Skip to main content
SpringerLink
Log in

The oligosaccharyl transferase subunit STT3 mediates fungal development and is required for virulence in Verticillium dahliae

  • Original Article
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

Verticillium dahliae is the most overwhelming plant pathogen, causing Verticillium wilt in a number of economic crops. The molecular mechanism is still unclear and identification of the genes involved in the pathogenicity or virulence of this fungus would benefit to uncover such mechanism. STT3 is a catalytic subunit of the multi-subunit oligosaccharyl transferase (OST) and plays an essential role in glycoprotein modification. Here, we characterized STT3 gene (VDAG_03232.1) of V. dahliae to explore its regulatory role in the development and virulence by deletion and complementation of this gene, as well as its silence in transgenic plants. The expression of the STT3 gene increased at the stage of conidia germination and reached its peak level with germ tube formation and elongation. We generated the knockout mutants (ΔSTT3) using protoplast transformation. Mycelial growth, sporulation ability and glycoprotein secretion were impaired when ΔSTT3 mutants were grown on media supplemented with different carbon sources. Moreover, ΔSTT3 mutants exhibited distinctly decreased germination ratio and reduction in virulence compared with the wild type (Vd wt) and complementary (ΔSTT3-C) strains. We also generated transgenic Nicotiana benthamiana (Trans-1 and -2) plants by expressing dsRNA against the STT3 gene. Transgenic plants showed significant reduction in the disease index and fungal biomass resulting in elevated resistance to V. dahliae compared with the wild-type plants when inoculated with Vd wt. Our results indicated that STT3 mediates the full virulence through the regulation in fungal development, hyphal growth, glycoprotein secretion of V. dahliae and merits further study as a potential RNAi target to control this fungus.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

OST:

Oligosaccharyl transferase

ER:

Endoplasmic reticulum

MS:

Murashige and Skoog

Vta2 :

Transcription activator of adhesion 2

VDH1 :

Hydrophobin gene

HPT:

Hygromycin B phosphotransferase

Vd wt:

Wild-type Verticillium dahliae

Nb wt:

Wild-type Nicotiana benthamiana

References

  • Aguiar TQ, Maaheimo H, Heiskanen A, Wiebe MG, Penttila M, Domingues L (2013) Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway. Carbohydr Res 381:19–27

    Article  CAS  PubMed  Google Scholar 

  • Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Biochem 55:611–622

    CAS  Google Scholar 

  • Chavan M, Rekowicz M, Lennarz W (2003a) Insight into functional aspects of Stt3p, a subunit of the oligosaccharyl transferase. Evidence for interaction of the N-terminal domain of Stt3p with the protein kinase C cascade. J Biol Chem 278:51441–51447

    Article  CAS  PubMed  Google Scholar 

  • Chavan M, Suzuki T, Rekowicz M, Lennarz W (2003b) Genetic, biochemical, and morphological evidence for the involvement of N-glycosylation in biosynthesis of the cell wall beta1,6-glucan of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 100:15381–15386

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Davis DA, Low PS, Heinstein P (1998) Purification of a glycoprotein elicitor of phytoalexin formation from Verticillium dahliae. Physiol Mol Plant Pathol 52:259–273

    Article  CAS  Google Scholar 

  • Dempski RE, Imperiali B (2002) Oligosaccharyl transferase: gatekeeper to the secretory pathway. Curr Opin Chem Biol 6:844–850

    Article  CAS  PubMed  Google Scholar 

  • Deshmukh R, Purohit HJ (2014) siRNA mediated gene silencing in Fusarium sp HKF15 for overproduction of bikaverin. Bioresour Technol 157:368–371

    Article  CAS  PubMed  Google Scholar 

  • Duressa D, Anchieta A, Chen D, Klimes A, Garcia-Pedrajas MD, Dobinson KF, Klosterman SJ (2013) RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae. BMC Genomics 14:498

    Article  Google Scholar 

  • Faridmoayer A, Fentabil MA, Haurat MF, Yi W, Woodward R, Wang PG, Feldman MF (2008) Extreme substrate promiscuity of the Neisseria oligosaccharyl transferase involved in protein O-glycosylation. J Biol Chem 283:34596–34604

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fradin EF, Thomma BP (2006) Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo-atrum. Mol Plant Pathol 7:71–86

    Article  CAS  PubMed  Google Scholar 

  • Garber R, Houston B (1966) Penetration and development of Verticillium albo-atrum in cotton plant. Phytopathology 56:1121–1128

    Google Scholar 

  • Ghag SB, Shekhawat UK, Ganapathi TR (2014) Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana. Plant Biotechnol J 12:541–553

    Article  CAS  PubMed  Google Scholar 

  • Gianinazzi-Pearson V, Dumas-Gaudot E, Gollotte A, Tahiri-Alaoufand A, Gianinazzi S (1996) Cellular and molecular defence-related root responses to invasion by Arbuscular mycorrhizal fungi. New Phytol 133:45–57

    Article  Google Scholar 

  • Gonzalez-Fernandez R, Jorrin-Novo JV (2012) Contribution of proteomics to the study of plant pathogenic fungi. J Proteome Res 11:3–16

    Article  CAS  PubMed  Google Scholar 

  • Helenius A, Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73:1019–1049

    Article  CAS  PubMed  Google Scholar 

  • Hese K, Otto C, Routier FH, Lehle L (2009) The yeast oligosaccharyltransferase complex can be replaced by STT3 from Leishmania major. Glycobiology 19:160–171

    Article  CAS  PubMed  Google Scholar 

  • Hu G, Linning R, McCallum B, Banks T, Cloutier S, Butterfield Y, Liu J, Kirkpatrick R, Stott J, Yang G, Smailus D, Jones S, Marra M, Schein J, Bakkeren G (2007) Generation of a wheat leaf rust, Puccinia triticina, EST database from stage-specific cDNA libraries. Mol Plant Pathol 8:451–467

    Article  CAS  PubMed  Google Scholar 

  • Hu D, Wang C, Tao F, Cui Q, Xu X, Shang W, Hu X (2014) Whole genome wide expression profiles on germination of Verticillium dahliae microsclerotia. PLoS One 9:e100046

    Article  PubMed  PubMed Central  Google Scholar 

  • Huang C, Mohanty S, Banerjee M (2010) A novel method of production and biophysical characterization of the catalytic domain of yeast oligosaccharyl transferase. Biochemistry 49:1115–1126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huisman OC (1982) Interrelations of root growth dynamics to epidemiology of root-invading fungi. Annu Rev Phytopathol 20:303–327

    Article  Google Scholar 

  • Jonkers W, Rodrigues CD, Rep M (2009) Impaired colonization and infection of tomato roots by the Deltafrp1 mutant of Fusarium oxysporum correlates with reduced CWDE gene expression. Mol Plant Microbe Interact 22:507–518

    Article  CAS  PubMed  Google Scholar 

  • Karimi M, Inzé D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  CAS  PubMed  Google Scholar 

  • Kim H, von Heijne G, Nilsson I (2005) Membrane topology of the STT3 subunit of the oligosaccharyl transferase complex. J Biol Chem 280:20261–20267

    Article  CAS  PubMed  Google Scholar 

  • Klimes A, Dobinson KF (2006) A hydrophobin gene, VDH1, is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae. Fungal Genet Biol 43:283–294

    Article  CAS  PubMed  Google Scholar 

  • Klosterman SJ, Atallah ZK, Vallad GE, Subbarao KV (2009) Diversity, pathogenicity, and management of Verticillium species. Annu Rev Phytopathol 47:39–62

    Article  CAS  PubMed  Google Scholar 

  • Klosterman SJ, Subbarao KV, Kang S, Veronese P, Gold SE, Thomma BP, Chen Z, Henrissat B, Lee YH, Park J, Garcia-Pedrajas MD, Barbara DJ, Anchieta A, de Jonge R, Santhanam P, Maruthachalam K, Atallah Z, Amyotte SG, Paz Z, Inderbitzin P, Hayes RJ, Heiman DI, Young S, Zeng Q, Engels R, Galagan J, Cuomo CA, Dobinson KF, Ma LJ (2011) Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathog 7:e1002137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Knauer R, Lehle L (1999) The oligosaccharyltransferase complex from yeast. BBA Biochimica et Biophysica Acta 1426:259–273

    Article  CAS  PubMed  Google Scholar 

  • Koch A, Kumar N, Weber L, Keller H, Imani J, Kogel KH (2013) Host-induced gene silencing of cytochrome P450 lanosterol C14alpha-demethylase-encoding genes confers strong resistance to Fusarium species. Proc Natl Acad Sci USA 110:19324–19329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Koiwa H, Li F, McCully MG, Mendoza I, Koizumi N, Manabe Y, Nakagawa Y, Zhu J, Rus A, Pardo JM, Bressan RA, Hasegawa PM (2003) The STT3a subunit isoform of the Arabidopsis oligosaccharyltransferase controls adaptive responses to salt/osmotic stress. Plant Cell 15:2273–2284

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar S, Nei M, Dudley J, Tamura K (2008) Mega: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform 9:299–306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li K, Ouyang H, Lu Y, Liang J, Wilson IB, Jin C (2011) Repression of N-glycosylation triggers the unfolded protein response (UPR) and overexpression of cell wall protein and chitin in Aspergillus fumigatus. Microbiology 157:1968–1979

    Article  CAS  PubMed  Google Scholar 

  • Liu SY, Chen JY, Wang JL, Li L, Xiao HL, Adam SM, Dai XF (2013) Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae. Gene 529:307–316

    Article  CAS  PubMed  Google Scholar 

  • Mao Y, Cai W, Wang J, Hong G, Tao X, Wang L, Huang Y, Chen X (2007) Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol 25:1307–1313

    Article  CAS  PubMed  Google Scholar 

  • Maruthachalam K, Klosterman SJ, Anchieta A, Mou B, Subbarao KV (2013) Colonization of spinach by Verticillium dahliae and effects of pathogen localization on the efficacy of seed treatments. Phytopathology 103:268–280

    Article  CAS  PubMed  Google Scholar 

  • Mumbanza FM, Kiggundu A, Tusiime G, Tushemereirwe WK, Niblett C, Bailey A (2013) In vitro antifungal activity of synthetic dsRNA molecules against two pathogens of banana, Fusarium oxysporum f sp cubense and Mycosphaerella fijiensis. Pest Manag Sci 69:1155–1162

    Article  CAS  PubMed  Google Scholar 

  • Nakayashiki H, Hanada S, Nguyen BQ, Kadotani N, Tosa Y, Mayama S (2005) RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet Biol 42:275–283

    Article  CAS  PubMed  Google Scholar 

  • Nasab FP, Schulz BL, Gamarro F, Parodi AJ, Aebi M (2008) All in one: Leishmania major STT3 proteins substitute for the whole oligosaccharyltransferase complex in Saccharomyces cerevisiae. Mol Biol Cell 19:3758–3768

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nekrasov V, Li J, Batoux M, Roux M, Chu ZH, Lacombe S, Rougon A, Bittel P, Kiss-Papp M, Chinchilla D, van Esse HP, Jorda L, Schwessinger B, Nicaise V, Thomma BP, Molina A, Jones JD, Zipfel C (2009) Control of the pattern-recognition receptor EFR by an ER protein complex in plant immunity. EMBO J 28:3428–3438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nilsson I, Kelleher DJ, Miao Y, Shao Y, Kreibich G, Gilmore R, von Heijne G, Johnson AE (2003) Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex. J Cell Biol 161:715–725

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nouroozi RV, Noroozi MV, Ahmadizadeh M (2015) Determination of protein concentration using Bradford microplate protein quantification assay. Int Electron J Med 4:11–17

    Google Scholar 

  • Obrepalska-Steplowska A, Wieczorek P, Budziszewska M, Jeszke A, Renaut J (2013) How can plant virus satellite RNAs alter the effects of plant virus infection? A study of the changes in the Nicotiana benthamiana proteome after infection by peanut stunt virus in the presence or absence of its satellite RNA. Proteomics 13:2162–2175

    Article  CAS  PubMed  Google Scholar 

  • Pang J, Zhu Y, Li Q, Liu J, Tian Y, Liu Y, Wu J (2013) Development of agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense. PLoS One 8:e73211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qi X, Su X, Guo H, Qi J, Cheng H (2016) VdThit, a thiamine transport protein, is required for pathogenicity of the vascular pathogen Verticillium dahliae. Mol Plant Microbe Interact 29:545–559

    Article  CAS  PubMed  Google Scholar 

  • Rangel DE, Alder-Rangel A, Dadachova E, Finlay RD, Kupiec M, Dijksterhuis J, Braga GU, Corrochano LM, Hallsworth JE (2015) Fungal stress biology: a preface to the fungal stress responses special edition. Curr Genet 61:231–238

    Article  CAS  PubMed  Google Scholar 

  • Rehman L, Su X, Guo H, Qi X, Cheng H (2016) Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae. BMC Biotechnol 16:57–65

    Article  PubMed  PubMed Central  Google Scholar 

  • Samuelson J, Banerjee S, Magnelli P, Cui J, Kelleher DJ, Gilmore R, Robbins PW (2005) The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases. Proc Natl Acad Sci USA 102:1548–1553

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shalaby S, Horwitz BA (2015) Plant phenolic compounds and oxidative stress: integrated signals in fungal-plant interactions. Curr Genet 61:347–357

    Article  CAS  PubMed  Google Scholar 

  • Shams-Eldin H, Blaschke T, Anhlan D, Niehus S, Müller J, Azzouz N, Schwarz RT (2005) High-level expression of the Toxoplasma gondii STT3 gene is required for suppression of the yeast STT3 gene mutation. Mol Biochem Parasitol 143:6–11

    Article  CAS  PubMed  Google Scholar 

  • Singh S, Braus-Stromeyer SA, Timpner C, Tran VT, Lohaus G, Reusche M, Knufer J, Teichmann T, von Tiedemann A, Braus GH (2010) Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem. Appl Microbiol Biotechnol 85:1961–1976

    Article  CAS  PubMed  Google Scholar 

  • Su X, Rehman L, Guo H, Li X, Zhang R, Cheng H (2017) AAC as a potential target gene to control Verticillium dahliae. Genes (Basel) 8:25–41

    Article  Google Scholar 

  • Tran VT, Braus-Stromeyer SA, Kusch H, Reusche M, Kaever A, Kuhn A, Valerius O, Landesfeind M, Asshauer K, Tech M, Hoff K, Pena-Centeno T, Stanke M, Lipka V, Braus GH (2014) Verticillium transcription activator of adhesion Vta2 suppresses microsclerotia formation and is required for systemic infection of plant roots. New Phytol 202:565–581

    Article  CAS  PubMed  Google Scholar 

  • Tsror L, Levin AG (2003) Vegetative compatibility and pathogenicity of Verticillium dahliae Kleb. isolates from Olive in Israel. J Phytopathol 151:451–455

    Article  Google Scholar 

  • Tzima AK, Paplomatas EJ, Tsitsigiannis DI, Kang S (2012) The G protein beta subunit controls virulence and multiple growth- and development-related traits in Verticillium dahliae. Fungal Genet Biol 49:271–283

    Article  CAS  PubMed  Google Scholar 

  • Vallad GE, Subbarao KV (2008) Colonization of resistant and susceptible lettuce cultivars by a green fluorescent protein-tagged isolate of Verticillium dahliae. Phytopathology 98:871–885

    Article  CAS  PubMed  Google Scholar 

  • van Els CA, Corbiere V, Smits K, van Gaans-van den Brink JA, Poelen MC, Mascart F, Meiring HD, Locht C (2014) Toward understanding the essence of post-translational modifications for the Mycobacterium tuberculosis immunoproteome. Front Immunol 5:361–370

    PubMed  PubMed Central  Google Scholar 

  • Wacker M, Linton D, Hitchen PG, Nitalazar M, Haslam SM, North SJ, Panico M, Morris HR, Dell A, Wren BW (2002) N-Linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298:1790–1793

    Article  CAS  PubMed  Google Scholar 

  • Wang HM, Lin ZX, Zhang XL, Chen W, Guo XP, Nie YC, Li YH (2008) Mapping and quantitative trait loci analysis of verticillium wilt resistance genes in cotton. J Integr Plant Biol 50:174–182

    Article  PubMed  Google Scholar 

  • Wang Y, Liang C, Wu S, Zhang X, Tang J, Jian G, Jiao G, Li F, Chu C (2016) Significant improvement of cotton Verticillium wilt resistance by manipulating the expression of gastrodia antifungal proteins. Mol Plant 9:1436–1439

    Article  CAS  PubMed  Google Scholar 

  • Xiong D, Wang Y, Tang C, Fang Y, Zou J, Tian C (2015) VdCrz1 is involved in microsclerotia formation and required for full virulence in Verticillium dahliae. Fungal Genet Biol 82:201–212

    Article  CAS  PubMed  Google Scholar 

  • Xiong D, Wang Y, Tian L, Tian C (2016) MADS-Box Transcription factor VdMcm1 regulates conidiation, microsclerotia formation, pathogenicity, and secondary metabolism of Verticillium dahliae. Front Microbiol 7:1192–1206

    PubMed  PubMed Central  Google Scholar 

  • Yang X, Ben S, Sun Y, Fan X, Tian C, Wang Y (2013) Genome-wide identification, phylogeny and expression profile of vesicle fusion components in Verticillium dahliae. PLoS One 8:e68681

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang Z, Song Y, Liu CM, Thomma BP (2014) Mutational analysis of the Ve1 immune receptor that mediates Verticillium resistance in tomato. PLoS One 9:e99511

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang YL, Li ZF, Feng ZL, Feng HJ, Zhao LH, Shi YQ, Hu XP, Zhu HQ (2015) Isolation and functional analysis of the pathogenicity-related gene VdPR3 from Verticillium dahliae on cotton. Curr Genet 61:555–566

    Article  CAS  PubMed  Google Scholar 

  • Zhang T, Jin Y, Zhao JH, Gao F, Zhou BJ, Fang YY, Guo HS (2016) Host-induced gene silencing of target gene in fungal cells confers effective resistance to cotton wilt disease pathogen Verticillium dahliae. Mol Plant 9:939–942

    Article  CAS  PubMed  Google Scholar 

  • Zhao Z, Liu H, Wang C, Xu JR (2013) Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi. BMC Genomics 15:6–20

    Article  Google Scholar 

  • Zufferey R, Knauer R, Burda P, Stagljar I, te Heesen S, Lehle L, Aebi M (1995) STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo. EMBO J 14:4949–4960

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant from the National Natural Science Foundation of China 31372004.

Author information

Authors and Affiliations

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Xiaofeng Su, Latifur Rehman, Huiming Guo, Xiaokang Li & Hongmei Cheng

Authors
  1. Xiaofeng Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Latifur Rehman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huiming Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaokang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongmei Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hongmei Cheng.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Communicated by M. Kupiec.

Electronic supplementary material

Below is the link to the electronic supplementary material.

294_2017_729_MOESM1_ESM.tif

Supplementary material 1 (TIFF 1408 kb) Fig. S1 Conserved domains of STT3 and the phylogenetic tree for STT3 amino acid sequences in fungal species. a Conserved domains of STT3 were predicted by the NCBI Conserved Domain Search Service (CD Search). b Alignment of STT3 amino acid sequences of different fungal species. The phylogenetic tree was constructed by MEGA software (version 6.06; bootstraps: 1000). Fungal species and protein accession numbers: Verticillium alfalfae VaMs.102 (XP_003003631); Colletotrichum fioriniae PJ7 (XP_007599997); Colletotrichum sublineola (KDN70481); Colletotrichum graminicola M1.001 (XP_008092879); Colletotrichum gloeosporioides Nara gc5 (XP_007281601); Colletotrichum orbiculare MAFF 240422 (ENH83564); Metarhizium anisopliae BRIP 53293 (KJK78990.1); Metarhizium robertsii (EXV02980); Eutypa lata UCREL1 (XP_007795041); Beauveria bassiana ARSEF 2860 (XP_008593447); Ustilaginoidea virens (KDB 12305); Grosmannia clavigera kw1407 (EFX03520); Gaeumannomyces graminis var. tritici R3-111a-1 (XP_009217935); Neurospora crassa ORA74A (XP_001728408.1); Trichoderma harzianum (KKP03854); Cordyceps militaris CM01 (XP_006671571); Metarhizium anisopliae (KFG87554.1); Metarhizium guizhouense ARSEF 977 (KID90219.1); Metarhizium acridum CQMa 102 (XP_007809515); Ophiocordyceps sinensis CO18 (EQL03686.1)

294_2017_729_MOESM2_ESM.tif

Supplementary material 2 (TIFF 6637 kb) Fig. S2 Construction of stt3 mutant vectors and confirmation of mutants. a Knockout fragment construction for the deletion of STT3. The hygromycin resistance (HPT) cassette was fused with ~1.2 kb upstream and downstream of the STT3 gene to generate the knockout fragment. b Expression cassettes of GFP and neomycin resistance (neoR) were cloned into pCAMBIA1302 to yield the pCAMBIA1302::Neo::GFP. Then, GFP ORF was replaced with STT3 ORF via ScaI and PstI restriction sites to produce pCAMBIA1302::Neo::STT3 for complementation. c Expression of targeted genes was confirmed in the disruption mutants, complementation and ΔSTT3-GFP transformants via RT-PCR. Vdactin gene was used as a housekeeping gene

294_2017_729_MOESM3_ESM.tif

Supplementary material 3 (TIFF 533 kb) Fig. S3 Colony diameters of ΔSTT3, ΔSTT3-C and wild-type V. dahliae (Vd wt) strains after 2 weeks on Czapek Dox agar supplemented with different carbon sources. Conidia (106/mL) were cultured in the center of each plate and the colony diameter was measured after 2 weeks. Data represent mean ± SD (n = 3 plates) for three experiments

294_2017_729_MOESM4_ESM.tif

Supplementary material 4 (TIFF 539 kb) Fig. S4 Virulence and germination analysis of ΔSTT3, ΔSTT3-C and wild-type V. dahliae (Vd wt) strains. a Disease index for N. benthamiana seedlings at 10 to 12 days post inoculation with Vd wt, STT3 disruption and complementation strains of V. dahliae. b Percentage germination of conidia produced by Vd-GFP or ΔSTT3-GFP after 48 h on PDA. Data represent mean ± SD (the experiment was repeated twice). Significant differences between Vd-GFP and ΔSTT3-GFP (P < 0.05) are marked by different letters as determined by the Duncan’s multiple range test

294_2017_729_MOESM5_ESM.tif

Supplementary material 5 (TIFF 1286 kb) Fig. S5 Construction of pK7GW1WG2(I)-STT3 for transgenic plants. a Region (562-1239 bp) of STT3 gene used in RNAi experiment. Numbers indicate nucleotide positions. b Schematic representation of the pK7GWIWG2(I)-STT3 construction containing the sense and antisense partial ORF of STT3

294_2017_729_MOESM6_ESM.tif

Supplementary material 6 (TIFF 1201 kb) Fig. S6 Characterization of N. benthamiana plants transformed with dsSTT3. a Confirmation of positive plants. Genomic DNA was extracted from the putative transformed plant and detected by PCR. The wild-type seedling served as negative control. b Disease index for seedlings of wild-type N. benthamiana and transgenic lines from 10 to 12 days post inoculation with wild-type V. dahliae. Data points represent mean ± SD of three experiments

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Su, X., Rehman, L., Guo, H. et al. The oligosaccharyl transferase subunit STT3 mediates fungal development and is required for virulence in Verticillium dahliae . Curr Genet 64, 235–246 (2018). https://doi.org/10.1007/s00294-017-0729-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-017-0729-0

Keywords

Search

Navigation