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. 2026 Jun 5;27(6):e70289. doi: 10.1111/mpp.70289

The DENN Protein SMD1 Forms a Signalling Hub With Rab GTPase YPT1 to Regulate Diverse Biological Processes in Phytopathogenic Fungi and Serves as a HIGS Target for Crop Protection

Cheng Zhang 1,2, Lin Li 1, Sunhong Liu 1, Yan Xu 1, Xin Li 3,4,✉, Yuelin Zhang 1,2,3,✉
PMCID: PMC13241621  PMID: 42249639

ABSTRACT

Sclerotinia sclerotiorum is a notorious necrotrophic plant pathogen that causes severe yield losses in many important crops. Despite its profound economic impact, the molecular mechanisms underpinning its development and virulence processes remain largely elusive. In this study, we characterized a mutant with reduced virulence through a forward genetic screen. Genetic analysis confirmed that mutations in a gene encoding a DENN (Differentially Expressed in Normal cells and Neoplasia) domain‐containing protein SsSMD1 (Sclerotia Maturation Deficient 1) led to impaired sclerotia formation and reduced virulence. SsSMD1 affects cell wall integrity and stress tolerance. Notably, SsSMD1 seems to act as a Guanine Nucleotide Exchange Factor (GEF) that interacts with the Rab GTPase SsYPT1. While the knockout of SsYPT1 was lethal, a single amino acid substitution (A136D) in SsYPT1 significantly impaired vegetative growth, sclerotia formation, and virulence. Similarly, knockout analysis in different phytopathogenic fungi revealed that SMD1 homologues were essential for their growth and virulence. We employed host‐induced gene silencing (HIGS) targeting SsSMD1 and SsYPT1, which effectively attenuated the virulence of S. sclerotiorum in Nicotiana benthamiana and Arabidopsis thaliana . Collectively, our findings demonstrate that the DENN domain‐containing protein is indispensable for development and virulence in major phytopathogenic fungi, and the SsSMD1‐SsYPT1 modules can be used as practical HIGS targets against necrotrophic fungal pathogens.

Keywords: DENN, GEF, host‐induced gene silencing (HIGS), Rab GTPase YPT1, Sclerotinia sclerotiorum, SMD1

The DENN domain‐containing protein SsSMD1 is highly conserved across phytopathogenic fungi. Functioning as a putative GEF for RAS GTPase SsYPT1, SsSMD1 orchestrates its activation to regulate cell wall integrity, sclerotia development, and virulence in Sclerotinia sclerotiorum.

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1. Introduction

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen that can infect over 400 plant species, causing Sclerotinia stem rot or white mould diseases (Liu et al. 2025). Sclerotia are overwintering structures crucial for its life cycle, capable of surviving in soil for extended periods (Sousa Melo et al. 2019; Ordóñez‐Valencia et al. 2015). Sclerotia can initiate infection through two primary routes: carpogenic germination, which forms ascocarps that release ascospores; or myceliogenic germination, which produces infective hyphae (Bolton et al. 2006; Zhu et al. 2024). The infection mechanism of S. sclerotiorum is complex, involving coordinated secretion of cell wall‐degrading enzymes (CWDEs), oxalic acid (OA), and a broad array of effectors. For example, SsPG1 (polygalacturonase 1) and SsCUTA (cutinase) contribute to early infection and lesion expansion, and at least five distinct endopolygalacturonases are expressed during various infection stages and conditions (Bashi et al. 2012). A novel secreted cellobiohydrolase, SsdchA, is indispensable for the pathogenicity of S. sclerotiorum by facilitating the enzymatic degradation of host cellulose (Chen et al. 2024). OA is a key pathogenicity determinant, acidifying the environment and impairing plant guard cell function through preventing stomatal closure during early infection (Daniel et al. 2024; Guimarães and Stotz 2004). OA also modulates host redox homeostasis, effectively quenching plant reactive oxygen species (ROS) bursts and suppressing callose deposition and programmed cell death (PCD) (Williams et al. 2011). In S. sclerotiorum, disruption of OAH1, which encodes oxaloacetate acetylhydrolase, eliminates OA biosynthesis and leads to a partial attenuation of virulence (Liang and Liberti 2015). Effector proteins such as SsSSP1 (small secreted virulence‐related protein) (Lyu et al. 2016), SsCP1 (Cerato‐platanin proteins) (Yang et al. 2018), SsPINE1 (PGIP‐inactivating effector 1) (Wei et al. 2022), and SsCm1 (chorismate mutase 1) (Xiao et al. 2025) are secreted to facilitate infection.

Host‐induced gene silencing (HIGS) is a powerful and effective method for protecting plants against pathogens or pests (Ali et al. 2024). By transiently or stably expressing target genes in plants, small interfering RNAs (siRNAs) are produced, inducing post‐transcriptional gene silencing (PTGS) of complementary pathogen or pest transcripts (Koch and Wassenegger 2021). Using virulence factor genes of S. sclerotiorum as HIGS targets, including SsPG1 (endo‐polygalacturonase), SsCBH (cellobiohydrolase) and SsOAH1 (oxaloacetate acetylhydrolase), can effectively control Sclerotinia blight in rapeseed (Wu et al. 2022). The RAS signalling components (SsGAP1, SsRAS1 and SsRAS2) are involved in growth and virulence, and SsGAP1 has been proven to be an effective HIGS target to prevent S. sclerotiorum and Botrytis cinerea (Xu, Tan, et al. 2023). SsSte50‐SsSte11‐SsSte7‐Smk1 forms a conserved mitogen‐activated protein kinase cascade in S. sclerotiorum that is required for sclerotia development and virulence. Silencing of SsSte50 by HIGS provides a powerful method to disrupt pathogen growth (Tian et al. 2023). The GDP‐mannose pyrophosphorylases SsMPG1 and SsMPG2 form a complex that regulates GDP‐mannose synthesis, controlling the formation of compound appressoria and virulence. HIGS‐targeting SsMPG1 and SsMPG2 via hairpin RNA expression in Nicotiana benthamiana and Arabidopsis thaliana significantly attenuates lesion expansion (Zhang et al. 2025). SsPDE2 encodes a cAMP phosphodiesterase that can serve as a HIGS target for disease control (Xu, Qiu, et al. 2023). In addition to these, the transcriptional module SsSnf5‐SsHsf1‐SsHsp70 (Xiao et al. 2024), the Magnaporthe appressoria‐specific (MAS) protein homologues Sscnd1 (Ding et al. 2021), the pH‐responsive transcription factor SsPac1, SsSmk1 (Pant and Kaur 2024), and SsChsI–VII‐SsCas1‐SsPG1‐SsOAH1 (Ren et al. 2026) have all been shown as effective HIGS targets against S. sclerotiorum. Nevertheless, more effective HIGS target genes are needed for controlling S. sclerotiorum.

In this study, we identified a mutant S1635 with reduced virulence by screening Ssoah1 ascospores through UV mutagenesis (Li et al. 2018; Xu et al. 2022; Zhang et al. 2025). The candidate gene for S1635 is sscle_09g072210, encoding a DENN domain‐containing protein SMD1. SsSMD1 exerts a multifaceted regulatory role in the development and virulence of S. sclerotiorum. Interestingly, SsSMD1 may act as a GEF for SsYPT1 (Yeast Protein Transport 1), and a single amino acid substitution (A136D) in SsYPT1 significantly affects growth and virulence. The DENN domain amino acid sequence is highly conserved among diverse pathogenic fungi, and the SsSMD1 orthologs in phytopathogenic fungi are also crucial for growth and virulence. Moreover, expression of SsSMD1 or SsYPT1 dsRNA in N. benthamiana and A. thaliana significantly inhibits S. sclerotiorum lesion expansion, indicating that the SsSMD1‐SsYPT1 module members are effective HIGS targets for disease control.

2. Results

2.1. Identification of a DENN‐Domain Protein Essential for Virulence in the S. sclerotiorum UV‐Mutant S1635

We previously performed UV mutagenesis on the OA‐deficient mutant Ssoah1 to identify OA‐independent pathogenicity factors (Zhang et al. 2025). Mutant S1635 was identified with reduced virulence on N. benthamiana leaves compared to Ssoah1 (Figure 1a). Interestingly, pre‐inoculation wounding treatment had little impact on the size of the lesions, indicating its virulence defect is post‐penetration. To verify whether the virulence defect is related to the formation of the compound appressorium, we observed that S1635 could still form compound appressoria (Figure 1b). The mycelial growth rate of S1635 was slightly slower than Ssoah1 on potato dextrose agar (PDA) plates, and it was still able to form black sclerotia like Ssoah1 after 14 days (Figure 1c and Figure S1a).

FIGURE 1.

FIGURE 1

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Phenotypic characterization and next‐generation sequencing (NGS) analysis of S1635. (a) Virulence assay of Ssoah1 and S1635 on unwounded and wounded Nicotiana benthamiana leaves. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains. (b) Microscopic observation of compound appressoria formation of Ssoah1 and S1635 on glass slides. Bar = 100 μm. (c) Comparative colony growth of Ssoah1 and S1635 on potato dextrose agar plates. (d) Structural diagram of sscle_09g072210 depicting the point mutation and predicted amino acid substitution in S1635.

To pinpoint the causal mutation in the S1635 mutant, we employed whole‐genome sequencing and found that three genes had significant mutations in their respective coding regions. Further analysis revealed that the base C to G change in sscle_09g072210 leads to a premature stop codon (Figure 1d and Figure S1b). Intriguingly, sscle_09g072210 is putatively identified as a DENN (Differentially Expressed in Normal and Neoplastic cells) domain‐containing protein. As DENNs are harboured by various signalling proteins that orchestrate Rab‐mediated membrane trafficking or modulate MAPK signalling cascades in humans (Levivier et al. 2001; Miyoshi and Takai 2004), sscle_09g072210 was prioritized as the candidate gene for S1635.

2.2. sscle_09g072210 Is Vital for the Growth and Virulence of S. sclerotiorum

To examine whether sscle_09g072210 is the causal gene for the S1635 phenotype, we knocked it out in the wild‐type (WT) S. sclerotiorum 1980 background by homologous recombination‐based gene replacement. Due to the multinucleate nature of fungal mycelia (Ford et al. 1995; Xu et al. 2025), we used PCR to confirm that the selectable marker gene HPH (hygromycin B phosphotransferase) successfully replaced the sscle_09g072210 and that sscle_09g072210 was absent in two independent knockout alleles (Figure S2a,b). Both mutants exhibited abnormal mycelial growth and a slightly reduced growth rate on PDA compared to WT (Figure 2a and Figure S2c). Surprisingly, neither of the mutants formed sclerotia on PDA at 7 or 14 days post‐inoculation (dpi), but hyphae intertwining and sclerotia initials were observed (Figure 2a). Hence, we named sscle_09g072210 Sclerotia Maturation Deficient 1 (SsSMD1). Similar to S1635, both Sssmd1 mutants formed compound appressoria on glass slides (Figure 2b). Virulence assays were performed by inoculating the two Sssmd1 mutants on N. benthamiana and Arabidopsis leaves. In comparison with the WT, both knockout lines exhibited significantly attenuated virulence. Furthermore, the pre‐inoculation wound treatment did not significantly affect the size of the lesion (Figure 2c,d).

FIGURE 2.

FIGURE 2

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Phenotypic analysis of the two Sssmd1 deletion knockout mutants. (a) Comparative colony growth of wild‐type (WT) and Sssmd1 deletion mutants on potato dextrose agar. (b) Microscopic observation of compound appressoria formation of the WT and Sssmd1 deletion mutants on glass slides. Bar = 100 μm. (c) Virulence assay of WT and Sssmd1 mutants on unwounded and wounded Nicotiana benthamiana leaves. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains. (d) Virulence assay of WT and Sssmd1 mutants on unwounded and wounded Arabidopsis leaves. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains.

To further confirm that the candidate gene in the S1635 mutant is an allele of SsSMD1, we performed hyphal fusion, using Sssmr1‐1 (Sclerotial melanogenesis regulatory gene 1–1) as a control, which forms pink sclerotia (Xu et al. 2022). Mycelia germinating from the fused black sclerotia were then used for inoculation on N. benthamiana leaves (Figure S3a,b). Consistent with our prediction, Sssmd1 did not regain its virulence after fusing with S1635, while its virulence was restored after fusing with Sssmr1‐1 (Figure S3c). These results demonstrate that the disruption of SsSMD1 is the underlying genetic cause for the various developmental and virulence defects manifested by the S1635 mutant. Although S1635 carries a stop codon mutation that would yield a truncated protein deleting about 1/3 of the protein, it can still form sclerotia, suggesting that it is likely a hypomorphic allele of SsSMD1 and the truncated protein retains residual functions of SsSMD1.

2.3. The Roles of SsSMD1 in Response to Different Stresses

The fungal cell wall underpins key biological processes, including cell viability, morphogenesis and pathogenesis (Gow et al. 2017). To investigate whether SsSMD1 is involved in the synthesis of the cell wall associated with hyphal development, we added three cell wall synthesis inhibitors (sodium dodecyl sulphate [SDS], calcofluor white [CFW], and Congo red [CR]) to PDA. In each case, the growth of the two Sssmd1 mutants was remarkably inhibited compared with WT (Figure 3a,b). These findings suggest that SsSMD1 plays a pivotal role in maintaining cell wall homeostasis, as its disruption leads to compromised structural integrity.

FIGURE 3.

FIGURE 3

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The role of SsSMD1 in response to cell wall stress and oxidative stress. (a) Comparative colony growth of wild‐type (WT) and Sssmd1 mutants on potato dextrose agar (PDA) added with SDS, calcofluor white (CFW) or Congo red (CR). (b) The colony diameter of WT and Sssmd1 mutants was measured at 48 h post‐inoculation on PDA. (c) Comparative colony growth of WT and Sssmd1 mutants under different concentrations of H2O2. (d) Statistical comparison of the growth inhibition rates between the WT strain and Sssmd1 mutants. Student's t test (*p ≤ 0.05, **p ≤ 0.01).

Successful plant pathogens are able to evade the plant's defence systems during host colonization (Fones and Preston 2012). To evaluate the involvement of SsSMD1 in oxidative stress tolerance, the Sssmd1 mutants were subjected to PDA containing 5, 10, 15 or 20 mM H2O2. The results revealed that Sssmd1 mycelial expansion was profoundly compromised under oxidative insult, with the mutants exhibiting a significantly elevated growth inhibition rate compared to the WT strain (Figure 3c,d). These results suggest that the attenuated virulence of the Sssmd1 mutant may be related to its lack of reactive oxygen detoxification mechanism. It is worth noting that Sssmd1 mutants also exhibit attenuated osmotic stress responses (Figure S4a,b). These results indicate that SsSMD1 plays an important role in a broad spectrum of stress resistance responses in S. sclerotiorum.

2.4. SsSMD1 Interacts With the GDP‐Binding Form of SsYPT1

SsSMD1 (1151 amino acids) contains the tripartite DENN domains (Figure 4a): uDENN (residues 261–360 amino acids [aa]), DENN (residues 465–662 aa), and dDENN (residues 964–1036 aa). SsSMD1 is highly conserved across filamentous fungi (Figure S5), and there is another DENN domain‐containing protein that has only 36.4% sequence similarity to SsSMD1. To distinguish between these two DENN proteins, we constructed a phylogenetic tree of DENN proteins from the filamentous fungi, which shows that SMD1 and its paralog diverged early in evolution and belong to different clades (Figure 4b).

FIGURE 4.

FIGURE 4

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SsSMD1 can interact with SsYPT1S22N. (a) Protein sequence diagram of SsSMD1. The information of SsSMD1 was analysed by SMART (SMART: Main page). (b) Phylogenetic tree illustrating the orthologous relationships of DENN‐domain proteins among Sclerotinia sclerotiorum and other related fungi. Protein accession numbers have been marked on the phylogenetic tree. (c) The interaction model prediction between SsSMD1 and SsYPT1 based on AlphaFold 3. Visualization of key interacting amino acid residues was achieved through PyMOL analysis. (d) Yeast two‐hybrid analysis between SsSMD1 and SsYPT1 or SsYPT1S22N. Yeast transformants were serially diluted and spotted (5 μL per spot, OD600 = 100, 10− 1, 10− 2) onto SD−L−T and SD−L−T−H−A plates to confirm protein–protein interactions. X‐α‐gal was dropped onto yeast colonies at a concentration of 10 mg/mL. (e) In planta interaction between SsSMD1 and SsYPT1S22N by split‐luciferase complementation in Nicotiana benthamiana. Representative images show bioluminescence signals captured 48 h post‐agroinfiltration and 5 min after 1 mM luciferin treatment.

The function of DENN proteins in fungi remains elusive. Recent reports indicate that the DENN domain can directly interact with small GTPases in the Rab family, potentially functioning as Rab‐specific guanine nucleotide exchange factors (GEF) (Marat et al. 2011). To verify whether SsSMD1 can interact with Rabs, we firstly identified corresponding Rabs in S. sclerotiorum using the 11 major Rabs from yeast (Figure S6). We then used AlphaFold 3 to predict the binding affinity of SsSMD1 with these 11 Rabs. Interestingly, SsSMD1 only exhibited high confidence interaction scores with SsYPT1 (ipTM = 0.82), SsSEC4 (ipTM = 0.83), and SsYPT31 (ipTM = 0.8) (Figure S7a).

Rab GTPases function as molecular switches, cycling between active GTP‐bound and inactive GDP‐bound conformations to orchestrate intracellular signalling. For example, the GDP‐binding form of Vps21 can interact specifically with its GEF in fungi (Li et al. 2015; Zhu et al. 2018). To verify the AlphaFold's predictions (Figure 4c), we used yeast two‐hybrid (Y2H) assay to examine the interactions between SsSMD1 and SsYPT1, SsSEC4 or SsYPT31 of the GDP‐binding form. Only SsYPT1S22N interacted with SsSMD1 (Figure S7b). X‐α‐gal staining further confirmed that SsSMD1 only interacted with SsYPT1S22N (GDP‐bound form), but not with WT SsYPT1 (Figure 4d). Consistent with the Y2H results, the association between SsSMD1 and SsYPT1S22N was further validated through a split‐luciferase complementation (SLC) assay in N. benthamiana, providing independent evidence for their interaction (Figure 4e). These results suggest that SsSMD1 can interact directly with the GDP‐bound form of SsYPT, probably to assist its GEF function.

2.5. SsYPT1 Plays a Pivotal Role in the Survival of S. sclerotiorum

The biological function of Rab GTPase SsYPT1 in S. sclerotiorum has not been reported. To investigate its function, we first knocked out SsYPT1 using homologous recombination. However, despite multiple rounds of protoplast purification from the hyphal tips, we failed to obtain homokaryotic knockout mutants, suggesting that SsYPT1 knockout is lethal. Previous studies have reported that a single amino acid mutation (A136D) in YPT1 in yeast and Magnaporthe oryzae can affect fungal growth at permissive temperatures (Lynch‐Day et al. 2010; Wu et al. 2024). We obtained two independent Ssypt1 A136D mutants through homologous knock‐in (Figure S8a). Under 25°C incubation conditions, both mutants exhibited slow hyphal growth (Figure 5a and Figure S8b). Interestingly, after 14 days of growth on PDA plates, both Ssypt1 A136D mutants, like the Sssmd1 mutant, did not form sclerotia but showed intertwined hyphae and sclerotia initials (Figure 5a). Similar to Sssmd1, both Ssypt1 A136D mutants formed compound appressoria on glass slides (Figure 5b). Subsequently, the virulence of two Ssypt1 A136D mutants was evaluated on N. benthamiana and Arabidopsis leaves; the necrotic lesions elicited by these mutants were significantly reduced in size relative to those of the WT (Figure 5c,d). These results indicate that SsYPT1 is a critical determinant of vegetative growth, sclerotial development and virulence in S. sclerotiorum.

FIGURE 5.

FIGURE 5

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SsYPT1 affects fungal growth and virulence. (a) Comparative colony growth of wild‐type (WT) and Ssypt1 A136D mutants on potato dextrose agar. (b) Microscopic observation of compound appressoria formation of WT and Ssypt1 A136D mutants on glass slides. Bar = 100 μm. (c) Virulence assay of WT and Ssypt1 A136D mutants on Nicotiana benthamiana leaves. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains. (d) Virulence assay of WT and Ssypt1 A136D mutants on Arabidopsis leaves. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains.

2.6. Functional Conservation of SsSMD1 Orthologs in Diverse Phytopathogenic Fungi

Because SMD1 is highly conserved across different fungi, we further investigated whether SMD1 has similar roles among various phytopathogenic fungi, such as Botryis cinerea, M. oryzae and Fusarium graminearum. We first knocked out BcSMD1 in B. cinerea , obtaining two independent knockout mutants, Bcsmd1‐1 and Bcsmd1‐2 (Figure S9a). Compared with the B. cinerea WT, the two Bcsmd1 mutants exhibited significantly more severe growth defects, and their growth rates were severely reduced on PDA (Figure 6a). Like the Sssmd1 mutants, the two Bcsmd1 mutants were also capable of forming compound appressoria on glass slides further demonstrates that BcSMD1 does not affect the formation of the infection structure (Figure 6b). Subsequently, inoculation assays on Arabidopsis leaves revealed that both Bcsmd1 mutants exhibited significantly attenuated virulence compared to the WT, confirming that BcSMD1 is essential for virulence (Figure 6c).

FIGURE 6.

FIGURE 6

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Knocking out SMD1 in other phytopathogenic fungi affects their development and virulence. (a) Comparative colony growth of Botrytis cinerea wild‐type (WT) strain and Bcsmd1 mutants on potato dextrose agar (PDA). (b) Microscopic observation of compound appressoria formation of WT and Bcsmd1 mutants on glass slides. Bar = 100 μm. (c) Virulence assay of WT and Bcsmd1 mutants on Arabidopsis leaves. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains. (d) Comparative colony growth of Magnaporthe oryzae WT strain Guy11 and two Mosmd1 mutants on complete medium (CM) plates. (e) Virulence assay of M. oryzae WT and Mosmd1 mutants on wounded leaves of rice. Bar = 0.5 cm. Lesion length quantification and statistical significance (p value) for the indicated strains. (f) Comparative colony growth of Fusarium graminearum PH‐1 and Fgsmd1 mutants on PDA. (g) Virulence assay of F. graminearum PH‐1 and Fgsmd1 mutants on wheat leaves. Bar = 0.5 cm. Lesion length quantification and statistical significance (p value) for the indicated strains.

To further investigate the functional conservation of SMD1, we generated deletion mutants in M. oryzae and F. graminearum (Figure S9b,c). Both Mosmd1 and Fgsmd1 mutants exhibited significant vegetative growth retardation on plates, effectively recapitulating the developmental defects observed in S. sclerotiorum and B. cinerea . These consistent phenotypes across multiple fungi reinforce the notion that SMD1 serves as a phylogenetically conserved regulator of fungal morphogenesis and vegetative fitness (Figure 6d,f). Through inoculation experiments, we found that the virulence of the Mosmd1 and Fgsmd1 mutants was significantly reduced on their hosts (Figure 6e,g). In summary, SMD1 plays a fundamental and evolutionarily conserved role in modulating the development and virulence of diverse phytopathogenic fungi.

2.7. HIGS of SsSMD1 and SsYPT1 Reduces S. sclerotiorum Virulence in N. benthamiana and A. thaliana

Given the pivotal role of SsSMD1 and SsYPT1 in fungal survival and pathogenicity of S. sclerotiorum, we evaluated their potential as effective host‐induced gene silencing (HIGS) targets for enhancing crop resistance against fungal infection. Specifically, a 514‐bp fragment from the first exon of SsSMD1 or a 487‐bp sequence from the SsYPT1 transcript were fused with an A. thaliana malate synthase intron to generate hairpin RNA (hpRNA) constructs (Figure S10). Upon transient expression in N. benthamiana via Agrobacterium‐mediated infiltration, pre‐expressing SsSMD1‐RNAi or SsYPT1‐RNAi significantly attenuated the necrotic lesions caused by S. sclerotiorum at 48 h post‐inoculation (hpi) compared to the empty vector (pC1300‐E9) control (Figure 7a,b).

FIGURE 7.

FIGURE 7

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Attenuation of Sclerotinia sclerotiorum virulence in Nicotiana benthamiana and Arabidopsis thaliana via host‐induced gene silencing (HIGS) targeting SsSMD1 and SsYPT1. (a) Virulence assay of S. sclerotiorum wild‐type (WT) on N. benthamiana leaves transiently expressing empty vector (EV) or SsSMD1‐RNAi. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains. (b) Virulence assay of S. sclerotiorum WT on N. benthamiana leaves transiently expressing EV or SsYPT1‐RNAi. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains. (c) Virulence assays of S. sclerotiorum WT on T2 transgenic A. thaliana lines expressing SsSMD1‐RNAi. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains. (d) Virulence assays of S. sclerotiorum WT on T2 transgenic A. thaliana lines expressing SsYPT1‐RNAi. Bar = 0.5 cm. Lesion area quantification and statistical significance (p value) for the indicated strains.

To further investigate whether HIGS‐conferred protection could be stably maintained, A. thaliana WT plants were subjected to Agrobacterium‐mediated transformation with SsSMD1‐RNAi and SsYPT1‐RNAi vectors, respectively. In SsSMD1‐RNAi T2 generation, 4 of T1 transgenic lines (#21, #25, #26, #31) exhibited enhanced resistance to S. sclerotiorum infection. In SsYPT1‐RNAi T2 generation, 4 of T1 transgenic lines (#3, #5, #6, #17) exhibited a markedly elevated resistance to S. sclerotiorum infection (Figure 7c,d). These results demonstrate that SsSMD1 and SsYPT1 can serve as HIGS targets to prevent S. sclerotiorum infection.

3. Discussion

In our study, we revealed the function of DENN domain‐containing proteins in plant pathogenic fungi for the first time, establishing the essential role of SMD1 in the growth and pathogenicity of S. sclerotiorum and other pathogenic fungi. Our studies demonstrate that mutant screens using the OA‐deficient mutant Ssoah1 are efficient in identifying novel components involved in virulence. SsSMD1 seems to interact with YPT1 to exert its function. Finally, SsSMD1 and SsYPT1 can serve as effective targets for HIGS.

Gene knockout and hyphal fusion demonstrated that mutations in SsSMD1 affected the virulence of S1635. The Sssmd1 mutant was significantly more sensitive to cell wall inhibitors (Figure 3a,b), indicating that SsSMD1 is essential for maintaining cell wall integrity (CWI) in S. sclerotiorum. The cell wall is not only the basis for fungal morphology, but also a barrier against the host's ROS burst (Gow et al. 2017). Our study further found that the tolerance of the Sssmd1 mutant to H2O2 stress was severely impaired (Figure 3c,d), suggesting that its ROS scavenging mechanism may fail due to cell wall defects or dysregulated expression of antioxidant genes (Yaakoub et al. 2022). In addition, although the Sssmd1 mutants formed compound appressoria normally (Figure 2b), the expansion of lesions after infection was still inhibited, suggesting that SsSMD1 may be involved in regulating the secretion of effector factors, such as CWDEs and effector proteins, or the expansion of hyphae.

The DENN domain is an evolutionarily ancient and conserved protein module that can interact with small GTPases of the Rab family, functioning enzymatically as a Rab‐specific guanine nucleotide exchange factor (GEF) (Marat et al. 2011; Zhang et al. 2012). GEFs initiate signalling by catalysing the exchange of G protein‐bound GDP for GTP (Bos et al. 2007). The uDENN (for upstream DENN) and dDENN (for downstream DENN) are part of the tripartite DENN domain. DENN domain‐containing proteins participate in various signalling pathways, including Rab‐mediated processes or MAPK signalling regulation (Levivier et al. 2001). For example, in humans, there are 18 genes encoding proteins containing DENN domains (Marat et al. 2011; Yoshimura et al. 2010). DENND1B can interact with Rab35 and participate in regulating the development of childhood asthma and other immune disorders (Yang et al. 2016; Wu et al. 2011). DENND2B harbours a C‐terminal DENN domain that specifically recruits Rab13, a modular interaction essential for driving cell migration and invasive potential (Ioannou et al. 2015). The connecdenn/DENND1A DENN domain is a lipid‐binding module that functions as a GEF for Rab35 to regulate endosomal membrane trafficking (Allaire et al. 2010). FAM45A is a non‐classical DENN domain that co‐localizes with Rab27a and Rab27b in the multivesicular endosomes, to regulate endosome positioning, maturation, and secretion (Zhang et al. 2019). In Arabidopsis, SCD1 (STOMATAL CYTOKINESIS DEFECTIVE1) and SCD2 can form a protein complex and interact with the RabE1 family protein Sec4p/Rab8 to participate in post‐Golgi trafficking to the plasma membrane and cell plate (Mayers et al. 2017). In addition, SCD1 plays a role not only in plant growth and development but also in the immune response to bacteria and may serve as a negative regulator of the immune response (Korasick et al. 2010). RME‐4 is a conserved DENN domain protein that binds to RAB‐35 to regulate yolk endocytosis in Caenorhabditis elegans (Sato et al. 2008). In yeast, YPT1 is known to be a key regulator of endoplasmic reticulum‐to‐Golgi transport and is regulated by the TRAPP III complex (Lipatova et al. 2013; Thomas et al. 2018). Phylogenetic analysis revealed that SsSMD1 contains a typical tripartite DENN domain (uDENN‐DENN‐dDENN), and its sequence is highly conserved in filamentous fungi.

Alphafold 3 prediction revealed that SsSMD1 has a high affinity for Rab GTPase SsYPT1. Our Y2H and SLC assays confirmed that SsSMD1 can interact with the GDP‐bound form of SsYPT1, but not with wild‐type SsYPT1, indicating that SsSMD1 probably acts as a GEF for SsYPT1 to regulate the conversion of its GDP‐bound form to the GTP‐bound form. Whether SsYPT1 is regulated by the TRAPP III complex or whether SsSMD1 can regulate SsYPT1 in place of the TRAPP III complex is unclear in S. sclerotiorum. Knockout of YPT1 is lethal not only in S. sclerotiorum, but also in F. graminearum and M. oryzae . Constitutive activation and dominant inactivation mutants of YPT1 in F. graminearum affect vesicle transport and autophagy, thereby impacting fungal growth, conidia formation and pathogenicity. In M. oryzae , the temperature‐sensitive mutant ypt1 A136D exhibits growth defects and reduced pathogenicity at higher temperatures (Wu et al. 2024). In our study, a single amino acid mutation (A136D) in SsYPT1 affected hyphal growth, sclerotia formation and virulence in S. sclerotiorum. These findings provide compelling evidence that YPT1 plays a crucial role in fungi. However, it is still unclear whether SsSMD1 affects vesicle transport and translocation processes through SsYPT1, and further verification is needed.

HIGS is an emerging strategy for controlling fungal diseases. Our study demonstrated that expression of SsSMD1 or SsYPT1 dsRNA in N. benthamiana and A. thaliana significantly attenuated the virulence of S. sclerotiorum. These results are consistent with the HIGS attempts targeting the RAS signalling pathway (SsGAP1), the MAP kinase cascade (SsSte50), and the GDP‐mannose synthesis pathway (SsMPG1‐SsMPG2), further expanding the effective HIGS targets library (Xu, Tan, et al. 2023; Tian et al. 2023; Zhang et al. 2025). Because SMD1 is highly conserved in many pathogenic fungi and its knockout results in severe growth defects, HIGS technology targeting this gene is expected to develop into a broad‐spectrum disease resistance strategy (Ghag 2017), especially against soil‐borne pathogens that are difficult to control chemically.

4. Experimental Procedures

4.1. Fungal Strain and Culture Conditions

The Ssoah1 mutant and S. sclerotiorum 1980 WT served as backgrounds for UV mutagenesis and targeted gene knockouts, respectively. Putative knockout mutants were selected and purified on PDA amended with 50 μg/mL hygromycin B. All S. sclerotiorum strains were grown on PDA at room temperature.

All B. cinerea strains (B05.10 background) were cultured on PDA at room temperature.

All M. oryzae strains (Guy11 background) were cultured on complete medium (CM) at 25°C (12 h‐light/12 h‐dark photoperiod). Tomato‐oat medium was used to induce spore production.

All F. graminearum strains (PH‐1 background) were cultured on PDA at a constant temperature of 25°C.

4.2. Next‐Generation Sequencing Analysis

Fungal genomic DNA was extracted using the Genomic DNA Kit (Tsingke Biotechnology) or the CTAB method. Next‐generation sequencing (NGS) data analysis was performed as previously described (Xu, Tan, et al. 2023; Zhang et al. 2025). Candidate genes were analysed by UniProt and the NCBI BLAST.

4.3. Assessment of Mycelial Growth Rates and Colony Morphology

For the growth rate assays, fungal isolates were precultured on 90 mm PDA plates for 2 days. From the peripheral expansion zone of each colony, a 4‐mm mycelial disc was extracted with a sterile tip and subcultured onto the centre of a new PDA plate. These plates were then incubated at room temperature. The growth rate of each colony was quantified by measuring the diameter of the mycelium every 12 h over 48 h. Colony morphology images were taken at 3, 7 and 14 dpi for morphological observation.

4.4. Target Gene Knockout and Knock‐In

Gene deletion cassettes were constructed via overlapping PCR, and the corresponding knockout mutants were generated through homologous recombination‐mediated transformation of fungal protoplasts, as previously described (Zhang et al. 2025). The knock‐in procedure is similar as the knockout method, which involves introducing a point mutation and replacing the original sequence, and then using a selection marker to replace the non‐functional sequence after the termination sequence (Tian et al. 2023). All primers used for PCR are listed in Table S1.

4.5. Compound Appressoria Observation

To examine compound appressoria formation, fresh mycelial plugs were excised from the colony margins using a sterile pipette tip and inoculated onto glass slides. These slides were incubated in a humidified chamber (Petri dishes lined with moistened filter paper) at 25°C for 24 h. The formation of compound appressoria was visualized under a Zeiss microscope.

4.6. Plant Infection Assay

All experimental methods for fungal infection of hosts were performed according to the methods previously described (Zhang et al. 2025).

4.7. Cell Wall Integrity and Stress Sensitivity Assays

Cell wall integrity of the strains was determined by culturing on PDA with calcofluor white (CFW; 20 μg/mL), 0.01% sodium dodecyl sulphate (SDS) or Congo red (CR; 10 μg/mL). The colony diameters were measured 48 hpi. For oxidative stress analysis, strains were inoculated on PDA with different H2O2 concentrations (5, 10, 15, 20 mM) (Jiao et al. 2022). The colony diameters were measured after 48 hpi, and the mycelial growth inhibition rate was calculated as follows. Inhibition rate (%) = [(Colony diameter without H2O2 − Colony diameter with H2O2)/Colony diameter without H2O2] × 100. For osmotic stress analysis, strains were inoculated on PDA with 1 M sorbitol, 1 M KCl or 1 M NaCl. The colony diameters were measured after 48 hpi.

4.8. Phylogenetic Tree Construction and Protein Sequence Analysis

Protein sequences were analysed and phylogenetic trees were constructed using Geneious, raxmlGUI and FigTree software. Sequence alignment analysis and visualization were performed using ESPript. Adobe Illustrator was used to modify and enhance the results.

4.9. Y2H Interaction Assays

The SsSMD1 coding sequence was cloned into the bait vector pGBKT7, while SsYPT1 and SsYPT1 S22N were integrated into the prey vector pGADT7 via homologous recombination. The resulting bait and prey constructs were co‐transformed into the yeast strain Y2HGold and selected on SD/−Leu/−Trp medium. Positive transformants were grown in liquid SD/−Leu/−Trp medium to an OD600 of 1, followed by serial dilution and spotting onto SD/−Leu/−Trp and SD/−Leu/−Trp/−His/−Ade plates. After culturing at 28°C for 3–5 days, the Petri dishes were photographed to record the interaction results.

4.10. SLC Assay

Vectors for SLC assay were generated following established protocols (Zhang et al. 2025). The SsSMD1 coding sequence was cloned into the p35S‐Nluc vector, while SsYPT1 S22N was inserted into p35S‐Cluc, resulting in the fusion constructs p35S‐SsSMD1‐nLUC and p35S‐SsYPT1 S22N ‐cLUC, respectively.

4.11. HIGS Construct and Expression in N. benthamiana and A. thaliana

The construction of RNAi vectors and the methods for their expression in plants were carried out as previously described (Xu, Tan, et al. 2023; Zhang et al. 2025). For SsSMD1 interference, a 514‐bp exonic region was amplified, while a 487‐bp cDNA fragment was used for SsYPT1. These target sequences were structured into inverted repeats separated by a malate synthase intron‐3 spacer.

Author Contributions

Sunhong Liu: methodology, writing – review and editing. Lin Li: methodology, writing – review and editing. Yan Xu: methodology, validation, writing – review and editing. Xin Li: formal analysis, project administration, writing – review and editing, writing – original draft, supervision, conceptualization. Yuelin Zhang: conceptualization, formal analysis, supervision, resources, project administration, funding acquisition, writing – review and editing. Cheng Zhang: methodology, data curation, investigation, validation, formal analysis, writing – original draft.

Funding

This work was supported by Fundamental Research Funds for the Central Universities (YJ202255 to Y.Z.), Natural Science Foundation of Sichuan Province (2025ZNSFSC1116 to Y.X.) and the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants to X.L.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Mycelial growth rate and next‐generation sequencing (NGS) result of S1635. (a) Growth rate of Ssoah1 and S1635 was measured on potato dextrose agar plates every 12 h for 48 h of Ssoah1 and S1635. (b) List of candidate genes of S1635 from NGS data analysis. Possible candidate genes are marked in red.

MPP-27-e70289-s009.docx (121.7KB, docx)

Figure S2: Generation and analysis of two independent Sssmd1 knockout alleles. (a) Targeted gene knock‐out by homologous recombination. The target gene and HPH gene are shown as yellow and green rectangles, respectively. This strategy is used to knock out all genes in this article. (b) PCR verification of the two Sssmd1 knockdown alleles. Genomic DNAs from wild‐type (WT) Sclerotinia sclerotiorum and two Sssmd1 mutants were used as PCR templates. (c) Growth rate was measured on potato dextrose agar plates every 12 h for 48 h of WT and the two Sssmd1 mutants.

MPP-27-e70289-s004.docx (294.9KB, docx)

Figure S3: Hyphae fusion demonstrates that SsSMD1 is the gene responsible for the S1635 mutant phenotype. (a) The diagram illustrates the experimental design for inoculation after the mutant's mycelia fusion. (b) Colony morphology of two Sssmd1 mutants fused with S1635 or Sssmr1‐1 on potato dextrose agar plates. (c) Virulence assay of wild‐type (WT) and fusion mutants on Nicotiana benthamiana leaves at 24 h post‐inoculation. Lesion area quantification and statistical significance (p value) for the indicated strains.

MPP-27-e70289-s011.docx (798KB, docx)

Figure S4: The role of SsSMD1 in response to osmotic stress. (a) Comparative colony growth of wild‐type (WT) and Sssmd1 mutants on potato dextrose agar (PDA) added with sorbitol, KCl, or NaCl. (b) The colony diameter of WT and Sssmd1 mutants was measured 48 h post‐inoculation on PDA.

MPP-27-e70289-s006.docx (498.3KB, docx)

Figure S5: Alignments of the conserved amino acid sequences of SMD1 in filamentous fungi by ESPript.

MPP-27-e70289-s010.docx (1MB, docx)

Figure S6: Phylogenetic analysis of 11 Rab GTPases from different fungi. The tree was built using Geneious, RaxmlGUI, and FigTree methods and evaluated by Bootstrap. The bootstrap values from 1000 replicates are labelled above the branches.

MPP-27-e70289-s003.docx (734.3KB, docx)

Figure S7: The interactions between SsSMD1 and Rab GTPase. (a) The table of binding ability analysis of SsSMD1 with 11 Rabs by AlphaFold (https://alphafoldserver.com). The accuracy of the prediction could be collectively provided by pTM (Predicted Template Modelling Score) and ipTM (Interface Predicted Template Modelling Score) values. (b) Yeast two‐hybrid analysis of interactions between SsSMD1 and the GDP‐bound form of SsYPT1, SsSEC4, or SsYPT31.

MPP-27-e70289-s007.docx (431.3KB, docx)

Figure S8: Generation and analysis of two independent Ssypt1 A136D alleles. (a) The representative DNA sequencing chromatograms for A136D in two Ssypt1 A136D mutants. (b) Growth rate was measured on potato dextrose agar plates every 12 h for 48 h of wild‐type (WT) and the two Ssypt1 A136D mutants.

MPP-27-e70289-s002.docx (409.6KB, docx)

Figure S9: Generation of independent SMD1 deletion alleles by homologous recombination in Botrytis cinerea, Magnaporthe oryzae, and Fusarium graminearum. (a) PCR verification of the two Bcsmd1 knockdown alleles. Genomic DNAs from wild‐type (WT) B. cinerea and two Bcsmd1 mutants were used as PCR templates. (b) PCR verification of the MoSMD1 deletion alleles. Genomic DNAs from WT M. oryzae Guy11 and two Mosmd1 mutants were used as PCR templates. (c) PCR verification of the FgSMD1 deletion alleles. Genomic DNAs from WT F. graminearum PH‐1 and two Fgsmd1 mutants were used as PCR templates.

MPP-27-e70289-s008.docx (448.2KB, docx)

Figure S10: Schematic architecture of the RNAi vectors pC1300‐SsSMD1RNAi‐E9 and pC1300‐SsYPT1RNAi‐E9 designed for host‐induced gene silencing.

MPP-27-e70289-s001.docx (90.6KB, docx)

Table S1: The list of primers used in this study.

MPP-27-e70289-s005.docx (21.8KB, docx)

Acknowledgements

We are grateful to Jeffrey A. Rollins for the S. sclerotiorum strains and oah1 mutant; Matthias Hahn, Amir Sharon, Min He, and Jin‐Rong Xu for kindly providing the B. cinerea B05.10, M. oryzae Guy11, and F. graminearum PH‐1 strains, respectively.

Contributor Information

Xin Li, Email: xinli@msl.ubc.ca.

Yuelin Zhang, Email: yuelin.zhang@scu.edu.cn.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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

Supplementary Materials

Figure S1: Mycelial growth rate and next‐generation sequencing (NGS) result of S1635. (a) Growth rate of Ssoah1 and S1635 was measured on potato dextrose agar plates every 12 h for 48 h of Ssoah1 and S1635. (b) List of candidate genes of S1635 from NGS data analysis. Possible candidate genes are marked in red.

MPP-27-e70289-s009.docx (121.7KB, docx)

Figure S2: Generation and analysis of two independent Sssmd1 knockout alleles. (a) Targeted gene knock‐out by homologous recombination. The target gene and HPH gene are shown as yellow and green rectangles, respectively. This strategy is used to knock out all genes in this article. (b) PCR verification of the two Sssmd1 knockdown alleles. Genomic DNAs from wild‐type (WT) Sclerotinia sclerotiorum and two Sssmd1 mutants were used as PCR templates. (c) Growth rate was measured on potato dextrose agar plates every 12 h for 48 h of WT and the two Sssmd1 mutants.

MPP-27-e70289-s004.docx (294.9KB, docx)

Figure S3: Hyphae fusion demonstrates that SsSMD1 is the gene responsible for the S1635 mutant phenotype. (a) The diagram illustrates the experimental design for inoculation after the mutant's mycelia fusion. (b) Colony morphology of two Sssmd1 mutants fused with S1635 or Sssmr1‐1 on potato dextrose agar plates. (c) Virulence assay of wild‐type (WT) and fusion mutants on Nicotiana benthamiana leaves at 24 h post‐inoculation. Lesion area quantification and statistical significance (p value) for the indicated strains.

MPP-27-e70289-s011.docx (798KB, docx)

Figure S4: The role of SsSMD1 in response to osmotic stress. (a) Comparative colony growth of wild‐type (WT) and Sssmd1 mutants on potato dextrose agar (PDA) added with sorbitol, KCl, or NaCl. (b) The colony diameter of WT and Sssmd1 mutants was measured 48 h post‐inoculation on PDA.

MPP-27-e70289-s006.docx (498.3KB, docx)

Figure S5: Alignments of the conserved amino acid sequences of SMD1 in filamentous fungi by ESPript.

MPP-27-e70289-s010.docx (1MB, docx)

Figure S6: Phylogenetic analysis of 11 Rab GTPases from different fungi. The tree was built using Geneious, RaxmlGUI, and FigTree methods and evaluated by Bootstrap. The bootstrap values from 1000 replicates are labelled above the branches.

MPP-27-e70289-s003.docx (734.3KB, docx)

Figure S7: The interactions between SsSMD1 and Rab GTPase. (a) The table of binding ability analysis of SsSMD1 with 11 Rabs by AlphaFold (https://alphafoldserver.com). The accuracy of the prediction could be collectively provided by pTM (Predicted Template Modelling Score) and ipTM (Interface Predicted Template Modelling Score) values. (b) Yeast two‐hybrid analysis of interactions between SsSMD1 and the GDP‐bound form of SsYPT1, SsSEC4, or SsYPT31.

MPP-27-e70289-s007.docx (431.3KB, docx)

Figure S8: Generation and analysis of two independent Ssypt1 A136D alleles. (a) The representative DNA sequencing chromatograms for A136D in two Ssypt1 A136D mutants. (b) Growth rate was measured on potato dextrose agar plates every 12 h for 48 h of wild‐type (WT) and the two Ssypt1 A136D mutants.

MPP-27-e70289-s002.docx (409.6KB, docx)

Figure S9: Generation of independent SMD1 deletion alleles by homologous recombination in Botrytis cinerea, Magnaporthe oryzae, and Fusarium graminearum. (a) PCR verification of the two Bcsmd1 knockdown alleles. Genomic DNAs from wild‐type (WT) B. cinerea and two Bcsmd1 mutants were used as PCR templates. (b) PCR verification of the MoSMD1 deletion alleles. Genomic DNAs from WT M. oryzae Guy11 and two Mosmd1 mutants were used as PCR templates. (c) PCR verification of the FgSMD1 deletion alleles. Genomic DNAs from WT F. graminearum PH‐1 and two Fgsmd1 mutants were used as PCR templates.

MPP-27-e70289-s008.docx (448.2KB, docx)

Figure S10: Schematic architecture of the RNAi vectors pC1300‐SsSMD1RNAi‐E9 and pC1300‐SsYPT1RNAi‐E9 designed for host‐induced gene silencing.

MPP-27-e70289-s001.docx (90.6KB, docx)

Table S1: The list of primers used in this study.

MPP-27-e70289-s005.docx (21.8KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Molecular Plant Pathology are provided here courtesy of Wiley

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