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A complete MAP kinase cascade controls hyphopodium formation and virulence of Verticillium dahliae

A complete MAP kinase cascade controls hyphopodium formation and virulence of Verticillium dahliae aBIOTECH https://doi.org/10.1007/s42994-023-00102-y aBIOTECH RESEARCH ARTICLE A complete MAP kinase cascade controls hyphopodium formation and virulence of Verticillium dahliae 1,2 3 1,2 1,4 1,2 Ziqin Ye , Jun Qin , Yu Wang , Jinghan Zhang , Xiaoyun Wu , 5 1,2 1,2& Xiangguo Li , Lifan Sun , Jie Zhang State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing 100049, China State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling 712100, China School of Life Sciences, Hebei University, Baoding 710023, China College of Agronomy, Shanxi Agricultural University, Taigu 030801, China Received: 4 November 2022 / Accepted: 2 March 2023 Abstract Phytopathogens develop specialized infection-related structures to penetrate plant cells during infec- tion. Different from phytopathogens that form appressoria or haustoria, the soil-borne root-infecting fungal pathogen Verticillium dahliae forms hyphopodia during infection, which further differentiate into penetration pegs to promote infection. The molecular mechanisms underlying the regulation of hyphopodium formation in V. dahliae remain poorly characterized. Mitogen-activated protein kinases (MAPKs) are highly conserved cytoplasmic kinases that regulate diverse biological processes in eukaryotes. Here we found that deletion of VdKss1, out of the five MAPKs encoded by V. dahliae, significantly impaired V. dahliae hyphopodium formation, in vitro penetration, and pathogenicity in cotton plants. Constitutive activation of MAPK kinase (MAPKK) VdSte7 and MAPK kinase kinase (MAPKKK) VdSte11 specifically activate VdKss1. Deletion of VdSte7 or VdSte11 resulted in a phenotype similar to that of the mutant with VdKss1 deletion. Thus, this study demonstrates that VdSte11-VdSte7- VdKss1 is a core MAPK cascade that regulates hyphopodium formation and pathogenicity in V. dahliae. Keywords Verticillium, MAPK, Hyphopodium, Pathogenicity INTRODUCTION MAPK kinases (MAPKKs, MAPK/ERK- kinases/MEKs), and MAPKs that are sequentially phosphorylated In eukaryotes, mitogen-activated protein kinase (MAPK) (Widmann et al. 1999). In general, MAPKKKs phos- cascades are highly conserved cytoplasmic kinases that phorylate Ser and/or Thr residues located within the integrate extracellular signaling and transduce to activation loop of MAPKKs, which in turn trigger dual downstream substrates to regulate a wide range of phosphorylation of a highly conserved TXY motif located biological processes (Bigeard and Hirt 2018; Dixon et al. within the activation loop of MAPKs to activate MAPKs 1999; Gustin et al. 1998). The MAPK cascades consist of (Bigeard and Hirt 2018; Dixon et al. 1999; Gustin et al. MAPK kinase kinases (MAPKKKs, MAPK/extracellular 1998; Segmuller et al. 2007). signal-regulated kinase (ERK)-kinase kinases/MEKKs), In fungi, MAPKs regulate growth and development, maintenance of cellular integrity, and responses to stress (Gustin et al. 1998; Jiang et al. 2018; Wang and & Correspondence: zhangjie@im.ac.cn (J. Zhang) The Author(s) 2023 aBIOTECH Dohlman 2002; Zhao et al. 2005). In pathogenic fungi 7 days in the dark. To harvest spores, hyphae block was that infect plants, MAPK cascades regulate the patho- cultured in potato dextrose broth (PDB) liquid medium genesis and development of infection-related structures with shaking at 150 rpm at 25 C for 4 days. Cotton (Jiang et al. 2018;Lietal. 2019; Zhao et al. 2005). For plants (‘Xinluzao No. 16’) were used in this study for instance, in the ascomycete Magnaporthe oryzae, which virulence assessment (Zhou et al. 2017). All primers infects rice, barley, and other crops causing disease, used in this study are listed in Table. S1 (see in Sup- pathogenicity MAP kinase 1 (Pmk1) is crucial for plementary Information). appressorium formation. MEKK MST11-MEK MST7- MAPK PMK1 constitutes a MAPK cascade regulating Generation of the HopAI1 expression construct infection-related morphogenesis (Jiang et al. 2018; Zhao and transformant et al. 2005). In Colletotrichum gloeosporioides, Cgl-Slt2 MAPK plays an important role in the early stage of The HopAI1 gene was amplified from Pseudomonas appressorium formation (Yong et al. 2013). syringae DC3000 strain and cloned into the pSULPH-Tef- Verticillium dahliae infects a broad range of plants GFP vector to generate the pSULPH-Tef-HopAI1-GFP and causes severe wilt disease and agricultural losses. plasmid. The HopAI1 expression construct then was The development of infection-related structure is crucial transformed into Agrobacterium tumefaciens EHA105 for successful infection of host plants. Unlike M. oryzae strain, used for Agrobacterium tumefaciens mediated and C. gloeosporioides, which form appressoria during transformation (ATMT), as described previously, to infection, V. dahliae adheres tightly around the plant generate the HopAI1-expressing transformant (Wang root surface and forms hyphopodia, which further et al. 2016a). develop into penetration pegs during infection (Zhao et al. 2014; Zhou et al. 2017). This process involves Generation of gene deletion mutants calcium accumulation, NADPH oxidase-dependent reac- and complementary strains tive oxygen species burst, and cAMP-mediated signaling (Sun et al. 2019; Zhao et al. 2014; Zhou et al. 2017); To generate the target gene deletion plasmids, upstream however, additional signaling components as well as the and downstream genomic sequences of corresponding molecular mechanisms regulating these events remain genes were amplified from V. dahliae V592 and cloned elusive. The MAPKKK VdSte11, but not VdSsk2, plays a into the pGKO-HPT vector respectively. The resulting major role in regulating V. dahliae penetration (Yu et al. plasmids were used for ATMT to generate the deletion 2019). However, the core MAPK and complete MAPK mutants (Wang et al. 2016a). To obtain the comple- cascade regulating hyphopodium formation in V. dahliae mentary strains of target genes, the genomic coding remain uncharacterized. regions of target genes were PCR amplified and cloned In this study, we investigate the functions of MAPKs, into the pSULPH-Tef-GFP vector, respectively. The MAPKKs, and MAPKKKs in hyphopodium development resulting constructs were transformed into Agrobac- in V. dahliae. Mutations in VdKss1, VdSte7,or VdSte11, terium tumefaciens EHA105 strain respectively, and respectively, significantly disrupted hyphopodium for- used for ATMT. mation, in vitro penetration, and pathogenicity in cotton plants. The constitutive active forms of VdSte11 and Penetration assay and hyphopodium VdSte7 specifically induced the phosphorylation of visualization VdKss1. Thus, VdSte11-VdSte7-VdKss1 constitutes a complete MAPK cascade that regulates hyphopodium Sterilized cellophane membrane (DINGGUO, Beijing, formation and pathogenicity of V. dahliae in host plants. China), used to simulate plant root surface to induce formation of hyphopodium, was covered on MM med- ium (Zhao et al. 2016). Equal amounts of conidia col- MATERIALS AND METHODS lected from V. dahliae strains as indicated were incubated on the cellophane membrane and grown at Fungal strains, plant materials and culture 25 C for 5 days. The cellophane membrane was then conditions removed and observed under the Leica SP8 confocal laser scanning microscope system for determining Colonies of V. dahliae V592 (Gao et al. 2010) strain, hyphopodium formation. Further culture for an addi- HopAI1-expressing strain and mutation strains of target tional 3 days to observe whether there were hyphae genes generated in this study were routinely cultured grew on the underlying medium. on potato dextrose agar (PDA) medium at 25 C for The Author(s) 2023 aBIOTECH Pathogenicity assay activation and appressorium formation (Zhang et al. 2017). To examine whether MAPK activation con- The conidia of V. dahliae strains as indicated were col- tributes to hyphopodium formation in V. dahliae, HopAI1 lected and resuspended in sterilized water at the con- was expressed in V. dahliae. The expression of HopAI1 in centration of 10 /mL. Cotton plants were infected by the HopAI1-expressing strain was confirmed by anti-GFP the root-dipping inoculation method (Gao et al. 2010). immunoblot (Fig. S1). Similar to that in M. oryzae, Cotton plants then were photographed and subjected to expression of HopAI1 in V. dahliae can inhibit MAPK disease index analyses 3–4 weeks post inoculation. The activity (Fig. S2). Compared with the wild-type (WT) V. disease index was classified as follows: 0 (no symp- dahliae strain, hyphopodium formation was significantly toms), 1 (0% – 25% wilted leaves), 2 (25% – 50% reduced in the HopAI1-expressing V. dahliae strain wilted leaves), 3 (50% – 75% wilted leaves) and 4 (75% (Fig. 1A, B), suggesting that MAPK activation is crucial – 100% wilted leaves). The disease index was calculated for V. dahliae hyphopodium development. WT and as 100 9 [sum (number of plants 9 disease grade)]/ HopAI1-expressing V. dahliae strains were cultured on [(total number of plants) 9 (maximal disease grade)] minimal medium (MM) covered with cellophane and (Xu et al. 2014). examined for in vitro penetration (Zhao et al. 2016). Consistent with the deficiency of hyphopodium forma- Detection of MAPK phosphorylation tion, the HopAI1-expressing strain displayed defects in in vitro penetration compared to the WT strain V. dahliae protoplasts were isolated from 36 h vegeta- (Fig. 1C). WT and HopAI1-expressing V. dahliae strains tive hyphae grown in CM medium and transfected with were inoculated into cotton plants. Four weeks post plasmid as described (Rehman et al. 2016). Fourteen inoculation, cotton plants infected with HopAI1-ex- hours after transfection, proteins of transfected proto- pressing strain exhibited much weaker disease symp- plasts were extracted with extraction buffer containing toms than those infected with WT V592 (Fig. 1D). 50 mM HEPES (4-(2-Hydroxyethyl)-1-piperazi- Disease index analyses indicated reduced pathogenicity neethanesulfonic acid sodium salt) (pH 7.5), 150 mM of the HopAI1-expressing strain relative to the WT strain KCl, 1 mM EDTA (ethylenediaminetetraacetic acid), (Fig. 1E). These results strongly suggested that sup- 1 mM DTT (dithiothreitol), 0.2% Triton X-100, and pression of MAPK compromises hyphopodium forma- 1 9 proteinase inhibitor cocktail and subjected to IP tion and penetration, indicating an important role of and immunoblot assay with anti-pERK, anti-HA or anti- MAPK activation in regulating hyphopodium formation FLAG. of V. dahliae. The presence of VdKss1-HA, VdMAPKKs-FLAG and VdMAPKKKs-FLAG was detected by anti-HA or anti- Deletion of a specific MAPK compromises V. FLAG immunoblot. For anti-HA IP, total proteins were dahliae hyphopodium formation and penetration incubated with 2 lg of anti-HA antibody together with protein A agarose at 4 C for 4 h. The agarose beads Based on its homology with yeast homologs, V. dahliae were collected and boiled for 5 min with 1 9 protein encodes the following five putative MAPKs (Hamel et al. loading buffer. Total proteins were separated in a 10% 2012): the Kss1 type VdKss1 (VDAG_09461), Hog1 SDS-PAGE gel and transferred to the PVDF membrane. types VdHog1-1 (VDAG_08982) and VdHog1-2 The phosphorylation of VdKss1 TEY motif was detected (VDAG_02354), Ime type VdIme2 (VDAG_06935), and by anti-pERK immunoblot. Slt2 type VdSlt2 (VDAG_02584). To further confirm the requirement of MAPK activation for hyphopodium for- mation, V. dahliae mutants with gene deletion of indi- RESULTS vidual MAPKs in the V592 WT strain were generated by homologous recombination (Fig. S3) using the ATMT- Expression of a MAPK inhibitor compromises DS-vector (Wang et al. 2016a). hyphopodium formation in V. dahliae The VdDkss1,VdDhog1-1,VdDhog1-2,VdDime2, VdDslt2, and WT V. dahliae strains were grown on PDA MAPK cascades are conserved signaling kinases that medium, transferred to MM covered with cellophane, regulate diverse biological processes. HopAI1, a Pseu- and tested for their ability to penetrate artificial mem- domonas syringae effector protein, has a unique phos- brane. At 4 days after growth, hyphal growth of all phothreonine lyase activity towards plant, animal and mutants and the WT strain, except for the VdDkss1 fungal MAPKs (Li et al. 2007a; Zhang et al. 2007, 2017). mutant, was observed on the medium when the cello- Expression of HopAI1 in M. oryzae inhibits MAPK phane membrane was removed (Fig. 2A), suggesting The Author(s) 2023 aBIOTECH A B V592/ V592 HopAI1 -GFP Above Below V592/ V592 HopAI1-GFP V592/ Mock V592 HopAI1-GFP Fig. 1 Expression of a MAPK inhibitor in Verticillium dahliae compromises hyphopodium formation. A Hyphopodium formation is suppressed by HopAI1. V592 and HopAI1-expressing strains were grown on MM covered with a cellophane for 5 days. Hyphopodium formation (red arrow) on the cellophane was observed by confocal laser scanning microscopy (CLSM). B Hyphopodium quantification of V592 and HopAI1-expressing strains. The number of hyphopodium in three fields was counted for each strain. Error bars indicate the standard deviation of three fields. Student’s t-test was carried out to determine the significance of difference. **Indicates significant difference at P-value of \ 0.01. C The HopAI1-expressing strain displays a defect in penetration. The spores of V592 and HopAI1- expressing strains were grown on minimal medium (MM) covered with a cellophane for 5 days and photographed (above). The cellophane was removed and the culture was continued for 3 days and photographed (below). D Suppression of MAPK compromises V. dahliae virulence. Disease symptoms of upland cotton plants infected with the V592 and HopAI1-expressing strains were photographed and subjected to disease index analyses 3 weeks post inoculation. E Disease index analyses of upland cotton infected with the indicated strains. The disease indexes were evaluated with three replicates generated from 18 plants for each inoculum. Error bars indicate the standard deviation of three biological replicates. Student’s t-test was carried out to determine the significance of difference. **Indicates significant difference at P-value of \ 0.01 The Author(s) 2023 aBIOTECH Above Below Vd∆kss1/ Vd∆hog Vd∆hog V592 Vd∆kss1 Vd∆ime2 Vd∆slt2 Kss1-GFP 1-1 1-2 B C Vd∆kss1/ V592 Vd∆kss1 Kss1-GFP Vd∆hog1-1 Vd∆hog1-2 Vd∆ime2 Vd∆slt2 Fig. 2 Deletion of a specific MAPK significantly compromises Verticillium dahliae hyphopodium formation and in vitro penetration. A Penetration phenotypes of VdMAPKs deletion mutants and VdDkss1/VdKss1-GFP complementary strain. The spores of indicated strains were grown on MM covered with a cellophane for 5 days and photographed (above). The cellophane was removed and the culture was continued for 3 days and photographed (below). B Hyphopodium formation of VdMAPKs deletion mutants and VdDkss1/VdKss1-GFP complementary strain. Indicated strains were grown on MM covered with a cellophane for 5 days. Hyphopodium formation (red arrow) on the cellophane was observed by confocal laser scanning microscopy (CLSM). C Hyphopodium quantification of VdMAPKs deletion mutants and VdDkss1/VdKss1-GFP complementary strain. The number of hyphopodium in three fields was counted for each strain. Error bars indicate the standard deviation of three fields. Student’s t-test was carried out to determine the significance of difference. *Indicates significant difference at P-value of \ 0.05. **Indicates significant difference at P-value of \ 0.01 that mutation of VdKss1 compromised the capacity for VdSte7 phosphorylates VdKss1 and regulates membrane penetration. Next, we examined hyphopo- hyphopodium formation dium formation in these mutants. Mutation of VdKss1 impaired hyphopodium formation (Fig. 2B, C). The Within the MAPK cascade, MAPKKK, MAPKK, and MAPK complementation of VdKss1 in the VdDkss1 mutant are sequentially phosphorylated. To determine the restored membrane-penetration (Fig. 2A) and hypho- upstream MAPKKs responsible for phosphorylating podium formation (Fig. 2B, C). Expression of VdKss1- VdKss1, V. dahliae MAPKK genes were cloned and ana- lyzed for their ability to activate VdKss1. The genome of GFP in the complementary strain was confirmed by anti- GFP immunoblot (Fig. S4A). Mutation of VdIme2 and V. dahliae contains three putative MAPKK genes, VdSte7 (VDAG_08626), VdMKK1 (VDAG_09823), and VdPbs2 VdSlt2 exhibited reduced hyphopodium formation (Fig. 2B, C), suggesting a contribution of VdIme2 and (VDAG_02783) (Hamel et al. 2012). Double mutation of VdSlt2 to that process. These results indicated that the conserved serine and/or threonine residues located VdKss1 plays a major role, whereas VdIme2 and VdSlt2 in the kinase-activation loop converts MAPKKs into play minor roles, in regulating hyphopodium formation. their constitutive active (CA) forms (Asai et al. 2002; Liu The VdDkss1 mutant and WT strains were tested for and Zhang 2004; Ren et al. 2002). CA mutants of each of pathogenicity in cotton plants. The VdDkss1 mutant the above MAPKKs were generated and co-expressed with VdKss1 in V. dahliae protoplasts. The phosphory- displayed much less severe disease symptoms than the WT strain (Fig. 3A, B). The virulence of the VdDkss1 lation of VdKss1 TEY motif was examined using an anti- CA CA pERK immuno-blot. VdSte7 , but not VdMKK1 or mutant was restored upon complementation with CA VdKss1 (Fig. 3A, B). These results indicate that VdKss1 VdPbs2 , specifically induces the phosphorylation of VdKss1 (Fig. 4A), indicating that VdSte7 can phospho- plays an important role in regulating the development of hyphopodium and is required for the full rylate VdKss1. pathogenicity of V. dahliae. We next generated mutants with gene deletion of individual MAPKK genes (Fig. S5) and examined the penetration and development of hyphopodium in these The Author(s) 2023 aBIOTECH (Fig. 4B) and hyphopodium formation (Fig. 4C, D). Expression of VdSte7-GFP in the complementary strain was confirmed by anti-GFP immunoblot (Fig. S4B). The results indicating that VdSte7 acts as the upstream MAPKK to phosphorylate VdKss1 and contributes to the development of hyphopodium in V. dahliae. VdSte11 activates VdKss1 and regulates hyphopodium formation Mock V592 VdSte11 (VDAG_05822), VdBck1 (VDAG_00874), and VdSsk2 (VDAG_08787) are three putative MAPKKKs encoded by V. dahliae (Hamel et al. 2012). MAPKKK usually contains an N-terminal auto-inhibitory domain that negatively regulates kinase activity. Removal of the N-terminal inhibitory domain results in activation of MAPKKKs in the absence of upstream kinases (Asai et al. 2002; Bergmann et al. 2004). Deletion of VdSte11, a V. dahliae MAPKKK, has been reported to impair hyphopodium formation in V. dahliae (Yu et al. 2019), Vd∆kss1/ suggesting an important role of VdSte11 in regulating V. Vd∆kss1 Kss1-GFP dahliae hyphopodium development. Whether VdSte11 B B activates VdKss1 and whether additional MAPKKKs are involved in hyphopodium development remain unde- termined. The truncated C-terminal forms of the indi- CA CA CA vidual MAPKKKs, VdSte11 , VdBck1 , and VdSsk2 , were then constructed and co-expressed with VdKss1 in CA V. dahliae protoplasts. Co-expression of VdSte11 , but CA CA not VdBck1 or VdSsk2 , induced phosphorylation of VdKss1 (Fig. 5A), indicating that VdSte11 acts as the upstream MAPKKK that induces VdKss1 phosphorylation. To further examine the role of MAPKKKs in the regu- lation of hyphopodium development, mutants with deletion of individual MAPKKKs were generated (Fig. S5). While the VdDbck1 and VdDssk2 mutants exhibited nor- mal penetration, the VdDste11 mutant exhibited deficient Fig. 3 VdKss1 contributes to Verticillium dahliae virulence in cellophane penetration (Fig. 5B). Expression of VdSte11- cotton plants. A Disease symptoms of upland cotton. Plants GFP in the complementary strain was confirmed by anti- infected with indicated strains were photographed and subjected GFP immunoblot (Fig. S4C). All three mutants generated to disease index analyses 3–4 weeks post inoculation. B Disease fewer hyphopodia than the WT strains, among which the index analyses of upland cotton infected with the indicated VdDste11 mutant was almost impaired in hyphopodium strains. The disease indexes were evaluated with three replicates generated from 24 plants for each inoculum. Error bars indicate formation (Fig. 5C, D). The results indicated that VdSte11, the standard deviation of three biological replicates. Student’s t- VdBck1, and VdSsk2 are all involved in regulating test was carried out to determine the significance of difference. hyphopodium formation, and that VdSte11 plays a **Indicates significant difference at P-value of \ 0.01 greater role than VdBck1 and VdSsk2. mutants. Consistent with the specific phosphorylation of CA VdKss1 induced by VdSte7 , the VdDste7 mutant, but VdSte7 and VdSte11 are required for full neither the VdDmkk1 nor the VdDpbs2 mutants, exhib- pathogenicity of V. dahliae ited compromised membrane penetration (Fig. 4B) and significantly reduced hyphopodium formation (Fig. 4C, To determine the role of VdSte7 and VdSte11 in D). The complementation of VdSte7 in the VdDste7 pathogenicity, VdDste7,VdDste11 mutants and WT mutant restored membrane penetration in the medium strains were subjected to pathogenicity assays in cotton The Author(s) 2023 aBIOTECH Above Below Vd∆ste7/ Vd∆ste7 V592 Vd∆mkk1 Vd∆pbs2 Ste7-GFP Vd∆ste7/ V592 Vd∆ste7 Vd∆mkk1 Vd∆pbs2 Ste7-GFP CA Fig. 4 VdSte7 phosphorylates VdKss1 and regulates hyphopodium formation. A The constitutive active (CA) mutant VdSte7 specifically CA phosphorylates VdKss1. Verticillium dahliae protoplasts were transfected with VdKss1-HA alone or together with VdSte7 -FLAG, CA CA VdMKK1 -FLAG, or VdPbs2 -FLAG. Proteins were extracted 16 h post transfection and subjected to Co-immunoprecipitation, then followed by anti-pERK or anti-HA immunoblot. The experiments were repeated three times with similar results. B Penetration phenotypes of VdMAPKKs deletion mutants and VdDste7/VdSte7-GFP complementary strain. The spores of indicated strains were grown on MM covered with a cellophane for 5 days and photographed (above). The cellophane was removed and the culture was continued for 3 days and photographed (below). C Hyphopodium formation of VdMAPKKs deletion mutants and VdDste7/VdSte7-GFP complementary strain. Indicated strains were grown on MM covered with a cellophane for 5 days. Hyphopodium formation (red arrow) on the cellophane was observed by confocal laser scanning microscopy (CLSM). D Hyphopodium quantification of VdMAPKKs deletion mutants and VdDste7/VdSte7-GFP complementarystrain. The number of hyphopodium in three fields was counted for each strain. Error bars indicate the standard deviation of three fields. Student’s t-test was carried out to determine the significance of difference. **Indicates significant difference at P-value of \ 0.01 plants. Reduced pathogenicity was observed in both the DISCUSSION VdDste7 (Fig. 6A, B) and VdDste11 mutants (Fig. 6C, D). Complementation of VdSte7 in VdDste7 mutant (Fig. 6A, V. dahliae infects more than 200 kinds of plants, causing B) and VdSte11 in VdDste11 (Fig. 6C, D) restored their Verticillium wilt, leading to severe yield losses world- pathogenicity to the level of the WT strain. These results wide. Formation of hyphopodium is crucial for the suggest that both VdSte7 and VdSte11 are required for establishment of V. dahliae infection, and the underlying full pathogenicity of V. dahliae. Thus, the results indicate regulatory mechanisms remain poorly characterized. that VdSte11-VdSte7-VdKss1 constitutes a complete We have previously reported the cAMP-mediated regu- MAPK cascade that regulates the hyphopodium forma- lation of hyphopodium formation (Sun et al. 2019). tion and pathogenicity of V. dahliae. VdSho1, a tetraspan transmembrane protein, regulates V. dahliae cellophane penetration and virulence in The Author(s) 2023 aBIOTECH A D Above Below Vd∆ste11/ V592 Vd∆ste11 Vd∆bck1 Vd∆ssk2 Ste11-GFP Vd∆ste11/ V592 Vd∆ste11 Vd∆bck1 Vd∆ssk2 Ste11-GFP CA Fig. 5 VdSte11 activates VdKss1 and regulates hyphopodium formation. A The CA mutant VdSte11 activates VdKss1. Verticillium CA CA CA dahliae protoplasts were transfected with VdKss1-HA alone or together with VdSte11 -FLAG, VdBck1 -FLAG, or VdSsk2 -FLAG. Proteins were extracted 16 h post transfection and subjected to Co-immunoprecipitation, then followed by anti-pERK or anti-HA immunoblot. The experiments were repeated three times with similar results. B Penetration phenotypes of VdMAPKKKs deletion mutants and VdDste11/VdSte11-GFP complementary strain. The spores of indicated strains were grown on MM covered with a cellophane for 5 days and photographed (above). The cellophane was removed and the culture was continued for 3 days and photographed (below). C Hyphopodium formation of VdMAPKKKs deletion mutants and VdDste11/VdSte11-GFP complementary strain. Indicated strains were grown on MM covered with a cellophane for 5 days. Hyphopodium formation (red arrow) on the cellophane was observed by confocal laser scanning microscopy (CLSM). D Hyphopodium quantification of VdMAPKKKs deletion mutants and VdDste11/Ste11-GFP complementary strain. The number of hyphopodium in three fields was counted for each strain. Error bars indicate the standard deviation of three fields. Student’s t-test was carried out to determine the significance of difference. **Indicates significant difference at P- value of \ 0.01 plants via the downstream MAPK signaling adaptor 2019), indicating the involvement of MAPK cascades in Vst50 (Li et al. 2019). Saccharomyces cerevisiae trans- regulating hyphopodium formation. However, a com- membrane mucin Msb2, which is widely conserved in plete MAPK cascade has not been characterized yet. fungi, functions upstream of the Kss1 MAPK cascade to In this study, we explored the function of the MAPK regulate filamentous growth (Cullen et al. 2004; Perez- pathway and identified VdSte11-VdSte7-VdKss1 as a Nadales and Di Pietro 2011). In V. dahliae, VdMsb, has complete MAPK cascade that regulates V. dahliae been reported to be required for plant infection and hyphopodium formation and virulence. Among the five microsclerotia formation (Jiang et al. 2018; Tian et al. MAPKs encoded by V. dahliae, VdKss1 and VdHog1-1 2014). Moreover, mutation of VdSte11 has also been have been shown to regulate microsclerotia formation shown to impair hyphopodium formation (Yu et al. and pathogenicity (Rauyaree et al. 2005; Wang et al. The Author(s) 2023 aBIOTECH oryzae (Dixon et al. 1999), Bipolaris oryzae (Moriwaki et al. 2006), Botrytis cinerea (Segmuller et al. 2007), and the oomycete Phytophthora sojae (Li et al. 2010). Hog pathway is vital for the accumulation of osmo-protec- tant molecules and thus required for responses to Vd∆ste7/ Mock V592 Vd∆ste7 environmental stresses in fungi (Hamel et al. 2012). Hog Ste7-GFP pathway also regulates virulence in some phy- topathogens but not in M. oryzae (Dixon et al. 1999; Wang et al. 2016b). Our study showed that VdHog1-1 and VdHog1-2 are dispensable for hyphopodium for- mation in V. dahliae. The evidence indicates the con- Vd∆ste11/ vergence of MAPK cascades in M. oryzae and V. dahliae, Mock V592 Vd∆ste11 Ste11-GFP although different infection-related structures are B D developed by the two pathogens during infection. In phytopathogenic fungi M. oryzae, the appresso- rium formation is regulated by the conserved MAPK pathway Mst11-Mst7-Pmk1. As the major intracellular MAPK that is targeted by HopAI in M. oryzae, Mps1 (the ortholog of Slt2 in S. cerevisiae) is important for appressorium penetration and plant infection but is not necessary for appressorium formation (Xu et al. 1998; Fig. 6 Deletion of VdSte7 or VdSte11 compromises virulence of Zhang et al. 2017). In M. oryzae, G protein-coupled Verticillium dahliae in cotton plants. A, C. Disease symptoms of upland cotton. Plants infected with indicated strains were receptors (GPCRs) (Li et al. 2012, 2007b) and cyclic photographed and subjected to disease index analyses 3–4 weeks adenosine monophosphate (cAMP)-protein kinase A post inoculation. B, D Disease index analyses of upland cotton (PKA) signaling pathways are involved in the regulation infected with the indicated strains. The disease indexes were of appressoria formation (Jin et al. 2013; Zhao et al. evaluated with three replicates generated from 24 plants for each inoculum. Error bars indicate the standard deviation of three 2007). biological replicates. Student’s t-test was carried out to determine We have showed that VdKss1 plays an important role the significance of difference. **Indicates significant difference at in regulating the development of hyphopodium and is P-value of \ 0.01 required for the full pathogenicity of V. dahliae.In addition, the VdSlt2 also plays an important role in 2016b). Our study indicated that VdKss1, but not hyphopodium formation and pathogenicity of V. dahliae. VdHog1-1, is essential for hyphopodium formation. In Our results suggested that both VdKss1 and VdSlt2 addition, VdBck1 and VdSsk2 are involved in hyphopo- contribute to V. dahliae hyphopodium formation and dium formation. However, the CA forms of VdBck1 and pathogenicity, with VdKss1 plays a greater role. Whether VdSsk2 did not induce VdKss1 phosphorylation, sug- and how GPCRs and cAMP pathways interplay with the gesting the existence of additional regulatory mecha- VdSte11-VdSte7-VdKss1 cascade to regulate hyphopo- nisms of hyphopodium formation that are mediated by dium formation in V. dahliae remains to be further VdBck1 and VdSsk2. investigated. Although MAPK cascades are highly conserved in fungi, differential MAPK cascade specificities also occurs Supplementary InformationThe online version contains in different fungi. In S. cerevisiae, the high osmolarity supplementary material available at https://doi.org/10.1007/ s42994-023-00102-y. glycerol (HOG) pathway activates the Ste11/Ssk2/ Ssk22-Pbs2-Hog1 MAPK cascades (Hamel et al. 2012). Acknowledgements This work was supported by grants from In V. dahliae, however, VdSsk2, but not VdSte11, induces the National Key R&D Program of China (2022YFD1400800), the the phosphorylation of VdHog1 in response to stress. Chinese Natural Science Foundation (32172504), the CAS Projects for Young Scientists in Basic Research (YSBR-080), the Chinese Also, a differential contribution between VdSsk2 and Natural Science Foundation (32200241), the Strategic Priority VdSte11 in V. dahliae pathogenesis has been reported Research Program of Chinese Academy of Sciences (Grant No. (Yu et al. 2019). XDPB16) and the Youth Innovation Promotion Association of the The MAPK cascade regulating V. dahliae hyphopo- Chinese Academy of Sciences. dium formation is highly homologous to that utilized by Data availability All data generated or analyzed during this M. oryzae in the regulation of appressorium formation. study are included in this published article and its supplementary Hog homologs are widely found in fungi including M. information files. The Author(s) 2023 aBIOTECH Declarations Li CM, Hienonen E, Haapalainen M, Kontinen VP, Romantschuk M, Taira S (2007a) Type III secretion system-associated pilus of Conflict of interest The authors declare no conflicts of interest. Pseudomonas syringae as an epitope display tool. 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A complete MAP kinase cascade controls hyphopodium formation and virulence of Verticillium dahliae

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aBIOTECH https://doi.org/10.1007/s42994-023-00102-y aBIOTECH RESEARCH ARTICLE A complete MAP kinase cascade controls hyphopodium formation and virulence of Verticillium dahliae 1,2 3 1,2 1,4 1,2 Ziqin Ye , Jun Qin , Yu Wang , Jinghan Zhang , Xiaoyun Wu , 5 1,2 1,2& Xiangguo Li , Lifan Sun , Jie Zhang State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing 100049, China State Key Laboratory of Crop Stress Biology for Arid Areas, College of Plant Protection, Northwest A&F University, Yangling 712100, China School of Life Sciences, Hebei University, Baoding 710023, China College of Agronomy, Shanxi Agricultural University, Taigu 030801, China Received: 4 November 2022 / Accepted: 2 March 2023 Abstract Phytopathogens develop specialized infection-related structures to penetrate plant cells during infec- tion. Different from phytopathogens that form appressoria or haustoria, the soil-borne root-infecting fungal pathogen Verticillium dahliae forms hyphopodia during infection, which further differentiate into penetration pegs to promote infection. The molecular mechanisms underlying the regulation of hyphopodium formation in V. dahliae remain poorly characterized. Mitogen-activated protein kinases (MAPKs) are highly conserved cytoplasmic kinases that regulate diverse biological processes in eukaryotes. Here we found that deletion of VdKss1, out of the five MAPKs encoded by V. dahliae, significantly impaired V. dahliae hyphopodium formation, in vitro penetration, and pathogenicity in cotton plants. Constitutive activation of MAPK kinase (MAPKK) VdSte7 and MAPK kinase kinase (MAPKKK) VdSte11 specifically activate VdKss1. Deletion of VdSte7 or VdSte11 resulted in a phenotype similar to that of the mutant with VdKss1 deletion. Thus, this study demonstrates that VdSte11-VdSte7- VdKss1 is a core MAPK cascade that regulates hyphopodium formation and pathogenicity in V. dahliae. Keywords Verticillium, MAPK, Hyphopodium, Pathogenicity INTRODUCTION MAPK kinases (MAPKKs, MAPK/ERK- kinases/MEKs), and MAPKs that are sequentially phosphorylated In eukaryotes, mitogen-activated protein kinase (MAPK) (Widmann et al. 1999). In general, MAPKKKs phos- cascades are highly conserved cytoplasmic kinases that phorylate Ser and/or Thr residues located within the integrate extracellular signaling and transduce to activation loop of MAPKKs, which in turn trigger dual downstream substrates to regulate a wide range of phosphorylation of a highly conserved TXY motif located biological processes (Bigeard and Hirt 2018; Dixon et al. within the activation loop of MAPKs to activate MAPKs 1999; Gustin et al. 1998). The MAPK cascades consist of (Bigeard and Hirt 2018; Dixon et al. 1999; Gustin et al. MAPK kinase kinases (MAPKKKs, MAPK/extracellular 1998; Segmuller et al. 2007). signal-regulated kinase (ERK)-kinase kinases/MEKKs), In fungi, MAPKs regulate growth and development, maintenance of cellular integrity, and responses to stress (Gustin et al. 1998; Jiang et al. 2018; Wang and & Correspondence: zhangjie@im.ac.cn (J. Zhang) The Author(s) 2023 aBIOTECH Dohlman 2002; Zhao et al. 2005). In pathogenic fungi 7 days in the dark. To harvest spores, hyphae block was that infect plants, MAPK cascades regulate the patho- cultured in potato dextrose broth (PDB) liquid medium genesis and development of infection-related structures with shaking at 150 rpm at 25 C for 4 days. Cotton (Jiang et al. 2018;Lietal. 2019; Zhao et al. 2005). For plants (‘Xinluzao No. 16’) were used in this study for instance, in the ascomycete Magnaporthe oryzae, which virulence assessment (Zhou et al. 2017). All primers infects rice, barley, and other crops causing disease, used in this study are listed in Table. S1 (see in Sup- pathogenicity MAP kinase 1 (Pmk1) is crucial for plementary Information). appressorium formation. MEKK MST11-MEK MST7- MAPK PMK1 constitutes a MAPK cascade regulating Generation of the HopAI1 expression construct infection-related morphogenesis (Jiang et al. 2018; Zhao and transformant et al. 2005). In Colletotrichum gloeosporioides, Cgl-Slt2 MAPK plays an important role in the early stage of The HopAI1 gene was amplified from Pseudomonas appressorium formation (Yong et al. 2013). syringae DC3000 strain and cloned into the pSULPH-Tef- Verticillium dahliae infects a broad range of plants GFP vector to generate the pSULPH-Tef-HopAI1-GFP and causes severe wilt disease and agricultural losses. plasmid. The HopAI1 expression construct then was The development of infection-related structure is crucial transformed into Agrobacterium tumefaciens EHA105 for successful infection of host plants. Unlike M. oryzae strain, used for Agrobacterium tumefaciens mediated and C. gloeosporioides, which form appressoria during transformation (ATMT), as described previously, to infection, V. dahliae adheres tightly around the plant generate the HopAI1-expressing transformant (Wang root surface and forms hyphopodia, which further et al. 2016a). develop into penetration pegs during infection (Zhao et al. 2014; Zhou et al. 2017). This process involves Generation of gene deletion mutants calcium accumulation, NADPH oxidase-dependent reac- and complementary strains tive oxygen species burst, and cAMP-mediated signaling (Sun et al. 2019; Zhao et al. 2014; Zhou et al. 2017); To generate the target gene deletion plasmids, upstream however, additional signaling components as well as the and downstream genomic sequences of corresponding molecular mechanisms regulating these events remain genes were amplified from V. dahliae V592 and cloned elusive. The MAPKKK VdSte11, but not VdSsk2, plays a into the pGKO-HPT vector respectively. The resulting major role in regulating V. dahliae penetration (Yu et al. plasmids were used for ATMT to generate the deletion 2019). However, the core MAPK and complete MAPK mutants (Wang et al. 2016a). To obtain the comple- cascade regulating hyphopodium formation in V. dahliae mentary strains of target genes, the genomic coding remain uncharacterized. regions of target genes were PCR amplified and cloned In this study, we investigate the functions of MAPKs, into the pSULPH-Tef-GFP vector, respectively. The MAPKKs, and MAPKKKs in hyphopodium development resulting constructs were transformed into Agrobac- in V. dahliae. Mutations in VdKss1, VdSte7,or VdSte11, terium tumefaciens EHA105 strain respectively, and respectively, significantly disrupted hyphopodium for- used for ATMT. mation, in vitro penetration, and pathogenicity in cotton plants. The constitutive active forms of VdSte11 and Penetration assay and hyphopodium VdSte7 specifically induced the phosphorylation of visualization VdKss1. Thus, VdSte11-VdSte7-VdKss1 constitutes a complete MAPK cascade that regulates hyphopodium Sterilized cellophane membrane (DINGGUO, Beijing, formation and pathogenicity of V. dahliae in host plants. China), used to simulate plant root surface to induce formation of hyphopodium, was covered on MM med- ium (Zhao et al. 2016). Equal amounts of conidia col- MATERIALS AND METHODS lected from V. dahliae strains as indicated were incubated on the cellophane membrane and grown at Fungal strains, plant materials and culture 25 C for 5 days. The cellophane membrane was then conditions removed and observed under the Leica SP8 confocal laser scanning microscope system for determining Colonies of V. dahliae V592 (Gao et al. 2010) strain, hyphopodium formation. Further culture for an addi- HopAI1-expressing strain and mutation strains of target tional 3 days to observe whether there were hyphae genes generated in this study were routinely cultured grew on the underlying medium. on potato dextrose agar (PDA) medium at 25 C for The Author(s) 2023 aBIOTECH Pathogenicity assay activation and appressorium formation (Zhang et al. 2017). To examine whether MAPK activation con- The conidia of V. dahliae strains as indicated were col- tributes to hyphopodium formation in V. dahliae, HopAI1 lected and resuspended in sterilized water at the con- was expressed in V. dahliae. The expression of HopAI1 in centration of 10 /mL. Cotton plants were infected by the HopAI1-expressing strain was confirmed by anti-GFP the root-dipping inoculation method (Gao et al. 2010). immunoblot (Fig. S1). Similar to that in M. oryzae, Cotton plants then were photographed and subjected to expression of HopAI1 in V. dahliae can inhibit MAPK disease index analyses 3–4 weeks post inoculation. The activity (Fig. S2). Compared with the wild-type (WT) V. disease index was classified as follows: 0 (no symp- dahliae strain, hyphopodium formation was significantly toms), 1 (0% – 25% wilted leaves), 2 (25% – 50% reduced in the HopAI1-expressing V. dahliae strain wilted leaves), 3 (50% – 75% wilted leaves) and 4 (75% (Fig. 1A, B), suggesting that MAPK activation is crucial – 100% wilted leaves). The disease index was calculated for V. dahliae hyphopodium development. WT and as 100 9 [sum (number of plants 9 disease grade)]/ HopAI1-expressing V. dahliae strains were cultured on [(total number of plants) 9 (maximal disease grade)] minimal medium (MM) covered with cellophane and (Xu et al. 2014). examined for in vitro penetration (Zhao et al. 2016). Consistent with the deficiency of hyphopodium forma- Detection of MAPK phosphorylation tion, the HopAI1-expressing strain displayed defects in in vitro penetration compared to the WT strain V. dahliae protoplasts were isolated from 36 h vegeta- (Fig. 1C). WT and HopAI1-expressing V. dahliae strains tive hyphae grown in CM medium and transfected with were inoculated into cotton plants. Four weeks post plasmid as described (Rehman et al. 2016). Fourteen inoculation, cotton plants infected with HopAI1-ex- hours after transfection, proteins of transfected proto- pressing strain exhibited much weaker disease symp- plasts were extracted with extraction buffer containing toms than those infected with WT V592 (Fig. 1D). 50 mM HEPES (4-(2-Hydroxyethyl)-1-piperazi- Disease index analyses indicated reduced pathogenicity neethanesulfonic acid sodium salt) (pH 7.5), 150 mM of the HopAI1-expressing strain relative to the WT strain KCl, 1 mM EDTA (ethylenediaminetetraacetic acid), (Fig. 1E). These results strongly suggested that sup- 1 mM DTT (dithiothreitol), 0.2% Triton X-100, and pression of MAPK compromises hyphopodium forma- 1 9 proteinase inhibitor cocktail and subjected to IP tion and penetration, indicating an important role of and immunoblot assay with anti-pERK, anti-HA or anti- MAPK activation in regulating hyphopodium formation FLAG. of V. dahliae. The presence of VdKss1-HA, VdMAPKKs-FLAG and VdMAPKKKs-FLAG was detected by anti-HA or anti- Deletion of a specific MAPK compromises V. FLAG immunoblot. For anti-HA IP, total proteins were dahliae hyphopodium formation and penetration incubated with 2 lg of anti-HA antibody together with protein A agarose at 4 C for 4 h. The agarose beads Based on its homology with yeast homologs, V. dahliae were collected and boiled for 5 min with 1 9 protein encodes the following five putative MAPKs (Hamel et al. loading buffer. Total proteins were separated in a 10% 2012): the Kss1 type VdKss1 (VDAG_09461), Hog1 SDS-PAGE gel and transferred to the PVDF membrane. types VdHog1-1 (VDAG_08982) and VdHog1-2 The phosphorylation of VdKss1 TEY motif was detected (VDAG_02354), Ime type VdIme2 (VDAG_06935), and by anti-pERK immunoblot. Slt2 type VdSlt2 (VDAG_02584). To further confirm the requirement of MAPK activation for hyphopodium for- mation, V. dahliae mutants with gene deletion of indi- RESULTS vidual MAPKs in the V592 WT strain were generated by homologous recombination (Fig. S3) using the ATMT- Expression of a MAPK inhibitor compromises DS-vector (Wang et al. 2016a). hyphopodium formation in V. dahliae The VdDkss1,VdDhog1-1,VdDhog1-2,VdDime2, VdDslt2, and WT V. dahliae strains were grown on PDA MAPK cascades are conserved signaling kinases that medium, transferred to MM covered with cellophane, regulate diverse biological processes. HopAI1, a Pseu- and tested for their ability to penetrate artificial mem- domonas syringae effector protein, has a unique phos- brane. At 4 days after growth, hyphal growth of all phothreonine lyase activity towards plant, animal and mutants and the WT strain, except for the VdDkss1 fungal MAPKs (Li et al. 2007a; Zhang et al. 2007, 2017). mutant, was observed on the medium when the cello- Expression of HopAI1 in M. oryzae inhibits MAPK phane membrane was removed (Fig. 2A), suggesting The Author(s) 2023 aBIOTECH A B V592/ V592 HopAI1 -GFP Above Below V592/ V592 HopAI1-GFP V592/ Mock V592 HopAI1-GFP Fig. 1 Expression of a MAPK inhibitor in Verticillium dahliae compromises hyphopodium formation. A Hyphopodium formation is suppressed by HopAI1. V592 and HopAI1-expressing strains were grown on MM covered with a cellophane for 5 days. Hyphopodium formation (red arrow) on the cellophane was observed by confocal laser scanning microscopy (CLSM). B Hyphopodium quantification of V592 and HopAI1-expressing strains. The number of hyphopodium in three fields was counted for each strain. Error bars indicate the standard deviation of three fields. Student’s t-test was carried out to determine the significance of difference. **Indicates significant difference at P-value of \ 0.01. C The HopAI1-expressing strain displays a defect in penetration. The spores of V592 and HopAI1- expressing strains were grown on minimal medium (MM) covered with a cellophane for 5 days and photographed (above). The cellophane was removed and the culture was continued for 3 days and photographed (below). D Suppression of MAPK compromises V. dahliae virulence. Disease symptoms of upland cotton plants infected with the V592 and HopAI1-expressing strains were photographed and subjected to disease index analyses 3 weeks post inoculation. E Disease index analyses of upland cotton infected with the indicated strains. The disease indexes were evaluated with three replicates generated from 18 plants for each inoculum. Error bars indicate the standard deviation of three biological replicates. Student’s t-test was carried out to determine the significance of difference. **Indicates significant difference at P-value of \ 0.01 The Author(s) 2023 aBIOTECH Above Below Vd∆kss1/ Vd∆hog Vd∆hog V592 Vd∆kss1 Vd∆ime2 Vd∆slt2 Kss1-GFP 1-1 1-2 B C Vd∆kss1/ V592 Vd∆kss1 Kss1-GFP Vd∆hog1-1 Vd∆hog1-2 Vd∆ime2 Vd∆slt2 Fig. 2 Deletion of a specific MAPK significantly compromises Verticillium dahliae hyphopodium formation and in vitro penetration. A Penetration phenotypes of VdMAPKs deletion mutants and VdDkss1/VdKss1-GFP complementary strain. The spores of indicated strains were grown on MM covered with a cellophane for 5 days and photographed (above). The cellophane was removed and the culture was continued for 3 days and photographed (below). B Hyphopodium formation of VdMAPKs deletion mutants and VdDkss1/VdKss1-GFP complementary strain. Indicated strains were grown on MM covered with a cellophane for 5 days. Hyphopodium formation (red arrow) on the cellophane was observed by confocal laser scanning microscopy (CLSM). C Hyphopodium quantification of VdMAPKs deletion mutants and VdDkss1/VdKss1-GFP complementary strain. The number of hyphopodium in three fields was counted for each strain. Error bars indicate the standard deviation of three fields. Student’s t-test was carried out to determine the significance of difference. *Indicates significant difference at P-value of \ 0.05. **Indicates significant difference at P-value of \ 0.01 that mutation of VdKss1 compromised the capacity for VdSte7 phosphorylates VdKss1 and regulates membrane penetration. Next, we examined hyphopo- hyphopodium formation dium formation in these mutants. Mutation of VdKss1 impaired hyphopodium formation (Fig. 2B, C). The Within the MAPK cascade, MAPKKK, MAPKK, and MAPK complementation of VdKss1 in the VdDkss1 mutant are sequentially phosphorylated. To determine the restored membrane-penetration (Fig. 2A) and hypho- upstream MAPKKs responsible for phosphorylating podium formation (Fig. 2B, C). Expression of VdKss1- VdKss1, V. dahliae MAPKK genes were cloned and ana- lyzed for their ability to activate VdKss1. The genome of GFP in the complementary strain was confirmed by anti- GFP immunoblot (Fig. S4A). Mutation of VdIme2 and V. dahliae contains three putative MAPKK genes, VdSte7 (VDAG_08626), VdMKK1 (VDAG_09823), and VdPbs2 VdSlt2 exhibited reduced hyphopodium formation (Fig. 2B, C), suggesting a contribution of VdIme2 and (VDAG_02783) (Hamel et al. 2012). Double mutation of VdSlt2 to that process. These results indicated that the conserved serine and/or threonine residues located VdKss1 plays a major role, whereas VdIme2 and VdSlt2 in the kinase-activation loop converts MAPKKs into play minor roles, in regulating hyphopodium formation. their constitutive active (CA) forms (Asai et al. 2002; Liu The VdDkss1 mutant and WT strains were tested for and Zhang 2004; Ren et al. 2002). CA mutants of each of pathogenicity in cotton plants. The VdDkss1 mutant the above MAPKKs were generated and co-expressed with VdKss1 in V. dahliae protoplasts. The phosphory- displayed much less severe disease symptoms than the WT strain (Fig. 3A, B). The virulence of the VdDkss1 lation of VdKss1 TEY motif was examined using an anti- CA CA pERK immuno-blot. VdSte7 , but not VdMKK1 or mutant was restored upon complementation with CA VdKss1 (Fig. 3A, B). These results indicate that VdKss1 VdPbs2 , specifically induces the phosphorylation of VdKss1 (Fig. 4A), indicating that VdSte7 can phospho- plays an important role in regulating the development of hyphopodium and is required for the full rylate VdKss1. pathogenicity of V. dahliae. We next generated mutants with gene deletion of individual MAPKK genes (Fig. S5) and examined the penetration and development of hyphopodium in these The Author(s) 2023 aBIOTECH (Fig. 4B) and hyphopodium formation (Fig. 4C, D). Expression of VdSte7-GFP in the complementary strain was confirmed by anti-GFP immunoblot (Fig. S4B). The results indicating that VdSte7 acts as the upstream MAPKK to phosphorylate VdKss1 and contributes to the development of hyphopodium in V. dahliae. VdSte11 activates VdKss1 and regulates hyphopodium formation Mock V592 VdSte11 (VDAG_05822), VdBck1 (VDAG_00874), and VdSsk2 (VDAG_08787) are three putative MAPKKKs encoded by V. dahliae (Hamel et al. 2012). MAPKKK usually contains an N-terminal auto-inhibitory domain that negatively regulates kinase activity. Removal of the N-terminal inhibitory domain results in activation of MAPKKKs in the absence of upstream kinases (Asai et al. 2002; Bergmann et al. 2004). Deletion of VdSte11, a V. dahliae MAPKKK, has been reported to impair hyphopodium formation in V. dahliae (Yu et al. 2019), Vd∆kss1/ suggesting an important role of VdSte11 in regulating V. Vd∆kss1 Kss1-GFP dahliae hyphopodium development. Whether VdSte11 B B activates VdKss1 and whether additional MAPKKKs are involved in hyphopodium development remain unde- termined. The truncated C-terminal forms of the indi- CA CA CA vidual MAPKKKs, VdSte11 , VdBck1 , and VdSsk2 , were then constructed and co-expressed with VdKss1 in CA V. dahliae protoplasts. Co-expression of VdSte11 , but CA CA not VdBck1 or VdSsk2 , induced phosphorylation of VdKss1 (Fig. 5A), indicating that VdSte11 acts as the upstream MAPKKK that induces VdKss1 phosphorylation. To further examine the role of MAPKKKs in the regu- lation of hyphopodium development, mutants with deletion of individual MAPKKKs were generated (Fig. S5). While the VdDbck1 and VdDssk2 mutants exhibited nor- mal penetration, the VdDste11 mutant exhibited deficient Fig. 3 VdKss1 contributes to Verticillium dahliae virulence in cellophane penetration (Fig. 5B). Expression of VdSte11- cotton plants. A Disease symptoms of upland cotton. Plants GFP in the complementary strain was confirmed by anti- infected with indicated strains were photographed and subjected GFP immunoblot (Fig. S4C). All three mutants generated to disease index analyses 3–4 weeks post inoculation. B Disease fewer hyphopodia than the WT strains, among which the index analyses of upland cotton infected with the indicated VdDste11 mutant was almost impaired in hyphopodium strains. The disease indexes were evaluated with three replicates generated from 24 plants for each inoculum. Error bars indicate formation (Fig. 5C, D). The results indicated that VdSte11, the standard deviation of three biological replicates. Student’s t- VdBck1, and VdSsk2 are all involved in regulating test was carried out to determine the significance of difference. hyphopodium formation, and that VdSte11 plays a **Indicates significant difference at P-value of \ 0.01 greater role than VdBck1 and VdSsk2. mutants. Consistent with the specific phosphorylation of CA VdKss1 induced by VdSte7 , the VdDste7 mutant, but VdSte7 and VdSte11 are required for full neither the VdDmkk1 nor the VdDpbs2 mutants, exhib- pathogenicity of V. dahliae ited compromised membrane penetration (Fig. 4B) and significantly reduced hyphopodium formation (Fig. 4C, To determine the role of VdSte7 and VdSte11 in D). The complementation of VdSte7 in the VdDste7 pathogenicity, VdDste7,VdDste11 mutants and WT mutant restored membrane penetration in the medium strains were subjected to pathogenicity assays in cotton The Author(s) 2023 aBIOTECH Above Below Vd∆ste7/ Vd∆ste7 V592 Vd∆mkk1 Vd∆pbs2 Ste7-GFP Vd∆ste7/ V592 Vd∆ste7 Vd∆mkk1 Vd∆pbs2 Ste7-GFP CA Fig. 4 VdSte7 phosphorylates VdKss1 and regulates hyphopodium formation. A The constitutive active (CA) mutant VdSte7 specifically CA phosphorylates VdKss1. Verticillium dahliae protoplasts were transfected with VdKss1-HA alone or together with VdSte7 -FLAG, CA CA VdMKK1 -FLAG, or VdPbs2 -FLAG. Proteins were extracted 16 h post transfection and subjected to Co-immunoprecipitation, then followed by anti-pERK or anti-HA immunoblot. The experiments were repeated three times with similar results. B Penetration phenotypes of VdMAPKKs deletion mutants and VdDste7/VdSte7-GFP complementary strain. The spores of indicated strains were grown on MM covered with a cellophane for 5 days and photographed (above). The cellophane was removed and the culture was continued for 3 days and photographed (below). C Hyphopodium formation of VdMAPKKs deletion mutants and VdDste7/VdSte7-GFP complementary strain. Indicated strains were grown on MM covered with a cellophane for 5 days. Hyphopodium formation (red arrow) on the cellophane was observed by confocal laser scanning microscopy (CLSM). D Hyphopodium quantification of VdMAPKKs deletion mutants and VdDste7/VdSte7-GFP complementarystrain. The number of hyphopodium in three fields was counted for each strain. Error bars indicate the standard deviation of three fields. Student’s t-test was carried out to determine the significance of difference. **Indicates significant difference at P-value of \ 0.01 plants. Reduced pathogenicity was observed in both the DISCUSSION VdDste7 (Fig. 6A, B) and VdDste11 mutants (Fig. 6C, D). Complementation of VdSte7 in VdDste7 mutant (Fig. 6A, V. dahliae infects more than 200 kinds of plants, causing B) and VdSte11 in VdDste11 (Fig. 6C, D) restored their Verticillium wilt, leading to severe yield losses world- pathogenicity to the level of the WT strain. These results wide. Formation of hyphopodium is crucial for the suggest that both VdSte7 and VdSte11 are required for establishment of V. dahliae infection, and the underlying full pathogenicity of V. dahliae. Thus, the results indicate regulatory mechanisms remain poorly characterized. that VdSte11-VdSte7-VdKss1 constitutes a complete We have previously reported the cAMP-mediated regu- MAPK cascade that regulates the hyphopodium forma- lation of hyphopodium formation (Sun et al. 2019). tion and pathogenicity of V. dahliae. VdSho1, a tetraspan transmembrane protein, regulates V. dahliae cellophane penetration and virulence in The Author(s) 2023 aBIOTECH A D Above Below Vd∆ste11/ V592 Vd∆ste11 Vd∆bck1 Vd∆ssk2 Ste11-GFP Vd∆ste11/ V592 Vd∆ste11 Vd∆bck1 Vd∆ssk2 Ste11-GFP CA Fig. 5 VdSte11 activates VdKss1 and regulates hyphopodium formation. A The CA mutant VdSte11 activates VdKss1. Verticillium CA CA CA dahliae protoplasts were transfected with VdKss1-HA alone or together with VdSte11 -FLAG, VdBck1 -FLAG, or VdSsk2 -FLAG. Proteins were extracted 16 h post transfection and subjected to Co-immunoprecipitation, then followed by anti-pERK or anti-HA immunoblot. The experiments were repeated three times with similar results. B Penetration phenotypes of VdMAPKKKs deletion mutants and VdDste11/VdSte11-GFP complementary strain. The spores of indicated strains were grown on MM covered with a cellophane for 5 days and photographed (above). The cellophane was removed and the culture was continued for 3 days and photographed (below). C Hyphopodium formation of VdMAPKKKs deletion mutants and VdDste11/VdSte11-GFP complementary strain. Indicated strains were grown on MM covered with a cellophane for 5 days. Hyphopodium formation (red arrow) on the cellophane was observed by confocal laser scanning microscopy (CLSM). D Hyphopodium quantification of VdMAPKKKs deletion mutants and VdDste11/Ste11-GFP complementary strain. The number of hyphopodium in three fields was counted for each strain. Error bars indicate the standard deviation of three fields. Student’s t-test was carried out to determine the significance of difference. **Indicates significant difference at P- value of \ 0.01 plants via the downstream MAPK signaling adaptor 2019), indicating the involvement of MAPK cascades in Vst50 (Li et al. 2019). Saccharomyces cerevisiae trans- regulating hyphopodium formation. However, a com- membrane mucin Msb2, which is widely conserved in plete MAPK cascade has not been characterized yet. fungi, functions upstream of the Kss1 MAPK cascade to In this study, we explored the function of the MAPK regulate filamentous growth (Cullen et al. 2004; Perez- pathway and identified VdSte11-VdSte7-VdKss1 as a Nadales and Di Pietro 2011). In V. dahliae, VdMsb, has complete MAPK cascade that regulates V. dahliae been reported to be required for plant infection and hyphopodium formation and virulence. Among the five microsclerotia formation (Jiang et al. 2018; Tian et al. MAPKs encoded by V. dahliae, VdKss1 and VdHog1-1 2014). Moreover, mutation of VdSte11 has also been have been shown to regulate microsclerotia formation shown to impair hyphopodium formation (Yu et al. and pathogenicity (Rauyaree et al. 2005; Wang et al. The Author(s) 2023 aBIOTECH oryzae (Dixon et al. 1999), Bipolaris oryzae (Moriwaki et al. 2006), Botrytis cinerea (Segmuller et al. 2007), and the oomycete Phytophthora sojae (Li et al. 2010). Hog pathway is vital for the accumulation of osmo-protec- tant molecules and thus required for responses to Vd∆ste7/ Mock V592 Vd∆ste7 environmental stresses in fungi (Hamel et al. 2012). Hog Ste7-GFP pathway also regulates virulence in some phy- topathogens but not in M. oryzae (Dixon et al. 1999; Wang et al. 2016b). Our study showed that VdHog1-1 and VdHog1-2 are dispensable for hyphopodium for- mation in V. dahliae. The evidence indicates the con- Vd∆ste11/ vergence of MAPK cascades in M. oryzae and V. dahliae, Mock V592 Vd∆ste11 Ste11-GFP although different infection-related structures are B D developed by the two pathogens during infection. In phytopathogenic fungi M. oryzae, the appresso- rium formation is regulated by the conserved MAPK pathway Mst11-Mst7-Pmk1. As the major intracellular MAPK that is targeted by HopAI in M. oryzae, Mps1 (the ortholog of Slt2 in S. cerevisiae) is important for appressorium penetration and plant infection but is not necessary for appressorium formation (Xu et al. 1998; Fig. 6 Deletion of VdSte7 or VdSte11 compromises virulence of Zhang et al. 2017). In M. oryzae, G protein-coupled Verticillium dahliae in cotton plants. A, C. Disease symptoms of upland cotton. Plants infected with indicated strains were receptors (GPCRs) (Li et al. 2012, 2007b) and cyclic photographed and subjected to disease index analyses 3–4 weeks adenosine monophosphate (cAMP)-protein kinase A post inoculation. B, D Disease index analyses of upland cotton (PKA) signaling pathways are involved in the regulation infected with the indicated strains. The disease indexes were of appressoria formation (Jin et al. 2013; Zhao et al. evaluated with three replicates generated from 24 plants for each inoculum. Error bars indicate the standard deviation of three 2007). biological replicates. Student’s t-test was carried out to determine We have showed that VdKss1 plays an important role the significance of difference. **Indicates significant difference at in regulating the development of hyphopodium and is P-value of \ 0.01 required for the full pathogenicity of V. dahliae.In addition, the VdSlt2 also plays an important role in 2016b). Our study indicated that VdKss1, but not hyphopodium formation and pathogenicity of V. dahliae. VdHog1-1, is essential for hyphopodium formation. In Our results suggested that both VdKss1 and VdSlt2 addition, VdBck1 and VdSsk2 are involved in hyphopo- contribute to V. dahliae hyphopodium formation and dium formation. However, the CA forms of VdBck1 and pathogenicity, with VdKss1 plays a greater role. Whether VdSsk2 did not induce VdKss1 phosphorylation, sug- and how GPCRs and cAMP pathways interplay with the gesting the existence of additional regulatory mecha- VdSte11-VdSte7-VdKss1 cascade to regulate hyphopo- nisms of hyphopodium formation that are mediated by dium formation in V. dahliae remains to be further VdBck1 and VdSsk2. investigated. Although MAPK cascades are highly conserved in fungi, differential MAPK cascade specificities also occurs Supplementary InformationThe online version contains in different fungi. In S. cerevisiae, the high osmolarity supplementary material available at https://doi.org/10.1007/ s42994-023-00102-y. glycerol (HOG) pathway activates the Ste11/Ssk2/ Ssk22-Pbs2-Hog1 MAPK cascades (Hamel et al. 2012). Acknowledgements This work was supported by grants from In V. dahliae, however, VdSsk2, but not VdSte11, induces the National Key R&D Program of China (2022YFD1400800), the the phosphorylation of VdHog1 in response to stress. Chinese Natural Science Foundation (32172504), the CAS Projects for Young Scientists in Basic Research (YSBR-080), the Chinese Also, a differential contribution between VdSsk2 and Natural Science Foundation (32200241), the Strategic Priority VdSte11 in V. dahliae pathogenesis has been reported Research Program of Chinese Academy of Sciences (Grant No. (Yu et al. 2019). XDPB16) and the Youth Innovation Promotion Association of the The MAPK cascade regulating V. dahliae hyphopo- Chinese Academy of Sciences. dium formation is highly homologous to that utilized by Data availability All data generated or analyzed during this M. oryzae in the regulation of appressorium formation. study are included in this published article and its supplementary Hog homologs are widely found in fungi including M. information files. The Author(s) 2023 aBIOTECH Declarations Li CM, Hienonen E, Haapalainen M, Kontinen VP, Romantschuk M, Taira S (2007a) Type III secretion system-associated pilus of Conflict of interest The authors declare no conflicts of interest. Pseudomonas syringae as an epitope display tool. 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aBIOTECHSpringer Journals

Published: Jun 1, 2023

Keywords: Verticillium; MAPK; Hyphopodium; Pathogenicity

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