-SGs.	SG formation limits viral spread; G3BP upregulation strengthens antiviral response.	(Brown et al., 2023)
TYMaV	N, P	N and P co-assemble into condensates moving along microfilaments via myosin XI.	condensate growth and replication require microfilament-myosin XI.	(Liang et al., 2023)
TBSV	p33 (RdRp)	IDR-driven droplets sequester glycolytic/fermentation enzymes; recruit SUMO machinery to VROs.	VRO condensates use ATP and SUMOylation to enhance replication.	(Lin and Nagy, 2024; 2025)
CIRV	p36 (RdRp)	IDR-driven droplets with metabolic enzymes at VROs.	provide local ATP to support viral replication.	(Lin and Nagy, 2024)
RSV	SP	interacts with serrate to interfere with D-body LLPS.	disrupts miRNA processing and promotes infection.	(Zou et al., 2025)
RSMV	P	P undergoes LLPS, recruits ARF1C and PI4KB for PI4P production.	expands replication sites and enhances viral replication.	(Wang et al., 2025)
TSWV	N	N co-localizes with PBs, SGs, and RanGAP2; compound Z9 disrupts N-RNA condensates.	disruption of condensates reduces RNP stability and replication.	(Xu et al., 2022; Zan et al., 2025)
TuMV	HC-Pro	recruits HEN1 and ATG8a to HC-Pro bodies; associates with AGO1.	links autophagy, miRNA methylation, and RNA silencing control.	(Pan et al., 2025)
X. campestris	XopR (T3E)	IDR-mediated LLPS remodels host actin cytoskeleton.	hijacks actin dynamics to suppress host immunity.	(Sun et al., 2021)
Fol	FolSvp2	forms condensates sequestering SlISP via phase separation.	SlISP sequestration reduces ROS; SlPR1 opposes FolSvp2 entry.	(Li et al., 2024)

Despite these advances, most existing studies rely heavily on transient overexpression systems or in vitro droplet assays, raising concerns about whether the observed assemblies represent physiologically relevant LLPS or stress-induced artifacts. The existence and functional significance of effector condensates during natural infection therefore remain to be validated. It is also unclear whether effector condensation is mechanistically required for virulence or merely correlates with other infection-associated molecular changes. Moreover, host cellular parameters—including ionic strength, macromolecular crowding, and pH—are likely to strongly influence effector phase behavior in planta, yet these factors are rarely incorporated into experimental designs. Future studies integrating live-cell imaging, quantitative biophysics, and stage-specific genetic perturbations will be essential to determine whether condensates observed under overexpression conditions also form and function in native physiological contexts, thereby distinguishing mechanistic necessity from correlation.

Together, these examples illustrate that phase separation represents a versatile strategy employed by diverse pathogens to spatially reorganize virulence factors within host cells. In the following section, we shift to the host perspective and examine how plants themselves exploit biomolecular condensate formation to organize immune signaling networks.

Phase separation mechanisms in plant immunity

Plants, as sessile organisms, are continuously exposed to diverse environmental stresses and microbial pathogens. To cope with these challenges, they have evolved a multilayered immune system that is rapid, robust, and highly adaptable. The canonical immune framework consists of two interconnected layers: pattern-triggered immunity (PTI), which is activated by pattern recognition receptors (PRRs) at the plasma membrane upon perception of pathogen-associated molecular patterns (PAMPs), and effector-triggered immunity (ETI), which is activated when intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) recognize pathogen-secreted effectors. LLPS has recently emerged as a key mechanism contributing to both PTI and ETI by enabling the dynamic assembly or disassembly of immune signaling complexes during infection.

In addition to canonical PTI and ETI signaling, plants also rely on RNA-mediated immune mechanisms and cytoplasmic biomolecular condensates—such as SGs and PBs—to defend against pathogens. RNA silencing components—including SGS3, RDR6, and AGO1—can assemble into LLPS-driven bodies that amplify antiviral responses and maintain RNA homeostasis (Tan et al., 2023; Blagojevic et al., 2024). Likewise, SGs and PBs dynamically regulate mRNA storage and decay, acting as regulatory hubs that fine-tune immune gene expression under stress conditions (Kearly et al., 2024). Together, these multilayered defense strategies underscore the diverse ways in which biomolecular condensates coordinate plant immunity.

Biomolecular condensates in PTI and ETI

PTI is typically initiated when PRRs detect conserved microbial molecules, such as bacterial flagellin or fungal chitin, thereby triggering broad-spectrum defense responses including Ca²⁺ influx and rapid production of reactive oxygen species (ROS). ETI, in contrast, depends on intracellular NLRs that recognize specific pathogen effectors and elicit stronger defense outputs such as salicylic acid (SA)-mediated systemic acquired resistance (SAR) and localized programmed cell death (PCD; Wang et al., 2023a). Increasing evidence indicates that both PTI and ETI involve the formation of biomolecular condensates through LLPS, which modulates immune signaling strength and specificity (Tang et al., 2025a; Huang and Dong, 2025).

A well-characterized example involves Toll/interleukin-1 receptor (TIR)-type NLRs (TNLs). Upon activation, binding of the substrates NAD⁺ and ATP induces conformational rearrangements in the BB-loop of the TIR domain, promoting head-to-tail interactions and LLPS. These condensates markedly enhance NAD⁺ hydrolase activity, generating immune signaling molecules such as di-ADPR and pRib-AMP. These metabolites are then sensed by the EDS1–PAD4 and EDS1–SAG101 complexes, which activate the helper NLRs ADR1 and NRG1 to form resistosomes that function as Ca²⁺ channels and ultimately trigger hypersensitive response (HR)-mediated cell death. This multistep signaling cascade is shown in Figure 1A. Notably, TIR-driven condensates can also act as 2′3′-cNMP synthases, suggesting an additional regulatory layer within the EDS1 signaling axis (Li et al., 2025).

Figure 1.

Phase separation-mediated regulation of plant immune responses.

This schematic illustrates how biomolecular condensates formed through liquid–liquid phase separation (LLPS) modulate multiple layers of plant immunity during pathogen infection, including both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI).

(A) TIR-type NLR condensates in ETI signaling. Binding of NAD+ or ATP promotes condensation of TIR domains, enhancing NAD⁺ hydrolase activity and generating immune signaling molecules such as pRib-AMP, di-ADPR, and 2′3′-cNMP. These metabolites are sensed by the EDS1–PAD4 and EDS1–SAG101 complexes, which activate ADR1 and NRG1 resistosomes to induce Ca²⁺ influx and hypersensitive response (HR) cell death.

(B) NUP62 condensates facilitate nuclear import. The nucleoporin NUP62 undergoes LLPS to promote nuclear import of MPK3 and MPK6, thereby strengthening basal resistance. SINC-like nuclear condensates (SA-induced NPR1 condensates) help integrate cytoplasmic and nuclear immune signaling networks.

(C) MAPK-regulated P-body remodeling by TZF9. Phosphorylation of TZF9 disrupts P-bodies and releases defense-related mRNAs, alleviating translational repression and promoting PTI activation. These dynamic RNA–protein condensates fine-tune immune output.

(D) Fungal effector condensates in plastids. The Fusarium oxysporum effector FolSvp2 forms acetylation-dependent condensates that sequester the host iron–sulfur protein SlISP, reducing ROS production. The tomato defense protein PR1 (SlPR1) binds FolSvp2 and blocks its condensation, restoring ROS generation and immunity.

(E) SINC condensates in salicylic acid signaling. Accumulation of SA drives the conversion of NPR1 from oligomers to monomers that form CUL3-associated condensates. These structures sequester or ubiquitinate immune regulators such as EDS1 and WRKY factors to modulate HR intensity. Once in the nucleus, NPR1 undergoes SUMOylation and is degraded, amplifying ETI.

(F) HEM1 condensates balance defense and homeostasis. The plant-specific low-complexity domain (LCD) of HEM1 drives condensate formation with translation factors to buffer immune-gene translation and prevent excessive immune activation. During virulent infection, persistent HEM1 condensation at the ER traps lipid-metabolic enzymes and causes ER stress. BI-1- and ATG6-mediated autophagy clears HEM1 condensates, restoring lipid homeostasis and reducing tissue damage. Together, these panels show how phase-separated condensates organize immune signaling at multiple levels—from TIR-NLR activation and nucleocytoplasmic transport to translational control and autophagic clearance—, enabling plants to balance defense, tolerance, and cellular homeostasis.

Beyond TNLs, other immune regulators also exploit LLPS to fine-tune PTI and ETI. For instance, nucleoporin 62 (NUP62), a core component of the nuclear pore complex, undergoes LLPS to promote nuclear import of the immune-related kinase MPK3, thereby enhancing resistance to multiple pathogens including Botrytis cinerea (Wang et al., 2023b) (Figure 1B). Similarly, phosphorylation of the PB component TZF9 by MAPKs reduces its RNA-binding activity, leading to PB disassembly and the release of defense-related mRNAs. This relieves translational repression and promotes PTI activation (Maldonado-Bonilla et al., 2014; Tabassum et al., 2020) (Figure 1C).

Biomolecular condensates are also central to effector–host interactions. During fungal infection, the tomato defense protein PR1 binds the Fusarium oxysporum effector FolSvp2, blocking its endocytosis and preventing condensate formation. This restores access to the hijacked host target SlISP and reactivates ROS production (Li et al., 2024) (Figure 1D). Likewise, the SA receptor NPR1, which contains IDRs, forms SA-induced NPR1 condensates (SINCs) with the CULLIN3 (CUL3)–E3 ligase complex. These condensates sequester and ubiquitinate immune regulators such as NLRs, EDS1, and WRKY54/70, thereby restraining excessive HR (Spoel et al., 2009; Fu et al., 2012; Zavaliev et al., 2020). During ETI, however, increased SA levels trigger NPR1 dephosphorylation and conversion from oligomers to monomers, enabling its nuclear import. In the nucleus, NPR1 is SUMOylated and subsequently degraded by the CRL3^NPR3/NPR4 complex, a process that amplifies HR in infected cells (Spoel et al., 2009) (Figure 1E).

Finally, negative regulation by biomolecular condensates prevents runaway immune activation. HEM1, a conserved protein containing a plant-specific LCD, interacts with translation factors to form condensates that buffer the expression of immune-related genes. This translational checkpoint prevents excessive immune activation and mitigates ETI-associated programmed cell death (Zhou et al., 2023) (Figure 1F). However, this protective buffering creates a potential trade-off: by restraining immune overactivation, HEM1 condensates may allow limited pathogen proliferation, which could influence the establishment of disease tolerance (Huang and Dong, 2025). Moreover, persistent or excessive condensation can be detrimental. Tang et al. (2024) demonstrated that virulent pathogens trigger HEM1 condensation at the endoplasmic reticulum (ER), where unresolved condensates trap lipid-metabolic enzymes and disrupt lipid homeostasis, leading to ER stress and tissue damage. Controlled degradation of these condensates through BI-1-mediated autophagy alleviates such damage and enhances disease tolerance. Together, these observations highlight the context-dependent duality of biomolecular condensates and demonstrate that condensate dynamics must be precisely regulated to balance effective defense, disease tolerance, and recovery. Although LLPS has been proposed as a unifying mechanism underlying multiple immune modules, alternative explanations remain possible. Certain TIR-domain or NPR1 behaviors attributed to LLPS may also arise from concentration-dependent oligomerization or post-translational modifications that mimic condensate-like patterns. Future biochemical and imaging studies should therefore determine whether LLPS is required for immune activation or instead reflects downstream amplification events.

Biomolecular condensates in small RNA-mediated defense mechanisms

Beyond PTI and ETI, plants employ RNA silencing pathways as a powerful layer of antiviral defense. Viral infection triggers the production of virus-derived small interfering RNAs (vsiRNAs), which guide sequence-specific degradation of viral RNAs and thereby suppress pathogen replication. Recent studies show that LLPS supports several key steps in this process. An integrated overview of sRNA-related condensates and their interactions with SGs and PBs is presented in Figure 2.

Figure 2.

Phase separation of small RNA pathways, stress granules (SGs), and processing bodies (PBs) in antiviral immunity.

This schematic illustrates how plant viruses hijack or remodel host condensates—including small RNA (sRNA) bodies, SGs, and PBs—to modulate antiviral defense.

(A) sRNA-body condensates in antiviral RNA silencing. During cucumber mosaic virus (CMV) infection, SGS3 undergoes phosphorylation-dependent phase separation, forming condensates with RDR6 and DCL2/4 that generate vsiRNAs. Dynamic regulation of SGS3 condensation by kinases determines siRNA body assembly and antiviral strength.

(B) RSV SP disrupts D-bodies to suppress miRNA processing. During rice stripe virus (RSV) infection, the viral SP protein exploits its intrinsic LLPS capacity to disassemble SERRATE (SE)-containing D-bodies, preventing miRNA biogenesis and weakening antiviral defenses.

(C) SUMOylation-driven VRO condensates promote viral replication. The p33 and p36 proteins of tombusviruses (TBSV and CIRV) form SUMO1/SCE1b-modified condensates that assemble into viral replication organelles (VROs). These condensates concentrate host factors to accelerate viral RNA replication and recombination while evading post-transcriptional gene silencing.

(D) PEMV2 p26 hijacks SGs through G3BP. The movement protein p26 of pea enation mosaic virus 2 (PEMV2) interacts with G3BP to form SG-like condensates, modulating SG assembly and function during infection. SGs, along with PBs and NUP62-associated structures, help integrate antiviral signaling and stress responses.

(E) BV1–AS2 condensates repress PB-mediated gene silencing. The geminiviral BV1 protein forms nuclear condensates with AS1 and AS2, which relocalize to DCP2-containing PBs to inhibit post-transcriptional gene silencing (PTGS) and jasmonic acid responses, thereby facilitating viral infection.

(F) Multiple endogenous condensates—including SGs, PBs, siRNA bodies, D-bodies, Cajal bodies, and virus-induced replication factories—are spatially coordinated around trafficking hubs such as NUP62 and MPK3. Viral infection dynamically reshapes these condensates to modulate antiviral signaling, RNA processing, organelle communication, and immune activation. This panel summarizes the interconnected condensate network that orchestrates antiviral defense and pathogen manipulation.

The suppressor of gene silencing protein (SGS3) plays a central role in RDR6-dependent vsiRNA biogenesis by stabilizing dsRNA precursors through condensate formation. The LLPS capacity of SGS3 is dynamically regulated by phosphorylation, which not only influences vsiRNA accumulation and antiviral efficiency but also affects reproductive development (Mergner et al., 2020; Han et al., 2023; Tan et al., 2023) (Figures 2A and 2F).

MicroRNAs (miRNAs) also contribute to immunity by fine-tuning defense-related gene expression. The SERRATE (SE) protein undergoes LLPS to assemble dicing bodies (D-bodies), where it associates with DCL1 and HYL1 to promote miRNA biogenesis. However, pathogens can disrupt this process. The rice stripe virus (RSV)-encoded SP protein exploits its own phase-separation capacity to disassemble SE condensates, thereby preventing D-body formation and weakening antiviral defenses (Zou et al., 2025) (Figures 2B and 2F). Similarly, the RNA helicases RH6, RH8, and RH12 not only contribute to D-body assembly and miRNA processing but also interact with viral proteins to form mixed condensates, potentially coordinating substrate RNA delivery and processing (Li et al., 2021).

LLPS also intersects with RNA quality control (RQC). The core RQC factor Pelota recognizes defective viral RNAs and promotes their degradation through SUMOylation (Garcia et al., 2014). However, viruses can hijack this system. Viral replication proteins such as p33 (from TBSV) and p36 (from CIRV) recruit host SUMOylation machinery to assemble viral replication organelles (VROs) via IDR-mediated LLPS. These condensates concentrate host factors to promote viral RNA replication and recombination while simultaneously evading host surveillance (Lin and Nagy, 2024, 2025) (Figures 2C and 2F). Together, these findings illustrate how condensates dynamically regulate small RNA pathways, functioning both as platforms for antiviral defense and as targets of viral countermeasures. A major remaining question is whether the formation and turnover of sRNA-related condensates strictly follow LLPS principles or instead use additional scaffold-based mechanisms. Moreover, viral proteins such as RSV SP and pea enation mosaic virus 2 (PEMV2) p26 may perturb condensates not only through direct interference with phase separation but also by altering host translational status or RNA metabolism. Disentangling these overlapping effects will be essential for determining the specific contribution of LLPS to antiviral defense.

Biomolecular condensates formed by stress granules (SGs) and processing bodies (PBs)

SGs and PBs are prototypical membraneless organelles assembled through LLPS of RNA and proteins. Core components—includingG3BP-like proteins, TTP-like proteins, and RNA helicases—drive their formation via IDRs or RNA-binding domains. In plants, as in other eukaryotes, SGs and PBs are rapidly induced by environmental stresses, including pathogen infection, and play central roles in reallocating cellular resources during immune activation (Brownsword and Locker, 2023). Functionally, SGs and PBs act as regulatory hubs, by sequestering translationally stalled mRNAs, conserving cellular energy and redirecting translation toward defense-related proteins. SGs also enrich immune signaling factors such as MAP kinases and resistance (R) proteins, helping amplify and transmit immune signals (Maldonado-Bonilla, 2014; Tabassum et al., 2020). These features position SGs and PBs as important contributors to antiviral defense. Key SG and PB nodes involved in antiviral responses are mapped in Figure 2.

Nevertheless, viruses have evolved strategies to circumvent or exploit these condensates. The movement protein p26 of PEMV2 forms high-viscosity condensates that hijack host SGs to promote infection. Overexpression of the SG core protein G3BP strongly inhibits PEMV2 replication, whereas G3BP mutants defective in condensate formation lose antiviral activity (Brown et al., 2023) (Figures 2D and 2F). Viruses also manipulate PBs; for example, the geminivirus nuclear shuttle protein BV1 activates the PB component AS2, thereby suppressing post-transcriptional gene silencing (PTGS) and weakening antiviral immunity (Ye et al., 2015) (Figures 2E and 2F).

In other cases, viruses directly co-opt SGs and PBs as replication platforms. The nucleocapsid (N) proteins of plant-infecting Bunyavirales members, including tomato spotted wilt virus (TSWV) and RSV, localize to both SGs and PBs. Silencing SG or PB core components significantly alters TSWV mini-replicon activity, with effects ranging from a ∼30% reduction to a fourfold increase, depending on the targeted factor (Xu et al., 2022). These findings illustrate the dual nature of SGs and PBs as both antiviral condensates and sites that viruses can repurpose for replication.

Biomolecular condensates formed by other host proteins

Beyond canonical immune regulators, an expanding set of host proteins exploit LLPS to fine-tune plant defense responses. These proteins act at multiple regulatory layers, including RNA processing, transcription, translation, and nuclear transport, highlighting the versatility of biomolecular condensates in immune modulation. Representative host immune regulators that undergo LLPS across these layers are summarized in Figure 3.

Figure 3.

Phase separation of additional host immune regulators and their roles in plant defense.

This schematic illustrates diverse host proteins that undergo LLPS during pathogen infection and regulate immune responses at multiple levels, including RNA splicing, transcription, and post-translational regulation.

(A) SR30 condensates regulate alternative splicing and disease susceptibility. In tomato, the splicing regulator SR30 forms nuclear condensates with SR46, RS29, SCL29, and SCL19 to modulate alternative splicing of defense-related genes. This condensation promotes the accumulation of susceptibility-associated transcripts, increasing vulnerability to Phytophthora infestans.

(B) Phase-separated HRC hubs determine Fusarium resistance or susceptibility. In wheat, the histidine-rich calcium-binding protein HRC forms distinct LLPS-driven hubs in response to the mycotoxin deoxynivalenol (DON) produced by Fusarium graminearum. The resistant allele (HRC-R) interacts with CXIP4 and ZFC10 to form an HRC-R hub that prevents DON-induced cell death, whereas the susceptible allele (HRC-S) condenses with SR45a to promote toxin-triggered cell death and Fusarium head blight susceptibility.

(C) GBPL3 defense-activated condensates (GDACs) drive transcriptional reprogramming. Upon immune activation, Arabidopsis GBPL3 and GBPL1 assemble GDACs in the nucleus, where they recruit RNA polymerase II and transcriptional co-activators to induce defense gene expression. These condensates amplify immune signaling and enhance disease resistance.

(D) KA120–MAC3 and TZF9 condensates maintain immune homeostasis. The nuclear transport receptor KA120 prevents spontaneous LLPS of the ubiquitin ligase MAC3, thereby maintaining basal immune quiescence. Upon pathogen infection, this inhibition is relieved, allowing MAC3 to form MAC3-dependent nuclear condensates (MDNCs) that activate immunity. Meanwhile, the tandem zinc-finger protein TZF9 forms cytoplasmic condensates that act as negative regulators, fine-tuning PTI signaling strength.

(E) Integrated host condensate network in immune regulation. Multiple immune-related condensates—including GDACs, MDNCs, SR30 condensates, HRC hubs, and organelle-associated bodies—converge within a shared nuclear–cytoplasmic LLPS network. These interconnected structures coordinate RNA metabolism, transcriptional activation, protein quality control, and organelle communication, forming an integrated phase-separation-based regulatory system that fine-tunes plant defense outputs in response to diverse pathogens.

(F) RIN4 condensates and XopR-mediated suppression of RPM1 activation. The bacterial effector XopR from Xanthomonas campestris disrupts plasma membrane-localized RIN4 condensates by altering their fluidity and preventing phosphorylation by the kinase RIPK. This interference blocks RPM1 activation and compromises bacterial resistance. Together, (A)–(F) illustrate how host-derived condensates integrate splicing regulation, toxin perception, transcriptional activation, protein turnover, and receptor signaling to coordinate immune defense and maintain homeostasis.

Alternative splicing (AS) is one such process that is strongly influenced by biomolecular condensates. In tomato, the splicing regulator SR30 forms nuclear condensates through IDR-driven LLPS, a property that is essential for its splicing activity and its role in disease susceptibility. During Phytophthora infestans infection, SR30 suppresses the splicing of defense-related genes, whereas CRISPR–Cas9-mediated knockout of SR30 enhances their splicing and confers resistance to P. infestans, P. capsici, and P. parasitica (Yan et al., 2025) (Figures 3A and 3E). In wheat, the effector TaHRC recruits splicing factors such as TaSR45a via LLPS. The susceptible allele (TaHRC-S) undergoes deoxynivalenol (DON)-induced condensation, forming stable complexes that promote cell death, whereas the resistant allele (TaHRC-R) remains insensitive to DON and suppresses toxin-mediated cell death (He et al., 2024) (Figures 3B and 3E).

At the transcriptional level, the GTPase GBPL3 undergoes LLPS in response to immune-related cues such as salicylic acid and defense-associated phosphorylation, assembling defense-activated condensates (GDACs) in the nucleus. These condensates localize to defense gene promoters and recruit transcriptional co-activators including RNA polymerase II, driving large-scale reprogramming of immune gene expression (Huang et al., 2021) (Figures 3C and 3E). Similarly, the nuclear transport receptor KA120 prevents spontaneous LLPS of the ubiquitin ligase MAC3, maintaining immune homeostasis under normal conditions. Upon infection, this inhibition is lifted, allowing MAC3 to form MAC3-dependent nuclear condensates (MDNCs) that directly activate defense gene expression (Jia et al., 2023) (Figures 3D and 3E). Pathogens can also manipulate these host condensates; for instance, the type III effector XopR from Xanthomonas campestris alters the fluidity of RIN4 condensates through LLPS, preventing their phosphorylation by the kinase RIPK and weakening RPM1-mediated resistance (Zhu et al., 2025) (Figure 3F). Together, these findings show that LLPS provides plants with a flexible mechanism to regulate immunity across multiple molecular layers while simultaneously creating vulnerabilities that pathogens can exploit. However, many of the reported condensates are inferred primarily from fluorescent puncta or co-localization, without direct biophysical validation of liquid-like behavior. Whether transitions between dispersed and condensed states actively drive immune reprogramming or simply reflect changes in protein abundance remains unresolved. Integrating in vivo biophysical approaches such as FRAP, rheology, and quantitative imaging will be essential for confirming the functional relevance of these condensates.

These findings as a whole underscore the dual nature of biomolecular condensates in plant immunity. In the next section, we explore how insights into condensate dynamics can be translated into practical strategies and examine key challenges in plant–pathogen interactions.

Prospective strategies and challenges

Research on biomolecular condensates in plant immunity reveals a dynamic arms race between plants and pathogens. Viruses use condensates to assemble replication factories, whereas fungal, bacterial, and oomycete effectors hijack host condensates to suppress immunity. In turn, plants deploy condensates—such as SGs and those formed by TIR-domain proteins, GBPL3, and PR1—to mount counter-defenses. However, dysregulation of host condensates caused by mutations that either enhance or weaken LLPS may lead to pathogenic condensates or even autoimmune-like disorders. Conversely, targeting pathogen-derived condensates, particularly their IDRs or LCDs, represents a promising strategy for limiting infection. Dissecting the molecular basis of condensate dynamics in plant–pathogen interactions therefore deepens our understanding of coevolution and opens the door to precision crop protection.

Application strategies in plant immunity

Targeting biomolecular condensates offers novel opportunities for engineering broad-spectrum and durable plant resistance. One promising approach involves the development of small-molecule modulators that target conserved IDRs within viral or microbial effectors. For example, the LLPS inhibitors Z9 and vanillin derivatives (compound 29) show potential as cross-species antiviral agents by preventing nucleocapsid protein aggregation in TSWV (Zan et al., 2025). Although several small molecules capable of modulating viral condensates have been identified in human systems, their application in plants remains limited, underscoring the need to establish plant-specific discovery pipelines. Together, these emerging strategies highlight the promise of condensate-centered antiviral approaches while revealing substantial gaps in translating such tools into crop protection.

Another strategy involves the direct degradation of harmful condensates. Luo et al. identified the self-degradable E3 ligase E3TCD1 and designed X–E3TCD1 fusion proteins as genetically encoded degraders. This targeted condensation-prone protein degradation (TCD) system selectively recognizes proteins undergoing misregulated or deleterious condensation, ubiquitinates them, and promotes their degradation. In rice, pathogen-inducible expression of OsELF3–E3TCD1 under the TBF1p::uORFs_TBF1 cassette significantly improved resistance to rice blast without affecting flowering time (Luo et al., 2025). Recent reviews have further discussed ongoing advances in uORF-mediated translational regulation and TCD, providing a valuable framework for applying these technologies to crop improvement (Mou, 2024; Wang et al., 2024; Luo et al., 2025; Nazarian-Firouzabadi et al., 2025; Niu et al., 2025; Xiang and Dong, 2025).

Beneficial condensates may also be strengthened through pharmacological or genetic interventions. For instance, enhancing SG formation could broadly increase stress resilience across diverse pathogens. Targeted reprogramming of host condensates provides another avenue: modifying the LLPS capacity of the wheat resistance protein TaHRC and its TaSR45a complex to improve Fusarium resistance (He et al., 2024); engineering MAC3 condensation to fine-tune immune activation (Jia et al., 2023); or mutating host cofactors such as NbRANL or myosin XI to block viral condensate maturation (Zan et al., 2025). Together, these approaches demonstrate how condensate manipulation could be leveraged for next-generation crop improvement.

Although the concept of LLPS has reshaped our understanding of cellular organization, several conceptual ambiguities remain in plant systems. First, the physiological boundary between functional condensates and pathological aggregates remains poorly defined. Second, commonly used criteria for identifying LLPS—such as droplet fusion or FRAP recovery—may not fully capture the unique biophysical constraints of plant cells, including high turgor pressure and dense cytoplasm. Third, plant-specific features such as cell wall mechanics may fundamentally alter condensate behavior compared with animal systems. Clarifying these boundaries will be essential for establishing a coherent framework for condensate biology in plants.

Challenges and future directions

Despite exciting progress, research on biomolecular condensates in plant immunity remains at an early stage and faces several technical and conceptual challenges. Biomolecular condensates are highly sensitive to local parameters such as protein concentration, temperature, and pH (Gao et al., 2022). Many pathogen effectors are transiently expressed during specific infection stages, and their condensates may form only within narrow cellular niches, making them difficult to detect and reproduce in vitro. Moreover, effector families such as RxLR effectors often include hundreds of variants with redundant or divergent condensation behaviors, further complicating systematic analysis (Seong and Krasileva, 2023). Functional validation therefore requires integrated strategies that combine genetic engineering approaches, such as allele swapping of TaHRC, with direct perturbation of condensate formation, such as inhibition of TaSR45a LLPS (He et al., 2024). This level of complexity extends well beyond traditional protein interaction assays.

Advancing this field will rely heavily on improved imaging and structural tools. Live-cell dynamic imaging coupled with nanoscale labeling will allow real-time visualization of condensate assembly and dissolution (Zhang et al., 2023b). APEX-based proximity labeling combined with cryo-electron microscopy (cryo-EM) or cryo-electron tomography (cryo-ET) can resolve condensate composition and architecture at near-atomic resolution (Zhu et al., 2017; Markmiller et al., 2018; Huang et al., 2021). In parallel, single-cell omics and super-resolution microscopy can reveal the spatiotemporal heterogeneity of condensate behaviors across cell types. Recent advances in proximity labeling and ligation-based methods now permit high-resolution mapping of molecular interaction networks around specific RNAs or genomic loci, not only in animal systems (Gao et al., 2018; Myers et al., 2018; Ramanathan et al., 2018) but increasingly in plants (Yang et al., 2021; Wen et al., 2024). Additionally, ADAR-based in vivo RNA-editing strategies, recently adapted for plant systems, provide a powerful means to identify RNAs associated with specific condensates or RNA-binding proteins (Zhou et al., 2021; Yin et al., 2023). Together, these emerging approaches offer a more comprehensive and precise framework for dissecting condensate organization and functional diversity in plant immunity.

Another major obstacle lies in computational prediction. Current sequence-based algorithms for LLPS prediction struggle to detect LCDs or multivalent interactions and often produce false positives (Gao et al., 2022). Some effectors achieve functional convergence through “sequence-unrelated structural similarity” (SUSS), which evades detection by standard bioinformatic tools (Seong and Krasileva, 2023). To overcome these limitations, Zhang et al. constructed a multi-species condensate proteome atlas using b-isox precipitation, covering model and crop plants such as Arabidopsis, maize, wheat, and Chinese cabbage. This work provides valuable insights into conserved phase-separation-prone proteins across plant species (Zhang et al., 2023a). Integrating such empirical datasets with AI-driven models that incorporate structural features and sequence context could enable high-throughput identification of pathogen effectors with condensate-forming potential.

Ultimately, meaningful progress will require interdisciplinary integration. Bridging plant pathology with structural biology and biophysics will be essential for elucidating how biomolecular condensates regulate immunity. Understanding condensate-mediated defense will depend on structural insights into condensate assemblies, quantitative modeling of phase transition dynamics, and in vivo validation of infection phenotypes. For example, elucidation of TIR-mediated condensate-driven immune activation depended on the combined application of biochemical reconstitution and live-cell imaging (Song et al., 2024). Such interdisciplinary strategies will be vital for addressing outstanding questions and translating condensate biology into practical tools for durable disease resistance.

Concluding remarks

In summary, biomolecular condensates represent a rapidly emerging frontier in plant–pathogen interactions. They serve as both battlegrounds and weapons in the host–pathogen arms race: pathogens reorganize virulence factors through LLPS, while plants harness condensate dynamics to coordinate defense signaling and maintain cellular homeostasis. This duality—protective yet potentially harmful—highlights the delicate balance required to achieve effective defense while preserving disease tolerance. While Huang and Dong (2025) provided a broad conceptual framework for condensate-mediated immunity, our review extends this perspective by integrating recent discoveries on pathogen-driven condensates and by exploring potential engineering strategies for crop protection. Elucidating the molecular principles governing condensate assembly, regulation, and subversion will not only deepen our understanding of plant immunity but also reveal new targets for crop improvement.

Despite rapid advances, the causal relationship between LLPS and immune outcomes remains to be rigorously established. It is still unclear whether condensate formation directly drives functional changes in immune signaling or instead arises as a by-product of immune activation. Moreover, much of the existing evidence is derived from overexpression systems or in vitro assays, raising concerns about whether LLPS behaves similarly under physiological conditions in planta. Future research combining live-cell imaging, quantitative biophysics, and genetic perturbations will be essential to distinguish LLPS events that merely correlate with immune activation from those that are mechanistically required. Ultimately, improving our understanding of condensate biology will enable precision, genome-informed, and sustainable crop protection strategies, thereby contributing to long-term agricultural resilience and global food security.

Data and code availability

All data needed to evaluate the conclusions of this study are included in the paper.

Funding

This research was supported by grants from the National Natural Science Foundation of China (32025031 and 32300149) and the Natural Science Foundation of Fujian Province of China (2024J01378 and 2025J010022).

Acknowledgments

We apologize to colleagues whose original research and review articles could not be cited in this review due to space constraints. No conflict of interest declared.

Author contributions

J.W. conceived the project; J.Z. and S.Z. wrote the initial draft of the manuscript; B.Z., J.C., and J.W. thoroughly edited the paper; and J.Z., X.Z., Z.Z., and S.Z. generated the figures. All authors contributed to reviewing and proofreading the manuscript.

Published: December 4, 2025