Hypothetical Protein VDAG_07742 Is Required for Verticillium dahliae Pathogenicity in Potato
Abstract
:1. Introduction
2. Results
2.1. Differentially Expressed Proteins (DEPs) Identified during V. dahliae Infection of Potato Plants
2.2. Functional Cluster Analysis of Upregulated Proteins
2.3. Functional Enrichment Analysis of Upregulated Proteins
2.4. qRT-PCR Verification of Upregulated Proteins
2.5. Identification and Characterization of the VDAG_07742 Protein in V. dahliae
2.6. Vegetative Propagation, Conidial Production, and Spore Germination of Mutants
2.7. Conserved Hypothetical Protein VDAG_07742 Is Required for V. dahliae Pathogenicity to Potato Plants
2.8. Hypothetical Protein VDAG_07742 Impairs the Penetration Ability of V. dahliae
3. Discussion
4. Materials and Methods
4.1. Plant Material and Strains
4.2. V. dahliae Inoculation and Sample Preparation
4.3. Protein Extraction, Digestion, and LC-MS/MS Analysis
4.4. Functional Annotation and Enrichment Analysis
4.5. qRT-PCR Analysis
4.6. Bioinformatics Analysis
4.7. Generation of Knockout and Complementation Mutants, and Determination of Fungal Morphology
4.8. Pathogenicity Assays
4.9. Penetration Assays
4.10. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Klosterman, S.J.; Atallah, Z.K.; Vallad, G.E.; Subbarao, K.V. Diversity, pathogenicity, and management of Verticillium species. Annu. Rev. Phytopathol. 2009, 47, 39–62. [Google Scholar] [CrossRef]
- Inderbitzin, P.; Subbarao, K. Verticillium systematics and evolution: How confusion impedes Verticillium wilt management and how to resolve it. Phytopathology 2014, 104, 564–574. [Google Scholar] [CrossRef] [PubMed]
- Fan, R.; Klosterman, S.J.; Wang, C.H.; Subbarao, K.V.; Xu, X.M.; Shang, W.J.; Hu, X.P. Vayg1 is required for microsclerotium formation and melanin production in Verticillium dahliae. Fungal Genet. Biol. 2017, 98, 1–11. [Google Scholar] [CrossRef]
- Jiménez-Ruiz, J.; Leyva-Pérez, M.D.L.O.; Schilirò, E.; Barroso, J.B.; Bombarely, A.; Mueller, L.; Mercado-Blanco, J.; Luque, F. Transcriptomic analysis of Olea europaea L. roots during the Verticillium dahliae early infection process. Plant Genome 2017, 10, plantgenome2016-07. [Google Scholar] [CrossRef]
- Jin, Y.Y.; Fan, L.Q.; Zhang, Y.H.; Hu, W.; Han, X.; Yan, Q.D.; Yang, J.X.; Li, F.G.; Yang, Z.E. Functional divergence of GLP genes between G. barbadense and G. hirsutum in response to Verticillium dahliae infection. Genomics 2022, 114, 110470. [Google Scholar] [CrossRef] [PubMed]
- Zhang, J.; Hu, H.L.; Wang, X.N.; Yang, Y.H.; Zhang, C.J.; Zhu, H.Q.; Shi, L.; Tang, C.M.; Zhao, M.W. Dynamic infection of Verticillium dahliae in upland cotton. Plant Biol. 2020, 22, 90–105. [Google Scholar] [CrossRef]
- Wang, Y.L.; Xiao, S.X.; Xiong, D.G.; Tian, C.M. Genetic transformation, infection process and qPCR quantification of Verticillium dahliae on smoke-tree Cotinus coggygria. Australas. Plant Pathol. 2013, 42, 33–41. [Google Scholar] [CrossRef]
- Zhao, P.; Zhao, Y.L.; Jin, Y.; Zhang, T.; Guo, H.S. Colonization process of Arabidopsis thaliana roots by a green fluorescent protein-tagged isolate of Verticillium dahliae. Protein Cell 2014, 5, 94–98. [Google Scholar] [CrossRef]
- Yuan, L.; Su, Y.X.; Zhou, S.; Feng, Y.G.; Guo, W.Z.; Wang, X.Y. A RACK1-like protein regulates hyphal morphogenesis, root entry and in vivo virulence in Verticillium dahliae. Fungal Genet. Biol. 2017, 99, 52–61. [Google Scholar] [CrossRef]
- Qin, T.F.; Hao, W.; Sun, R.R.; Li, Y.Q.; Wang, Y.Y.; Wei, C.Y.; Dong, T.; Wu, B.J.; Dong, N.; Wang, W.P.; et al. Verticillium dahliae VdTHI20, involved in pyrimidine biosynthesis, is required for DNA repair functions and pathogenicity. Int. J. Mol. Sci. 2020, 21, 1378. [Google Scholar] [CrossRef]
- Yang, X.; Guo, C.M.; Chen, C.; Hu, Z.J.; Zheng, X.Y.; Xu, S.; Yang, X.Y.; Xie, C.J. A kinesin Vdkin2 required for vacuole formation, mycelium growth, and penetration structure formation of Verticillium dahliae. J. Fungi 2022, 8, 391. [Google Scholar] [CrossRef] [PubMed]
- Lin, L.L.; Cao, J.Y.; Du, A.Q.; An, Q.L.; Chen, X.M.; Yuan, S.S.; Batool, W.; Shabbir, A.; Zhang, D.M.; Wang, Z.H.; et al. EIF3k domain-containing protein regulates conidiogenesis, appressorium turgor, virulence, stress tolerance, and physiological and pathogenic development of Magnaporthe oryzae Oryzae. Front. Plant Sci. 2021, 12, 807845. [Google Scholar] [CrossRef] [PubMed]
- Guo, L.J.; Wang, J.; Liang, C.C.; Yang, L.Y.; Zhou, Y.; Liu, L.; Huang, J.S. Fosp9, a novel secreted protein, is essential for the full virulence of Fusarium oxysporum f. sp. cubense on Banana (Musa spp.). Appl. Environ. Microbiol. 2022, 88, e0060421. [Google Scholar] [CrossRef] [PubMed]
- Bauernfeind Amy, L.; Babbitt Courtney, C. The predictive nature of transcript expression levels on protein expression in adult human brain. BMC Genom. 2017, 18, 322. [Google Scholar] [CrossRef] [PubMed]
- Wang, Y.M.; Wu, J.N.; Park, Z.Y.; Kim, S.G.; Rakwal, R.; Agrawal, G.K.; Kim, S.T.; Kang, K.Y. Comparative secretome investigation of Magnaporthe oryzae proteins responsive to nitrogen starvation. J. Proteome Res. 2011, 10, 3136–3148. [Google Scholar] [CrossRef]
- Chen, X.Y.; Pei, Z.X.; Li, P.P.; Li, X.B.; Duan, Y.H.; Liu, H.; Chen, X.L.; Zheng, L.; Luo, C.X.; Huang, J.B. Quantitative proteomics analysis reveals the function of the putative ester cyclase UvEC1 in the pathogenicity of the rice false smut fungus Ustilaginoidea virens. Int. J. Mol. Sci. 2021, 22, 4069. [Google Scholar] [CrossRef] [PubMed]
- Chen, J.Y.; Xiao, H.L.; Gui, Y.J.; Zhang, D.D.; Li, L.; Bao, Y.M.; Dai, X.F. Characterization of the Verticillium dahliae exoproteome involves in pathogenicity from cotton-containing medium. Front. Microbiol. 2016, 7, 1709. [Google Scholar] [CrossRef]
- Zhang, Y.; Gao, Y.B.; Liang, Y.B.; Dong, Y.J.; Yang, X.F.; Yuan, J.J.; Qiu, D.W. The Verticillium dahliae snodprot1-like protein VdCP1 contributes to virulence and triggers the plant immune system. Front. Plant Sci. 2017, 8, 1880. [Google Scholar] [CrossRef]
- Paz, Z.; García-Pedrajas, M.D.; Andrews, D.L.; Klosterman, S.J.; Baeza-Montañez, L.; Gold, S.E. One step construction of agrobacterium-recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi. Fungal Genet. Biol. 2011, 48, 677–684. [Google Scholar] [CrossRef]
- Wang, S.; Xing, H.Y.; Hua, C.L.; Guo, H.S.; Zhang, J. An improved single-step cloning strategy simplifies the agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption method for Verticillium dahliae. Phytopathology 2016, 106, 645–652. [Google Scholar] [CrossRef]
- Chen, X.J.; Sopone, W.; Yuan, J.; Yang, X.H.; Wu, S.P.; Tai, Q.Q.; Wang, L.S.; Dusit, A.; Natthiya, B. In vitro inhibition of pathogenic Verticillium dahliae, causal agent of potato wilt disease in China by trichoderma isolates. Afr. J. Biotechnol. 2014, 13, 3402–3412. [Google Scholar] [CrossRef]
- Xiong, D.G.; Wang, Y.L.; Tang, C.; Fang, Y.L.; Zou, J.Y.; Tian, C.M. VdCrz1 is involved in microsclerotia formation and required for full virulence in Verticillium dahliae. Fungal Genet. Biol. 2015, 82, 201–212. [Google Scholar] [CrossRef] [PubMed]
- Li, J.J.; Zhou, L.; Yin, C.M.; Zhang, D.D.; Klosterman, S.J.; Wang, B.L.; Song, J.; Wang, D.; Hu, X.P.; Subbarao, K.V.; et al. The Verticillium dahliae Sho1-MAPK pathway regulates melanin biosynthesis and is required for cotton infection. Environ. Microbiol. 2019, 21, 4852–4874. [Google Scholar] [CrossRef]
- Zhao, Y.L.; Zhou, T.T.; Guo, H.S. Hyphopodium-specific VdNoxB/VdPls1-dependent ROS-Ca2+ signaling is required for plant infection by Verticillium dahliae. PLoS Pathog. 2016, 12, 1005793. [Google Scholar] [CrossRef] [PubMed]
- Tian, L.L.; Xu, J.; Zhou, L.; Guo, W.Z. VdMsb regulates virulence and microsclerotia production in the fungal plant pathogen Verticillium dahliae. Gene 2014, 550, 238–244. [Google Scholar] [CrossRef] [PubMed]
- Xiong, D.G.; Wang, Y.L.; Tian, L.Y.; Tian, C.M. MADS-Box transcription factor VdMcm1 regulates conidiation, microsclerotia formation, pathogenicity, and secondary metabolism of Verticillium dahliae. Front. Microbiol. 2016, 7, 1. [Google Scholar] [CrossRef]
- Zhou, T.T.; Zhao, Y.L.; Guo, H.S. Secretory proteins are delivered to the septin-organized penetration interface during root infection by Verticillium dahliae. PLoS Pathog. 2017, 13, e1006275. [Google Scholar] [CrossRef]
- Li, H.; Wang, D.; Zhang, D.D.; Geng, Q.; Li, J.J.; Sheng, R.C.; Xue, H.S.; Zhu, H.; Kong, Z.Q.; Dai, X.F.; et al. A polyketide synthase from Verticillium dahliae modulates melanin biosynthesis and hyphal growth to promote virulence. BMC Biol. 2022, 20, 125. [Google Scholar] [CrossRef]
- Tang, C.; Li, W.W.; Klosterman, S.J.; Wang, Y.L. Transcriptome variations in Verticillium dahliae in response to two different inorganic nitrogen sources. Front. Microbiol. 2021, 12, 712701. [Google Scholar] [CrossRef]
- Wang, H.; Chen, B.; Tian, J.; Kong, Z.S. Verticillium dahliae VdBre1 is required for cotton infection by modulating lipid metabolism and secondary metabolites. Environ. Microbiol. 2020, 23, 1991–2003. [Google Scholar] [CrossRef]
- Sarmiento-Villamil, J.L.; García-Pedrajas, N.E.; Cañizares, M.C.; García-Pedrajas, M.D. Molecular mechanisms controlling the disease cycle in the vascular pathogen Verticillium dahliae characterized through forward genetics and transcriptomics. Mol. Plant Microbe. Interact. 2020, 33, 825–841. [Google Scholar] [CrossRef] [PubMed]
- Zhang, X.Y.; Cheng, W.H.; Feng, Z.D.; Zhu, Q.H.; Sun, Y.Q.; Li, Y.J.; Sun, J. Transcriptomic analysis of gene expression of Verticillium dahliae upon treatment of the cotton root exudates. BMC Genom. 2020, 21, 155. [Google Scholar] [CrossRef] [PubMed]
- Klimes, A.; Dobinson, K. A hydrophobin gene, VDH1, is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae. Fungal Genet. Biol. 2006, 43, 283–294. [Google Scholar] [CrossRef] [PubMed]
- Chen, J.Y.; Liu, C.; Gui, Y.J.; Si, K.W.; Zhang, D.D.; Wang, J.; Short, D.P.G.; Huang, J.Q.; Li, N.Y.; Liang, Y.; et al. Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium. New Phytol. 2018, 217, 756–770. [Google Scholar] [CrossRef] [PubMed]
- El-Bebany, A.F.; Rampitsch, C.; Daayf, F. Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein expression in isolates that differ in aggressiveness. Proteomics 2010, 10, 289–303. [Google Scholar] [CrossRef]
- Klosterman, S.J.; Subbarao, K.V.; Kang, S.; Veronese, P.; Gold, S.E.; Thomma, B.P.H.J.; Chen, Z.H.; Henrissat, B.; Lee, Y.H.; Park, J.; et al. Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathog. 2011, 7, 1002137. [Google Scholar] [CrossRef]
- Mathioni, S.; Beló, A.; Rizzo, C.; Dean, R.; Donofrio, N. Transcriptome profiling of the rice blast fungus during invasive plant infection and in vitro stresses. BMC Genom. 2011, 12, 49. [Google Scholar] [CrossRef]
- O’Connell, R.J.; Thon, M.R.; Hacquard, S.; Amyotte, S.G.; Kleemann, J.; Torres, M.F.; Damm, U.; Buiate, E.A.; Epstein, L.; Alkan, N.; et al. Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses. Nat. Genet. 2012, 44, 1060–1065. [Google Scholar] [CrossRef]
- Gui, Y.J.; Chen, J.Y.; Zhang, D.D.; Li, N.Y.; Li, T.G.; Zhang, W.Q.; Wang, X.Y.; Short, D.P.G.; Li, L.; Guo, W.; et al. Verticillium dahliae manipulates plant immunity by glycoside hydrolase 12 proteins in conjunction with carbohydrate-binding module 1. Environ. Microbiol. 2017, 19, 1914–1932. [Google Scholar] [CrossRef]
- Zhou, J.L.; Wu, Y.J.; Zhang, X.J.; Zhao, L.H.; Feng, Z.L.; Wei, F.; Zhang, Y.L.; Feng, H.J.; Zhou, Y.; Zhu, H.Q. MPK homolog GhNTF6 was involved in cotton against Verticillium wilt by interacted with VdEPG1. Int. J. Biol. Macromol. 2021, 195, 456–465. [Google Scholar] [CrossRef]
- Sophien, K. A catalogue of the effector secretome of plant pathogenic oomycetes. Annu. Rev. Phytopathol. 2006, 44, 41–60. [Google Scholar]
- Hogenhout, S.A.; Vander, H.R.A.L.; Terauchi, R.; Kamoun, S. Emerging concepts in effector biology of plant-associated organisms. Mol. Plant. Microbe. Interact. 2009, 22, 115–122. [Google Scholar] [CrossRef]
- Chen, C.; Hu, Z.J.; Zheng, X.Y.; Yuan, J.J.; Zou, R.; Xu, S.; Yang, X.Y.; Xie, C.J. An auxiliary activity family 9 protein, VdAA91, is required for the penetration structure formation in Verticillium dahliae. Physiol. Mol. Plant Pathol. 2022, 122, 101921. [Google Scholar] [CrossRef]
- Kowalska, B. Management of the soil-borne fungal pathogen-Verticillium dahliae Kleb. causing vascular wilt diseases. J. Plant Pathol. 2021, 103, 1185–1194. [Google Scholar] [CrossRef]
- Zhao, Y.L.; Zhang, T.; Guo, H.S. Penetration assays, fungal recovery and pathogenicity assays for Verticillium dahliae. Bio-Protocol 2017, 7, e2133. [Google Scholar] [CrossRef] [PubMed]
- Bui, T.T.; Harting, R.; Braus-Stromeyer, S.A.; Tran, V.T.; Leonard, M.; Höfer, A.; Abelmann, A.; Bakti, F.; Valerius, O.; Schlüter, R.; et al. Verticillium dahliae transcription factors Som1 and Vta3 control microsclerotia formation and sequential steps of plant root penetration and colonisation to induce disease. New Phytol. 2019, 221, 2138–2159. [Google Scholar] [CrossRef] [PubMed]
- Zhu, X.H.; Sayari, M.; Islam, M.R.; Daayf, F. NoxA is important for Verticillium dahliae’s penetration ability and virulence. J. Fungi 2021, 7, 814. [Google Scholar] [CrossRef]
- Hu, X.P.; Bai, Y.W.; Chen, T.; Hu, D.F.; Yang, J.R.; Xu, X.M. An optimized method for in vitro production of Verticillium dahliae microsclerotia. Eur. J. Plant Pathol. 2013, 136, 225–229. [Google Scholar] [CrossRef]
- Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
- Liu, T.; Qin, J.; Cao, Y.H.; Subbarao, K.V.; Chen, J.Y.; Mandal, M.K.; Xu, X.M.; Shang, W.J.; Hu, X.P. Transcription factor VdCf2 regulates growth, pathogenicity, and the expression of a putative secondary metabolism gene cluster in Verticillium dahliae. Appl. Environ. Microbiol. 2022, 88, e01385-22. [Google Scholar] [CrossRef]
Protein ID a | Protein Description | Fold Change b | p-Value |
---|---|---|---|
VDAG_00499 | Endothiapepsin | 15.131 | 0.00639017 |
VDAG_07349 | Cysteine-rich-protein | 10.432 | 0.0109871 |
VDAG_09254 | Glutathione-independent formaldehyde dehydrogenase | 8.55 | 0.00033739 |
VDAG_10467 | Dicarboxylic amino acid permease | 7.929 | 0.00050033 |
VDAG_08662 | 4-coumarate-CoA ligase | 7.667 | 0.00182525 |
VDAG_05550 | General alpha-glucoside permease | 7.659 | 0.02286991 |
VDAG_10208 | Amino-acid permease inda1 | 7.505 | 7.8584 × 10−5 |
VDAG_07191 | High-affinity nicotinic acid transporter | 7.135 | 0.00014381 |
VDAG_07980 | Aminopeptidase Y | 7.004 | 0.0019787 |
VDAG_05967 | Alkaline proteinase | 6.903 | 0.00070141 |
VDAG_01172 | 4-coumarate:coenzyme a ligase(predicted) | 6.76 | 0.0044525 |
VDAG_05650 | Trypsin | 6.648 | 6.0437 × 10−5 |
VDAG_08248 | Hce2 domain-containing protein | 6.522 | 0.00073655 |
VDAG_05344 | Pectate lyase B | 6.385 | 0.02084166 |
VDAG_02304 | Aminopeptidase Y | 5.838 | 0.00167567 |
VDAG_01113 | EFG_II domain-containing protein(predicted) | 5.636 | 0.00489088 |
VDAG_10194 | Sodium/nucleoside cotransporter | 5.626 | 0.00236321 |
VDAG_10460 | Amino-acid permease inda1 | 5.549 | 0.00079813 |
VDAG_05115 | Zinc carboxypeptidase A | 5.42 | 0.00334736 |
VDAG_07742 | Podospora anserina S mat+ genomic DNA chromosome 6, supercontig 4 (predicted) | 5.4 | 0.00634638 |
VDAG_03907 | Peptide hydrolase | 5.35 | 0.00250297 |
VDAG_07373 | Serin endopeptidase | 5.188 | 2.0559 × 10−5 |
VDAG_07392 | Cutinase (predicted) | 5.154 | 0.00138373 |
Map | KEGG Pathway | Protein ID a | Fold Enrichment | p-Value |
---|---|---|---|---|
Map00040 | Pentose and glucuronate interconversions | VDAG_10081 VDAG_02904 VDAG_04977 VDAG_05799 VDAG_08495 VDAG_09741 VDAG_06080 VDAG_06523 VDAG_06079 VDAG_02886 VDAG_08496 VDAG_09536 VDAG_08929 VDAG_05344 | 10.37 | 11.3 |
Map00350 | Tyrosine metabolism | VDAG_04798 VDAG_03345 VDAG_07316 VDAG_07507 VDAG_07314 VDAG_07369 VDAG_06372 | 7.04 | 4.49 |
Map00600 | Sphingolipid metabolism | VDAG_05015 VDAG_05347 VDAG_01556 VDAG_07821 | 10.23 | 3.41 |
Map00561 | Glycerolipid metabolism | VDAG_09648 VDAG_04951 VDAG_09741 VDAG_02013 VDAG_07738 | 7.04 | 3.31 |
Map00010 | Glycolysis/Gluconeogenesis | VDAG_07446 VDAG_07507 VDAG_09741 VDAG_02013 VDAG_07057 VDAG_06372 | 4.33 | 2.69 |
Domain Description | Protein ID a | Fold Enrichment | p-Value |
---|---|---|---|
Zinc carboxypeptidase | VDAG_03588 VDAG_05115 VDAG_07183 VDAG_07290 | 14.67 | 4.25 |
Pectate lyase | VDAG_07238 VDAG_02904 VDAG_02886 VDAG_05344 | 14.67 | 4.25 |
GMC oxidoreductase | VDAG_00618 VDAG_08340 VDAG_05780 VDAG_07266 VDAG_05396 | 8.46 | 3.77 |
Nitronate monooxygenase | VDAG_01628 VDAG_07292 VDAG_00590 | 16.5 | 3.45 |
Glycosyl hydrolases family 2 | VDAG_05015 VDAG_05347 VDAG_05015 VDAG_05347 | 14.67 | 4.25 |
Copper amine oxidase, N3 domain | VDAG_07314 VDAG_07369 | 22 | 2.69 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Wang, D.; Wen, S.; Zhao, Z.; Long, Y.; Fan, R. Hypothetical Protein VDAG_07742 Is Required for Verticillium dahliae Pathogenicity in Potato. Int. J. Mol. Sci. 2023, 24, 3630. https://doi.org/10.3390/ijms24043630
Wang D, Wen S, Zhao Z, Long Y, Fan R. Hypothetical Protein VDAG_07742 Is Required for Verticillium dahliae Pathogenicity in Potato. International Journal of Molecular Sciences. 2023; 24(4):3630. https://doi.org/10.3390/ijms24043630
Chicago/Turabian StyleWang, Dahui, Shenglan Wen, Zhibo Zhao, Youhua Long, and Rong Fan. 2023. "Hypothetical Protein VDAG_07742 Is Required for Verticillium dahliae Pathogenicity in Potato" International Journal of Molecular Sciences 24, no. 4: 3630. https://doi.org/10.3390/ijms24043630