A conserved fungal effector disturbs Ca2+ sensing and ROS homeostasis to induce plant cell death

Lin, Yunlong; Xu, Chan; Li, Lili; Fan, Liqin; Li, Rui; He, Jiaxin; Li, Hongli; Deng, Wei; Kang, Zhensheng; Li, Zhengguo; Cheng, Yulin

doi:10.1038/s41467-025-58833-z

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Published: 14 April 2025

A conserved fungal effector disturbs Ca²⁺ sensing and ROS homeostasis to induce plant cell death

Yunlong Lin^1,2^na1,
Chan Xu¹^na1,
Lili Li²,
Liqin Fan^1,2,
Rui Li¹,
Jiaxin He¹,
Hongli Li²,
Wei Deng¹,
Zhensheng Kang ORCID: orcid.org/0000-0001-5575-0122³,
Zhengguo Li ORCID: orcid.org/0000-0001-7019-2560¹ &
…
Yulin Cheng ORCID: orcid.org/0000-0001-9049-7740¹

Nature Communications volume 16, Article number: 3523 (2025) Cite this article

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Abstract

Acting as a major Ca²⁺ sensor, calmodulin (CaM) activates target proteins to regulate a variety of cellular processes. Here, we report that CaM–target binding is disturbed by a fungal virulence effector PdCDIE1 (Penicillium digitatum Cell Death-Inducing Effector 1), which results into reactive oxygen species (ROS)-dependent plant cell death. PdCDIE1 is an evolutionarily conserved fungal effector that exhibits plant cell death-inducing activity and contributes significantly to pathogen virulence. PdCDIE1 interacts with a plant heat shock protein Hsp70 that is antagonistic to ROS-dependent plant cell death. Hsp70 is a bona fide target of CaM and its CaM-binding domain also interacts with N-terminal PdCDIE1. The interaction between CaM and Hsp70 in citrus fruit is disturbed during pathogen infection but recovered during ΔPdCDIE1 mutant infection. Application of a CaM inhibitor and silencing of CaM genes induce plant cell death and high levels of ROS as PdCDIE1 does. These results reveal a molecular framework of effector-triggered susceptibility which integrates Ca²⁺ sensing and ROS homeostasis to induce plant cell death.

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Introduction

Cytosolic calcium (Ca²⁺) is one of the most important second messengers in plants, and Ca²⁺ signals are shown to be a core regulator of many plant biological processes, especially for plant responses to environmental stresses¹. Ca²⁺ signals, which are shaped through the concerted activity of ion channels and exchangers, are decoded by an array of Ca²⁺ sensors². Calmodulin (CaM) is a highly conserved and the most prominent Ca²⁺ sensor in eukaryotic cells, and CaM activates target proteins to regulate a variety of cellular processes³. Plant diseases caused by pathogenic microbes have resulted in famines throughout human history and continue to threaten global food security, and increasing evidence indicates the important contribution of Ca²⁺ signaling in plant disease resistance^2,4. Diverse Ca²⁺ signaling components mainly including Ca²⁺ channels and Ca²⁺ sensors play important roles in regulating PAMP-triggered immunity (PTI), effector-triggered immunity (ETI), and systemic defense responses⁴. Noticeably, recent researches showed that some Ca²⁺ signaling components were also involved in plant immune suppression or plant disease susceptibility^5,6. The CNGC2–CNGC4 calcium channel during Flg22-triggered PTI immunity was blocked by a calmodulin CaM7 in Arabidopsis⁵. Rice ROD1, which was a C2 domain Ca²⁺ sensor, was proven to suppress plant immunity by mediating the scavenging of reactive oxygen species (ROS)⁶. Further investigation of the involvement of Ca²⁺ signaling in plant disease susceptibility will provide an important foundation for comprehensively understanding Ca²⁺ signaling in plant–pathogen interactions.

Plant disease susceptibility is not only due to the loss of resistance genes but also due to the manipulation by pathogens, and dissection of plant disease susceptibility is crucial for developing novel strategies for controlling plant diseases⁷. To complete their life cycles, plant pathogens produce an arsenal of virulence-associated “effector” proteins to interfere with plant immune signaling or manipulate other plant physiological processes, thus enabling effector-triggered susceptibility (ETS)⁸. Effectors secreted by pathogens are thought to be major determinants of pathogen virulence on host plants and their molecular functions have become the central questions for studying plant–pathogen interactions⁹. Numerous plant immunity-suppressing effectors from different pathogens have been identified, and pathogen effectors can target and disrupt any phase of the plant immune response¹⁰. Compared with understanding how pathogen effectors interfere with plant immune signaling, understanding how pathogen effectors manipulate non-immunity-related plant processes is just beginning to emerge¹¹. Pathogen effectors can also manipulate plant metabolisms, such as the transport of sugars and the production of gamma-aminobutyric acid (GABA), to support pathogen nutrition^12,13. In contrast to biotrophic pathogens that feed on living plant cells, necrotrophic pathogens kill host tissues and then acquire nutrients from dead plant cells to promote infection¹⁴. Plant cell death is essential for plant infection of necrotrophic pathogens and many cell death-inducing effectors (CDIEs) have been identified from necrotrophic fungi¹⁵. Understanding how CDIEs from necrotrophic fungi promote plant cell death mainly comes from the well-known host-selective toxin (HST) effectors, and a series of HST effectors were proven to manipulate plant cell death by hijacking R-mediated resistance in an inverse gene-for-gene manner¹⁵.

Fruit contains abundant nutrients and is an essential part of the human diet. Postharvest diseases caused by plant pathogens, most of which are necrotrophic fungi, are the main cause of fruit decay during storage and transportation, resulting in enormous economic losses worldwide¹⁶. As one of the most cultivated and consumed fruit crops worldwide, citrus fruit is subjected to a series of postharvest fungal pathogens¹⁷. The necrotrophic fungus Penicillium digitatum (Pd) causes the notorious citrus postharvest green mold disease, which is the major factor (can account for up to 90%) resulting in citrus fruit decay, and also serves as a model fungus to understand the mechanisms of pathogen infection in fruit^18,19. Noticeably, molecular aspects in fruit–pathogen interactions, especially the role of virulence effectors in postharvest diseases, remain relatively unexplored^16,19. Identification and functional analysis of putative effectors were reported in several postharvest fungal pathogens and a minority of these putative effectors were proven to be required for pathogen virulence²⁰. However, nothing is known about how virulence effectors promote pathogen infection in fruit. In this study, we identified an evolutionarily conserved effector PdCDIE1 from Pd and investigated the specific mechanism of PdCDIE1 regulating plant cell death. PdCDIE1 contributes significantly to Pd virulence and disturbs the CaM–target binding to induce ROS-dependent plant cell death. Our results reveal a mechanism of pathogen interaction with fruit at an unprecedented level of detail.

Results

Identification of the cell death-inducing effector PdCDIE1

To identify candidate effectors required for Pd infection in citrus fruit, we focused on these in planta induced Pd genes which contained at least three expressed sequence tags (ESTs) or showed at least two-fold induction in the cDNA library of Pd-infected citrus fruit²¹. Annotation as a hypothetical protein and the presence of an N-terminal signal peptide predicted by SignalP 5.0 were the criteria for selecting candidate effectors, and four Pd genes encoding candidate effector proteins (Fig. 1a) were selected. Agrobacterium-mediated transient expression of these four Pd genes in the model plant Nicotiana benthamiana showed that only PDIP_06490 exhibited obvious plant cell death-inducing activity (Fig. 1b; Supplementary Fig. 1a–d) as the positive control BAX, which is a well-known mammalian pro-apoptotic factor. Transient expression of PDIP_06490 exhibited an obvious increase in electrolyte leakage than transient expression of the negative control GFP (Fig. 1c), which was consistent with the cell death phenotype (Fig. 1b). The predicted mature protein of PDIP_06490 is rich in cysteine residues (up to 10), which is a common feature for many pathogen effectors and may be required for effector stability by forming multiple disulfide bonds²². Thus, PDIP_06490 was termed PdCDIE1 (Penicillium digitatum Cell Death-Inducing Effector 1). Moreover, Agrobacterium-mediated transient expression of PdCDIE1, but not GFP, in tomato leaf also induced obvious cell death (Supplementary Fig. 2a). Agrobacterium-mediated transient expression can also be utilized for evaluating gene function in fruits²³, and thus the putative cell death-inducing activity of PdCDIE1 in citrus fruit, the host of Pd, was checked. Noticeably, Agrobacterium-mediated transient expression of PdCDIE1 in citrus fruit induced cell death (Fig. 1d; Supplementary Fig. 1f). To increase the visibility of cell death phenotype on fruit, citrus fruit were stained with a cell death indicator, propidium iodide (PI) that can pass through damaged cell membranes and stain the nucleus²⁴. As expected, transient expression of PdCDIE1 resulted into obvious PI staining on citrus fruit (Fig. 1d). Meanwhile, transient expression of PdCDIE1 in citrus fruit exhibited an obvious increase in electrolyte leakage (Fig. 1f). In addition, Agrobacterium-mediated transient expression of PdCDIE1 in apple fruit also induced cell death (Fig. 1e; Supplementary Fig. 1g), which was confirmed by PI staining (Fig. 1e) and electrolyte leakage (Fig. 1g) assays. These results indicate that PdCDIE1 shows cell death-inducing activity in different plant species.

**Fig. 1: PdCDIE1 and its homologs from two other postharvest fungal pathogens exhibit plant cell death-inducing activity.**

PdCDIE1 is conserved among diverse fungal pathogens and its homologs from two other postharvest fungal pathogens also exhibit cell death-inducing activity

Although most known pathogen effectors are unique to specific pathogens, some important effectors are evolutionarily conserved among diverse pathogens²⁵. BLAST analyses suggest that PdCDIE1 has homologs in many other plant fungal pathogens and even insect or human fungal pathogens (Fig. 1h). Noticeably, PdCDIE1 homologs are also observed in hemibiotrophic plant pathogens, such as Fusarium oxysporum, and biotrophic plant pathogens, such as Claviceps purpurea (Fig. 1h). These results indicate the evolutionary conservation of PdCDIE1 among diverse fungal pathogens.

P. italicum and P. expansum are two other important necrotrophic fungal pathogens which infect citrus fruit and a wide range of pome fruits, respectively²⁶. There is one PdCDIE1 homolog in both P. italicum and P. expansum (Fig. 1h), and high sequence conservation is observed between PdCDIE1 and the two homologs including PITC_010880 from P. italicum and PEX2_090640 from P. expansum (Supplementary Fig. 2b). Noticeably, the two homologs also contain an N-terminal signal peptide (Supplementary Fig. 2b), inferring that they are also putative effectors. Agrobacterium-mediated transient expression of PITC_010880 and PEX2_090640 in N. benthamiana showed that they also exhibited obvious cell death-inducing activity (Fig. 1i, j; Supplementary Fig. 1e). In addition, the two homologs also induced cell death on citrus fruit and apple fruit (Fig. 1d, e; Supplementary Fig. 1f, g), which was confirmed by PI staining (Fig. 1d, e) and electrolyte leakage (Fig. 1f, g) assays. These data highlight the conserved cell death-inducing activity among PdCDIE1 and its homologs from other postharvest fungal pathogens.

PdCDIE1 is a cytoplasmic effector

There is an N-terminal signal peptide (1–21 aa) in PdCDIE1 (Fig. 1a), and a yeast signal trap assay²⁷ was performed to validate the secretion function of the signal peptide (SP) of PdCDIE1. Compared with YTK12 strain carrying the pSUC2 empty vector, YTK12 strain carrying pSUC2:PdCDIE1^SP was able to grow on the YPRAA medium (with raffinose as the sole carbon source) and reduce triphenyltetrazolium chloride (TTC) to insoluble red-colored triphenylformazan (Supplementary Fig. 3a), which supports the functionality of the SP of PdCDIE1. To confirm the secretion of PdCDIE1 during Pd infection in citrus fruit, total proteins from in vitro-grown fungus, uninfected fruit tissues, and Pd-infected fruit tissues were immunoblotted with an anti-PdCDIE1 antibody. As shown in Supplementary Fig. 3b, the specific band of PdCDIE1 was observed in Pd-infected fruit tissues, but not uninfected fruit tissues and in vitro-grown fungus. Pd-infected fruit tissues were ground by a homogenizer and then filtrated by a microfilter to remove fungus, and total proteins from filtered fluids were immunoblotted with the anti-PdCDIE1 antibody. As expected, the specific band of PdCDIE1 was also observed in filtered fluids (Supplementary Fig. 3b). These results support the secretion of PdCDIE1 during Pd infection in citrus fruit.

To investigate the site of PdCDIE1 functions in plant, subcellular localization assays in the model plant N. benthamiana were performed. As shown in Supplementary Fig. 4, PdCDIE1, PdCDIE1^ΔSP (PdCDIE1 without its SP), and SP^PR3-PdCDIE1^ΔSP that replaced its native SP with the SP of Arabidopsis pathogenesis-related protein 3 (PR3) were all localized in plant cytoplasm. Meanwhile, cell death assays showed that PdCDIE1^ΔSP still induced cell death in N. benthamiana and citrus fruit (Supplementary Fig. 3c–f). These data suggest that PdCDIE1 is a cytoplasmic effector. To identify the putative uptake signal of PdCDIE1 that probably mediates effector translocation into plant cells²⁸, we performed Agroinfiltration assays of N. benthamiana leaves with subcellular localization constructs containing the SP along with various regions of the predicted mature protein of PdCDIE1 (Supplementary Fig. 5). As expected, SP^PdCDIE1 that only contained the SP of PdCDIE1 was localized in apoplastic spaces. Noticeably, SP^PdCDIE1-PdCDIE1^22-63 that contained the SP along with PdCDIE1^22-63 was translocated into plant cytoplasm (Supplementary Fig. 5). Thus, a 42-residue region of the N-terminus (PdCDIE1^22-63) probably mediates the translocation of PdCDIE1 into plant cells.

PdCDIE1 contributes significantly to Pd virulence in citrus fruit

Although PdCDIE1 was proven to be induced during Pd infection in citrus fruit²¹, quantitative reverse transcription-PCR (qRT-PCR) analysis was performed to characterize the specific transcript profiles of PdCDIE1 in different Pd infection stages. As shown in Fig. 2a, the abundance of PdCDIE1 transcripts was significantly induced during Pd infection in citrus fruit and peaked at 3 days post inoculation (dpi). To verify the role of PdCDIE1 in Pd pathogenicity, we knocked out PdCDIE1 in Pd genome by using the Agrobacterium-mediated transformation system (Supplementary Fig. 6a, b). There was no significant difference in germination and growth between ΔPdCDIE1 mutant and wild-type (WT) strain (Supplementary Fig. 6c–e). Whereas, knock-out of PdCDIE1 significantly attenuated Pd virulence in citrus fruit (Fig. 2b, c). PI staining (Fig. 2d) and electrolyte leakage (Fig. 2e) assays showed that plant cell death was attenuated in ΔPdCDIE1 mutant-infected citrus fruit compared with WT strain-infected citrus fruit, which is consistent with the disease phenotype (Fig. 2b). Phytophthora NPP1 (Nep1-like Protein 1) is a marker of necrotrophic growth during pathogen infection²⁹, and the expression of PdNPP1 (GenBank accession no. XM_014676454.1) encoding the homolog of Phytophthora NPP1 during Pd infection was assessed by qRT-PCR. As shown in Supplementary Fig. 7, PdNPP1 was significantly induced in WT strain infection compared with in vitro-grown fungus, indicating the involvement of PdNPP1 to necrotrophic growth during Pd infection. Noticeably, the abundance of PdNPP1 transcripts was decreased in ΔPdCDIE1 mutant infection compared with WT strain infection (Supplementary Fig. 7), which suggests that knock-out of PdCDIE1 probably has negative impact on necrotrophic growth during Pd infection. Collectively, these results indicate that PdCDIE1 contributes significantly to Pd virulence in citrus fruit.

**Fig. 2: PdCDIE1 is required for Pd virulence in citrus fruit.**

PdCDIE1 physically associates with a plant heat shock protein Hsp70

Because PdCDIE1 can induce plant cell death in N. benthamiana, we used the immunoprecipitation-mass spectrometry (IP-MS) method (Supplementary Fig. 8a) to identify N. benthamiana proteins potentially targeted by PdCDIE1. Agrobacterium-mediated transient expression of HA-tagged PdCDIE1 in N. benthamiana was performed, and then putative plant interactors were identified by using anti-HA-probe monoclonal-antibody-conjugated agarose beads at 3 dpi when PdCDIE1 was highly expressed (Supplementary Fig. 8b). Among six identified N. benthamiana proteins from the IP-MS assay (Supplementary Table 1), heat shock protein Hsp70 was inferred as the potential target of PdCDIE1 because heat shock proteins play important roles in plant–pathogen interactions and some of them were proven to regulate plant cell death³⁰. Yeast two-hybrid (Y2H) assays indicated the interaction between PdCDIE1 and N. benthamiana Hsp70 (NbHsp70) (Supplementary Fig. 9a, b). In addition, in vitro pull-down assays confirmed the direct interaction between PdCDIE1 and NbHsp70 (Supplementary Fig. 9c, d).

Noticeably, Hsp70 is highly evolutionarily conserved in diverse plant species and sequence identity between NbHsp70 and the orthologous protein (CsHsp70) from Citrus sinensis is up to 98% (Supplementary Fig. 10). Y2H assays showed that PdCDIE1 interacted with CsHsp70 (Fig. 3a). Both NbHsp70 and CsHsp70 contain a C-terminal EEVD motif (Supplementary Fig. 10), which is a characteristic of cytosolic Hsp70³¹, and subcellular localization assays indicated their cytoplasmic localization (Supplementary Fig. 4). To determine the putative interaction between PdCDIE1 and CsHsp70, PdCDIE1-GFP and mCherry-CsHsp70 were co-expressed in plant. As shown in Fig. 3b, PdCDIE1 and CsHsp70 colocalized in plant cytoplasm. Moreover, the interaction between PdCDIE1 and CsHsp70 at plant cytoplasm was proven by bimolecular fluorescence complementation (BiFC) assays (Fig. 3c). To further confirm the interaction of PdCDIE1 and CsHsp70 in planta, co-immunoprecipitation (Co-IP) assays in citrus fruit were performed. As shown in Fig. 3d, e, PdCDIE1 interacted with CsHsp70 in citrus fruit. Collectively, our results indicate that PdCDIE1 physically associates with CsHsp70.

**Fig. 3: PdCDIE1 interacts with citrus CsHsp70.**

Hsp70 is antagonistic to ROS-dependent plant cell death

High level of ROS has been proven to trigger plant cell death, thus contributing to plant infection of necrotrophic pathogens³². As shown in Fig. 4a, b, a noticeable increase in hydrogen peroxide (H₂O₂) production was observed during PdCDIE1-triggered cell death in N. benthamiana. Besides, exogenous treatment of dimethylthiourea (DMTU), an effective H₂O₂ scavenger³³, greatly inhibited PdCDIE1-triggered cell death in N. benthamiana (Fig. 4c, d). Meanwhile, H₂DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) staining assays (Fig. 4e) and quantitative determination of H₂O₂ production (Fig. 4f) confirmed the ROS-scavenging activity of DMTU. Thus, ROS is required for PdCDIE1-triggered plant cell death.

**Fig. 4: PdCDIE1-triggered plant cell death is dependent on ROS and Hsp70 is antagonistic to plant cell death.**

To investigate the putative role of the interactor Hsp70 in plant cell death, tobacco rattle virus (TRV)-mediated gene silencing of NbHsp70 in N. benthamiana was performed. NbHsp70 was specifically silenced in TRV:NbHsp70-treated plants compared with TRV:00-treated (control) plants (Supplementary Fig. 11a, b). Noticeably, NbHsp70-silenced (TRV:NbHsp70-treated) plants showed impaired plant growth (Supplementary Fig. 11c) and also showed cell death in leaves (Fig. 4g, h). H₂O₂ accumulation was higher in NbHsp70- silenced plants than control plants (Fig. 4i). In addition, Agrobacterium-mediated transient expression of NbHsp70 or CsHsp70 in N. benthamiana showed that both NbHsp70 and CsHsp70 did not trigger plant cell death (Supplementary Fig. 11d) but could suppress PdCDIE1-triggered plant cell death (Fig. 4j). These data confirm that Hsp70 is antagonistic to ROS-dependent plant cell death.

Hsp70 contains a CaM-binding domain and is a bona fide target of CaM

CaM-binding proteins act as the downstream targets of CaM during Ca²⁺ signaling³, and some plant cytosolic Hsp70 proteins were proven to be CaM-binding proteins which contain a conserved CaM-binding domain (CBD) of 21 amino acids and bind to CaM in a Ca²⁺-dependent manner^34,35. Noticeably, the CaM-binding domain was observed in CsHsp70 (Fig. 5a) and NbHsp70 (Supplementary Fig. 12a), and three 30aa-peptides intercepted in CsHsp70 including peptide A containing the CaM-binding domain, N-terminal peptide B, and C-terminal peptide C (Fig. 5a) were used for subsequent studies. Y2H assays were performed to investigate the putative interactions between the three peptides of CsHsp70 and a citrus CaM protein CsCaM (GenBank accession no. KAH9675065.1) that is a homolog of potato CaM protein PCM6³⁴. As shown in Fig. 5b, only peptide A containing the CaM-binding domain interacted with CsCaM. Moreover, in vitro peptide binding assays³⁴ were also performed and the association of CsCaM with peptides will result in a change in electrophoretic mobility. Compared with the CsCaM band in the gel in the absence of peptide, the CsCaM band was shifted upward in the presence of peptide A and Ca²⁺ (Fig. 5c), and the change in the CsCaM band was eliminated by adding the specific Ca²⁺ chelator EGTA (ethylene glycol tetraacetic acid) (Fig. 5c). By contrast, the CsCaM band in the gel was not shifted upward in the presence of peptide B or peptide C (Fig. 5c). More importantly, Co-IP assays also showed that CsCaM interacted with CsHsp70, but not CsHsp70^ΔCBD (CsHsp70 without its CaM-binding domain) in planta (Fig. 5d). To investigate whether NbHsp70 also shows similar functions, Y2H assays were performed to investigate the putative interactions between NbHsp70 and a N. benthamiana CaM protein NbCaM (Nbv6.1trP1436) that is also a homolog of potato CaM protein PCM6³⁴. As shown in Supplementary Fig. 12b, NbCaM interacted with NbHsp70, but not NbHsp70^ΔCBD (NbHsp70 without its CaM-binding domain). Collectively, these results indicate that plant Hsp70 targeted by PdCDIE1 is also a bona fide target of CaM.

**Fig. 5: CsHsp70 contains a CaM-binding domain that interacts with CsCaM and also PdCDIE1.**

N-terminal PdCDIE1 interacts with the CaM-binding domain of Hsp70

To check which specific regions of PdCDIE1 interact with CsHsp70, the predicted mature protein of PdCDIE1 was divided into two regions, including N-terminal PdCDIE1 (PdCDIE1^22-144, termed PdCDIE1^N) and C-terminal PdCDIE1 (PdCDIE1^145-270, termed PdCDIE1^C). Y2H (Fig. 5b) and in vitro pull-down (Fig. 5e) assays showed CsHsp70 interacted with PdCDIE1^N, but not PdCDIE1^C. In addition, Agrobacterium-mediated transient expression of PdCDIE1^N, but not PdCDIE1^C, triggered plant cell death (Supplementary Fig. 13a, b), which is consistent with the interaction of between N-terminal PdCDIE1 and Hsp70. To investigate whether the CaM-binding domain of Hsp70 is the specific region that interacts with PdCDIE1, Y2H assays were performed. Noticeably, both PdCDIE1 and PdCDIE1^N only interacted with peptide A of CsHsp70 that contains the CaM-binding domain, but not peptide B of CsHsp70 and peptide C of CsHsp70 (Fig. 5a, b). More importantly, Co-IP assays also showed that PdCDIE1 interacted with CsHsp70, but not CsHsp70^ΔCBD in planta (Fig. 5d). The similar phenomenon was also observed in NbHsp70, and PdCDIE1 interacted with NbHsp70, but not NbHsp70^ΔCBD (Supplementary Fig. 12b). Moreover, in vitro peptide binding assays showed that the PdCDIE1^N band was shifted upward in the presence of peptide A of CsHsp70 and Ca²⁺ (Fig. 5f), but the change in the PdCDIE1^N band was not eliminated by adding the specific Ca²⁺ chelator EGTA (Fig. 5f). By contrast, the PdCDIE1^C band was not shifted upward in the presence of peptide A of CsHsp70 (Fig. 5f). Taken together, we can conclude that N-terminal PdCDIE1 interacts with the CaM-binding domain of CsHsp70 in a Ca²⁺-independent manner.

The interaction between CaM and Hsp70 in citrus fruit is disturbed during Pd infection but recovered during ΔPdCDIE1 mutant infection

Because both CsCaM and PdCDIE1 interact with the CaM-binding domain of CsHsp70 (Fig. 5), we want to check whether PdCDIE1 competes with CsCaM for CsHsp70 binding during Pd infection in citrus fruit. The elevation of intracellular Ca²⁺ levels is usually observed in plants upon pathogen infection², and there was also an increase in intracellular Ca²⁺ concentration in Pd-infected citrus fruit compared with uninfected citrus fruit (Supplementary Fig. 14a). A weak interaction between CsCaM and CsHsp70 was observed in uninfected citrus fruit (Supplementary Fig. 14b) due to low level of intracellular Ca²⁺, but an obvious interaction between CsCaM and CsHsp70 was observed in uninfected citrus fruit adding Ca²⁺ (Fig. 6a). Although there was high level of intracellular Ca²⁺ concentration in Pd-infected citrus fruit (Supplementary Fig. 14a), only a weak interaction between CsCaM and CsHsp70 was observed in Pd-infected citrus fruit (Fig. 6b). By contrast, there was an obvious interaction between PdCDIE1 and CsHsp70 (Fig. 3d, e). Moreover, the obvious interaction between CsCaM and CsHsp70 was recovered in citrus fruit inoculated by the ΔPdCDIE1 mutant (Fig. 6c). Overall, these data indicate that the interaction between CaM and Hsp70 in citrus fruit was disturbed during Pd infection probably due to the competitive binding of PdCDIE1 to Hsp70.

**Fig. 6: The interaction between CsCaM and CsHsp70 in citrus fruit was checked by Co-IP assays.**

Application of a CaM inhibitor and silencing of CaM genes induce plant cell death and high levels of ROS

To investigate the role of CaM–Hsp70 binding in plant cell death, application of trifluoperazine, an important CaM inhibitor that induces conformational change in Ca²⁺-CaM to disrupt the CaM–target binding³⁶, was performed to disturb the CaM–Hsp70 binding. Noticeably, trifluoperazine treatment induced obvious plant cell death and a noticeable increase in H₂O₂ production in N. benthamiana (Fig. 7a, b). Meanwhile, plant cell death (Fig. 7c), which was confirmed by electrolyte leakage (Fig. 7d) and PI staining (Fig. 7e) assays, and high levels of H₂O₂ (Fig. 7f) were also observed in citrus fruit upon trifluoperazine treatment. Moreover, silence of CaM genes in N. benthamiana was performed to further define the role of CaM in plant cell death. There were seven CaM genes in N. benthamiana (Supplementary Fig. 15a), and the pre-mentioned NbCaM (Nbv6.1trP1436) and other two randomly selected CaM genes including Nbv6.1trP41917 and Nbv6.1trP52729 were used for silencing (Supplementary Fig. 15b). Both Nbv6.1trP41917 and Nbv6.1trP52729 interacted with NbHsp70 as Nbv6.1trP1436 did (Supplementary Fig. 16a), and the three CaM proteins did not interact with each other (Supplementary Fig. 16b). qRT-PCR analysis showed that silencing efficiencies of the three CaM genes in their corresponding silenced plants were all higher than 70% (Supplementary Fig. 15c–e), although their homologous genes were also slightly silenced due to the strong sequence conservation (Supplementary Fig. 15b) of CaM genes³⁷. Noticeably, silencing of the three CaM genes also induced plant cell death (Fig. 7g, h) and high levels of H₂O₂ (Fig. 7i). Collectively, our results suggest that both application of a CaM inhibitor and silencing of CaM genes, which will result into the disturbance of the CaM–Hsp70 binding, induce plant cell death and high levels of ROS as PdCDIE1 does.

**Fig. 7: Application of trifluoperazine and silencing of *CaM* genes induced cell death and high ROS levels in plants.**

Discussion

How pathogens manipulate host plant cells to establish plant diseases is one of the most important questions in plant pathology⁹. Identification of virulence-associated effector proteins and their plant targets is the key to answer the question, and this study is the first investigation of how virulence effectors promote pathogen infection in fruit. Pd is a typical necrotrophic fungal pathogen¹⁸, and PdCDIE1 is a plant cell death-inducing effector that contributes significantly to Pd virulence. Compared with WT strain-infected citrus fruit, ΔPdCDIE1 mutant-infected citrus fruit showed a lower degree of cell death, indicating PdCDIE1 contributes significantly to Pd-induced cell death in citrus fruit. Notably, PdCDIE1 is not the sole factor required for Pd-induced cell death because ΔPdCDIE1 mutant-infected citrus fruit still showed cell death (Fig. 2d, e), and the well-known necrosis and ethylene-inducing peptide-like proteins (NLPs)³⁸ may also contribute to Pd-induced cell death. Mitochondria and chloroplasts play important roles in plant cell death and some known cell death-inducing effectors (CDIEs) from necrotrophic pathogens were proven to directly manipulate plant mitochondrial and chloroplast functions³⁹. Unlike these CDIEs, PdCDIE1 interacted with a cytosolic Hsp70 at plant cytoplasm to induce plant cell death, which provides new insights into the understanding of how pathogens manipulate plant cell death. Some host-selective toxin (HST) effectors were shown to interact with host specific leucine-rich repeat (NLR) receptor proteins in plant cytoplasm, resulting into host-specific plant cell death¹⁴. On the contrary, PdCDIE1 showed a broad-spectrum cell death-inducing activity in different plant species, probably because Hsp70, the plant interactor of PdCDIE1, is highly evolutionarily conserved in diverse plant species. In addition, PdCDIE1 can be regarded as an important effector because it is evolutionarily conserved among diverse fungal pathogens. PdCDIE1 homologs in hemibiotrophic pathogens may also contribute to their necrotrophic lifestyle at later stages, and some cell death-inducing effectors from hemibiotrophic pathogens have been identified^40,41.

Ca²⁺ signaling plays important roles in plant response to pathogen infection and some plant Ca²⁺ signaling components were proven to be targeted by pathogen effectors^4,10. Several reports showed that pathogen effectors interacted with plant CaM proteins to inhibit plant immunity or hijack plant metabolism to support pathogen nutrition^13,42,43. In this study, plant cytosolic Hsp70, the target of PdCDIE1, is proven to be a bona fide target of CaM, suggesting CaM-binding protein during Ca²⁺ signaling is also an important battleground encountered by plant pathogenic fungi. PdCDIE1 interacted with NbHsp70, but not NbHsp70 without its CaM-binding domain (Supplementary Fig. 12b). The similar phenomenon was also observed in the interaction between citrus CsHsp70 and PdCDIE1 (Fig. 5d). There are twelve cytosolic Hsp70 proteins containing a C-terminal EEVD motif in N. benthamiana (Supplementary Fig. 12a), and we showed that another cytosolic Hsp70, Nbv6.trP36254 lacking a CaM-target binding domain, did not interact with PdCDIE1 (Supplementary Fig. 12b). These results indicate the PdCDIE1 specifically interact with the CaM-binding domain of Hsp70. Noticeably, the interaction of CaM and Hsp70 in citrus fruit was disturbed during Pd infection but recovered during ΔPdCDIE1 mutant infection. Thus, we can infer that PdCDIE1 competitively binds to the CaM-binding domain of Hsp70, thus disturbing the CaM–Hsp70 binding in citrus fruit during Pd infection (Fig. 8). The mechanism of interaction between PdCDIE1 and plant Hsp70 is different from a previous study which showed that pathogen effector HopI1 interacted with Arabidopsis cytosolic Hsp70 to stimulate its ATP hydrolysis activity and increase its protein abundance⁴⁴. The protein abundance of CsHsp70 was not changed upon co-expression of PdCDIE1 and CsHsp70 (Supplementary Fig. 17), suggesting PdCDIE1 does not affect the stability of CsHsp70. In addition, heat shock proteins are well-known for their chaperone activity that prevents protein aggregation during heat stress. Thermal denaturation assays of Escherichia coli proteins showed that CsHsp70 but not PdCDIE1 improved the protein thermostability (Supplementary Fig. 18a), indicating the probable chaperone activity of CsHsp70. A previous report showed that fungal effector CSEP0105 interacted with plant small heat shock protein Hsp16.9 to inhibit its chaperone activity⁴⁵. PdCDIE1 was proven to prevent CsHsp70 from improving the thermostability of E. coli proteins (Supplementary Fig. 18b), suggesting PdCDIE1 may also inhibit the chaperone activity of CsHsp70 (Fig. 8). Noticeably, two homologs from two other postharvest fungal pathogens also exhibit plant cell death-inducing activity, and plant interactor Hsp70 in this study is highly evolutionarily conserved in plant species. Thus, the two homologs may show a similar cell death-inducing mechanism as PdCDIE1.

**Fig. 8: Working model of how PdCDIE1 induces plant cell death during Pd infection.**

ROS at low levels acts as an important second messenger to regulate many signaling pathways in plants, but high levels of ROS can induce plant cell death⁴⁶, which contributes to plant infection of necrotrophic pathogens¹⁴. Although many organelles, such as chloroplasts, mitochondria, and peroxisomes produce ROS, plants can utilize their ROS-scavenging systems to maintain ROS homeostasis^46,47. In this study, ROS is required for PdCDIE1-triggered plant cell, and Hsp70, the plant interactor of PdCDIE1, is antagonistic to ROS-dependent plant cell death. Ca²⁺ is an important second messenger that mediates plant responses to pathogen infection, and Ca²⁺ signals can stimulate ROS production at the early stage of pathogen infection by activating plant PTI immune pathways^48,49. Thus, we can infer that plant Hsp70 was activated by CaM to inhibit excessive ROS production in absence of pathogen effector PdCDIE1, thus maintaining ROS homeostasis (Fig. 8). However, the disruption of CaM–Hsp70 binding caused by PdCDIE1 resulted in high levels of ROS, thus triggering plant cell death (Fig. 8). Our results reveal a molecular framework of effector-triggered susceptibility which integrates Ca²⁺ sensing and ROS homeostasis. A recent report showed that Ca²⁺ sensing and ROS homeostasis were also manipulated by a Magnaporthe oryzae virulence effector⁶. These results highlight the importance of interaction between ROS and Ca²⁺ signaling in plant–pathogen interactions.

Plant Hsp70 proteins were proven to be important regulators in plant cell death upon pathogen infection or abiotic stress^50,51,52,53 and plant developmental programmed cell death⁵⁴, but little is known the specific mechanisms of cytosolic Hsp70 proteins in regulating plant cell death. Cytosolic Hsp70 proteins were shown to regulate ETI in plants by interacting with SGT1 that is critical for NLR-triggered cell death⁵³. However, NLR-triggered cell death is usually involved in plant response to biotrophic and hemibiotrophic pathogens, but not necrotrophic pathogens⁵⁵. NLR-triggered cell death usually belongs to host-specific plant cell death⁵⁵, which is different from the broad-spectrum cell death-inducing activity of PdCDIE1 in different plant species. There are cytosolic ROS-scavenging mechanisms in plants^46,47 and some heat shock proteins were proven to interact with cytosolic ROS-scavenging enzymes^56,57. Thus, Hsp70, the plant target of PdCDIE1, in the study inhibits ROS-dependent plant cell death probably by interacting and regulating cytosolic ROS-scavenging enzymes.

In conclusion, we identified a virulence-associated effector PdCDIE1 that targets plant Hsp70 and disturbs the CaM–Hsp70 binding, which results into ROS-dependent plant cell death. Our study may provide a template for studying effector proteins from postharvest pathogens on fruits and highlights the utilizability of heterologous plant systems, such as the model plant N. benthamiana, in studying effector proteins from pathogens of non-model crop plants.

Methods

Plant and fungal materials

In this study, Gannan navel orange (Citrus sinensis), ‘Fuji’ apple (Malus domestica), N. benthamiana, and tomato (Solanum lycopersicum) cv. Micro-Tom were used as plant materials. Fruit samples of uniform size were purchased at the same supermarket, and used fruits were from the same citrus or apple cultivar and from orchards in similar areas by communicating with the fruit supplier. Pd strain CQU-1⁵⁸ was used for pathogen inoculation in this study. Citrus fruits were wounded by making punctures and then were inoculated with 5-μL drops of Pd conidia (10⁵ spores mL⁻¹) at each wound. Inoculated citrus fruits were placed in a 25 °C incubator and diseases phenotypes were monitored.

Sequence analysis and phylogenetic analysis

SignalP 5.0 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0) was used to predict the presence of signal peptide in candidate effector proteins. Homologous proteins from different organisms were identified using the BLAST algorithm on NCBI. Phylogenetic analysis showing the phylogenetic relationships between PdCDIE1 and their homologous was carried out with the MEGA5 software using the neighbor-joining method. Sequence alignment was generated by MEGA5 and then was visualized using the SnapGene Viewer (https://www.snapgene.com/snapgene-viewer).

Agrobacterium-mediated transient expression in plants

For transient expression of genes in N. benthamiana or tomato leaves and citrus or apple fruits, Agrobacterium-mediated transient expression assays^59,60 were performed in plants. The coding sequences (CDSs) of target genes were PCR-amplified using cDNA templates derived from Pd strain CQU-1, N. benthamiana, or Gannan navel orange (C. sinensis). The fragments were cloned into pGR107 (HA tag in C-terminus) and recombinant constructs were introduced into A. tumefacient strain GV3101. Then, 50 μL of A. tumefaciens suspensions at an OD600 of 0.8 were injected into leaves or fruits for transient expression. Finally, phenotypes of injected leaves and fruits were photographed using a digital camera (Canon Inc., Tokyo, Japan).

Western blotting analysis

For verifying protein expression in N. benthamiana leaves, and citrus or apple fruits, western blotting analysis was performed. The total proteins in leaves or fruits were extracted using the protein extraction kit (Cwbiotech, Beijing, China) and proteins were detected using the anti-HA-antibody (Beyotime, AF2305, 1:2000 dilution). For verifying PdCDIE1 expression in in vitro-grown fungus, uninfected fruit, and Pd-infected fruit, western blotting analysis was performed using the customized anti-PdCDIE1 monoclonal antibody (ABclonal, China, 1:2000 dilution).

PI staining assay

For increasing the visibility of cell death phenotype on fruits, PI staining assay⁶¹ was performed using a PI assay kit (Yuanye Bio-Technology, Shanghai, China). In brief, sliced peels from citrus or apple fruits were stained by immersing in 0.2 mL PI solution (1.25 mg/mL) for 20 min at 4 °C. After washing with phosphate buffered saline, stained samples were observed under a confocal fluorescence microscope (TCS SP8, Leica, Germany; excitation/emission: 535/617 nm).

Electrolyte leakage assay

Electrolyte leakage from leaves or fruits²⁴ was measured to quantify plant cell death, and the conductivity was measured using a DDSJ-307F conductometer (INESA Scientific Instrument Co. Ltd., Shanghai, China). Six discs (1 cm diameter) from leaves or fruits were immersed in 5 ml distilled water for 5 h and the initial conductivity was measured. Subsequently, samples were boiled for 20 min to release all ions. When the solution cooled to room temperature, the total conductivity was measured. Data were expressed as percent leakage ((initial conductivity/total conductivity) × 100).

qRT-PCR analysis

To characterize the specific transcript profiles of PdCDIE1, RNA was isolated from infected citrus fruit at different Pd infection stages and in vitro-grown fungus (control) using the Ultrapure RNA Kit (Cwbiotech, Beijing, China). qRT-PCR reactions were conducted with a CFX Connect Real-Time System (Bio-Rad, CA, USA) and the GenStar RealStar Green Mixture (GenStar, Beijing, China). The Pd housekeeping gene actin (ID: PDIP_15850) was used as the endogenous control.

Transformation of Pd and characterization of these transformants

For knocking out PdCDIE1 in Pd genome, A. tumefaciens-mediated transformation of Pd with hygromycin resistance as the selectable marker was performed according to published protocols⁶². PdCDIE1 deletion plasmid pzp-hph-ΔPdCDIE1 was introduced to A. tumefaciens strain AGL1 and then co-cultured with Pd conidia to achieve the deletion of PdCDIE1. Finally, these transformants were confirmed by PCR and southern blot analysis. For checking the germinate rate of WT or ΔPdCDIE1 conidia, Pd conidial suspensions (10⁶ spores mL⁻¹) were inoculated onto potato dextrose agar (PDA) plates and cultured at 25 °C, and the germinate rate of Pd conidia were counted by choosing at least three random fields under the microscope (Nikon Co. Ltd., Japan). For checking the growth of WT or ΔPdCDIE1 conidia on PDA plates, colony morphology was photographed using a digital camera. For checking the growth of WT or ΔPdCDIE1 conidia on liquid media, Pd conidia were added into potato Dextrose Broth (PDB) and fungal growth was photographed by the microscope (Nikon Co. Ltd., Japan). For checking the virulence of WT strain or ΔPdCDIE1 mutant in citrus fruit, at least twenty fruits were used in each measurement, and disease symptoms were observed every day.

Fungal biomass by qPCR

Relative Pd biomass in inoculated citrus fruit was measured by qPCR with specific primers (Supplementary Data 1). 1 cm-diameter citrus peels at the infection site were collected and their genomic DNA was extracted as a template to confirm the relative biomass of Pd in citrus. The Pd actin gene (ID: PDIP_15850) was used to estimate Pd biomass by comparison to the citrus actin gene (ID: LOC102609791).

IP-MS assay

IP-MS assay in N. benthamiana leaves⁶³ was performed. A tagged protein PdCDIE1-HA was expressed in N. benthamiana leaves by using Agrobacterium-mediated transient gene expression. Then, putative plant interactors were screened using anti-HA-probe monoclonal-antibody-conjugated agarose beads (Beyotime, Suzhou, China), and non-specific binding of protein-beads complex were reduced by incubating with a solution of 200 μL HA peptide (5 mg/mL in TBS) for 2 h. Identification of these interactors was performed with mass spectrometry at Sangon Biotech (Sangon Biotech, Shanghai, China). The TripleTOF 5600 mass spectrometry system was utilized and three technical replicates were conducted. Processed samples were initially bound to a C18 capture column (5 µm, 5 × 0.3 mm) and subsequently eluted onto an analytical column (75 µm × 150 mm, 3 µm particle size) for efficient separation. A 60-minute analysis gradient (0 min in 3% B, 0.1 min of 3–7% B, 39.9 min of 7–23% B, 3 min of 23–50% B, 2 min of 50–80% B, 80% B for 5 min, 0.1 min of 80–5% B, 5% B for 9.9 min) was established with two mobile phases (A: H₂O, 0.1% formic acid; B: ACN, 0.1% formic acid). The flow rate of the liquid phase was set at 300 nL min⁻¹. The MS/MS acquisition criteria were set to select precursor ions exhibiting a signal intensity exceeding 120 counts per second (CPS) and charge states between +2 and +5. The mass spectrometry data were analyzed using the ProteinPilot software (version 4.5), with the Paragon algorithm for database searching. Information related to mass spectrometry was shown in Supplementary Table 2.

Yeast two-hybrid assay

Yeast two-hybrid assay was performed according to the Matchmaker GAL4 yeast two-hybrid system. The bait vector pGBKT7 and prey vector pGADT7 with target fragments were co-transformed into yeast strain AH109, and the positive protein interactions were assessed on the SD/-Ade/-His/-Leu/-Trp deficient media supplemented with X-α-gal.

Subcellular localization and BiFC assays

For subcellular localization assay, genes were cloned into the pGreen (p35S: GFP) vector and these recombinant plasmids were introduced into A. tumefacient cells which were injected into N. benthamiana leaves. For BiFC assay, genes were cloned into the pXY106 vector carrying N-terminal YFP and the pXY104 vector carrying C-terminal YFP, respectively. The two vectors were co-transformed into N. benthamiana leaves by agroinfiltration. Fluorescence in subcellular localization and BiFC assays was observed under a confocal fluorescence microscope (TCS SP8, Leica, Germany) at 3 dpi.

Pull-down assay

NbHsp70 (Nbv6.1trP74609, http://benthgenome.qut.edu.au/) and CsHsp70 (GenBank accession no. XP_052288226.1) were cloned into the modified pET32a vector (with HA-tag) for prokaryotic expression, and these recombinant plasmids were mobilized in E. coli strain BL21 (DE3) to produce HA-NbHsp70 and HA-CsHsp70 proteins. PdCDIE1, PdCDIE1^N, and PdCDIE1^C were cloned into the vector pCMV-N-GST for eukaryotic expression, and these recombinant plasmids were mobilized in HEK293T cells⁶⁴ to produce GST-PdCDIE1, GST-PdCDIE1^N, and GST-PdCDIE1^C proteins. Purified HA-tagged and GST-tagged recombinant proteins were mixed and then bound to an affinity column filled with anti-HA affinity gel (Beyotime, Suzhou, China) or anti-GST affinity gel (Smart Lifesciences, Changzhou, China). After washing three times, protein samples eluted from the beads were used for western blotting analysis with the anti-HA antibody (Beyotime, AF2305, 1:2000 dilution) or anti-GST antibody (Beyotime, AF0174, 1:2000 dilution).

Co-IP assay

For checking the putative interaction of PdCDIE1 or CsCaM and CsHsp70 or CsHsp70^ΔCBD, Agrobacterium-mediated transient gene expression in N. benthamiana leaves or citrus fruit was performed. Sequences encoding PdCDIE1 or CsCaM were cloned into pGR107 (HA tag in C-terminus), and sequences encoding CsHsp70 or CsHsp70^ΔCBD were cloned into pEAQ-HT (His tag in N-terminus). After protein expression in plants, mixed protein solutions were incubated with anti-His or anti-HA affinity gel (Smart Lifesciences, Changzhou, China). Finally, eluted protein samples were used for western blotting analysis with the anti-His antibody (Beyotime, AG8061, 1:2000 dilution) or anti-HA antibody (Beyotime, AF2305, 1:2000 dilution).

For checking the putative interaction of CsCaM and CsHsp70 in uninfected and infected citrus fruit, purified CsCaM proteins (with HA-tag) by prokaryotic expression were added to the protein solutions of uninfected citrus fruits, inoculated citrus fruits by WT strain or inoculated citrus fruits by ΔPdCDIE1 mutant. Mixed protein solutions were incubated with anti-HA affinity gel (Smart Lifesciences, Changzhou, China). Finally, eluted protein samples were used for western blotting analysis with the anti-HA antibody (Beyotime, AF2305, 1:2000 dilution).

In vitro protein binding assay

In vitro protein binding assay was performed as previously described³⁴. Tested protein and peptide-encoding sequences were cloned into the pGEX4T-1 vector and these recombinant plasmids were mobilized in E. coli strain BL21 (DE3) for prokaryotic expression. GST fusion proteins/peptides were produced and purified with the anti-GST affinity gel (Smart Lifesciences, Changzhou, China). GST tag was then removed from fusion proteins/peptides by thrombin digestion. Finally, proteins and peptides were mixed in the reaction mixture and incubated at 25 °C for 1 h. These samples were run on a 10% native polyacrylamide gel electrophoresis for checking whether the electrophoretic migration of proteins is affected.

Detection and measurement of H₂O₂ production

For detecting the production of H₂O₂ in plant tissues, histochemical analysis by staining with 3-3′diaminobe-benzidine (DAB)⁶⁵ was performed. N. benthamiana leaves were cut and subsequently immersed in a solution containing 1 mg ml⁻¹ DAB for 12 h. The leaves were treated in a bleaching solution (ethanol/acetic acid/glycerol, volume ratio of 3:1:1) to remove chlorophyll and were finally photographed using a digital camera. In addition, spectrophotometric determination of H₂O₂ content in N. benthamiana leaves or citrus fruit was performed based on the reaction between H₂O₂ and titanium, and experimental steps refer to a previous study⁶⁶. Specifically, 0.1% titanium chloride in 50% (v/v) sulfuric acid was used and the content of H₂O₂ was determined by measuring absorbance at 410 nm.

H₂DCFDA staining assay

ROS production in N. benthamiana leaves was visualized by H₂DCFDA staining assay⁶⁷. Small pieces cutting from leaves were staining with 20 µM H₂DCFDA (Biosharp, Hefei, China) for 30 min. After staining, samples were washed three times with distilled water and images were visualized under a confocal fluorescence microscope (TCS SP8, Leica, Germany).

Gene silencing in N.benthamiana

Tobacco rattle virus (TRV)-mediated gene silencing in N. benthamiana was performed using the Agrobacterium-mediated infiltration method⁶⁸. Specific primers (Supplementary Data 1) were designed using with the online SGN VIGS tool (https://vigs.solgenomics.net/), and a 300 bp fragment of target genes (Supplementary 11a; Supplementary 15b) was PCR-amplified using cDNA templates derived from N. benthamiana. Then, A. tumefaciens was transformed with plasmid vectors pTRV1 and pTRV2 with target genes. Silenced lines were generated by injecting these A. tumefaciens suspensions.

Detection of intracellular Ca²⁺ by staining with Fluo-3-AM

For the detection of intracellular Ca²⁺ in citrus fruit, citrus peels were cut into thin strips of 0.2 mm in thickness and 2–3 mm in length, and these samples were stained with the Ca²⁺-sensitive fluorescent dye Fluo-3/AM⁶⁹. These samples were placed in an MS liquid medium and then added with 20 μM Fluo-3/AM for incubation at 4 °C for 2 h in the dark. After washing three times, fluorescent probes were observed under a confocal fluorescence microscope (TCS SP8, Leica, Germany).

Exogenous dimethylthiourea (DMTU) or trifluoperazine treatment

For checking the effect of DMTU on plant cell death or ROS accumulation triggered by PdCDIE1, exogenous DMTU (Macklin, Shanghai, China) treatment in plants⁷⁰ was performed. The DMTU solution (50 mM) was added into N. benthamiana leaves before or after Agrobacterium-mediated transient expression of PdCDIE1. For checking the effect of trifluoperazine on plant cell death or ROS accumulation, exogenous trifluoperazine (Macklin, Shanghai, China) treatment in plants⁷¹ was performed. A range of concentrations (0–5 mM) of trifluoperazine were injected into N. benthamiana leaves or citrus fruits.

Protein stability assay

Sequences encoding PdCDIE1 or GFP (control) were cloned into pGR107 (HA tag in C-terminus), and sequences encoding CsHsp70 were cloned into pEAQ-HT (His tag in N-terminus). The two recombinant plasmids were co-transformed into N. benthamiana leaves by agroinfiltration. After 3 d or 4 d of infiltration, total proteins were extracted and immunoblot analysis was conducted using the anti-His antibody (Beyotime, AG8061, 1:2000 dilution) or anti-HA antibody (Beyotime, AF2305, 1:2000 dilution). The relative intensity of protein bands of CsHsp70 was quantified using the ImageJ software.

Thermal denaturation assay of E. coli proteins

To investigate whether CsHsp70 shows chaperone activity, a thermal denaturation assay of E. coli proteins⁴⁵ was performed. E. coli strain BL21 was cultured in Luria-Bertani (LB) medium to harvest its total proteins which were placed on the ice and acted as a blank control. CsHsp70, GFP, and PdCDIE1 sequences were cloned into pGEX4T-1 and these recombinant plasmids were mobilized in BL21 to produce corresponding proteins. These protein solutions were separated into 1.5 mL clean centrifuge tubes and then placed into a water bath with different temperatures (40, 50, 60, 70, and 80 °C) for 30 min. After centrifugation at 4 °C, protein concentration of the supernatant was measured. Different combinations, including GFP + CsHsp70, GFP + PdCDIE1, and PdCDIE1 + CsHsp70, were sent to test the effect of interaction on the thermal stability of E. coli proteins.

Reporting summary

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

Data availability

Data supporting the findings of this work are available in the main text and its Supplementary Information files. The raw mass spectrometry data for IP-MS assay have been deposited in ProteomeXchange under an accession number IPX0011111000. Source data are provided with this paper.

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Acknowledgements

We thank Qin Deng from the Analytical and Testing Center of Chongqing University for technological guidance. This study was supported by the National Key Research and Development Program of China (2022YFD2100104), the National Natural Science Foundation of China (32272381 and 31972123), the Project of Chongqing Science and Technology Commission (cstc2021jcyj-msxmX0160), the Department of Science and Technology Planning Project of Henan Province (222102110102 and 222102110015), and the Chongqing Talents: Exceptional Young Talents Project (cstc2021ycjh-bgzxm0042).

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These authors contributed equally: Yunlong Lin, Chan Xu.

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Key Laboratory of Plant Hormones and Molecular Breeding of Chongqing, School of Life Sciences, Chongqing University, Chongqing, China
Yunlong Lin, Chan Xu, Liqin Fan, Rui Li, Jiaxin He, Wei Deng, Zhengguo Li & Yulin Cheng
Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, Zhoukou, China
Yunlong Lin, Lili Li, Liqin Fan & Hongli Li
State Key Laboratory of Crop Stress Resistance and High-Efficiency Production, College of Plant Protection, Northwest A&F University, Shaanxi, Yangling, China
Zhensheng Kang

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Yunlong Lin
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Chan Xu
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Lili Li
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Liqin Fan
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Hongli Li
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Wei Deng
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Contributions

Y.C., Z.L., and Z.K. designed the experiments and provide resources; Y.L., C.X., L.L., L.F., R.L., J.H., H.L., W.D., and Y.C. conducted the experiments; Y.C., Y.L., and Z.K. wrote and revised this paper.

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Correspondence to Zhensheng Kang, Zhengguo Li or Yulin Cheng.

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Lin, Y., Xu, C., Li, L. et al. A conserved fungal effector disturbs Ca²⁺ sensing and ROS homeostasis to induce plant cell death. Nat Commun 16, 3523 (2025). https://doi.org/10.1038/s41467-025-58833-z

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Received: 13 July 2023
Accepted: 29 March 2025
Published: 14 April 2025
DOI: https://doi.org/10.1038/s41467-025-58833-z