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Verticillium dahliae secreted protein Vd424Y is required for full virulence, targets the nucleus of plant cells, and induces cell death

Abstract Fungal pathogens secrete effector proteins that regulate host immunity and can suppress basal defence mechanisms against colonization in plants. Verticillium dahliae is a widespread and destructive soilborne fungus that can cause vascular wilt disease and reduces plant yields. However, little is currently known about how the effectors secreted by V. dahliae function. In this study, we analysed and identified 34 candidate effectors in the V. dahliae secretome and found that Vd424Y, a glycoside hydrolase family 11 protein, was highly upregulated during the early stages of V. dahliae infection in cotton plants. This protein was located in the nucleus and its deletion compromised the virulence of the fungus. The transient expression of Vd424Y in Nicotiana benthamiana induced BAK1‐ and SOBIR1‐dependent cell death and activated both salicylic acid and jasmonic acid signalling. This enhanced its resistance to the oomycetes Phytophthora capsici in a way that depended on its nuclear localization signal and signal peptides. Our results demonstrate that Vd424Y is an important effector protein targeting the host nucleus to regulate and activate effector‐triggered immunity in plants.

. Plants have acquired a second layer of immune response known as effector-triggered immunity (ETI), a process through which host cells detect the presence of a pathogen's effectors via R proteins. These two kinds of immunity are involved in the accumulation of reactive oxygen species (ROS), callose deposition, and the regulation of hormone signalling genes (Dodds & Rathjen, 2010;Tsuda & Katagiri, 2010). Immune signalling contains PTI and ETI and is initiated from either the extracellular space or the cytoplasm of the cell through the interaction of the pathogen molecule and plant receptor, though this signalling typically must be transduced into the plant nucleus to activate the expression of defence genes (Eulgen & Somssich, 2007). The barley MLA10 protein can recognize the effector AVR A10 and induce nuclear association between the receptor and WRKY transcription factors, which is needed to activate ETI (Shen et al., 2007). In Fusarium oxysporum, the effector AVR2 triggers ETI via recognition by the resistance protein I-2 and subsequent translocation into the plant nucleus (Ma et al., 2013). In Arabidopsis, the PTI signalling pathway also relies on the function of transcription factors (Eulgen & Somssich, 2007).
The fungus Verticillium dahliae is a hemibiotrophic pathogen that progresses through biotrophic and necrotrophic stages during infection. V. dahliae is a soilborne vascular pathogen that infects plants through the roots and colonizes the xylem vessels of different plants species, such as cotton, potato, pepper, Arabidopsis, tomato, and Nicotiana benthamiana (Lo Presti et al., 2015;Pantelides et al., 2010;Wang, Cai, et al., 2004). V. dahliae is similar to other pathogens in that it uses apoplastic and intracellular effector proteins to combat plant immunity and successfully colonize the host (Lo Presti et al., 2015). Examples include the following: Ave 1 is the first cloned V. dahliae effector and is recognized by a membrane-bound receptor-like protein in tomatoes (de Jonge et al., 2012); Vdlcs1 is an isochorismate synthase able to suppress salicylic acid (SA) signalling during host colonization (Liu et al., 2014); VdSCP41 is located in the nucleus and can suppress plant immunity by disrupting the transcriptional activity of host genes (Qin et al., 2018); VdCUT11 is an apoplastic effector that induces cell death and triggers defence responses in N. benthamiana, cotton, and tomato (Gui et al., 2018).
These studies demonstrated that V. dahliae effectors have evolved to target host regulatory proteins and disrupt the host defence signal network during the infection process.
The secretome of V. dahliae is composed of nearly 800 proteins (Klosterman et al., 2011). However, relatively few of these secreted proteins have been functionally characterized, and a better understanding of how V. dahliae facilitates plant immunity could provide insights into ways to improve host plant resistance. In this study, we assessed the variation of the Vd991 strain and compared the published Vd991 genome with our de novo assembly of the V. dahliae 991 genome (unpublished data), finding that they were highly conserved. We also analysed 34 candidate effectors from secreted proteins and identified a potential secreted protein, Vd424Y, that induces cell death in tobacco, Arabidopsis, and pepper leaves. Vd424Y is a member of the glycoside hydrolase family 11 (GH11) and targeted the nucleus of the host cells. Its presence was necessary for V. dahliae to be fully virulent in cotton. Moreover, we observed that whether or not Vd424Y triggered cell death depended on its nuclear localization signal (NLS) and signal peptide (SP), and that its ability to induce cell death was dependent on BAK1 and SOBIR1. Finally, we demonstrated that a Vd424Y-induced immune response in N. benthamiana leaves could depend on its translocation from the plant cell apoplastic space to the nucleus.

| Identification of a V. dahliae secreted protein that elicits plant cell death
Few proteins secreted by V. dahliae capable of inducing cell death have been reported. We analysed the genomes of V. dahliae strain 991 (Vd991) and 34 effectors with a signal peptide (SP), which were individually cloned into potato virus X (PVX) vector pGR107 (Table   S1). We then performed transient expression in N. benthamiana to screen potential candidate V. dahliae effectors capable of inducing cell death. Green fluoresecnt protein (GFP) and Bcl-2-associated X protein (BAX) were used as negative and positive controls, respectively. Agrobacterium carrying each of the effectors was injected into the leaves of 4-week-old N. benthamiana plants. Seven days after infiltration, we identified a candidate effector EVM0004916 (hereafter designated Vd424Y) that induced intense cell death (Figure 1a).

Western blot analysis demonstrated that both GFP and Vd424Y
were expressed (Figure 1c), with a significant increase in the electrolyte leakage around the region of Vd424Y expression compared to regions of GFP expression. This is consistent with the cell death phenotype (Figure 1b). Additionally, the Agrobacterium-mediated transient expression of Vd424Y in pepper and Arabidopsis thaliana induced cell death, which did not occur in the GFP control ( Figure   S1). These findings highlighted the ability of Vd424Y to induce cell death in various plant species.
Vd424Y encodes a 223 amino acid protein with a conserved glycoside hydrolase domain ( Figure S2), and has a signal peptide (SP) encoded by the first 60 nucleotides ( Figure S2) along with a predicted nuclear localization signal (NLS) (Dingwall & Laskey, 1991) encoded by 33 nucleotides ( Figure S3). This indicates that Vd424Y could have secretory functioning and target the host protein in the nucleus. As shown in Figure S3, only Avr1b SP and Vd424Y SP constructs grew well on the YPRAA medium. However, the empty vector pSUC2 and the YTK12 strain did not grow well. These results indicate that the SP of Vd424Y is essential for secretory functioning.

| The NLS and SP are required for Vd424Yinduced cell death
Subcellular localization demonstrated that Vd424Y is predominantly located in the plant nuclei (Figures 2c and S4a). This suggests it could be closely related to the presence of a putative NLS ( Figure S2). To investigate whether the NLS had a significant effect on the Vd424Yinduced cell death due to its location in the nucleus, we performed NLS mutation analysis and fused a nuclear export signal (NES) to the effector (Vd424Y-nls). Three vectors were individually expressed in N. benthamiana via Agrobacterium tumefaciens-mediated infiltration, and yellow fluorescent protein (YFP) fluorescence was detected in the infiltrated leaves using a confocal microscope at 48 hr postinfiltration (hpi). These vectors included Vd424Y-nls-YFP, in which the NLS residues were mutated to alanines (Figures 2a and S4), Vd424Ynls-NES-YFP, in which Vd424Y-nls was fused with a NES (Du, Berg, et al., 2015) at the C-terminus (Figure 2a), and Vd424Y-NES-YFP, in which Vd424Y was fused with an NES at the C-terminus ( Figure 2a). Figure 2c, the YFP signal intensity in the nuclei expressing Vd424Y-nls was weaker than the signalling intensity in the nuclei expressing Vd424Y-YFP. Vd424Y-YFP also displayed a significantly stronger YFP signal intensity in the nucleus than both Vd424Y-nls-NES and Vd424Y-NES. These results indicate that the predicted NLS in Vd424Y affects nuclear localization, and that the NES is capable of guiding Vd424Y out of the nucleus. Additionally, we performed a transient expression assay demonstrating that Vd424Y-nls, Vd424Ynls-NES, and Vd424Y-NES were unable to induce intense cell death ( Figure 2b). This suggests that NLS-mediated nuclear localization is required for Vd424Y to induce cell death.

As shown in
To determine whether or not Vd424Y-induced cell death depends on Vd424Y secretion, we constructed an SP-deleted (amino acids 1-20; Figure Figure S5). We found that Vd424N did not induce cell death in N. benthamiana, while the expression of Vd424N-SP NbPR1 did ( Figure 2b). This suggests that SP is required for Vd424Y-induced cell death and that Vd424Y must target the extracellular space for cell death to occur. These results demonstrate that NLS and SP are necessary for Vd424Y to induce intense cell death, and that a Vd424Y-induced immune response in N. benthamiana leaves depends on its translocation from the plant's apoplastic space to the nucleus.

| Vd424Y triggers plant immunity responses
To determine whether Vd424Y-activated cell death was associated with plant immune response, we examined a hypersensitive response (HR)-specific marker gene (NbHIN1) (Takahashi et al., 2004) in N. benthamiana leaves following Vd424Y infiltration via Agrobacterium. Analysis of expression levels demonstrated that 3 days after infiltration NbHIN1 was significantly activated by Vd424Y but not by Vd424N or Vd424Y-nls ( Figure 3). To confirm whether Vd424Y-trigged immunity involved hormone signalling pathways, we used quantitative reverse transcription PCR (RT-qPCR) analysis to detect the SA-dependent immunity marker genes NbPR1a and NbPR2 (Dean et al., 2005), and the jasmonic acid (JA)-dependent immunity gene NbPR4 (Asai & Yoshioka, 2009;Rodriguez et al., 2014).
These marker genes were significantly induced in N. benthamiana  Figure 3). Furthermore, we demonstrate that Vd424Y (but not its variants Vd424N and Vd424Y-nls) was able to induce the expression of PTI-related marker genes, including NbCYP71D20 (Heese et al., 2007), NbPti5, NbWRKY7, and NbWRKY8 (Ishihama et al., 2011;Nguyen et al., 2010) (Figure 4). This confirms that Vd424Y is a microbial PAMP. These results suggest that Vd424Y can be recognized by N. benthamiana and activate plant immunity in a manner dependent on its NLS and SP.  Figure S6).

F I G U R E 3
Vd424Y induces plant defence responses in Nicotiana benthamiana. Relative expression of hypersensitive-response-specific and defence-related marker genes in N. benthamiana infiltrated with Agrobacterium tumefaciens carrying Vd424Y, Vd424N, and Vd424Y-nls. At 3 days postinfiltration (dpi), total RNA was extracted and transcript levels were detected by quantitative reverse transcription PCR. NbActin was used as the internal reference gene. The data shown represents the mean across three independent experiments. Bars indicate ± SE. Significance levels p < 0.05 and p < 0.01 are represented by * and **, respectively F I G U R E 4 Vd424Y promotes transcription of PAMP-PTI marker genes in Nicotiana benthamiana. Relative transcript levels of NbCYP71D20, NbPti5, NbWRKY7, and NbWRKY8 were analysed in N. benthamiana infiltrated with Agrobacterium tumefaciens carrying Vd424Y, Vd424N, and Vd424Y-nls. At 3 days postinfiltration, total RNA was extracted and transcript levels were detected by quantitative reverse transcription PCR. NbActin was used as the internal reference gene. The data shown represent the mean across three independent experiments. Significance levels p < 0.05 and p < 0.01 are represented by * and **, respectively

| Vd424Y enhances N. benthamiana resistance to oomycete pathogens
To investigate whether Vd424Y modulates plant resistance to the oomycete pathogen Phytophthora capsici, we transiently expressed Vd424Y, Vd424N, and Vd424Y-nls in N. benthamiana leaves. P. capsici zoospores were inoculated onto the infiltrated area 24 hr after Agrobacterium infiltration (Figure 6a), and the size of the resulting lesions was recorded at 48 hr postinoculation (hpi). As shown in Figure 6b,c, the biomass and lesion size of Phytophthora in leaves expressing Vd424Y were both significantly lower than in leaves expressing Vd424N, Vd424Y-nls, and an empty vector control. These results strongly demonstrate that Vd424Y triggers plant defence responses and alters plant immunity, enhancing N. benthamiana resistance to oomycete pathogens. We also found that both SP and NLS are essential for enhancing Vd424Y's resistance in N. benthamiana. To determine whether Vd424Y is involved in fungal virulence, we generated V. dahliae mutants by replacing the Vd424Y gene with a hygromycin resistance gene ( Figure S8) according to methods previously described (Gao et al., 2010;Gui et al., 2017). There were similarities in the growth rate and colony morphology of Vd424Y knockout mutants (ΔVd424Y) and the wild type Vd991 (Figure 7a,b).

| Vd424Y contributes to V. dahliae virulence
However, compared to the wild type, ΔVd424Y mutants caused fewer disease symptoms and had a lower disease index 26 days after pathogen inoculation in cotton plants (Figure 7c,d). We found that complementing ΔVd424Y mutants resulted in disease symptoms and colony morphology similar to the wild type Vd991 (Figure 7a,d), highlighting the important role that Vd424Y plays in V. dahliae virulence during cotton infection.

| D ISCUSS I ON
V. dahliae is a soilborne fungal pathogen that destroys the xylem of host cells and causes injury to plants (Fradin & Thomma, 2006;Klosterman et al., 2009). Relatively few secreted effector proteins have been found to play a role in virulence. Therefore, it is necessary to identify virulence-related effectors and determine their potential roles and pathogenic mechanisms. In this study, we analysed transient expression in N. benthamiana leaves to identify the toxic effectors that induced intense cell death. One of the effectors secreted by V. dahliae, Vd424Y, can trigger strong cell death in N. benthamiana, pepper, and Arabidopsis (Figures 1 and   S1). This indicates that Vd424Y could act as a PAMP during host plant infection.
After assessing the yeast signal trap assay system, we found that the fusion of the signal peptide of Vd424Y into the invertase gene resulted in the secretion of invertase in yeast ( Figure S3).  (Kloppholz et al., 2011;Szurek et al., 2001). PSR1 was identified in oomycetes and targeted the RNAi component PINP1 to disturb the defence mechanism in plants (Qiao et al., 2013(Qiao et al., , 2015. A VdSCP7-induced immune response was dependent on its nuclear localization . Similar research found that Vd424Y-induced immunity depends on its nu-  (Nie et al., 2019). We observed that SP and NLS are essential for Vd424Y-trigged immune responses, because they activate the expression of marker genes involved in SA and JA signalling and PTI, while mutations in the SP and the NLS weaken expression of the above marker genes (Figures 3 and 4). These results demonstrated that immune responses in N. benthamiana leaves that are induced by the Vd424Y effector protein depend on its translocation from the plant's apoplatic space to the nucleus. Phylogenetic analysis demonstrated that Vd424Y is conserved in different Verticillium species and that its homologs are present in several fungi species (Figure S9), suggesting that Vd424Y may have a conserved function in triggering plant immunity.

This indicates that
We also performed V. dahliae infection assays in cotton plants and found that Vd424Y was highly expressed at the early stages of cotton infection by V. dahliae ( Figure S7). This indicates that Vd424Y is closely related to the virulence of V. dahliae when it has infected cotton. Vd424Y knockout mutants did not affect the growth rate and colony morphology of Vd991 but weakened its virulence. Similar findings were reported in Fusarium graminearum (Yang et al., 2021), where the secreted ribonuclease Fg12 was significantly induced at the early stages of F. graminearum infection in soybean. Deleting the secreted ribonuclease Fg12 reduced the virulence of F. graminearum. In conclusion, our results demonstrate that Vd424Y can efficiently activate of plant immunity.

| Strains and plant growth conditions
The wildtype V. dahliae strain 991 (Vd991) was grown in the dark on potato dextrose agar (PDA) at 25 ℃. The P. capsici strain 35 (Table S2) was also grown in the dark in 10% V8 juice agar at 25 ℃. Escherichia coli DH5α, which was cultured in a Luria-Bertani (LB) medium at 37 ℃, was used for plasmid construction, whereas A. tumefaciens

| Yeast signal sequence trap
A yeast secretion system was performed to validate the function of the predicted SP (Yin et al., 2018). Specifically, Vd424Y's predicted SP was cloned into pSUC2T7M13ORI (pSUC2) using specific primers (Table S2), and the resulting plasmid was then transformed into the YTK12, an invertase mutant yeast strain (Oh et al., 2009). The positive colonies were screened on a CMD−W medium (0.67% yeast N base without amino acids, 0.075% W dropout supplement, 2% sucrose, 0.1% glucose, 2% agar) and incubated on YPRAA medium (1% yeast extract, 2% peptone, 2% raffinose, 2 mg/ml antimycin A) to assay invertase secretion. The empty pSUC2 and pSUC2-Avr1b SP vectors were used as negative and positive controls, respectively.

| Infection assays
To infect cotton plants with V. dahliae, 4-week-old Jimian 11 plants were inoculated with 10 ml conidial suspensions following previously published methods (Gong et al., 2017). The disease index (DI) was calculated according to previously described protocols (Gong et al., 2017). The infected plants were classified into five grades (grade 0, 1, 2, 3, and 4) according to the symptoms on the cotyledons and new leaves (Wang, Chen, et al., 2004;Zhang et al., 2012).
This procedure was repeated twice for validation and provided similar results.  Table S2.

| Subcellular localization assays
Recombinant plasmids were transformed into A. tumefaciens GV3101. The resulting strains were then expressed in 4-week-old N. benthamiana leaves using a previously described protocol (Qiao et al., 2015). Forty-eight hours after Agrobacterium-infiltration, yellow fluorescence from YFP was detected using a LSM780 confocal microscope.

| Protein extraction and western blotting
For transient expression analysis, the protein was extracted from the leaves of 4-week-old N. benthamiana plants after Agrobacteriuminfiltration. The infiltrated leaves were subsequently collected after 48 hr, ground in liquid nitrogen, and mixed with an equal volume of cold protein isolation buffer (1 mM EDTA pH 8.0, 20 mM Tris-HCl pH 7.5, 5 mM dithiothreitol, 150 mM NaCl, 0.1% sodium dodecyl sulphate [SDS], 10% glycerol, and 1 × protease inhibitor cocktail [Sigma-Aldrich]). The mixture was centrifuged at 4 ℃ for 10 min at 13,000 × g, transferred to a new tube, and boiled in protein sample buffer for 5 min. Proteins were analysed by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare).

ACK N OWLED G EM ENTS
The authors thank Professor Suomeng Dong (Nanjing Agriculture University) for providing the pGR107 vector and Professor Yongli Qiao F.G.L., and Y.J.L. wrote the paper. All authors commented on the article before submission.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are available from the corresponding author upon reasonable request.