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.
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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
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This work was supported by a grant from the National Natural Science Foundation of China 31372004.
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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
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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
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DOI: https://doi.org/10.1007/s00294-017-0729-0