The pathogen effector BcSSP2 suppresses the NPC phase separation to facilitate Botrytis cinerea infection

Wang, Jiaojiao; Wu, Dewei; Pei, Gaofeng; Wang, Yupei; Liu, Xiaokang; Tian, Shiping; Zhang, Zhanquan; Zhang, Xiaolin; Li, Pilong; Xie, Daoxin; Shan, Xiaoyi

doi:10.1038/s41467-025-63632-7

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Published: 29 September 2025

The pathogen effector BcSSP2 suppresses the NPC phase separation to facilitate Botrytis cinerea infection

Nature Communications volume 16, Article number: 8610 (2025) Cite this article

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Abstract

Biomolecular phase separation-mediated immunity was recently uncovered as an important strategy of plants and animals for their survival in the challenging environment. Effectors are powerful weapons evolved by pathogens to overcome host defense response for effective infection. However, plant pathogen effectors were rarely identified to suppress the phase separation-mediated host defense response. Here, we report a pathogen effector that disrupts phase separation process in host cells to suppress host defense response and facilitate pathogen infection. We identify a host-internalized effector protein BcSSP2 from a disastrous pathogen Botrytis cinerea targets the central barrier of plant nuclear pore complex (NPC). BcSSP2 disrupts the phase separation of NUP62 and attenuates phase separation-dependent nuclear transport of immune regulator MPK3 to suppress plant defense response for effective pathogen infection. Intriguingly, BcSSP2 homologs are widely distributed among necrotrophic ascomycetes and also interfere with NUP62 phase separation, suggesting that disrupting plant NPC phase separation might be an evolutionarily conserved strategy evolved by these necrotrophic pathogens to facilitate their infection.

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Introduction

Plants and animals have developed effective immune systems to protect them from a variety of aggressive pathogens for their survival in nature¹. These host immune systems were regulated by a multitude of mechanisms including RNAi-based immunity², resistance gene-regulated immunity³ and autophagy-triggered immunity⁴. Phase separation, a driving force for the formation of various membraneless organelles, deeply involves in the health maintenance of mammals^5,6,7, including the innate immune signaling activated by the viral DNA-induced phase separation of cGAS^8,9,10, and severe neurodegenerative diseases caused by abnormal phase separation of FUS, tau and TDP-43¹¹. In plants, phase separation of plant nuclear pore complex (NPC) was uncovered to regulate diverse plant defense responses against fungal and bacterial infection and insect attack¹², phase separation of NPR1, GBPL1/3, HEM1 and Toll/interleukin-1 receptor were found to regulate plant resistance to bacterial infection^13,14,15,16. These results suggest that phase separation-mediated defense response has emerged as a general mechanism to regulate plant defense against diverse biotic stresses.

To achieve successful infection, pathogens also have evolved sophisticated mechanisms to fight against host defense response. Secreting effector proteins is a powerful strategy to counter host immune system, which is commonly adopted by almost all types of pathogens including bacteria, fungi and viruses^17,18,19,20. Enormous efforts made by biologists worldwide have uncovered a huge amount of pathogen effectors that suppress host defense response via different mechanisms, including targeting host immune regulators to affect their transcription, translation or posttranslational modifications^21,22,23,24, disturbing cellular organelles²⁵, and binding with host small RNAs²⁶. However, effectors from plant pathogens have rarely been shown to suppress the phase separation-mediated host defense response.

In this study, we reported the effector protein BcSSP2 from Botrytis cinerea (B. cinerea), one of the most disastrous plant pathogens causing severe economic losses worldwide^27,28, is secreted into plant cells to target the central barrier of plant NPC, interfere with phase separation of host NPC central barrier, and consequently suppress the phase separation-dependent nuclear transport of a key immune regulator MPK3, leading to attenuated plant defense response and thus facilitating pathogen infection.

Results

BcSSP2 is a putative effector from B. cinerea

B. cinerea is generally regarded as a necrotrophic pathogen which usually secrets lytic enzymes, oxalic acid and phytotoxins to extracellular spaces of host cells for infection^27,28. It is so far unknown whether B. cinerea delivers effector proteins into plant cells to disturb host immunity for successful infection. We collected B. cinerea hyphae from the in vitro culture or the infected Arabidopsis leaves to compare the hyphal transcriptomic profiles. Among the genes upregulated in B. cinerea infection process (Supplementary Data 1, 2), 15 genes were shortlisted as effector candidates due to the typical effector features of other well-studied pathogens such as RXLR motif and cysteine-rich region in their encoding proteins (Fig. 1a; Supplementary Data 3)²⁹.

**Fig. 1: BcSSP2 is an effector protein.**

Recent studies revealed that suppression of host immunity in biotrophic stage is required for necrotrophic pathogens to establish colonization^30,31. As shown in Supplementary Fig. 1, B. cinerea takes a considerable period to establish colonization and rot plant tissues. We separately collected B. cinerea-infected Arabidopsis tissues from “annular edge area” (where the plant cells remained alive and a limited amount of B. cinerea hyphae were growing within apoplastic spaces) and “central lesion area” (where the plant cells already died and a high density of hyphae were growing) (Fig. 1b), which then were subjected to quantitative real-time PCR (qRT-PCR) analysis of the 15 candidate effector genes. Among them, BCIN_05g03680, which has been reported as B. cinerea small secreted protein 2 (BcSSP2)^32,33, was induced in the infection process and showed the most elevated expression at the edge area (Fig. 1c, d, and Supplementary Fig. 1c), implying a possible role for BcSSP2 in suppressing host defense response for B. cinerea infection.

Prediction by SignalP server showed that the N-terminal region (amino acid 1–20) of BcSSP2 has the typical characteristic of secretory signal peptide (Supplementary Fig. 2a), which is essential for effector secretion from pathogens³⁴. Yeast secretion assay showed that invertase fused with the BcSSP2 N-terminal signal peptide could be secreted to reduce colorless 2,3,5-triphenyltetrazolium chloride to red formazan, confirming the secretory characteristic of the BcSSP2 N-terminal signal peptide (Fig. 1e and Supplementary Fig. 2b), consistent with previous findings³². We further found that Alexa 546-labeled BcSSP2, but not control protein, was located in plant cells when incubated with Arabidopsis roots (Fig. 1f), suggesting that BcSSP2 could be internalized by plant cells. Moreover, we used a split GFP system for tracing translocation of pathogen effector in an infection context³⁵, in which tobacco leaves transiently expressing GFP_1-10 (engineered GFP protein consists of strands 1–10 with a specific exclusion of strand 11) was incubated with B. cinerea strain containing GFP₁₁-tagged BcSSP2. Notably, GFP signals were accumulated in cytoplasm and nuclei of cells in the edge area of infected leaves (Fig. 1g), validating secretion of BcSSP2 by B. cinerea into plant cells. Taken together, these results indicate that BcSSP2 has typical effector features and is translocated into living plant cells.

BcSSP2 contributes to B. cinerea virulence

To verify the function of BcSSP2 in B. cinerea virulence, we generated B. cinerea BcSSP2-knockout mutant strains (termed as bcssp2-4, bcssp2-10 and bcssp2-12) through homologous recombination (Supplementary Fig. 3), and subsequently assessed the virulence of these mutant strains. Disease symptoms caused by the bcssp2 mutant strains on Arabidopsis were significantly attenuated, as indicated by smaller lesions compared with wild-type strain (B05.10) (~55%, 68% and 63% for bcssp2-4, bcssp2-10 and bcssp2-12, respectively) (Fig. 2a, b). To confirm that the reduced virulence was specifically due to the loss of BcSSP2, we generated two independent complementation strains (com-2 and com-3) by reintroducing BcSSP2 gene into the bcssp2-10 mutant. Real-time PCR analysis confirmed the restored expression of BcSSP2 in both complemented strains (Supplementary Fig. 4a), and both com-2 and com-3 fully rescued the virulence phenotype, resulting in lesion sizes comparable to the wild-type strain (Supplementary Fig. 4b–e). These data collectively demonstrate that BcSSP2 is required for full virulence of B. cinerea.

**Fig. 2: BcSSP2 contributes to *B. cinerea* pathogenicity.**

Since B. cinerea has a very broad host range, we further examined the effect of BcSSP2 on B. cinerea virulence in other host plants including lettuce, strawberry and rose. Similarly, the virulence of BcSSP2 mutants is significantly declined in all tested plant species, indicating a conserved role of BcSSP2 in facilitating B. cinerea infection on diverse host plants (Fig. 2c, d).

We then generated transgenic Arabidopsis with estradiol-inducible expression of BcSSP2 (iBcSSP2) (Supplementary Fig. 5) to further verify the BcSSP2 function in B. cinerea pathogenicity. Estradiol treatment led to more severe disease symptoms in iBcSSP2 plants but not in the control wild-type (WT) plants upon bcssp2 strain infection, as indicated by increased B. cinerea biomass (~2.1-fold) and plant disease severity (~2.1-fold dead plants) (Fig. 2e, f), demonstrating that the ectopic overexpression of BcSSP2 promotes plant susceptibility to B. cinerea infection. These data further define BcSSP2 as an effector secreted by B. cinerea into host cells to promote its virulence.

BcSSP2 interacts with NUP62 and partitions into NUP62 condensates

As BcSSP2 is translocated into plant cells, we wondered whether BcSSP2 targets some protein(s) in plant cells to suppress host immunity. To test this hypothesis, we screened potential BcSSP2 targets from a panel of over a dozen plant proteins known to participate in defense against B. cinerea, including coronatine insensitive 1, ethylene insensitive 2, transcription factor WRKY33, and nucleoporins using luciferase (LUC) complementation-imaging (LCI) assay. Intriguingly, strong LUC activity was detected when BcSSP2 was co-expressed with all the components of the center barrier of nuclear pore complex (NUP62, NUP58, NUP54), but not with other nuclear pore components such as NUP88 and NUP96 (Fig. 3a and Supplementary Fig. 6a, b). Such interaction was verified by co-immunoprecipitation (Co-IP) assay in tobacco leaves transiently co-expressing Flag-NUP62 and BcSSP2-GFP (Fig. 3b), and by pull-down assay using purified GST-NUP62 and BcSSP2-His proteins (Fig. 3c). Moreover, BcSSP2 could co-localize with NUP62 in the nuclear envelop (Supplementary Fig. 6c). These results collectively suggest that the effector BcSSP2 targets plant NPC central barrier.

Furthermore, we identified the responsible domain of NUP62 for its interaction with BcSSP2. We truncated NUP62 into a C-terminal domain (CTD) harboring the coil-coil motif and an N-terminal domain (NTD) containing the FG-rich domain. In the pull-down assay, the NTD, but not the CTD, of NUP62 could efficiently pull down BcSSP2 (Fig. 3d), indicating that NUP62 interacts with BcSSP2 via its NTD.

As the central barrier undergoes phase separation to form condensates in vitro through the NTD¹², we investigated whether BcSSP2 partitions into NUP62 condensates. BcSSP2, but not the control protein, could efficiently partition into NUP62 condensates when incubated with NUP62 (Fig. 3e and Supplementary Fig. 7a, b). Meanwhile, BcSSP2 was unable to undergo phase separation alone (Supplementary Fig. 7c). These results further verify the interaction between BcSSP2 and NUP62, and suggest that BcSSP2 is able to interact with NUP62 in its phase-separated conformation.

BcSSP2 interferes with NUP62 phase separation

Since BcSSP2 interacts with the NTD of NUP62 (Fig. 3d) and NUP62 phase separation is crucial for host defense against B. cinerea¹², we wonder whether BcSSP2 affects NUP62 phase separation to facilitate B. cinerea infection. To address this question, we first tested whether BcSSP2 disturbs phase separation of NUP62 in vitro. We found that the formation of NUP62 condensates was disturbed significantly when incubated with BcSSP2 (Fig. 4a, b).

Furthermore, we used atomic force microscopy (AFM) to characterize the impact of BcSSP2 on the mechanical properties of NUP62 hydrogel which is formed through phase separation. The hydrogels constituted of NUP62 alone, NUP62 with BcSSP2 or NUP62 with control protein, were prepared and subjected to AFM analysis. We measured the stiffness of the hydrogels at multiple points, which is indicated by Young’s modulus. The stiffness of NUP62 + BcSSP2 hydrogel decreased dramatically (by about 67% in Median and 70% in Mean of Young’s modulus) compared with that of NUP62 alone or NUP62+control hydrogels (Fig. 4c, d), suggesting that BcSSP2 evidently modifies the property of NUP62 hydrogels. Taken together, these results demonstrate that BcSSP2 interferes with NUP62 phase separation in vitro.

To gain insights into the effect of BcSSP2 on NUP62 phase separation in vivo, we inoculated B05.10 strain or bcssp2-12 mutant strain onto tobacco leaves transiently expressing NUP62-GFP for confocal imaging. In the bcssp2-infected edge area, the total fluorescent intensity of NUP62 condensates was significantly higher (~1.4-fold) compared with that of B05.10-infected edge area (Fig. 4e, f) while the overall protein level of NUP62 presented no significant difference (Supplementary Fig. 8a). Consistently, estradiol-induced BcSSP2 reduced NUP62 condensates by 81% in tobacco expressing NUP62-GFP, with no change of NUP62 total protein level (Fig. 4g, h, and Supplementary Fig. 8b, c). These results collectively demonstrate that BcSSP2 suppresses the phase separation of NUP62 in vivo.

Homologs of BcSSP2 in necrotrophic ascomycetes interfere with NUP62 phase separation

Phylogenetic analysis showed that BcSSP2 homologs were widely distributed among ascomycetes and highly conserved in necrotrophic ascomycetes (Supplementary Fig. 9a). Representative analysis of the BcSSP2 homologs from S. sclerotiorum (a broad host range necrotrophic pathogen that causes one of the most prevalent diseases in oilseed rape) and Aspergillus flavus (an extremely common fungal pathogen that produces carcinogenic aflatoxins in the infected crops) showed that the SsSSP2 (S. sclerotiorum SSP2) and AfSSP2 (A. flavus SSP2) were able to interact with NUP62 (Supplementary Fig. 9b), indicating an evolutionarily conserved strategy adopted by various necrotrophic ascomycetes to secret BcSSP2-like effectors that target plant NPCs to facilitate pathogen infection.

Furthermore, we wondered if SsSSP2 and AfSSP2 suppress NUP62 phase separation. To test this hypothesis, NUP62-GFP together with estradiol-inducible SsSSP2 (iSsSSP2) or estradiol-inducible AfSSP2 (iAfSSP2) were transiently co-expressed in tobacco leaves for NUP62 puncta observation. The formation of NUP62 condensate decreased evidently with estradiol-induced expression of SsSSP2 and AfSSP2, respectively (Supplementary Fig. 10a, b). Meanwhile, the protein abundance of NUP62 presented no significant difference without (Mock) or with estradiol provision (ES) (Supplementary Fig. 10c). Thus, SsSSP2, as well as AfSSP2, could interfere with NUP62 phase separation, suggesting a conserved feature of necrotrophic BcSSP2-like effectors for interfering with the phase separation of NPC central barrier.

BcSSP2 attenuates the NUP62 phase separation-dependent nuclear transport of MPK3

As phase separation of the NPC central barrier mediates selective nucleocytoplasmic transport which is facilitated by the nuclear transport receptor (NTR)¹², we next investigated whether BcSSP2-mediated perturbation on NUP62 phase separation affects the selective nucleocytoplasmic transport. Firstly, we adopted in vitro hydrogel transport assay to detect the influence of BcSSP2 on the transport efficiency of NUP62 using the surface-redesigned GFP protein (GFP8W, eight amino acids located in GFP surface were mutated to tryptophans) to mimic the NTR-mediated selective transport³⁶. The translocation speed of MBP-GFP8W in NUP62 + BcSSP2 hydrogel decreased by nearly half, compared with that of NUP62 or NUP62+control hydrogel (Fig. 5a, b). Consistently, the translocation speed of MBP-GFP8W in NUP62 hydrogel pre-incubated with BcSSP2 was obviously decreased (Supplementary Fig. 11), suggesting that BcSSP2 attenuates the NUP62 phase separation-mediated MBP-GFP8W transport.

**Fig. 5: BcSSP2 suppresses NUP62 phase separation-mediated selective nucleocytoplasmic transport.**

Since phase separation of the NPC central barrier mediates nuclear import of defense-related cargo MPK3¹², we then tested whether BcSSP2 suppresses MPK3 nuclear transport. The in vitro assays showed that the partition of MPK3 (with Arabidopsis crude extracts) in NUP62 hydrogel was significantly inhibited by BcSSP2 (Fig. 5c, d). As assayed in vivo, nuclear proportion of MPK3 in plants infected by wild-type B. cinerea B05.10 was obviously lower (by ~61.5%) in comparison with that infected by BcSSP2 knockout strain bcssp2-12, though the overall amount of MPK3 kept unchanged in plants infected by either B05.10 or bcssp2-12 (Fig. 5e, f). In line with nuclear attenuation of MPK3, the expression level of defense marker gene PDF1.2 was significantly decreased (~50%) in B05.10-infected plants compared with that of bcssp2-infected plants (Fig. 5g). These results suggest that BcSSP2 of B. cinerea B05.10 inhibits nuclear transport of MPK3 in plant cells during pathogen infection.

Moreover, the ectopic expression of BcSSP2 in Arabidopsis plants significantly suppressed MPK3 nuclear transport. Though the total MPK3 protein level presented no discernible difference (Fig. 5h), the nuclear accumulation of MPK3 was remarkably impaired by 67% in estradiol-treated iBcSSP2 transgenic plants (Fig. 5i). Constantly, the expression level of PDF1.2 was declined by 88% in estradiol-treated iBcSSP2 transgenic plants (Fig. 5j).

Taken together, our results in Figs. 1–5 suggest that BcSSP2, secreted by B. cinerea into host cells, targets the NPC central barrier, interferes with its phase separation and consequently attenuates phase separation-dependent nucleocytoplasmic transport of MPK3, to suppress plant defense response for effective pathogen infection (Fig. 6).

**Fig. 6: A simplified model for BcSSP2 action.**

Discussion

Pathogens and their hosts are inevitably involved in endless arms race, while effector proteins act as the key weapons in pathogens’ arsenal^{3,17,18,19,20}. Previous studies uncovered various strategies used by pathogen effectors to combat host defense response, such as altering the expression of host genes^21,22, suppressing the host small RNA-based gene silencing^26,31, and interfering the degradation of host proteins²⁴. The phase separation of effector protein has recently emerged as a novel strategy for pathogens to interfere with host defense response³⁷. In this study, we identify an effector from a plant pathogen that, rather than undergoing phase separation itself, suppresses host phase separation-mediated defense processes to promote infection. These findings highlight phase separation as a new and critical battleground in pathogen-host interactions.

NPC serves as the sole exchange channel between nucleus and cytoplasm^38,39, and plays important roles in diverse biological processes^{40,41,42,43,44}. We previously showed that the integrity of NPC central barrier is required for plant defense and that the NPC central barrier undergoes phase separation to facilitate the active transport of cargoes essential for plant defense¹². In the current study, we further report a pathogen effector, BcSSP2, that targets plant NPCs. BcSSP2 binds to FG-nucleoporins of the central barrier and interferes with their phase separation to disrupt nucleocytoplasmic transport of important immune regulators, including MPK3, to inhibit host defense for effective pathogen infection. In addition to MPK3, the nucleocytoplasmic transport of other immune components involved in resistance to B. cinerea may also be affected, a possibility that warrants further investigation. These data support the notion that phase separation of NPC central barrier is crucial for plant defense as demonstrated in our previous work¹², and further uncover that some pathogens encode specific effectors that can interfere with phase separation of host NPCs to promote infection.

B. cinerea is one of the most devastating plant pathogens, which has a broad host range²⁷, and causes huge economic losses worldwide. Direct killing host cells with the secreted lytic enzymes, oxalic acid and phytotoxins were thought to be the major infection strategies adopted by B. cinerea²⁸. Recent studies revealed that B. cinerea secrets small RNAs into plant cells to suppress RNAi-based plant defense response for successful infection³¹. Here, we found that B. cinerea secrets BcSSP2 to attenuate phase separation of plant NPC to suppress host defense response for effective pathogen invasion. Previous studies have shown that the phase separation of NUP62 is mediated by weak, multivalent hydrophobic interactions among its N-terminal FG repeat domains¹². Consistently, our results demonstrate that BcSSP2 interacts with the N-terminal region of NUP62 (Fig. 3d), suggesting that BcSSP2 may disrupt NUP62 phase separation by interfering with FG-FG interactions of NUP62. Elucidating the precise mechanism by which BcSSP2 interferes with NUP62 condensate formation will pave the way for engineering the nuclear pore complex to enhance plant resistance against B. cinerea.

Interestingly, previous studies have revealed that prolonged ectopic expression of BcSSP2 or infiltration of a physiological relevant concentration of purified BcSSP2 proteins in plant tissues could cause chlorosis and slow development of cell death (visible around 15 days after agroinfiltration/treatment)^32,33, which differs from typical CDIPs (cell death inducing proteins) that induce instant cell death (within 5 days). It was hypothesized that BcSSP2 protein might not cause direct damage to the plant cell but affect some unknown pathways that eventually lead to cell death³². Considering the crucial function of NPC in plant cell physiology, our work provides a possible mechanistic explanation (e.g., through inhibiting plant NPC function) for the previously reported slow phytotoxicity of BcSSP2. However, it is also plausible that BcSSP2 may target other components beyond NPC to induce slow plant cell death, as pathogen effectors are known to promote infection with multiple mechanisms¹⁹.

As BcSSP2 homologs are widely distributed among ascomycetes (Supplementary Fig. 9a), an evolutionarily conserved strategy might be evolved by these necrotrophic pathogens to facilitate their colonization and infection. It would be exciting to explore whether targeting the NPC central barrier is a general mechanism evolved by necrotrophic pathogens to suppress the NPC phase separation-mediated host defense response for their effective invasion.

Methods

Plants

Arabidopsis thaliana ecotype Col-0 was used as wild-type (WT) control. To generate the transgenic plant iBcSSP2, BcSSP2 CDS region was cloned into pER8, whose expression was driven by an estradiol-inducible promoter, and then transformed into WT plants using the Agrobacterium-mediated floral dip method. Seeds were sterilized with bleach buffer (20% [v/v] bleach, 0.1% [v/v] triton-100), washed with sterile water, and chilled at 4 °C in dark for 3 days. After vernalization, seeds were plated on MS medium (Murashige and Skoog medium, Sigma-Aldrich), and then cultured in a growth room (22 °C with a 16 h/8 h light/dark photoperiod, long day, LD). 8-day-old seedlings were transplanted into soil and grown under the same condition. For pathogen inoculation, plants were grown in soil for 3 to 4 weeks at 22 °C with a 10 h/14 h light/dark photoperiod (short day, SD).

Nicotiana benthamiana (N. benthamiana) seedlings were grown at 22 °C with a 16 h/8 h light/dark photoperiod.

Botrytis cinerea

The wild-type Botrytis cinerea (B. cinerea) strain B05.10 was previously described⁴⁵. PDA medium (Potato Dextrose Agar medium, BD) was used to cultivate B. cinerea at 22 °C.

B. cinerea inoculation

Conidiospores were collected from B. cinerea plates and diluted to 1 × 10⁶ conidiospores/mL in PDB medium (Potato Dextrose Broth, BD). For drop-inoculation assay, 5 µL conidiospores were dripped on the central of detached leaves, and the inoculated leaves were kept in high humidity for 48 h and photographed for lesion size measurement. For spray-inoculation assay, the transgenic plants iBcSSP2 and WT plants were transplanted into MS medium supplemented with 1 μM estradiol or mock (0.002% DMSO) for 24 h, and then sprayed evenly with B. cinerea conidiospores. The inoculated seedlings were kept in high humidity for 36 h. The disease severity was classified into four grades: healthy (H, green), light symptoms (L, pale green), severe symptoms (S, yellow), or completely dead (D, red). For B. cinerea biomass detection, quantitative real-time PCR (qRT-PCR) was applied with primers for B. cinerea-specific ITS gene and Actin of the responding hosts (Arabidopsis, lettuce, strawberry and rose) served as internal control (Supplementary Data 4).

Construction of B. cinerea mutants and complementation strains

To generate B. cinerea knockout mutants, ~600 bp upstream and downstream flanking sequences of BcSSP2 were cloned into pLOB7 which carrying a hygromycin B resistance cassette as a selectable marker. The resulting plasmids were introduced into B. cinerea protoplasts prepared by enzymatic digestion of young mycelia with lysing enzymes. Protoplast transformation was performed using polyethylene glycol (PEG)-mediated transfoamation⁴⁶. Transformants were selected with hygromycin and confirmed by PCR amplification. Primers are listed in Supplementary Data 4.

For complementation, the coding sequence of BcSSP2 were amplified from B05.10 cDNA and cloned into the pNAN-OGG vector, where it was placed under the control of the fungal Polic promoter and linked to nourseothricin resistance. The resulting plasmid was amplified, and the PCR products were used to transform the bcssp2-10 mutant strain. The transformants were initially selected on nourseothricin-containing plates and subsequently verified by PCR-based genotyping. All primer pairs are listed in Supplementary Data 4.

BcSSP2 signal peptides validation

Functional validation of BcSSP2 predicted signal peptides was conducted using the yeast secretion system⁴⁷. The coding sequence of BcSSP2 signal peptide (1-20Aa, SP) was cloned into pSUC2 vector to generate pSUC2-BcSSP2SP, in which the SP of BcSSP2 was fused with truncated SUC2 invertase lacking its original SP. pSUC2-BcSSP2SP, pSUC2-Mg87 and pSUC2-Avr1b⁴⁸ were transformed into yeast strain YTK12 (SUC2-minus), respectively. The transformants were selected by CMD-W plates (0.67% [v/v] yeast nitrogen base without amino acids, 2% [v/v] sucrose, 0.1% [v/v] glucose, 0.075% [v/v] tryptophan dropout supplement, and 2% [v/v] agar). The selected transformants were then coated on YPRAA plates (1% [v/v] yeast extract, 2% [v/v] peptone, 2% [v/v] raffinose, 2 µg/L Antimycin A and 2% [v/v] agar), only strains with secreting SUC2 invertase could consume raffinose and grow well.

Invertase secretion was also confirmed with 2,3,5-triphenyltetrazolium chloride (TTC) assay since invertase could reduce colorless TTC to red-colored 2,3,5-triphenylformazan. Transformants cultured in liquid medium were collected. After washing, the transformants were resuspended in sterile water. Subsequently, 0.1% [v/v] TTC was added into the transformants suspension, and the mixture was incubated at 35 °C for 30 min. The resulting mixture was transferred into cuvettes for photographing.

Effector internalization assay

For the root incubation assay, about 1 cm root tips were detached from plate-grown Arabidopsis Col-0 seedlings and incubated with 50 μM Alexa 546-labeled effector BcSSP2 or TrxA-His (negative control). After incubation for 12–16 h at 23 °C, the roots were washed 4 times with MS medium before confocal imaging⁴⁹.

For the GFP strand assay³⁵, BcSSP2 or signal peptide of BcSSP2 (SP, negative control) were tagged with the 11^th strand of GFP and transformed into Botrytis cinerea strain B05.10 to generate the transgenic stain BcSSP2-GFP₁₁ or SP-GFP₁₁. Tobacco expressing GFP_1-10 (strands 1–10) for 24 h were infected with the transgenic stain BcSSP2-GFP₁₁ and SP-GFP₁₁. The edge area of the infected leaves (24 hpi) was subjected to confocal imaging.

N. benthamiana transient expression

Freshly cultured Agrobacterium was collected and resuspended in infiltration buffer (10 mM MES, 10 mM MgCl₂, 0.2 mM acetosyringone) and kept in dark for 3 h. Leaves of 4-week-old N. benthamiana plants were infiltrated with indicated combinations of Agrobacterium strains. Plants were maintained at 22 °C for 36 h before LCI and subcellular localization assay, respectively. In the assay to detect BcSSP2, SsSSP2 and AfSSP2 effect on NUP62 phase separation, discs of N. benthamiana detached leaves were soaked in 10 μM estradiol or mock buffer (0.02% [v/v] DMSO) for 6 h to induce BcSSP2, SsSSP2 and AfSSP2 expression before confocal imaging.

Co-IP assay

Total proteins were extracted from 5 to 10 g N. benthamiana leaves co-expressing indicated protein combinations with extraction buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 0.5% [v/v] Triton X-100, 0.5% [v/v] Nonidet P-40, 10 μM MG132, 0.1 mM PMSF, Roche inhibitor cocktail). After centrifugation at 16,000 × g, 4 °C for 10 min to remove the debris, 20 μL of pre-washed anti-GFP magnetic agarose beads (GFP-TrapMA, Chromo Tek) were added to the supernatants. The mixture was incubated for 4 hours at 4 °C. The beads were then washed with extraction buffer for 5 times, and the precipitated proteins were eluted by boiling with SDS loading buffer for 5 min. The final samples were analyzed by Western Blot. The concentrated total proteins before adding anti-GFP beads were also detected for indicating the amount of the total input proteins. For Western blotting, anti-Flag (Abmart, M20008, 2000-fold dilution) and Mouse monoclonal anti-GFP (Abmart, M20004, 2000-fold dilution) were used.

In vitro pull-down assay

Purified GST-NUP62, GST-NUP62-NTD, GST-NUP62-CTD and GST proteins were used as baits to incubate with 50 µL pre-washed Glutathione Sepharose 4B affinity chromatography (GST beads, GE Healthcare). 20 µg purified BcSSP2-His and TrxA-His proteins (purified from pET32a as control) were incubated with GST-NUP62, GST-NUP62-NTD, GST-NUP62-CTD or GST at 4 °C for 1 h in reaction buffer (20 mM Tris·HCl, pH 7.5, 500 mM NaCl, 10% [v/v] glycerol and 0.1% [v/v] Triton-X 100), respectively. After extensive washing with reaction buffer, the complexes were released from GST beads by boiling with SDS loading buffer for 5 min.

BcSSP2-His and control proteins were detected by Western blot using Anti-His antibody (Anti-His Mouse mAb, Abmart), and GST-NUP62, GST-NUP62-NTD, GST-NUP62-CTD or GST was showed by Ponceau S staining of the polyvinylidene difluoride membranes.

Protein labeling

The purified proteins were labeled by Alexa Fluor 488 carboxylic acid (ThermoFisher) or Alexa Fluor 546 carboxylic acid (ThermoFisher) at room temperature for 1 h. Then, the free dye was removed by illustra™ Microspin G-50 Columns (GE Healthcare). To minimalize the impact of Alexa dye, less than 1% total proteins were labeled to perform partitioning experiments in our study.

Protein condensate assays

Protein condensate assay was performed in the buffer containing 20 mM HEPES, pH7.4, 150 mM NaCl, 10% [v/v] glycerol, 5 mM DTT. For MBP-fused proteins, additional TEV protease (the molar concentration of TEV was 1% of MBP-tagged proteins) was added to the system to cleave MBP-tag during the condensate assembly^50,51. For detecting the effect of BcSSP2 on NUP62 phase separation, 2 μM NUP62 protein (NUP62 is cleaved from MBP-NUP62 and labeled with Alexa 546) was mixed with 40 μM dye-free BcSSP2 (BcSSP2-His) or control (TrxA-His) protein. For detecting the colocalization of MBP-GFP8W with NUP62 condensate, MBP-GFP8W and MBP-GFP were added to the NUP62 condensate pool. All the experiments were performed on 384-low-binding multi-well 0.17 mm microscopy plates (Cellvis, P384-1.5H-N). The images were captured by a Laser Scanning Confocal Microscopy (Nikon A1R HD25). NIS-Element AR5.2 software was used to analyze the data.

Hydrogel’s preparation

Hydrogels were prepared according to previously described procedure^36,52 with some modifications. Purified MBP-NUP62 was precipitated by 4 M (NH4)₂SO₄ solution and subjected to resuspension in the 0.1% [v/v] trifluoroacetic acid (TFA) to final concentration about 1 mM. 1 µL protein mixture was dripped on glass bottom dishes (Cellvis) and were allowed for 2 days to complete gelation. For MBP-NUP62 + BcSSP2 or MBP-NUP62+control hydrogels, MBP-NUP62 incubated with BcSSP2 or control protein in 4 °C for 1 h before precipitating with (NH4)₂SO₄. The final concentration was 1 mM for MBP-NUP62 and 0.1 mM for BcSSP2 and control protein.

AFM assay

The AFM experiment was performed on the home-built atomic force microscope (AFM)⁵³. Prior to each measurement, in situ spring constant and sensitivity of the probe was calibrated in PBS solution. Probe was moved vertically on the top of hydrogels dipping in PBS solution. The force generated by the downward-moving probe was measured. The signal curve of the tip deflection was recorded. The slope value κ (pN/nm) of the approaches curve was analyzed, adjusted with spring constant and sensitivity of the probe. And the Young’s modulus E (kPa) was calculated. The histograms of the Young’s modulus were fitted with Gauss distribution with the model of GaussAmp in OriginPro 9.1 software.

Translocation assay

For assays in the translocation experiments of MBP-GFP8W, MBP-GFP8W (to final concentration of 4 μM) were added into hydrogels of NUP62, NUP62 + BcSSP2 and NUP62 + control. For the translocation experiments of GFP-MPK3, 10 μL target proteins solutions (4 μM GFP-MPK3, 4 μM MBP-GFP, 4 μM GFP-MPK3 along with 10 μg/μL Col-3 plant crude, 4 μM GFP-MPK3 along with 10 μg/μL Col-3 plant crude) were added to cover the hydrogels. For assays in Supplementary Fig. 10, MBP-NUP62 hydrogels were pre-incubated with 8 μM dye-free BcSSP2, control protein (TrxA-His) or protein storage buffer for 1 h before adding MBP-GFP8W (to final concentration of 4 μM).

After samples loading, images were captured immediately using the Laser Scanning Confocal Microscopy (Nikon A1R HD25). Since sample loading and instrument commissioning (including location marking and samples focusing) are time-consuming, it is hard to capture the first frame within 10 min. Translocation speed was analyzed with NIS-Elements AR. We focused on the bottom of the hydrogel solutions and measured the inside fluorescence of the hydrogels (within dotted circles as shown in the Figure). Half of the mean fluorescence intensity outside the hydrogel was set as the cutoff in the translocation speed assays.

Protein purification

The recombinant vectors (see as Supplementary Data 5) of MBP-NUP62-His, MBP-GFP-His, MBP-GFP8W-His, GFP-MPK3-his, TrxA-His, GST-NUP62, GST-NUP62-NTD and GST-NUP62-CTD were transformed into Escherichia coli BL21 (DE3), respectively. The cells were cultured in LB broth (25 μg/mL ampicillin) at 37 °C until they reached at the logarithmic phase (OD₆₀₀ = 0.8). The expression of proteins was induced by 0.5 mM IPTG at 16 °C for 16 h. Then, the cells were harvested through centrifugation at 4000 × g for 30 min, and resuspended in lysis buffer (40 mM Tris·HCl, pH 8.0, 500 mM NaCl, 10% [v/v] glycerol), and lysed by ultrasonication before centrifugation (16000 × g, 30 min at 4 °C). The target proteins were purified through Ni-NTA Agarose (Thermo Fisher Scientific) for MBP-NUP62-His, MBP-GFP-His, MBP-GFP8W-His, GFP-MPK3-His and TrxA-His, proteins and Glutathione Sepharose 4B affinity chromatography (GE Healthcare) for GST-NUP62, GST-NUP62-NTD and GST-NUP62-CTD. Finally, Gel-filtration chromatography (SD200, Superdex200 Increase 10/300, GE Healthcare) was performed for further purification with the buffer: 20 mM HEPES, pH 7.4, 500 mM NaCl, 10% [v/v] glycerol.

The BcSSP2-His recombinant plasmid (pFastBac-BcSSP2-His) was transformed into DH10Bac competent cells to produce recombinant baculovirus of BcSSP2-His Bacmid. Subsequently, BcSSP2-His protein was expressed within Sf9 insect cells (Gibco) which were infected by recombinant baculovirus of BcSSP2-His Bacmid. After cultured at 28 °C in Sf-900™ II SFM medium for 48 h, the cells were collected and re-suspended in the TBS buffer: 20 mM Tris·HCl, pH 8.0, 150 mM NaCl. Cell lysis was centrifuged at 16000 × g for 30 min to remove cell debris after sonication. The proteins in the supernatant were purified by Ni-NTA Agarose (Thermo Fisher Scientific) and followed by gel-filtration chromatography (SD200, Superdex200 Increase 10/300, GE Healthcare) for further purification with the HEPES buffer: 20 mM HEPES, pH7.4, 150 mM NaCl.

Each purification step was identified by SDS-PAGE, only the high-quality proteins were concentrated and frozen in liquid nitrogen and stored at −80 °C in storage buffer.

Subcellular fraction assay

Seedlings were harvested and ground to a fine powder in liquid nitrogen. 0.5 g powder was suspended in 3 mL precooled extraction buffer (10 mM Tris·HCl, pH 7.4, 10 mM MgCl₂, 10 mM NaCl, 10 mM β-mercaptoethanol, 10% [v/v] glycerol and Roche protease inhibitor cocktail). The homogenate was filtered through double layers of Miracloth twice. The filtrate was spun at 1500 × g, 4 °C for 5 min to pellet the nuclei. The supernatant was then centrifuged at 10,000 × g, 4 °C for 10 min and the new supernatant was collected as the cytoplasmic fraction. The pellets were washed 7 times with 2 mL of washing buffer consisting of 10 mM Tris, pH 7.5, 10 mM MgCl₂, 10 mM NaCl, 1 M hexylene glycol, 0.5% [v/v] Triton X-100, 10 mM β-mercaptoethanol and Roche protease inhibitor cocktail. The final pellets were resuspended in 200 μL 8 M urea and sonicated. The homogenate was centrifuged at 16,000 × g for 10 min and 4 °C to remove debris and collected as the nuclear fraction. For Western blotting, the following primary antibodies were used: rabbit polyclonal anti-AtMPK3 (Sigma-Aldrich, M8318; 1000-fold dilution), rabbit polyclonal anti-PEP Carboxylase (Abcam, ab34793; 10000-fold dilution), and rabbit polyclonal anti-Histone H3 (Sigma-Aldrich, H0164; 10000-fold dilution).

Confocal imaging of plant materials

Detached roots and leaf discs were fixed in microslides filling with liquid MS medium. The fluorescence signal was detected on a Nikon A1R HD25 with the galvano scanner. For puncta quantification, the 3D model was used. All the layers with signals were scanned and 30 layers (0.5 μm/layer) with the strongest signals were selected for analysis. Analysis of puncta number and intensity was performed by the software of NIS-Element AR5.2 and the presented images were processed with deconvolution.

In the assay to detect BcSSP2, SsSSP2 and AfSSP2 effect on NUP62 phase separation, discs of tobacco detached leaves were soaked in 10 μM estradiol or mock buffer (0.02% [v/v] DMSO) for 6 h to induce BcSSP2, SsSSP2 and AfSSP2 expression and then washed 4 times with MS medium before sample loading. In the assay with pathogen infection, tobacco leaves expressing NUP62-GFP for 24 h were subsequently infected with bcssp2-12 or B05.10 strains for 24 h, and the edge area of the infected leaves was subjected for confocal imaging and relative fluorescent intensity analysis.

RNA seq analysis

For transcriptome analysis of B. cinerea genes during infection, total RNAs were extracted from Arabidopsis (Col-0) leaves after inoculation with B. cinerea for 24 h, or from B. cinerea hypha grown on PDA medium (control group). Qualified RNAs were sent to BGI for RNA-seq by BGISEQ-500 platform. The clean reads were mapped to B. cinerea genome using HISAT2 and differently expressed genes were filtered out by DESeq2. The cut-offs for fold-change and FDR were set at 2 and 0.05, respectively. The whole sequencing data were deposited in the NCBI database (GSE217021; token: cpcdiukqfrydrct).

Phylogenetic analysis of BcSSP2

For constructing the phylogenetic tree for BcSSP2, the protein sequence of BcSSP2 (XP_001559885.1) was blasted against the NCBI Reference protein database via BlastP. Among the 85 protein sequences that showed significant alignments (E-value < 0.05) with BcSSP2, the best-aligned sequences from each species were selected for the phylogenetic tree construction. The phylogenetic tree was constructed by MEGA-X using the maximum likelihood method (Whelan and Goldman model) with the default parameters.

Statistics and reproducibility

No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq data generated in this study have been deposited in the NCBI GEO under accession numbers GSE217021. The processed RNA-seq data are available at Supplementary Data. All other data supporting the findings of this study are available in the main text, Supplementary Information or Source Data files. Source data are provided with this paper.

References

Jones, J. D., Vance, R. E. & Dangl, J. L. Intracellular innate immune surveillance devices in plants and animals. Science 354, aaf6395 (2016).
Guo, Z., Li, Y. & Ding, S. W. Small RNA-based antimicrobial immunity. Nat. Rev. Immunol. 19, 31–44 (2019).
Article CAS PubMed Google Scholar
Jones, J. D. & Dangl, J. L. The plant immune system. Nature 444, 323–329 (2006).
Article ADS CAS PubMed Google Scholar
Leary, A. Y., Savage, Z., Tumtas, Y. & Bozkurt, T. O. Contrasting and emerging roles of autophagy in plant immunity. Curr. Opin. Plant Biol. 52, 46–53 (2019).
Article CAS PubMed Google Scholar
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
Article CAS PubMed PubMed Central Google Scholar
Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).
Article CAS PubMed PubMed Central Google Scholar
Protter, D. S. W. & Parker, R. Principles and properties of stress granules. Trends Cell Biol. 26, 668–679 (2016).
Article CAS PubMed PubMed Central Google Scholar
Du, M. & Chen, Z. J. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science 361, 704–709 (2018).
Article ADS CAS PubMed PubMed Central Google Scholar
Slavik, K. M. et al. cGAS-like receptors sense RNA and control 3′2′-cGAMP signaling in Drosophila. Nature 597, 109–113 (2021).
Xu, G. et al. Viral tegument proteins restrict cGAS-DNA phase separation to mediate immune evasion. Mol. Cell 81, 2823–2837 e2829 (2021).
Article CAS PubMed Google Scholar
Mathieu, C., Pappu, R. V. & Taylor, J. P. Beyond aggregation: Pathological phase transitions in neurodegenerative disease. Science 370, 56–60 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Wang, J. et al. Phase separation of nuclear pore complex facilitates selective nuclear transport to regulate plant defense against pathogen and pest invasion. Mol. Plant 16, 1016–1030 (2023).
Huang, S., Zhu, S., Kumar, P. & MacMicking, J. D. A phase-separated nuclear GBPL circuit controls immunity in plants. Nature 594, 424–429 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Zavaliev, R., Mohan, R., Chen, T. & Dong, X. Formation of NPR1 condensates promotes cell survival during the plant immune response. Cell 182, 1093–1108 e1018 (2020).
Article CAS PubMed PubMed Central Google Scholar
Zhou, Y. et al. Plant HEM1 specifies a condensation domain to control immune gene translation. Nat. Plants 9, 289–301 (2023).
Article CAS PubMed Google Scholar
Song, W. et al. Substrate-induced condensation activates plant TIR domain proteins. Nature 627, 847–853 (2024).
Article ADS CAS PubMed PubMed Central Google Scholar
Lo Presti, L. et al. Fungal effectors and plant susceptibility. Annu. Rev. Plant Biol. 66, 513–545 (2015).
Article CAS PubMed Google Scholar
Wu, Q., Wang, X. & Ding, S. W. Viral suppressors of RNA-based viral immunity: host targets. Cell Host Microbe 8, 12–15 (2010).
Article CAS PubMed PubMed Central Google Scholar
Xin, X. F. & He, S. Y. Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants. Annu Rev. Phytopathol. 51, 473–498 (2013).
Article CAS PubMed Google Scholar
Figueroa, M., Ortiz, D. & Henningsen, E. C. Tactics of host manipulation by intracellular effectors from plant pathogenic fungi. Curr. Opin. Plant Biol. 62, 102054 (2021).
Article CAS PubMed Google Scholar
Fu, Z. Q. et al. A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity. Nature 447, 284–288 (2007).
Article ADS CAS PubMed Google Scholar
Kong, L. et al. A phytophthora effector manipulates host histone acetylation and reprograms defense gene expression to promote infection. Curr. Biol. 27, 981–991 (2017).
Article CAS PubMed Google Scholar
Ma, Z. et al. A paralogous decoy protects Phytophthora sojae apoplastic effector PsXEG1 from a host inhibitor. Science 355, 710–714 (2017).
Article ADS CAS PubMed Google Scholar
Wu, D. et al. Viral effector protein manipulates host hormone signaling to attract insect vectors. Cell Res. 27, 402–415 (2017).
Article CAS PubMed PubMed Central Google Scholar
Jelenska, J., van Hal, J. A. & Greenberg, J. T. Pseudomonas syringae hijacks plant stress chaperone machinery for virulence. Proc. Natl Acad. Sci. USA 107, 13177–13182 (2010).
Article ADS CAS PubMed PubMed Central Google Scholar
Duan, C. G. et al. Suppression of Arabidopsis ARGONAUTE1-mediated slicing, transgene-induced RNA silencing, and DNA methylation by distinct domains of the Cucumber mosaic virus 2b protein. Plant Cell 24, 259–274 (2012).
Article CAS PubMed PubMed Central Google Scholar
Dean, R. et al. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 13, 414–430 (2012).
Article PubMed PubMed Central Google Scholar
Williamson, B., Tudzynski, B., Tudzynski, P. & van Kan, J. A. Botrytis cinerea: the cause of grey mould disease. Mol. Plant Pathol. 8, 561–580 (2007).
Article CAS PubMed Google Scholar
Heard, S., Brown, N. A. & Hammond-Kosack, K. An interspecies comparative analysis of the predicted secretomes of the necrotrophic plant pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS One 10, e0130534 (2015).
Article PubMed PubMed Central Google Scholar
Veloso, J. & van Kan, J. A. L. Many shades of grey in Botrytis-host plant interactions. Trends Plant Sci. 23, 613–622 (2018).
Article CAS PubMed Google Scholar
Weiberg, A. et al. Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science 342, 118–123 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Zhu, W. et al. Botrytis cinerea BcSSP2 protein is a late infection phase, cytotoxic effector. Environ. Microbiol. 24, 3420–3435 (2022).
Article CAS PubMed Google Scholar
Denton-Giles, M. et al. Conservation and expansion of a necrosis-inducing small secreted protein family from host-variable phytopathogens of the Sclerotiniaceae. Mol. Plant Pathol. 21, 512–526 (2020).
Article CAS PubMed PubMed Central Google Scholar
Petersen, T. N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786 (2011).
Article CAS PubMed Google Scholar
Henry, E., Toruno, T. Y., Jauneau, A., Deslandes, L. & Coaker, G. Direct and indirect visualization of bacterial effector delivery into diverse plant cell types during infection. Plant Cell 29, 1555–1570 (2017).
Article CAS PubMed PubMed Central Google Scholar
Frey, S. et al. Surface properties determining passage rates of proteins through nuclear pores. Cell 174, 202–217 e209 (2018).
Article CAS PubMed Google Scholar
Li, J. et al. Plant PR1 rescues condensation of the plastid iron-sulfur protein by a fungal effector. Nat. Plants 10, 1775–1789 (2024).
Article ADS CAS PubMed Google Scholar
Li, X. & Gu, Y. Structural and functional insight into the nuclear pore complex and nuclear transport receptors in plant stress signaling. Curr. Opin. Plant Biol. 58, 60–68 (2020).
Article CAS PubMed Google Scholar
Lin, D. H. & Hoelz, A. The structure of the nuclear pore complex. Annu. Rev. Biochem. 88, 725–783 (2019).
Article CAS PubMed PubMed Central Google Scholar
Beck, M. & Hurt, E. The nuclear pore complex: understanding its function through structural insight. Nat. Rev. Mol. Cell Biol. 18, 73–89 (2017).
Article CAS PubMed Google Scholar
Boeglin, M. et al. Reduced expression of AtNUP62 nucleoporin gene affects auxin response in Arabidopsis. BMC Plant Biol. 16, 2 (2016).
Article PubMed PubMed Central Google Scholar
Cheng, Y. T. et al. Nuclear pore complex component MOS7/Nup88 is required for innate immunity and nuclear accumulation of defense regulators in Arabidopsis. Plant Cell 21, 2503–2516 (2009).
Article CAS PubMed PubMed Central Google Scholar
Gu, Y. et al. Nuclear pore permeabilization is a convergent signaling event in effector-triggered immunity. Cell 166, 1526–1538 e1511 (2016).
Article CAS PubMed PubMed Central Google Scholar
Zhang, Y. & Li, X. A putative nucleoporin 96 Is required for both basal defense and constitutive resistance responses mediated by suppressor of npr1-1, constitutive 1. Plant Cell 17, 1306–1316 (2005).
Article CAS PubMed PubMed Central Google Scholar
Schumacher, J., Studt, L. & Tudzynski, P. The putative H3K36 demethylase BcKDM1 affects virulence, stress responses and photomorphogenesis in Botrytis cinerea. Fungal Genet Biol. 123, 14–24 (2019).
Article CAS PubMed Google Scholar
An, B. et al. Aquaporin8 regulates cellular development and reactive oxygen species production, a critical component of virulence in Botrytis cinerea. N. Phytologist 209, 1668–1680 (2016).
Article CAS Google Scholar
Oh, S.-K. et al. In Planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes, including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2. Plant Cell 21, 2928–2947 (2009).
Article CAS PubMed PubMed Central Google Scholar
Fang, A. et al. Identification and characterization of plant cell death-inducing secreted proteins from Ustilaginoidea virens. Mol. Plant Microbe Interact. 29, 405–416 (2016).
Article CAS PubMed Google Scholar
Kale, S. D. et al. External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells. Cell 142, 284–295 (2010).
Article CAS PubMed Google Scholar
Fang, X. et al. Arabidopsis FLL2 promotes liquid-liquid phase separation of polyadenylation complexes. Nature 569, 265–269 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Lin, Y., Protter, D. S., Rosen, M. K. & Parker, R. Formation and maturation of phase-separated liquid droplets by RNA-binding proteins. Mol. Cell 60, 208–219 (2015).
Article CAS PubMed PubMed Central Google Scholar
Frey, S. & Gorlich, D. A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell 130, 512–523 (2007).
Article CAS PubMed Google Scholar
Kong, F., Garcia, A. J., Mould, A. P., Humphries, M. J. & Zhu, C. Demonstration of catch bonds between an integrin and its ligand. J. Cell Biol. 185, 1275–1284 (2009).
Article CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We thank Paul Tudzynski (University of Münster, Germany) for providing B. cinerea stains; Chang He and Xiaokang Zhang (Institute of Botany, the Chinese Academy of Sciences) for help with B. cinerea transformation; Jizhong Lou, Hui Chen and Yanruo Zhang (Institute of Biophysics, Chinese Academy of Sciences) for help with AFM experiments; Huizhen Cao and Jinyu Wang (Center of Biomedical Analysis, Tsinghua University) for help with confocal imaging. This research was supported by funding from the National Natural Science Foundation of China (Grant no. 32250001 and Grant no. 32388101 to D.X., Grant no. 32125010 and 32330024 to P.L., Grant no. 32402306 to J.W., Grant no. 32400560 to G.P.); the National Key Research and Development Program of China (2022YFD1400800 and 2021YFA1300400 to D.X., 2023YFF1204703 to P.L.); the Beijing Natural Science Foundation (Grant no. Z230014 to P.L.); the Postdoctoral Fellowship Program of CPSF (Grant no. GZC20240859 to G.P.); the Shuimu Tsinghua Scholar Program (Grant no. 2023SM250 to G.P.); the Postdoctoral Fellowship of the Tsinghua-Peking University Joint Center for Life Sciences (G.P.); the Advanced Innovation Fellow Program (G.P.).

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These authors contributed equally: Jiaojiao Wang, Dewei Wu, Gaofeng Pei, Yupei Wang

Authors and Affiliations

College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, China
Jiaojiao Wang
MOE Key Laboratory of Bioinformatics, Tsinghua University-Peking University Joint Center for Life Sciences, and School of Life Sciences, Tsinghua University, Beijing, China
Jiaojiao Wang, Dewei Wu, Yupei Wang, Xiaokang Liu, Xiaolin Zhang, Daoxin Xie & Xiaoyi Shan
China-Uruguay Joint Laboratory on Soybean Research and Innovation, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
Dewei Wu
State Key Laboratory of Membrane Biology, Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University; Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China
Gaofeng Pei & Pilong Li
Institute of Botany, Chinese Academy of Sciences, Beijing, China
Shiping Tian & Zhanquan Zhang
University of Chinese Academy of Sciences, Beijing, China
Shiping Tian & Zhanquan Zhang

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Contributions

D.X., X.S. and P.L. conceived the study; D.X., J.W. designed the experiments; J.W., D.W., G.P., and Y.W. performed most of the experiments with assistance from X.L., S.T., Z.Z. and X.Z.; J.W. and D.W. wrote the manuscript; D.X., P.L., X.S., J.W., D.W., G.P., and Y.W. revised the paper.

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Correspondence to Jiaojiao Wang, Pilong Li, Daoxin Xie or Xiaoyi Shan.

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Wang, J., Wu, D., Pei, G. et al. The pathogen effector BcSSP2 suppresses the NPC phase separation to facilitate Botrytis cinerea infection. Nat Commun 16, 8610 (2025). https://doi.org/10.1038/s41467-025-63632-7

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Received: 18 May 2023
Accepted: 26 August 2025
Published: 29 September 2025
DOI: https://doi.org/10.1038/s41467-025-63632-7