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. 2025 Nov 10;26(11):e70166. doi: 10.1111/mpp.70166

Yeast‐Secreted Protein PgSCP and Citrus Transcription Factor CsMIKC Synergize to Activate Green Mould Resistance in Fruit

Ou Chen 1, Rui Huang 1, Yao Xu 1, Wenjun Wang 1, Jian Ming 1,2, Kaifang Zeng 1,2,3,✉
PMCID: PMC12602158  PMID: 41214411

ABSTRACT

Prior studies have shown that the secreted protein PgSCP from the biocontrol yeast Pichia galeiformis induces disease resistance responses in citrus fruit. However, the precise molecular basis of how PgSCP activates these disease resistance responses remains inadequately understood, particularly regarding direct interactions with host signalling components. Here, we demonstrate that PgSCP directly interacts with the citrus transcription factor CsMIKC through protein–protein interaction assays, including yeast two‐hybrid, bimolecular flourescence complementation and pull‐down experiments. Overexpression and gene‐silencing assays revealed that CsMIKC functions as a positive regulator of resistance to citrus green mould. Based on the JASPAR database, we predicted that CsMIKC likely binds to the promoters of PR1‐like and ATPase, two defence‐associated genes in citrus fruits. This was confirmed via yeast one‐hybrid and dual luciferase reporter assays, which showed that CsMIKC directly binds to and activates transcription of these genes. Importantly, co‐expression of PgSCP and CsMIKC significantly enhanced the transcriptional activation of PR1‐like and ATPase, suggesting a synergistic interaction between the microorganism‐derived protein and the host transcription factor. This study identifies the molecular mechanism by which the yeast secreted protein PgSCP interacts with citrus transcription factor CsMIKC to activate defence gene expression, thereby enhancing citrus fruit resistance. The findings establish a new model of cross‐kingdom signalling between biocontrol yeast and citrus fruit, offering a theoretical framework for engineering exogenous protein‐based strategies to improve postharvest disease resistance.

Keywords: citrus fruit, citrus‐yeast interaction, disease resistance, green mold, secreted protein, transcription factor

The yeast secreted protein PgSCP interacts with the citrus fruit transcription factor CsMIKC, enhancing its transactivation of disease‐resistance genes (PR1‐like and ATPase), thereby improving citrus resistance to green mould.

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1. Introduction

Microbial secreted proteins play crucial roles in mediating interactions with plants. Numerous plant proteins that interact with microbial secreted proteins have been identified and characterised (Galán et al. 2014; Gheysen and Mitchum 2011; Mejias et al. 2019; Rocafort et al. 2020). Among these plant proteins, transcription factors are particularly important, as microbial secreted proteins often interact with them to modulate host processes (Xiang et al. 2025). Transcription factors bind to regulatory sequences in DNA and directly regulate gene expression, playing central roles in plant growth, development and defence responses (Moore et al. 2011; Tsuda and Somssich 2015). Based on these interactions, microbial secreted proteins, particularly pathogen effector proteins, employ diverse strategies to manipulate plant transcription factors. These strategies include regulating transcription factor activity, modifying expression levels, enhancing their degradation via the ubiquitin‐proteasome pathway and triggering subcellular relocation of these proteins (Xiang et al. 2025).

During plant defence responses, the activation of transcription factors modulates genes involved in defence hormones, including salicylic acid, jasmonic acid and ethylene, as well as pathways involved in the production of reactive oxygen species (ROS), and the accumulation of defence‐related compounds (Contreras et al. 2020; Ishihama and Yoshioka 2012; Noman et al. 2019). Microbial secreted proteins can modulate plant defence by interacting with transcription factors, often affecting their DNA binding and/or transcriptional activity. These interactions subsequently modify the expression of downstream genes associated with defence, thereby modulating plant defence responses. For example, a recent study demonstrated that the secreted protein Vd6317 from Verticillium dahliae interacts with the transcription factor AtNAC53, inhibiting its DNA‐binding activity. AtNAC53 further interacts with AtUGT74E2, enhancing host resistance to V. dahliae (Liu et al. 2024). A separate study identified that the oomycete Phytophthora sojae effector PsCRN108 interacts with the plant transcription factor NbCAMTA2, leading to the suppression of HSP40 expression and the inhibition of the ROS burst (Yang, Ai, et al. 2024). These examples illustrate how microbes regulate plant defence by altering transcription factor binding or transcriptional activity through secreted proteins, thereby controlling downstream gene expression and defence responses. Despite significant progress in elucidating proteins secreted from pathogenic fungi–plant transcription factor interactions, there remains a paucity of knowledge regarding the mechanisms by which beneficial microorganisms, such as biocontrol yeast, leverage similar pathways to influence plant disease defence.

Given this gap, citrus provides a model system to explore such beneficial microbe–plant interactions. Citrus fruits are among the world's most important economically valuable orchard crops, renowned for their distinctive flavour, nutritional richness and high content of health‐promoting active components such as vitamin C and flavonoids (Lu et al. 2024). However, during postharvest storage and logistics, citrus is highly susceptible to pathogen infection. Among these pathogens, Penicillium digitatum, responsible for green mould, is the main cause of citrus fruit rot during storage and transport (Rovetto et al. 2024). Biocontrol yeasts can enhance postharvest disease resistance in citrus fruits. However, the molecular mechanisms underlying interactions between yeasts and citrus fruits—especially those involving yeast secreted proteins and citrus fruit responses—remain poorly explored.

Our previous studies have demonstrated that the secreted protein PgSCP from Pichia galeiformis effectively induces disease resistance in citrus fruits, thereby contributing to the control of postharvest green mould (Chen et al. 2023). However, the molecular mechanism of how PgSCP interacts with citrus fruits to confer resistance remains unclear. Our unpublished results from glutathione S‐transferase (GST) pull‐down assays combined with proteomic analysis revealed a potential interaction between PgSCP and CsMIKC. However, whether a direct physical interaction occurs between these proteins, whether PgSCP functions through CsMIKC to modulate green mould resistance in citrus fruit, and the underlying transcriptional regulatory mechanisms remain unexplored. In this study, we identified the citrus transcription factor CsMIKC that interacts with PgSCP, and elucidated that CsMIKC positively regulates citrus disease resistance by modulating the expression of defence‐related genes. Furthermore, the interaction between PgSCP and CsMIKC enhances the transcriptional activation capacity of CsMIKC on these defence‐related genes. Our findings offer novel insights into the molecular mechanisms governing yeast–citrus fruit interactions, thereby enhancing the understanding of these complex biological processes.

2. Results

2.1. Verification of the Interaction Between PgSCP and CsMIKC

Protein–protein interactions between PgSCP and CsMIKC were validated through multiple complementary assays. Yeast two‐hybrid (Y2H) screening identified a direct interaction between PgSCP and CsMIKC, as evidenced by colony growth on SD/−Ade−His−Leu−Trp medium and quantified β‐galactosidase activity (Figure 1A). This interaction was further validated in vitro through GST pull‐down assays, which revealed specific binding between PgSCP and CsMIKC (Figure 1B). Notably, bimolecular fluorescence complementation (BiFC) assays conducted in the epidermal cells of Nicotiana benthamiana leaves demonstrated the reconstitution of yellow fluorescent protein (YFP) upon the co‐expression of PgSCP‐YNE and CsMIKC‐YCE fusion proteins (Figure 1C), and this observation confirms that the interaction is localised specifically within the nucleus. Collectively, these orthogonal methodologies corroborate the interaction of PgSCP with CsMIKC in both recombinant and native systems.

FIGURE 1.

FIGURE 1

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Interaction analysis of yeast secreted protein PgSCP and CsMIKC. (A) PgSCP interacts with CsMIKC in yeast two‐hybrid assay. (B) Pull‐down assay shows interaction between PgSCP and CsMIKC. The proteins GST‐PgSCP, CsMIKC‐His and GST were successfully expressed in Escherichia coli Rosetta (DE3) cells. (C) Bimolecular fluorescence complementation (BiFC) assay confirming that PgSCP interacts with CsMIKC in Nicotiana benthamiana leaves.

2.2. The Effect of CsMIKC on the Resistance to Green Mould

Subcellular localisation assays showed that the CsMIKC‐GFP signal was detectable in both the nucleus and cytoplasm. The control plasmid pCAMBIA2300‐GFP displayed GFP fluorescence primarily in the cytoplasm and nucleus (Figure 2). The findings suggest that the CsMIKC‐GFP protein is localised within both the nucleus and the cytoplasm, implying a potential dual functional role in nuclear regulation as well as cytoplasmic processes.

FIGURE 2.

FIGURE 2

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Subcellular localisation of CsMIKC in Nicotiana benthamiana leaves.

As depicted in Figure 3A, transient overexpression of CsMIKC in citrus fruit significantly elevated its transcript abundance compared to controls, whereas virus‐induced gene silencing (VIGS) induced a marked downregulation of CsMIKC expression. Upon infection with P. digitatum , citrus fruits overexpressing CsMIKC exhibited a significant reduction in disease incidence (62% decrease) and lesion diameter (14% reduction) compared to empty‐vector controls at 7 days post‐inoculation (dpi). Conversely, fruits with VIGS‐mediated suppression of CsMIKC displayed heightened susceptibility, characterised by a 10% increase in disease incidence and a 16.5% expansion in lesion diameter relative to control fruits at 7 dpi (Figure 3B,C). These contrasting phenotypes underscore the role of CsMIKC as a positive regulator of citrus fruit resistance to green mould, with its activity directly correlated to reduced pathogen colonisation and lesion progression (Figure 3D).

FIGURE 3.

FIGURE 3

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Functional analysis of CsMIKC in modulating green mould resistance in citrus fruit. (A) Gene expression levels of CsMIKC in citrus fruits following transient overexpression or virus‐induced gene silencing (VIGS)‐mediated suppression. (B) Disease incidence of green mould in citrus fruit after transient overexpression or suppression of CsMIKC, followed by inoculation with Penicillium digitatum . (C) Lesion diameter of green mould in citrus fruit after transient overexpression or suppression of CsMIKC, followed by inoculation with P. digitatum . (D) Disease severity of green mould in citrus fruit after transient overexpression or suppression of CsMIKC, followed by inoculation with P. digitatum .

2.3. The Effect of PgSCP Synergized With CsMIKC on the Resistance to Green Mould

Transient overexpression of PgSCP or CsMIKC individually, or their co‐overexpression, significantly suppressed green mould development in citrus fruits compared to empty vector controls (Figure 4). Notably, fruits co‐overexpressing PgSCP and CsMIKC exhibited significantly lower disease incidence than those with single‐gene overexpression at 3–4 dpi. This suggests that the protein interactions between CsMIKC and PgSCP may augment the regulatory efficacy of the yeast secretory protein PgSCP in mitigating green mould in citrus fruits to a certain degree.

FIGURE 4.

FIGURE 4

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Effect of transient overexpression of CsMIKC in combination with PgSCP on green mould resistance in citrus fruit. (A) Disease incidence of green mould in citrus fruit following transient co‐overexpression of PgSCP and CsMIKC, followed by inoculation with Penicillium digitatum . (B) Lesion diameter of green mould in citrus fruit after transient co‐overexpression of PgSCP and CsMIKC, followed by inoculation with P. digitatum . (C) Disease severity of green mould in citrus fruit at 5 days post‐inoculation with P. digitatum , following transient co‐overexpression of PgSCP and CsMIKC.

2.4. CsMIKC ‐Mediated Transcriptional Regulation of Defence‐Related Target Genes in Citrus

Transient overexpression of the citrus transcription factor CsMIKC led to significant transcriptional reprogramming, with 1907 genes differentially expressed (DEGs) across all comparisons. Specifically, 1061 genes were upregulated and 846 genes downregulated (Figure 5A). Functional annotation through KEGG pathway enrichment analysis indicated that these DEGs were primarily associated with defence‐related metabolic pathways. Specifically, they were enriched in pathways such as phenylalanine, tyrosine and tryptophan biosynthesis (KEGG:00400), phenylpropanoid biosynthesis (KEGG:00940), flavonoid biosynthesis (KEGG:00941) and glutathione metabolism (KEGG:00480) (Figure 5B). These pathways are critical for pathogen defence in plants, including the synthesis of antimicrobial compounds and ROS scavenging via glutathione.

FIGURE 5.

FIGURE 5

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CsMIKC combines with promoters of disease resistance‐related genes in citrus fruit. (A) Volcano plot of differentially expressed genes (DEGs) after transient overexpression of CsMIKC. (B) KEGG enrichment analysis of DEGs. (C) Yeast one‐hybrid experiment analysis of CsMIKC binding to promoters of CsASA, CsFHY, Cssyf2, CsTF2‐11, CsPR1‐like and CsATPase.

In order to elucidate the mechanistic basis underlying the role of CsMIKC in disease resistance, we conducted a JASPAR motif analysis to identify potential binding sites of CsMIKC within the promoters of genes associated with defence responses. The predicted binding motifs corresponding to the DNA‐binding profile of CsMIKC were significantly enriched in the promoters of genes associated with the phenylalanine, tyrosine and tryptophan biosynthesis and glutathione metabolism, such as CsASA, CsFHY and Cssyf2. Notably, approximately 94.4% of DEGs contained CsMIKC‐binding motifs. This finding establishes a direct connection between the transcriptional activity of these motifs and the regulation of defence genes (Table S1).

To further validate this binding interaction, we conducted yeast one‐hybrid (Y1H) assays. Control transformations failed to support growth on SD/−His/−Leu/−Trp medium supplemented with 3‐amino‐1,2,4‐triazole (Figure 5C). Conversely, yeast colonies that were co‐transformed with CsMIKC and promoter fragments from all six target genes (CsASA, CsFHY, Cssyf2, CsTF2‐11, CsPR1‐like and CsATPase) successfully developed visible colonies on the selective medium (Figure 5C). These findings substantiate the direct DNA‐binding activity of CsMIKC to the promoter regions of the target genes.

2.5. CsMIKC ‐Mediated Transcriptional Activation of Disease Resistance‐Related Genes

To validate the transcriptional regulatory role of CsMIKC, we performed dual‐luciferase reporter (DLR) assays. Co‐transfection of CsMIKC and promoter fragments of CsASA1, CsFHY, Cssyf2, CsTf2‐11, CsPR1‐like or CsATPase resulted in a statistically significant increase in the LUC/REN ratio compared to the control group co‐transfected with empty pEAQ and the reporter (Figure 6A). Furthermore, overexpression of CsMIKC triggered a 1.07 to 1.53‐fold induction in transcript levels of all six target genes (Figure 6B), corroborating its role as a transcriptional activator. These findings collectively indicate that CsMIKC directly interacts with the promoter regions of these genes, thereby enhancing their transcription. This regulatory mechanism orchestrates the biosynthesis of phenylalanine, tyrosine and tryptophan, as well as glutathione metabolism, which are essential for disease resistance.

FIGURE 6.

FIGURE 6

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CsMIKC activates the expression of target genes associated with disease resistance in citrus fruit. (A) The regulatory effect of CsMIKC on the activity of promoters of disease resistance‐related target genes. *p < 0.05. (B) After transient overexpression of CsMIKC, analyse the regulatory effect of CsMIKC on disease resistance‐related target genes.

2.6. PgSCP Induces Disease Resistance in Citrus Fruit via CsMIKC

Compared to the single transient overexpression of PgSCP, the co‐expression of PgSCP and CsMIKC resulted in 1693 genes significantly upregulated and 1323 genes significantly downregulated. When compared to the single transient overexpression of CsMIKC, the combined co‐expression led to 2012 upregulated genes and 1540 downregulated genes (Figure 7A). KEGG enrichment analysis revealed that these DEGs were significantly enriched in several disease‐resistance‐related pathways, including phenylpropanoid biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, glutathione metabolism, and cysteine and methionine metabolism (Figure 7B). As shown in Figures 6B and 7C, the majority of genes were upregulated under single overexpression of PgSCP or CsMIKC. Co‐overexpression revealed distinct transcriptional responses, with PR1‐like and ATPase exhibiting significantly greater upregulation compared to single treatments. This finding is consistent with a synergistic effect between PgSCP and CsMIKC. In contrast, FHK and syf2 showed attenuated upregulation or even downregulation under co‐overexpression conditions. These results suggest that while PgSCP and CsMIKC synergistically enhance PR1‐like and ATPase expression, their combined action may suppress the transcriptional induction of FHK and syf2, indicating gene‐specific regulatory interactions.

FIGURE 7.

FIGURE 7

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Co‐expression of PgSCP and CsMIKC synergistically upregulates defence‐related genes. (A) Volcano plot of differentially expressed genes between transiently overexpressed PgSCP and co‐transiently overexpressed CsMIKC and PgSCP, as well as between transiently overexpressed CsMIKC and co‐transiently overexpressed CsMIKC and PgSCP. (B) KEGG enrichment analysis. (C) Analysis of the expression levels of disease resistance‐related target genes after transient overexpression of PgSCP and co‐transient overexpression of CsMIKC and PgSCP.

3. Discussion

Citrus fruit represents one of the most economically significant crops worldwide, leading in both global production and fresh consumption, and ranking third in international agricultural trade (Liu et al. 2025). However, postharvest fungal infections, particularly green mould caused by P. digitatum , severely threaten citrus postharvest quality (Wang et al. 2025). Although biocontrol agents have emerged as promising alternatives to chemical fungicides, the molecular mechanisms underlying their efficacy, especially how they interact with fruit‐associated biomolecules through their secreted proteins, remain poorly understood. Here, we previously identified that the yeast‐secreted protein PgSCP, produced by P. galeiformis, suppresses postharvest green mould disease in citrus; however, its mode of action remains unresolved (Chen et al. 2023). Gaining mechanistic insights into how PgSCP interacts with citrus fruit will be critical for developing targeted strategies to enhance postharvest disease resistance.

Small proteins secreted by microbes could modulate plant defence responses by interacting with host transcription factors (Xiang et al. 2025). Recent studies have revealed that these proteins often target transcription factors to reprogramme gene expression via activation or repression of downstream targets, thereby altering plant defence mechanisms (Bogino et al. 2025; Lovelace 2024; Marrero et al. 2024; Yang et al. 2025). For example, the oomycete Phytophthora secretes Pio3192, which specifically binds to NAC transcription factors NTP1/2 localised on the endoplasmic reticulum, preventing their nuclear translocation and subsequent regulation of defence‐related genes, thereby impairing Solanum tuberosum resistance to infection (Dong et al. 2024). Similarly, the Phytoplasma protein SJP39 directly interacts with nuclear‐localised transcription factor ZjbHLH87 in jujube cells, triggering developmental retardation (Yang et al. 2025). In our prior work, candidate interaction partners of the yeast‐secreted protein PgSCP, identified via GST pull‐down combined with mass spectrometry, included the citrus transcription factor CsMIKC. In this study, we provide evidence through Y2H, GST pull‐down and BiFC that PgSCP directly interacts with CsMIKC in citrus fruit.

CsMIKC belongs to the MIKC‐type MADS‐box transcription factor family. The MIKC‐type transcription factors are involved in plant developmental regulation and signalling, with emerging evidence suggesting their roles in biotic stress responses (Zhang et al. 2024). Genomic analysis has identified 136 MADS‐box genes in citrus, with 84% of their functions remaining uncharacterised (Yang, Zhang, et al. 2024). We demonstrated that transient overexpression of CsMIKC enhanced green mould disease resistance in citrus fruits, while VIGS‐mediated gene silencing of CsMIKC resulted in reduced resistance. Notably, recent studies highlight the role of transcription factors in fruit defence and disease resistance. Overexpression of tomato SlERF.C1 enhances resistance to Botrytis cinerea by directly activating pathogenesis‐related (PR) genes (Deng, Pei, et al. 2024). Papaya CpWRKY50 positively regulates resistance to anthracnose by activating CpMYC2 and CpPR4 to promote jasmonic acid signalling (Yang, Zhou, et al. 2024). Citrus CsWRKY76 elevates resistance via upregulating antimicrobial metabolites like scoparone and pentamethoxyflavones (Xu et al. 2025). In addition, suppression of NbMADS1 in tobacco reduces systemic resistance to Phytophthora nicotianae, indicating its critical role in disease resistance signalling (Zhang et al. 2016). MIKC‐TFs recognise CArG‐box motifs (CC(A/T)GG) in target promoters to regulate gene expression (Zhang et al. 2024). In this study, we combined JASPAR database predictions with Y1H and DLR assays to confirm that CsMIKC specifically binds and activates PR1‐like and ATPase.

Further, our study demonstrates that co‐overexpression of the yeast secretory protein PgSCP and the citrus transcription factor CsMIKC in citrus fruit significantly upregulated the expression of CsMIKC target genes PR1‐like and ATPase (Figure 7c). This synergistic interaction between PgSCP and CsMIKC enhances the transcriptional activation of these defence‐associated genes, thereby strengthening postharvest disease resistance in citrus. This discovery expands current understanding of microbial secreted protein mechanisms in host immunity regulation.

Previous studies have shown that pathogen‐secreted proteins can invade host plant cells and even the cell nucleus, affecting the function of host disease‐resistance‐related genes. Transcription factors that primarily function in the nucleus act as key elements in plants' responses to biotic stresses, transmitting signals to defence‐related genes and thereby activating or repressing their expression. Recent studies have demonstrated that microbial secreted proteins could target key transcription factors in plants and interfere with the activation or repression of downstream genes, enabling transcriptional reprogramming of genes (Dong et al. 2024). For example, pathogen effectors could prevent DNA binding and/or transcriptional activation of plant disease‐resistance‐related transcription factors, regulating the DNA binding or transcription of target transcription factors, thus modulating plant defence response (Duan et al. 2024; Yang, Ai, et al. 2024). The interaction between microbial secreted proteins and plant transcription factors can inhibit the DNA‐binding domain, alter transcription factor activity, and these interactions subsequently alter the expression of downstream genes (Singh et al. 2023). A recent report demonstrated that the V. dahliae effector protein Vd6317 targets Arabidopsis AtNAC53 and inhibits its DNA‐binding activity, as AtNAC53 directly targets the defence‐related gene AtUGT74E2; therefore Vd6317 blocks AtNAC53 transcriptional activity toward AtUGT74E2. In other words, Vd6317 directly inhibits the activity of the plant transcription factor AtNAC53, thereby suppressing the expression of AtUGT74E2 and the plant defence response (Liu et al. 2024).

Some secreted proteins influence the oligomerisation of target transcription factors, affecting their functionality and disrupting plant defence responses. For instance, the effector Pst21674 from Puccinia striiformis f. sp. tritici interacts with the wheat transcription factor TaSAR3, disrupting its oligomerisation and inhibiting its role in the transcriptional activation of defence‐related genes, ultimately diminishing the disease resistance of wheat (Zheng et al. 2023). Other secreted proteins also alter the subcellular localisation of transcription factors, or directly bind and modify their post‐translational modifications, suppressing their function and impacting fruit disease resistance (Bai et al. 2022; Nguyen et al. 2024). Inspired by these findings, we explored whether PgSCP might similarly modulate citrus immunity by targeting transcription factors. This study demonstrates that PgSCP interacts with CsMIKC through a synergistic mechanism, wherein PgSCP enhances CsMIKC's transcriptional activation of defence‐related genes to strengthen plant defence responses against pathogens.

In summary, the key findings of this study demonstrate that the yeast secretory protein PgSCP interacts with the citrus fruit transcription factor CsMIKC to activate defence‐related genes PR1‐like and ATPase, thereby enhancing disease resistance in citrus fruit. Notably, this work provides the first evidence that CsMIKC functions as a positive regulator of disease resistance in citrus fruit. Furthermore, PgSCP facilitates the transcriptional activation of defence genes by synergistically cooperating with CsMIKC, establishing an ‘exogenous protein‐transcription factor’ regulatory paradigm. Collectively, CsMIKC represents a promising candidate gene for engineering disease resistance within citrus fruit systems. Concurrently, PgSCP may function as an exogenously applied induced defence elicitor, offering potential applications. The yeast origin and proteinaceous characteristics of PgSCP indicate its potential for favourable biosafety and environmental compatibility. These findings lay the groundwork for translating the molecular mechanisms identified in this study into a sustainable biocontrol strategy for the storage and postharvest protection of citrus fruits.

4. Experimental Procedures

4.1. Verification of the Interaction Between PgSCP and CsMIKC

4.1.1. Y2H

The Y2H was performed following the method of Jiang et al. (2025). The coding sequences of PgSCP and CsMIKC were individually cloned into the pGADT7 and pGBKT7 vectors, respectively. Subsequently, these constructs were introduced into Y2H Gold yeast competent cells. The transformants were screened on selective media and subjected to X‐α‐Gal staining to evaluate the interaction between PgSCP and CsMIKC. The results were documented with photographic evidence.

4.1.2. BiFC

The BiFC assay was performed following the method described in the previous literature (Deng, Jiang, et al. 2024). The genes PgSCP and CsMIKC were individually subcloned into the nYFP and cYFP vectors, respectively. These constructs were then transformed into Agrobacterium tumefaciens GV3101. After co‐cultivating the two Agrobacterium strains, the mixture was infiltrated into the leaves of Nicotiana benthamiana. Subsequently, fluorescence signals were analysed using a confocal laser scanning microscope, and representative images were acquired 2–3 days after infiltration.

4.1.3. Pull Down

The pull‐down assay was performed according to the method described by Li et al. (2024). The genes encoding PgSCP and CsMIKC were respectively fused with His and GST tags, followed by transformation into competent Escherichia coli cells. Induction of expression generated the recombinant proteins PgSCP‐His and GST‐CsMIKC. The soluble proteins, PgSCP‐His and CsMIKC‐GST, were subsequently purified using GST agarose gel and a His‐tag protein purification kit, respectively. Finally, the purified proteins were used in pull‐down assays to confirm their interaction.

4.2. Subcellular Localisation of CsMIKC

The amino acid sequence of CsMIKC was retrieved from the Citrus Pan‐genome to Breeding Database (http://citrus.hzau.edu.cn/index.php). The subcellular localisation of CsMIKC was analysed. The subcellular localisation expression vector of CsMIKC‐GFP was constructed using a GFP fusion protein strategy. The CsMIKC sequence was amplified via PCR and subsequently cloned into the N‐terminal region of the green fluorescent protein (GFP) within the plant expression vector pCAMBIA‐GFP. The empty vector (pCAMBIA‐GFP) containing only GFP served as a negative control, while the nuclear localisation marker H2B‐mCherry was used as a co‐transformation reference to distinguish cytoplasmic and nuclear signals. The recombinant plasmid, CsMIKC‐GFP, and the nuclear marker plasmid, H2B‐mCherry, were individually introduced into A. tumefaciens GV3101. The bacterial cultures were diluted with sterile water to achieve an optical density at 600 nm (OD600) of 0.4, after which they were combined in equal volumes. This resultant mixture was subsequently infiltrated into the leaves of N. benthamiana. After 48 h, the subcellular localisation of CsMIKC was observed using the confocal laser microscope.

4.3. The Function of CsMIKC in Citrus Disease Resistance

To elucidate the function of CsMIKC in citrus disease resistance, both transient overexpression and gene‐silencing methodologies were utilised. For the transient overexpression, the CDS of CsMIKC was cloned into the pEAQ vector, resulting in the construction of pEAQ‐CsMIKC. Agrobacterium cultures harbouring either the pEAQ‐CsMIKC construct or the empty pEAQ vector (serving as the control) were harvested. The bacterial cells were resuspended in an infiltration buffer and adjusted to an OD600 of 0.5. These suspensions were then infiltrated into the equatorial region of citrus fruits at a volume of 0.5 mL per site. After 2 h, the fruits were inoculated with a spore suspension of P. digitatum , and the progression of symptoms was observed and recorded. For VIGS, a TRV2‐based vector containing a fragment of CsMIKC (TRV2‐CsMIKC) was developed. A. tumefaciens strains containing either TRV1 or TRV2/TRV2‐CsMIKC were combined in a 1:1 (vol/vol) ratio and incubated in darkness at 28°C for 2–3 h. Uniform citrus fruits were selected, and 0.5 mL of the bacterial suspension was injected into their equatorial region. After 2 h, the fruits were inoculated with a suspension of P. digitatum spores. The progression of the disease was recorded.

4.4. The Effect of PgSCP Collaborating With CsMIKC on the Resistance to Green Mould

The impact of co‐expressing PgSCP with CsMIKC on enhancing the green mould resistance of citrus fruits was examined, using P. digitatum as an indicator pathogen. The genes encoding PgSCP and CsMIKC were individually cloned into the plant dual expression vector pEAQ, and the resultant recombinant plasmids were introduced into A. tumefaciens GV3101. The experimental design comprises treatment groups characterised by the co‐expression of PgSCP with CsMIKC. Control groups include an empty vector, the singular expression of PgSCP, the singular expression of CsMIKC. Data on the incidence of the disease and the diameter of lesions in citrus fruits were systematically recorded.

4.5. Transcriptomic Analysis of the Induction of Disease Resistance Against Green Mould in Citrus Fruits Mediated by PgSCP and CsMIKC

Samples from the co‐transient overexpression of PgSCP and CsMIKC, as well as the transient overexpression of an empty vector, were collected 2 days post‐transfection for RNA‐seq analysis. Each experimental condition was represented by three biological replicates. The RNA‐seq analysis was conducted by Guangzhou Kidio Biotechnology Co. Ltd.

4.6. Y1H

Y1H was carried out as previously reported (Im et al. 2023), with slight adjustments. The pHIS2 vector was ligated to a 100 bp fragment surrounding the binding region of the target gene, and CsMIKC was cloned into the AD vector. The experimental group consisted of co‐transformation with pHIS2‐bait and AD‐CsMIKC plasmids, while the self‐activation control group consisted of co‐transformation with pHIS2‐bait and empty AD plasmids. Both groups were co‐transformed into Y187 chemically competent cells and spotted separately onto SD/−Leu/−Trp double‐dropout medium. Positive clones were selected and amplified using SD/−Leu−Trp liquid medium. They were then inoculated into SD/−His/−Leu/−Trp medium containing gradient concentrations of 3‐aminotriazole (3‐AT). Colonies formed on SD/−His/−Leu/−Trp medium indicated whether CsMIKC interacted with the target gene.

4.7. DLR

The downstream target genes were cloned into the pGreenII 0800‐LUC vector. pEAQ was used as the negative control, and pEAQ‐CsMIKC served as the effector (Wang et al. 2023). The promoters of the downstream target genes were used as reporter constructs. Following treatment with osmotic agents for 2–3 h, the constructs were injected into N. benthamiana plants. Measurements were taken after 48 h. The transcriptional activation or repression potential of CsMIKC on these genes was quantified using the LUC/REN ratio, with each experiment conducted a minimum of six times.

4.8. Statistical Analysis

Statistical comparisons between two groups were conducted using Student's t test. For multiple group comparisons, one‐way ANOVA with Tukey's post hoc test was applied. All analyses were performed using SPSS v. 23.0 (SPSS Inc.). A p‐value < 0.05 was deemed statistically significant. GraphPad Prism software was used to generate figures.

Author Contributions

All authors listed meet the criteria for authorship. Each author contributed sufficiently to the work to take public responsibility for its content, including involvement in conceptualisation, design, data analysis, writing and revision. Ou Chen designed the research, acted as the principal investigator, analysed the data and drafted and revised the manuscript. Rui Huang and Yao Xu conducted the experiments, contributed to the research implementation and revised the manuscript. Wenjun Wang collected the data and participated in manuscript revision. Jian Ming revised the manuscript, performed editing and proofreading, supervised the research conduct and ensured adherence to methodological standards. Kaifang Zeng conceived the study, coordinated its design and execution, provided financial and logistical support and revised the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Jaspar analyses the downstream target gene binding sites of CsMIKC.

MPP-26-e70166-s002.xlsx (19.7KB, xlsx)

Table S2: Primer sequences used in this study.

MPP-26-e70166-s001.xlsx (13KB, xlsx)

Acknowledgements

This research was supported by the National Natural Science Foundation of China (32272376).

Chen, O. , Huang R., Xu Y., Wang W., Ming J., and Zeng K.. 2025. “Yeast‐Secreted Protein PgSCP and Citrus Transcription Factor CsMIKC Synergize to Activate Green Mould Resistance in Fruit.” Molecular Plant Pathology 26, no. 11: e70166. 10.1111/mpp.70166.

Funding: This work was supported by the National Natural Science Foundation of China (32272376).

Data Availability Statement

All relevant data are available within the manuscript and its Supporting Information (Tables S1 and S2).

References

  1. Bai, B. X. , Zhang G. D., Li Y., et al. 2022. “The ‘Candidatus Phytoplasma tritici’ Effector SWP12 Degrades the Transcription Factor TaWRKY74 to Suppress Wheat Resistance.” Plant Journal 112, no. 6: 1473–1488. [DOI] [PubMed] [Google Scholar]
  2. Bogino, M. F. , Senz J. M. L., Kourdova L. T., et al. 2025. “Downy Mildew Effector HaRxL106 Interacts With the Transcription Factor BIM1 Altering Plant Growth, BR Signaling and Susceptibility to Pathogens.” Plant Journal 121, no. 1: e17159. [DOI] [PubMed] [Google Scholar]
  3. Chen, O. , Zhu R., Xu Y., Yao S. X., Yi L. H., and Zeng K. F.. 2023. “Genome‐Wide Discovery of Pichia galeiformis‐Secreted Proteins and Their Induction of Green Mold Resistance in Citrus Fruit.” Postharvest Biology and Technology 204: 112435. [Google Scholar]
  4. Contreras, G. , Shirdel I., Braun M. S., and Wink M.. 2020. “Defensins: Transcriptional Regulation and Function Beyond Antimicrobial Activity.” Developmental and Comparative Immunology 104: 103556. [DOI] [PubMed] [Google Scholar]
  5. Deng, H. , Pei Y. G., Xu X., et al. 2024. “Ethylene‐MPK8‐ERF.C1‐PR Module Confers Resistance Against Botrytis cinerea in Tomato Fruit Without Compromising Ripening.” New Phytologist 242, no. 2: 592–609. [DOI] [PubMed] [Google Scholar]
  6. Deng, S. , Jiang S., Liu B., et al. 2024. “ZmGDIα‐Hel Counters the RBSDV‐Induced Reduction of Active Gibberellins to Alleviate Maize Rough Dwarf Virus Disease.” Nature Communications 15, no. 1: 7576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dong, B. X. , Liu Y., Huang G., et al. 2024. “Plant NAC Transcription Factors in the Battle Against Pathogens.” BMC Plant Biology 24, no. 1: 985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duan, Y. H. , Yang G. G., Tang J. T., et al. 2024. “ Ustilaginoidea virens Secreted Effector UvSec117 Hijacks OsWRKY31‐OsAOC Module to Suppress Jasmonic Acid‐Mediated Immunity in Rice.” Plant Biotechnology Journal 22, no. 12: 3342–3344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Galán, J. E. , Lara‐Tejero M., Marlovits T. C., and Wagner S.. 2014. “Bacterial Type III Secretion Systems: Specialized Nanomachines for Protein Delivery Into Target Cells.” Annual Review of Microbiology 68: 415–438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gheysen, G. , and Mitchum M. G.. 2011. “How Nematodes Manipulate Plant Development Pathways for Infection.” Current Opinion in Plant Biology 14, no. 4: 415–421. [DOI] [PubMed] [Google Scholar]
  11. Im, J. H. , Son S., Kim W. C., et al. 2023. “Jasmonate Activates Secondary Cell Wall Biosynthesis Through MYC2‐MYB46 Module.” Plant Journal 117, no. 4: 1099–1114. [DOI] [PubMed] [Google Scholar]
  12. Ishihama, N. , and Yoshioka H.. 2012. “Post‐Translational Regulation of WRKY Transcription Factors in Plant Immunity.” Current Opinion in Plant Biology 15, no. 4: 431–437. [DOI] [PubMed] [Google Scholar]
  13. Jiang, X. , Li P., Li X., et al. 2025. “A Novel Small Cysteine‐Rich Protein, PbEL04, Identified From Plasmodiophora brassicae , Associates With AtSAMS2 to Regulate Arabidopsis Clubroot Resistance.” Plant Journal 122, no. 5: e70264. [DOI] [PubMed] [Google Scholar]
  14. Li, K. , Zhai L., Pi Y., et al. 2024. “Mitogen‐Activated Protein Kinase MxMPK3‐2 Mediated Phosphorylation of MxZR3.1 Participates in Regulating Iron Homoeostasis in Apple Rootstocks.” Plant, Cell & Environment 47, no. 7: 2508–2523. [DOI] [PubMed] [Google Scholar]
  15. Liu, L. S. , Li J. N., Wang Z. H., et al. 2024. “Suppression of Plant Immunity by Verticillium dahliae Effector Vd6317 Through AtNAC53 Association.” Plant Journal 119, no. 4: 1767–1781. [DOI] [PubMed] [Google Scholar]
  16. Liu, S. X. , Cai Y. L., Lou Y., et al. 2025. “Understanding Citrus Aroma: Changes Induced by Postharvest Treatments of Fresh Fruit.” Postharvest Biology and Technology 224: 113491. [Google Scholar]
  17. Lovelace, A. H. 2024. “A Tale of Two Functions: How the Effector PsCRN108 Hijacks Plant Transcription Factors to Suppress Immunity.” Molecular Plant–Microbe Interactions 37, no. 1: 4–5. [DOI] [PubMed] [Google Scholar]
  18. Lu, Y. Q. , Liu S. Q., Wang Y. Q., Yang F., Tian Z. H., and Long C. A.. 2024. “Postharvest Biocontrol Ability and Antagonistic Mechanism of Bacillus altitudins h217 Against Penicillium digitatum in Citrus.” Postharvest Biology and Technology 212: 112911. [Google Scholar]
  19. Marrero, M. C. , Capdevielle S., Huang W. J., et al. 2024. “Protein Interaction Mapping Reveals Widespread Targeting of Development‐Related Host Transcription Factors by Phytoplasma Effectors.” Plant Journal 117, no. 4: 1281–1297. [DOI] [PubMed] [Google Scholar]
  20. Mejias, J. , Truong N. M., Abad P., Favery B., and Quentin M.. 2019. “Plant Proteins and Processes Targeted by Parasitic Nematode Effectors.” Frontiers in Plant Science 10: 970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moore, J. W. , Loake G. J., and Spoel S. H.. 2011. “Transcription Dynamics in Plant Immunity.” Plant Cell 23, no. 8: 2809–2820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nguyen, Q. M. , Iswanto A. B. B., Kang H., et al. 2024. “The Processed C‐Terminus of AvrRps4 Effector Suppresses Plant Immunity via Targeting Multiple WRKYs.” Journal of Integrative Plant Biology 66, no. 8: 1769–1787. [DOI] [PubMed] [Google Scholar]
  23. Noman, A. , Aqeel M., Khalid N., et al. 2019. “Zinc Finger Protein Transcription Factors: Integrated Line of Action for Plant Antimicrobial Activity.” Microbial Pathogenesis 132: 141–149. [DOI] [PubMed] [Google Scholar]
  24. Rocafort, M. , Fudal I., and Mesarich C. H.. 2020. “Apoplastic Effector Proteins of Plant‐Associated Fungi and Oomycetes.” Current Opinion in Plant Biology 56: 9–19. [DOI] [PubMed] [Google Scholar]
  25. Rovetto, E. I. , La Spada F., El Boumlasy S., et al. 2024. “Biological Control of Green Mold in Simulated Post‐Harvest Chain of Citrus Fruit: Efficacy of Candida oleophila Strain O and Molecular Insight Into Elicitation of Host Immune System.” Biological Control 193: 105531. [Google Scholar]
  26. Singh, S. K. , Shree A., Verma S., et al. 2023. “The Nuclear Effector ArPEC25 From the Necrotrophic Fungus Ascochyta rabiei Targets the Chickpea Transcription Factor CaβLIM1a and Negatively Modulates Lignin Biosynthesis, Increasing Host Susceptibility.” Plant Cell 35, no. 3: 1134–1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tsuda, K. , and Somssich I. E.. 2015. “Transcriptional Networks in Plant Immunity.” New Phytologist 206, no. 3: 932–947. [DOI] [PubMed] [Google Scholar]
  28. Wang, D. M. , Wu F., Dong Y. W., et al. 2025. “Biocontrol Effects of Volatile Organic Compounds Released From Burkholderia sp. NAU16 Against Penicillium digitatum ACCC 30389 and Its Application in Postharvest Preservation of Citrus.” Food Microbiology 132: 104825. [DOI] [PubMed] [Google Scholar]
  29. Wang, W. , Li T., Chen Q., Yao S., and Zeng K.. 2023. “Transcriptional Regulatory Mechanism of a Variant Transcription Factor CsWRKY23 in Citrus Fruit Resistance to Penicillium digitatum .” Food Chemistry 413: 135573. [DOI] [PubMed] [Google Scholar]
  30. Xiang, H. , Stojilkovic B., and Gheysen G.. 2025. “Decoding Plant–Pathogen Interactions: A Comprehensive Exploration of Effector–Plant Transcription Factor Dynamics.” Molecular Plant Pathology 26, no. 1: e70057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Xu, S. Q. , Yang S. Z., Yang Y. T., et al. 2025. “Transcription Factor CsWRKY76 Potentially Involved in Accumulation of Scoparone and Pentamethoxyflavone in Citrus Fruit Stimulated by Salicylic Acid.” Postharvest Biology and Technology 228: 113672. [Google Scholar]
  32. Yang, M. , Zhou C. P., Kuang R. B., et al. 2024. “Transcription Factor CpWRKY50 Enhances Anthracnose Resistance by Promoting Jasmonic Acid Signaling in Papaya.” Plant Physiology 196, no. 4: 2856–2870. [DOI] [PubMed] [Google Scholar]
  33. Yang, S. , Lovelace A. H., Yuan Y., et al. 2025. “A Witches' Broom Phytoplasma Effector Induces Stunting by Stabilizing a bHLH Transcription Factor in Ziziphus jujuba Plants.” New Phytologist 247, no. 1: 249–264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yang, X. Y. , Zhang M. J., Xi D. X., et al. 2024. “Genome‐Wide Identification and Expression Analysis of the MADS Gene Family in Sweet Orange (Citrus sinensis) Infested With Pathogenic Bacteria.” PeerJ 12: e17001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yang, Z. T. , Ai G., Lu X. Y., et al. 2024. “ Phytophthora sojae Effector PsCRN108 Targets CAMTA2 to Suppress HSP40 Expression and ROS Burst.” Molecular Plant–Microbe Interactions 37, no. 1: 15–24. [DOI] [PubMed] [Google Scholar]
  36. Zhang, H. , Teng W., Liang J., et al. 2016. “MADS1, a Novel MADS‐Box Protein, Is Involved in the Response of Nicotiana benthamiana to Bacterial harpinXoo .” Journal of Experimental Botany 67, no. 1: 131–141. [DOI] [PubMed] [Google Scholar]
  37. Zhang, Z. H. , Zou W. H., Lin P. X., et al. 2024. “Evolution and Function of MADS‐Box Transcription Factors in Plants.” International Journal of Molecular Sciences 25, no. 24: 13278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zheng, P. J. , Liu M. X., Pang L. J., et al. 2023. “Stripe Rust Effector Pst21674 Compromises Wheat Resistance by Targeting Transcription Factor TaASR3.” Plant Physiology 193, no. 4: 2806–2824. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1: Jaspar analyses the downstream target gene binding sites of CsMIKC.

MPP-26-e70166-s002.xlsx (19.7KB, xlsx)

Table S2: Primer sequences used in this study.

MPP-26-e70166-s001.xlsx (13KB, xlsx)

Data Availability Statement

All relevant data are available within the manuscript and its Supporting Information (Tables S1 and S2).

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