The Verticillium dahliae Effector VdPHB1 Promotes Pathogenicity in Cotton and Interacts with the Immune Protein GhMC4

Abstract

Verticillium dahliae is a kind of pathogenic fungus that brings about wilt disease and great losses in cotton. The molecular mechanism of the effectors in V. dahliae regulating cotton immunity remains largely unknown. Here, we identified an effector of V. dahliae, VdPHB1, whose gene expression is highly induced by infection. The VdPHB1 protein is localized to the intercellular space of cotton plants. Knock-out of the VdPHB1 gene in V. dahliae had no effect on pathogen growth, but decreased the virulence in cotton. VdPHB1 ectopically expressed Arabidopsis plants were growth-inhibited and significantly susceptible to V. dahliae. Further, VdPHB1 interacted with the type II metacaspase GhMC4. GhMC4 gene-silenced cotton plants were more sensitive to V. dahliae with reduced expression of pathogen defense-related and programmed cell death genes. The accumulation of GhMC4 protein was concurrently repressed when VdPHB1 protein was expressed during infection. In summary, these results have revealed a novel molecular mechanism of virulence regulation that the secreted effector VdPHB1 represses the activity of cysteine protease for helping V. dahliae infection in cotton.

Introduction

Verticillium dahliae is an important phytopathogen that causes wilt disease in more than 400 crops including cotton (Fradin and Thomma 2006, Inderbitzin and Subbarao 2014). The pathogen mainly infects host plants through the injured parts of root tips or lateral roots (Zhang et al. 2020). Furthermore, they grow in the vascular tissues and generally cause the wilting of plant leaves, vascular discoloration and stunting of plant growths (Klimes et al. 2015, Tian and Kong 2020). Verticillium wilt disease frequently leads to great losses in cotton fiber yield and serious decreases in cotton fiber quality worldwide (Zhang et al. 2021, Zhu et al. 2023). Currently, there is no chemical or biological method to effectively control this disease (Klosterman et al. 2009, Lian et al. 2014).

Under natural conditions, the Verticillium pathogen produces large numbers of microsclerotia around infected areas of cotton plants (Klimes et al. 2015). When the microsclerotia of the V. dahliae pathogen sense root exudates, they start to germinate. The growing hyphae of the V. dahliae attack the injured cells and enter cotton plants from the root tips, root hairs and finally colonize in the root cortex and central cylinder of the cotton plants (Vallad and Subbarao 2008, Prieto et al. 2010). To successfully infect cotton plants, V. dahliae pathogens need to secrete a large number of effectors to circumvent the cotton immune system. Analysis of V. dahliae secretome showed that about 800 effectors are secreted into cotton plants during the process of pathogen infection (Klosterman et al. 2011, Seidl et al. 2015, Zhang et al. 2022, Li et al. 2023a).

According to their characterized functions, the secreted effectors in V. dahliae can disarm at least three different kinds of components in the cotton immune system. Firstly, the effector VdPDA1 could deacetylate chitin oligomers and evade host surveillance by inactivating the chitin-triggered host immune response (Gao et al. 2019, Liu et al. 2023). Secondly, the secretory protein VdSCP41 from V. dahliae could directly bind to the C-terminal of GhCBP60g to inhibit its activated-transcription activity for the cotton immunity response (Qin et al. 2018). Besides suppressing the transcriptional activation, the effectors still quench the production of regulatory microRNA in host plants. For example, VdSSR1 could disturb miRNA export and the function of the AGO1–microRNA complex in cotton (Zhu et al. 2022). As a result, the miRNAs for silencing virulence gene expression were correspondingly reduced, and the pathogenicity of V. dahliae was then enhanced. In addition to the above effectors, V. dahliae pathogens also secrete the effectors to suppress the production of immune signal molecules in cotton. Accordingly, VdISC1 could hydrolyze the isochorismate of cotton plants to disrupt the salicylate metabolism pathway, and then repress salicylate-mediated innate immunity (Liu et al. 2014). In summary, V. dahliae effectors are secreted to overcome cotton immunity from the beginning of invasion to the final colonization in cotton.

To confine pathogen proliferation, the hypersensitive response (HR) is widely adopted by host plants. HR is a defense response accompanied by rapid cell death at the invasion site of host plants (Coll et al. 2011). HR can send systemic signals to distal parts of host plants for resisting subsequent pathogen attack (Dalio et al. 2018, He et al. 2019). HR is generally stimulated by the effectors, in which at least five kinds of plant proteases are involved in pathogen perception (Cohen et al. 2019). Accumulating evidence suggests that apoplastic cysteine proteases are the most important immune components required for inducing effective local and systemic defense responses (Ziemann et al. 2018). For example, plant cysteine protease can cooperate with R protein to inhibit pathogen infection. Accordingly, Rcr3 constitutively binds to the kinase protein, acting as a receptor partner to sense the presence/absence of pathogens’ effectors (Dixon et al. 2000, Paulus et al. 2020). Furthermore, they can interact with HR inhibitors such as E-64 in the host plant–pathogen interaction system (Rooney et al. 2005). Due to their multiple roles in resistance to pathogens, cysteine proteases are regarded as the hubs of plant immunity and thus serve as important repressing targets of secreted effectors (Hotson and Mudgett 2004).

Though the importance of cysteine proteases in plant immunity has been characterized, there is limited information on how the Verticillium effectors influence the activity of cysteine proteases in cotton. To better understand the pathogenesis mechanism of V. dahliae, we previously analyzed the transcriptomics of V. dahliae strain Vd991 during cotton infection and found a number of effector candidates (Duan et al. 2016). Here we identified the function of the VdPHB1 gene, which encodes a prohibitin protein required for full pathogenicity of V. dahliae. Vdphb1 mutant strains reduced pathogenicity in cotton, whereas Arabidopsis plants ectopically expressing VdPHB1 had stunted growth and increased susceptibility to V. dahliae. Further analysis revealed that VdPHB1 reduces the resistance response of cotton plants by interacting with and promoting the protein degradation of metacaspase 4 (GhMC4) in cotton. Collectively, we characterized the function of VdPHB1 as a repressor of the cotton defense system.

Results

VdPHB1 encodes a prohibitin protein secreted to the intercellular space of host plants

Previous comparative transcriptomic analysis of Verticillium pathogens before and after inoculating cotton plants showed that a gene (named VdPHB1) homologous to prohibitin 1 in yeast was strongly induced after pathogen inoculation (Duan et al. 2016). Further analysis using qRT-PCR confirmed that the VdPHB1 gene was rapidly upregulated since the inoculation and remained at a high level for at least 48 h (Fig. 1A). The full length of the VdPHB1 gene is 906 bp with two exons (1–321 bp and 396–906 bp) and one intron (322–395 bp) (Fig. 1B). Comparative analysis of prohibitin proteins from V. dahliae, Arabidopsis thaliana, Oryza sativa, Saccharomyces cerevisiae and Caenorhabditis elegans showed that all of these contain a SPFH_prohibitin domain, which is located from 26 to 220 amino acids in the VdPHB1 protein (Fig. 1C, Supplementary Fig. S1A). A neighbor-joining phylogenetic tree of 41 PHB proteins from different species showed that the VdPHB1 had the highest homology with the PHB protein of Verticillium alfalfa (Supplementary Fig. S1B, Table S1). To analyze the localization of VdPHB1 protein at the subcellular level, the VdPHB1–YFP fusion protein was expressed in Nicotiana benthamiana leaves. As shown in Fig. 1D, the yellow fluorescence of the control protein (35S: YFP) was observed in both the nucleus and the cell membrane of N. benthamiana leaf cells, whereas the fluorescent signal of VdPHB1–YFP was distributed in dots out of the cell membrane, indicating that VdPHB1 may function in the intercellular space of host plants.

Knock-out of theVdPHB1 gene in V. dahliae decreases its pathogenicity

To gain insight into the function of the VdPHB1 gene, we generated VdPHB1 knock-out mutants by A. tumefaciens-mediated transformation (ATMT). Two independent knock-out mutants (Vdphb1-14, Vdphb1-16) were confirmed by PCR and transcription-level analysis (Supplementary Fig. S2, S3A). The colony diameters and growth curves of the mutant Vdphb1 (Vdphb1-14, Vdphb1-16) on PDA medium were not significantly different from Vd991, indicating that the VdPHB1 gene was not involved in the growth of V. dahliae (Supplementary Fig. S3B, C). However, fewer hyphae were attached to the colony surface of the mutant strains (Vdphb1-14, Vdphb1-16), and the statistics revealed that the number of spores produced by the mutant strains decreased by 73.43% compared with Vd991 (Supplementary Fig. S3B, S3D). The number of spores in Vdphb1 and Vd991 were 1.7 × 10⁷ and 6.4 × 10⁷, respectively (Supplementary Fig. S3D), suggesting that the VdPHB1 gene might be involved in the regulation of spore production. To test whether the VdPHB1 gene regulates the activity of extracellular enzymes in the V. dahlia, we determined the growth rates of Vdphb1 and Vd991 on media with the addition of starch, sucrose, or pectin as the carbon source. The Vdphb1 mutant showed no significant difference in both colony diameter and growth curves from the Vd991, indicating that the VdPHB1 gene may not regulate the activities of extracellular enzymes in V. dahliae (Supplementary Fig. S3E, F).

To analyze whether the VdPHB1 gene regulates pathogenicity, we inoculated tobacco leaves and cotton roots with the same number of spores of Vd991 and Vdphb1, respectively. The results showed that the yellow lesion spots on tobacco leaves inoculated with Vd991 were larger than Vdphb1, indicating that the pathogenicity of Vdphb1 was reduced (Supplementary Fig. S4). Cotton plants infected with Vd991 showed wilting symptoms on the leaf margins from14 d post-inoculation (DPI). In contrast, cotton plants inoculated with Vdphb1 spores grew normally (Fig. 2A). After 21 d, cotton plants inoculated with Vd991 spores showed severe disease symptoms, with most of the leaves turning yellow and wilted, whereas the plants inoculated with Vdphb1 (mutant) spores showed only mild disease symptoms (Fig. 2A). Moreover, the disease index (DI) caused by Vdphb1 infection (14 DPI) was significantly lower than that of Vd991 (Fig. 2B). The vascular bundles on transverse and longitudinal sections of the stems of cotton plants inoculated with Vd991 were deep brown compared with those of cotton plants inoculated with Vdphb1 (Fig. 2C). Furthermore, inoculation of cotton showed that the pathogenicity of the complementary strain in the vdphb1 mutant background [vdphb1 (35S:VdPHB1)] restored the pathogenicity level to that of the wild-type strain (Vd991) (Supplementary Fig. S5). In summary, the results indicate that the VdPHB1 gene is required for the pathogenicity of V. dahliae.

We further analyzed the transcriptional changes of resistance-related genes in cotton plants inoculated with Vd991 and Vdphb1 by qRT-PCR. The expression levels of PCD2-like and PCD4-like were significantly higher in the Vd991-infected cotton plants than those in the Vdphb1 mutant-infected cotton plants before 9 DPI. After that, their expression levels decreased and were significantly lower in the Vd991-infected cotton plants than in those of Vdphb1 mutant-infected plants (Fig. 2D). In addition, GhPR1 expression was lower in the Vdphb1-infected plants than that in Vd991-infected cotton plants (Fig. 2D). GhERF3 gene expression was higher in the Vdphb1-infected cotton plants than in those infected with Vd991 (Fig. 2D). The expression patterns of these genes showed that they quickly increased and later decreased after pathogen infection, which is similar to that of the resistance-related gene GbSBT1 (Fig. 2D, Duan et al. 2016). Given that pathogen-induced programmed cell death is an early response of the plant immune response (Coll et al. 2011), the reduced expression levels of defense-related genes were associated with the decreased pathogenicity of Vdphb1 mutant in cotton.

Ectopic expression of the VdPHB1 gene in Arabidopsis retards plant growth and enhances susceptibility to V. dahliae

Prohibitins have multiple functions in the control of mitochondrial organization and signal exchange between different organelles (Van Aken et al. 2010, Toki et al. 2021). To further analyze the function of VdPHB1 during V. dahliae infection, we ectopically expressed VdPHB1 in Arabidopsis. Compared with Col-0 plants, VdPHB1 transgenic Arabidopsis plants showed a significantly retarded vegetative growth, delayed flowering, severe plant dwarfing and smaller rosette leaves (Fig. 3A). These pleiotropic phenotype changes suggest that the VdPHB1 gene may affect plant development by inhibiting basic biological processes such as cell expansion and growth.

Further resistance analysis of VdPHB1 transgenic Arabidopsis plants revealed that visible sensitive symptoms appeared about 2 DPI after inoculation, which was earlier than that of wild-type plants (about 4 d on average) (Fig. 3B). Accordingly, yellow spots on the leaves of VdPHB1 transgenic Arabidopsis were enlarged compared with Col-0 plants. Ten days after inoculation, about 15% of VdPHB1 transgenic Arabidopsis plants wilted, which was significantly higher than Col-0 plants, and the disease index increased about two-fold compared with that of Col-0 plants (Fig. 3B). In conclusion, VdPHB1 expression in Arabidopsis increased the susceptibility to V. dahliae and inhibited plant growth.

VdPHB1 interacts with the cotton cysteine protease GhMC4

To identify potential interactors of VdPHB1 in cotton, we incubated the fusion protein VdPHB1–GFP with total protein from Vd991-infected cotton roots. Mass spectrometry analysis of pulled-down proteins showed that multiple proteins, including reported SBT1 and GhMC4, interracted with VdPHB1 (Duan et al. 2016) (Supplementary Table S2). The GhMC4 gene encodes a type II metacaspase with up to 65% identity to the Arabidopsis AtMC4 protein, containing a typical peptidase C14 caspase domain (3–146 aa) (Supplementary Fig. S6). We then confirmed the interaction between VdPHB1 and GhMC4 by yeast two-hybridization (Fig. 4A). The luciferase signals were captured only when VdPHB1-nLUC and GhMC4-cLUC were co-expressed (Fig. 4B). This interaction was further confirmed by GST-pull down assay using both GST-VdPHB1 and His-GhMC4 fusion proteins (Fig. 4C), suggesting a direct association between VdPHB1 and GhMC4. Meanwhile, the bimolecular fluorescence complementation (BiFC) assay further confirmed their interaction. BiFC also showed that the yellow fluorescence of VdPHB1 and GhMC4 interacted in the intercellular space of tobacco leaves (Fig. 4D). Collectively, these results provided evidence that VdPHB1 interacts with GhMC4 in vitro and in vivo.

Inhibition of GhMC4 in cotton increases susceptibility to V. dahliae infection

Plant metacaspases are involved in the regulation of programmed cell death in plant development and disease resistance (Basak and Kundu 2022, Wang et al. 2012). To analyze the function of GhMC4 during cotton defense, we firstly examined GhMC4 expression profiles after V. dahliae pathogen inoculation. The transcription level of GhMC4 had been significantly increased about four times 12 h post-inoculation (Fig. 5B). Subsequently, we silenced the GhMC4 gene in cotton using virus-induced gene silencing (VIGS). qRT-PCR identified two VIGS lines (pTRV: GhMC4-3 and pTRV: GhMC4-12) for subsequent analysis, which had 52.15% and 40.95% reduction in gene expression compared to pTRV:00, respectively (Supplementary Fig. S7). Fourteen days after inoculation, the GhMC4-silenced plants exhibited more severe wilting, and more chlorosis on the yellow leaves than the control plants (Fig. 5A). Furthermore, the expression of defense-related genes (GhPR1, GhPR2, and GhERF3) was significantly lower in GhMC4-silenced lines than those in pTRV:00 plants at 14 DPI. Meanwhile, the expression levels of PCD-related genes (GhPCD2 and GhPCD4) were also decreased in GhMC4-silenced lines (Fig. 5C). These results imply that inhibition of GhMC4 in cotton down-regulates the expression of defense-related genes, rendering the GhMC4-silenced plants more sensitive to Vd991 infection.

To investigate how the function of GhMC4 was affected by VdPHB1, we coexpressed YFP and VdPHB1–YFP, respectively, with the GhMC4-MYC protein in tobacco leaves. Forty-eight hours after infiltration, the accumulation of GhMC4-MYC protein was obviously decreased when it was coexpressed with VdPHB1–YFP, but GhMC4-MYC was not reduced when coexpressed with the YFP control (Fig. 5D). This result suggests that VdPHB1 protein can inhibit the accumulation of GhMC4 protein in plants.

To analyze whether GhMC4 can induce active oxygen production in plants, we injected tobacco leaves with agrobacterium containing GhMC4-MYC and MYC expression vector (Control), respectively. DAB staining showed that tobacco leaves expressing GhMC4-MYC showed more apoptotic spots compared with the control, whereas tobacco leaves co-expressing GhMC4-MYC and VdPHB1–YFP showed fewer apoptotic spots compared with GhMC4-MYC alone (Fig. 5E). In addition, the tobacco leaves expressing GhMC4-MYC had significantly higher active oxygen content (relative light unit) than the control, whereas the active oxygen content was significantly reduced in tobacco leaves co-expressing GhMC4-MYC and VdPHB1–YFP than expressing the GhMC4-MYC alone (Fig. 5F). Considering that active oxygen is known to trigger programmed cell death during HR, we deduce that the GhMC4 protein is involved in programmed cell death against pathogen infection.

Discussion

VdPHB1 is required for the full pathogenicity of V. dahliae

Pathogen infection and defense responses of host plants are in constant battle during evolution. To suppress the immune system of host plants, pathogens deliver hundreds of effectors into plant cells or the host apoplastic space to destroy systemic resistance signaling (Zhang et al. 2020, 2022). These effectors can circumvent different components of the cotton immune system for successful colonization, but how the effectors repress the cotton immune system remains largely unknown (Yang et al. 2015, Li et al. 2023b). In this study, we identified VdPHB1’s function in suppressing the cotton immune system. The VdPHB1 gene is rapidly upregulated by V. dahliae infection (Fig. 1A), sharing a similar expression pattern with the haustoria formation gene VdNOX but different to VdCrz and VdNLP (Zhou et al. 2012, Supplementary Fig. S8). Since VdNOX is required for the haustoria formation and pathogen colonization in cotton (Zhao et al. 2016), the similar expression pattern indicates that VdPHB1 may be involved in early infection of cotton. In addition, the VdPHB1 protein specifically targeted to the intercellular space of host plants (Fig. 1D). Given that the intercellular space is the initial communication site between invasive pathogens and host plant cells (Gupta et al. 2015, Zhou et al. 2022), and that VdPHB1 interacts with the subtilase protein GbSBT1 in the intercellular space (Duan et al. 2016), we hypothesized that VdPHB1 participates in recognizing immune components. Knocked out VdPHB1 gene in Verticillium pathogens decreased pathogenicity during cotton infection (Fig. 2A–C). Moreover, transgenic VdPHB1 Arabidopsis plants were significantly susceptible to V. dahliae attack (Fig. 3B). Differences in the expression of defense-related genes GhPR1, GhERF3 and GhPCDs, between cotton plants infected with Vd991 and Vdphb1 mutants further supported that VdPHB1 is required for the full pathogenicity of V. dahliae (Qin et al. 2006, Fig. 2D).

To analyze how VdPHB1 contributed to the virulence of V. dahliae, we screened the pulled-down proteins, and determined that GhMC4 is the interactor of VdPHB1 (Fig. 4). Our previous study also showed that the subtilase protein GbSBT1 could interact with VdPHB1 (Duan et al. 2016), indicating that VdPHB1 enhances pathogenicity probably by regulating the function of protease. Transgenic Arabidopsis plants ectopically expressing the VdPHB1 gene displayed both significantly susceptible to V. dahliae attack and remarkable growth dwarf (Fig. 3). It is known that the growth of sensitive cotton plants is usually retarded after V. dahliae infection, and decreased plant height may result from overaccumulation of reactive oxygen species (ROS) and abnormal apoptosis (Mo et al. 2015, Sharma et al. 2021). The accumulation of antioxidative enzymes and programmed cell death are correlated during the HR of host plants. Given that ROS production during the proper functioning of mitochondria in human cells is also regulated by prohibitin (Van Aken et al. 2010), we infer that VdPHB1 may block ROS production in cotton to assist pathogen invasion.

GhMC4 enhances cotton wilt resistance by activating immune response

Two kinds of cysteine proteinases have been characterized in activating the immune response of host plants (Sabale et al. 2019). The first kind of cysteine proteases such as tomato P69B proteolytically cleaves the effector PC2 to produce immune signaling peptides (Paulus et al. 2020, Wang et al. 2021). The type II cysteine proteases, metacaspases, are positive mediators of programmed cell death. Higher expressions of plant metacaspase protein could block pathogen invasion, resulting from programmed cell death at the sites of pathogen infection (Jones et al. 2001, Ameisen 2002, Vercammen et al. 2007, Fagundes et al. 2015). For instance, Arabidopsis MCP1b and MC1 knockout mutants showed significant alterations in sensitivity to P. syringae infection (Castillo-Olamendi et al. 2008, Coll et al. 2014). AtMC2d positively regulates programmed cell death in Arabidopsis in response to P. syringae attack (Watanabe and Lam 2011). Furthermore, structure analysis of the AtMC4 protein showed that it can cleave GST-Propep1 during immune defense in a Ca²⁺-dependent way (Zhu et al. 2020). Due to the conserved domain of type II cysteine proteases shared by GhMC4 (Supplementary Fig. S6), we deduce that GhMC4 also functions in the cotton immune system.

Here, we observed that expression of GhMC4 was strongly induced by pathogen infection (Fig. 5B). The importance of GhMC4 in cotton plant resistance to V. dahliae was later demonstrated. Accordingly, GhMC4-silenced cotton plants had decreased disease-resistance with lower expressions of PCD-related genes (Fig. 5A, C). Analysis of active oxygen content further showed that GhMC4 could strongly elicit active oxygen production and HR during pathogen infection (Fig. 5E, F). Given that the ROS increase aids to limit pathogen spreading (Liu et al. 2005, Tang et al. 2018), these results imply that GhMC4 positively regulates cotton resistance to V. dahliae by activating PCD response. In summary, our results show that VdPHB1 is an effector that represses GhMC4 function during the cotton defense response. Further analysis of the roles of GhMC4 in plant immunity will aid in elucidating the communication mechanism between the V. dahliae effector and plant metacaspases.

Materials and Methods

Plant materials and pathogen culture

Gossypium hirsutum variety Coker-312 was used in this study. To get sterile cotton seedlings, we soaked cotton seeds in the solution of corrosive sublimate (1/1,000, v/v) for 5 min and then washed them with sterilized water for three times. After that, we transferred them in the MS medium to grow. One week later, the healthy seedlings were used for pathogenicity analysis. To analyze the defense phenotype of Arabidopsis plants, Arabidopsis (ecotype Columbia, Col-0) and transgenic VdPHB1 Arabidopsis plants were grown in the chamber under the long-day condition (16/8 h light/dark) at 22°C.

The single defoliating isolate of V. dahliae Vd991 was grown on Czapek’s medium agar medium (NaNO₃, 0.3% w/v; MgSO₄, 0.1% w/v; KH₂PO₄, 0.1% w/v; FeSO₄, 0.0002% w/v; KCl, 0.1% w/v; sucrose, 3% w/v; and agar 3% w/v; pH 6.0) for 7 d (Qin et al. 2006, Duan et al. 2016). Conidial spores were harvested and adjusted to 1 × 10⁶ spores/mL with sterile distilled water.

VdPHB1 gene cloning and bioinformatics analysis

Based on the genomic sequence of V. dahliae, the full-length sequence of the VdPHB1 gene containing the entire ORF was cloned using ORF-specific primers (Supplementary Table S3). BLAST tools were used for similarity analyses and to identify homologs. The conserved domains of VdPHB1 were searched online through the domain Blast (http://www.ncbi.nlm.nih.gov). A neighbor-joining phylogenetic tree of different PHB proteins from different organisms was constructed using the software of MEGA 7.0 (http://www.megasoftware.net).

Subcellular localization of VdPHB1 protein

To analyze the subcellular localization of the VdPHB1 protein, we amplified the full length of the VdPHB1 gene and fused it with the YFP gene in the pEG101 vector. The construct expressing 35S::VdPHB1-YFP was then generated. The CaMV35S::YFP expression construct (empty vector) was used as the control. The expressing plasmids were respectively transformed into the Agrobacterium strain GV3101 (Song et al. 2023). Agrobacterium transformants were cultured and diluted into OD₆₀₀ of 0.6–0.8 with the solution (10 mM MES, pH 5.6; 10 mM MgCl₂, and 150 mM acetosyringone). The diluted bacterium cells were then injected into the expanded leaves of 3-week-old tobacco (Nicotiana benthamiana). After 2–4 d, the fluorescence signals of the VdPHB1 fusion protein were analyzed using confocal microscopy Leica TCS SP5. Meanwhile, the transfected tobacco leaves were cut into pieces for preparing the protoplasts following the reported method (Asai et al. 2002).

Generation of VdPHB1 deletion mutants

To generate VdPHB1 knock-out constructs, we amplified the DNA fragments located 1,000 bp upstream and downstream, respectively, of the VdPHB1 gene-coding sequence. The resulting DNA fragments were then integrated with the hygromycin gene as the described method (Rehman et al. 2016). The fusion DNA fragment was inserted into the pGKO-hyg vector (Maruthachalam et al. 2011, Qin et al. 2018). The construct was later transformed into Agrobacterium tumefaciens strain EHA105 to generate the VdPHB1 mutant strain (Vdphb1) by the method of ATMT (Duan et al. 2016). The transformants were selected on the PDA medium (potato, 200 g/L; glucose, 20 g/L; agar, 15 g/L) supplemented with hygromycin at 50 µg/mL (Zhang et al. 2018). The positive clones were confirmed by PCR with the internal and flanking primers of the VdPHB1 gene to characterize the knock-out mutants (Supplementary Table S3).

Growth activity assay of VdPHB1 deletion mutants

Single spore of V. dahliae Vd991 and Vdphb1 mutants were used in phenotypic analysis and inoculation experiment. To compare the growth assay between Vd991 and Vdphb1 mutants, 2 µL of the conidial suspension (2 × 10⁶ conidial/mL) were respectively inoculated in PDA medium and then grew at 25°C (Zhang et al. 2018). The colony diameter was measured after a 2-d incubation, and the fungal growths were investigated the 14th day after incubation. To evaluate the conidia production, agar plugs were collected from the edge of the fungal colony 9 d after incubation using a 5 mm-diameter cork borer, and the quantity of conidia was determined using a hemocytometer after the agar plugs had been shaken in 1 mL of sterile water for 5 min (Zhang et al. 2018). The V. dahliae conidia shapes (minor and major axes) were analyzed by the Leica microscope DM500. All the experiments were repeated three times with 16 conidia for each genotype.

Pathogenicity assay

The root-dipping method was used in the pathogenicity assay following the described method (Duan et al. 2016). Briefly, 2-week-old cotton seedlings (G. hirsutum cv. ‘coker312’) were gently pulled, washed and dipped into 15 mL fungus culture solution with 5 × 10⁶ conidia/mL for 5 min, and then transplanted into new pots. All the inoculated seedlings and the control plants were grown at 28°C under a 14 h light/10 h dark photoperiod (Zhang et al. 2017). The disease resistance symptom was assessed 2 weeks after inoculation. The disease grade was classified as following standard: grade 0 (no symptoms), grade 1 (0–25% wilted leaves), grade 2 (25–50%), grade 3 (50–75%) or grade 4 (75–100%) (Duan et al. 2016). The DIs were calculated based on the following equation: DI = (∑ (n × number of seedlings at level n)/(4 × number of total seedlings × 100), where n denotes the severity of the disease level of the cotton seedlings (Tong et al. 2021). Six pots of cotton seedlings with three replicates were applied for each transformant.

Generation of transgenic VdPHB1 Arabidopsis plants

The coding sequence of the VdPHB1 gene without a stop codon was recombined into the pDONR201 vector. The VdPHB1 gene was later recombined into the pEarlyGate101 vector by the Gateway LR recombination reaction (Invitrogen, CA, USA) to generate the expression cassette (35S::VdPHB1::NOS) (Duan et al. 2016). The vector was transformed into A. tumefaciens GV3101. After confirmation, the expression cassette was introduced into Arabidopsis (ecotype Col-0) plants by the floral dipping method (Mara et al. 2010). Herbicide-resistant plants (20 mg/L, BSATA) were screened and confirmed by PCR and DNA sequencing. The positive plants were self-crossed to generate homogenous T3 lines.

RT-qPCR analysis

Total RNAs were extracted from V. dahliae or plant samples following the manual of RNAprep Pure Plant Plus kit (Tiangen Biotech, Shanghai, China). After erasing DNA, mRNAs were reversely transcripted into the 1st cDNA with a PrimeScript RT reagent kit (TaKaRa, Japan). The RT-qPCR assays were performed following the method described by the kit of UltraSYBR Mixture (Cwbio, Shanghai, China). The cotton ubiquitin gene, the Arabidopsis AtUBC21 (Ubiquitin-Conjugating Enzyme 21) gene, and V. dahliae EF-1a (Elongation Factor-1a) genes were used as, respectively, the internal controls (Li et al. 2023b). PCR amplifications were completed by a Light Cycler 480 Real-Time PCR system (Roche). The relative expression levels were calculated by the 2^−∆∆Ct method (Michaelidou et al. 2013). The primer sequences were listed in Supplementary Table S3.

Pull-down assay

The coding DNA sequences of VdPHB1 and GhMC4 genes were cloned into pGEX-6P-1 and pET28a vectors, respectively, and then introduced into E. coli strain BL21 (DE3) cells. GST-VdPHB1 and His-GhMC4 recombinant proteins were further induced by 0.1 mM isopropyl-b-d-thiogalactopyrandoside (IPTG, Solabio, China) for 16 h at 25°C. The induced cells were collected by centrifugation at 8,000 g for 10 min (Duan et al. 2016, Li et al. 2023b). After discarding the supernatant, the pellets were resolved in the lysis buffer and lysed by an ultrasonic crusher (XM-650 T, China). Recombinant proteins were purified according to the protocols of GST-tag (Beyotime, Shanghai, China) and His-tag (Beyotime, China) protein purification kits. Equal amounts of GST-VdPHB1 and His-GhMC4 proteins were mixed with high-affinity GST resin (Invitrogen, USA) and incubated at 4°C overnight with gentle rotation (Yang et al. 2018). The GST-bound proteins were then eluted with fresh 10 mM glutathione elution buffer. Next, the eluted proteins were loaded and separated by 12% SDS-PAGE. After transferring to the nitrocellulose membrane, the target proteins were detected with anti-GST or anti-His antibody (Abmart, China).

Western blotting analysis

The coding sequences of VdPHB1 and GhMC4 were cloned into the vectors of pCAMBIA1302-YFP and pCAMBIA1302-MYC, respectively (Li et al. 2023b). The resulting constructs containing pCAMBIA1302-GhMC4-MYC, pCAMBIA1302-YFP and pCAMBIA1302-VdPHB1-YFP were introduced into Agrobacterium strain GV3101. After culturing, Agrobacterium cells carrying each construct were mixed and resuspended in the MES buffer (10 mM MgCl₂, 10 mM MES, 10 mM acetosyringone [pH 5.7]) to final OD₆₀₀ =1.0. The diluted bacterium was later injected into N. benthamiana leaves (Duan et al. 2016). After 3 d, the total proteins were extracted according to above method and immune-blotted with anti-YFP (Abmart, China) or anti-MYC (Abmart, Shanghai, China) antibodies. The hybridization signals were detected by the Pierce ECL western blotting substrate (Thermo Scientific, Waltham, Massachusetts, USA).

Bimolecular fluorescence complementation assay

To analyze the interaction between VdPHB1 and GhMC4 protein, VdPHB1 and GhMC4 proteins were cloned into the vectors of pEarlyGate201 and pEarlyGate202, respectively. The vectors were independently introduced into Agrobacterium GV3101. Bacterium cells harboring the above vectors were resuspended to a final concentration of OD₆₀₀ = 0.6 with the infiltration buffer (10 mM MgCl₂, 10 mM MES, and 150 μM acetosyringone, pH 5.8). The Agrobacterium cultures were then mixed at a ratio of 1:1 and infiltrated into N. benthamiana leaves. The fluorescence of the reconstituted YFP protein was visualized using confocal laser scanning microscopy with a TCS SP5 confocal laser scanning microscope 40–45 h after infiltration (Song et al. 2021).

Luciferase complementation assay

Luciferase complementation assay was performed following the described method (Li et al. 2023b , Song et al. 2023). The fusion proteins of nLUC-GhMC4 and cLUC-VdPHB1 in the pCAMBIA1300 vector were constructed. The vectors were then transformed into A. tumefaciens GV3101. The fusion protein transiently expressed in tobacco leaves followed the method described in the above section of Bimolecular fluorescence complementation (BiFC). Forty eight hours after Agrobacterium infiltration, the reaction substrate luciferin was added and put in the dark grow chamber; the fluorescent signals were recorded for 3 min using a PlantView100 plant in vivo imaging system (Photon Technology, China).

Yeast two hybridization analysis

Yeast two hybridization analysis was performed following the described method (Song et al. 2021, 2023). The GhMC4 gene was cloned into pGADT7 to generate the construct expressing the prey protein GhMC4-AD; while the VdPHB1 gene was cloned into the pGBKT7 vector to generate the vector BD-VdPHB1. The pGBKT7 (BD)-VdPHB1 and pGADT7 (AD)-GhMC4 constructs were co-transformed into the AH109 strain via the PEG/LiAc transformation procedures (Gietz and Schiestl 2007). After incubation on the SD/-Trp-Leu medium for 3–4 d at 30°C, a single yeast cell was picked and then screened on the solid medium SD-Trp/-Leu/-His/-Ade for the protein interaction test.

Active oxygen analysis

The method of 3, 3ʹ-diaminobenzidine (DAB) staining was used to analysis ROS accumulation (Yin et al. 2022). The infected tobacco leaves and control were treated with DAB, incubated on a shaker for 8 h in the dark, and later distaining with 95% ethanol. The treated samples were subsequently observed under light microscopy. Quantification of reactive oxygen species (ROS) levels using luminal methods (Jantean et al. 2022).

Supplementary Data

Supplementary Data are available at PCP online.

Data Availability

Gene accession number: V. dahliae VdPHB1 (XP_009649890.1), GhMC4 (XP_040964891.1).

Funding

This work was financially supported by the NSFC Project (32370267).

Acknowledgments

We thank Dr. Huishan Guo for kindly providing the pGKO plasmid.

Author Contributions

Z.K.J. designed and conducted all the experiments. S.Q.W., H.S., H.S. and X.Y.Y. performed the experiments. Z.K.J. and S.Q.W. wrote and revised the whole manuscript.

Disclosures

The authors have no conflicts of interest to declare.

References