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Ferric reductases are integral membrane proteins involved in the reduction of environmental ferric iron into the biologically available ferrous iron. In the most overwhelming phytopathogenic fungus, Verticillium dahliae, these ferric reductase are not studied in details. In this study we explored the role of FreB gene (VDAG_06616) in the ferric reduction and virulence of V. dahliae by generating the knockout mutants (ΔFreB) and complementary strains (ΔFreB-C) using protoplast transformation. When cultured on media supplemented with FeSO4, FeCl3 and no iron, ΔFreB exhibited significantly reduced growth and spore production especially on media with no iron. Transmembrane ferric reductase activity of ΔFreB was decreased up to 50% than wild type strains (Vd-wt). The activity was fully restored in ΔFreB-C. Meanwhile, the expression levels of other related genes (Frect-4, Frect-5, Frect-6 and Met) were obviously increased in ΔFreB. Compared with the Vd-wt and ΔFreB-C, ΔFreB-1 and ΔFreB-2 were impaired in colony diameter and spore number on different carbon sources (starch, sucrose, galactose and xylose). ΔFreB-1 and ΔFreB-2 were also highly sensitive to oxidative stress as revealed by the plate diffusion assay when 100 µM H2O2 was applied to the fungal culture. When Nicotiana benthamiana plants were inoculated, ΔFreB exhibited less disease symptoms than Vd-wt and ΔFreB-C. In conclusion, the present findings not only indicate that FreB mediates the ferric metabolism and is required for the full virulence in V. dahliae, but would also accelerate future investigation to uncover the pathogenic mechanism of this fungus.

Verticillium wilt, caused by the ascomycete fungus Verticillium dahliae (Vd), is a devastating disease of numerous plant species. However, the pathogenicity/virulence-related genes in this fungus, which may be potential targets for improving plant resistance, remain poorly elucidated. For the study of these genes in Vd, we used a well-established host-induced gene silencing (HIGS) approach and identified 16 candidate genes, including a putative adenylate kinase gene (VdAK). Transiently VdAK-silenced plants developed milder wilt symptoms than control plants did. VdAK-knockout mutants were more sensitive to abiotic stresses and had reduced germination and virulence on host plants. Transgenic Nicotiana benthamiana and Arabidopsis thaliana plants that overexpressed VdAK dsRNAs had improved Vd resistance than the wild-type. RT-qPCR results showed that VdAK was also crucial for energy metabolism. Importantly, in an analysis of total small RNAs from Vd strains isolated from the transgenic plants, a small interfering RNA (siRNA) targeting VdAK was identified in transgenic N. benthamiana. Our results demonstrate that HIGS is a promising strategy for efficiently screening pathogenicity/virulence-related genes of Vd and that VdAK is a potential target to control this fungus.

Verticillium wilt, caused by the ascomycete fungus Verticillium dahliae (Vd), is a devastating disease of numerous plant species. However, the pathogenicity/virulence-related genes in this fungus, which may be potential targets for improving plant resistance, remain poorly elucidated. For the study of these genes in Vd, we used a well-established host-induced gene silencing (HIGS) approach and identified 16 candidate genes, including a putative adenylate kinase gene (VdAK). Transiently VdAK-silenced plants developed milder wilt symptoms than control plants did. VdAK-knockout mutants were more sensitive to abiotic stresses and had reduced germination and virulence on host plants. Transgenic Nicotiana benthamiana and Arabidopsis thaliana plants that overexpressed VdAK dsRNAs had improved Vd resistance than the wild-type. RT-qPCR results showed that VdAK was also crucial for energy metabolism. Importantly, in an analysis of total small RNAs from Vd strains isolated from the transgenic plants, a small interfering RNA (siRNA) targeting VdAK was identified in transgenic N. benthamiana. Our results demonstrate that HIGS is a promising strategy for efficiently screening pathogenicity/virulence-related genes of Vd and that VdAK is a potential target to control this fungus.

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes a destructive vascular wilt, but details of the molecular mechanism behind its pathogenicity are not very clear. Here, we generated a red fluorescent isolate of V. dahliae by protoplast transformation to explore its pathogenicity mechanism, including colonization, invasion, and extension in Nicotiana benthamiana, using confocal microscopy. The nucleotide sequences of mCherry were optimized for fungal expression and cloned into pCT-HM plasmid, which was inserted into V. dahliae protoplasts. The transformant (Vd-m) shows strong red fluorescence and its phenotype, growth rate, and pathogenicity did not differ significantly from the wild type V. dahliae (Vd-wt). Between one and three days post inoculation (dpi), the Vd-m successfully colonized and invaded epidermal cells of the roots. From four to six dpi, hyphae grew on root wounds and lateral root primordium and entered xylem vessels. From seven to nine dpi, hyphae extended along the surface of the cell wall and massively grew in the xylem vessel of roots. At ten dpi, the Vd-m was found in petioles and veins of leaves. Our results distinctly showed the pathway of V. dahliae infection and colonization in N. benthamiana, and the optimized expression can be used to deepen our understanding of the molecular mechanism of pathogenicity.

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.

Verticillium dahliae invades the roots of host plants and causes vascular wilt, which seriously diminishes the yield of cotton and other important crops. The protein AAC (ADP, ATP carrier) is responsible for transferring ATP from the mitochondria into the cytoplasm. When V. dahliae protoplasts were transformed with short interfering RNAs (siRNAs) targeting the VdAAC gene, fungal growth and sporulation were significantly inhibited. To further confirm a role for VdAAC in fungal development, we generated knockout mutants (ΔVdACC). Compared with wild-type V. dahliae (Vd wt), ΔVdAAC was impaired in germination and virulence; these impairments were rescued in the complementary strains (ΔVdAAC-C). Moreover, when an RNAi construct of VdAAC under the control of the 35S promoter was used to transform Nicotiana benthamiana, the expression of VdAAC was downregulated in the transgenic seedlings, and they had elevated resistance against V. dahliae. The results of this study suggest that VdAAC contributes to fungal development, virulence and is a promising candidate gene to control V. dahliae. In addition, RNAi is a highly efficient way to silence fungal genes and provides a novel strategy to improve disease resistance in plants.

Verticillium dahliae invades the roots of host plants and causes vascular wilt, which seriously diminishes the yield of cotton and other important crops. The protein AAC (ADP, ATP carrier) is responsible for transferring ATP from the mitochondria into the cytoplasm. When V. dahliae protoplasts were transformed with short interfering RNAs (siRNAs) targeting the VdAAC gene, fungal growth and sporulation were significantly inhibited. To further confirm a role for VdAAC in fungal development, we generated knockout mutants (ΔVdACC), which were hypersensitive to stresses such as UV light and high concentrations of NaCl or sorbitol. Compared with wild-type V. dahliae(Vd wt), ΔVdAAC was impaired in germination and virulence; these impairments were rescued in the complementary strains (ΔVdAAC-C). Moreover, when an RNAi construct of VdAAC under the control of the 35S promoter was used to transform Nicotiana benthamiana, the expression of VdAAC was downregulated in the transgenic seedlings, and they had elevated resistance against V. dahliae. The results of this study suggest that VdAAC contributes to fungal development, virulence and response to stresses and is a promising candidate gene to control V. dahliae. In addition, RNAi is a highly efficient way to silence fungal genes and provides a novel strategy to improve disease resistance in plants.

Background Large efforts have focused on screening for genes involved in the virulence and pathogenicity of Verticillium dahliae, a destructive fungal pathogen of numerous plant species that is difficult to control once the plant is infected. Although Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used for gene screening, a quick and easy method has been needed to facilitate transformation. Results High-quality protoplasts, with excellent regeneration efficiency (65 %) in TB3 broth (yeast extract 30 g, casamino acids 30 g and 200g sucrose in 1L H20), were generated using driselase (Sigma D-9515) and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation. PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid; electroporation resulted in 29 transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid. To determine whether short interfering RNAs (siRNAs) can be delivered to the protoplasts and used for silencing genes, we targeted the GFP gene of Vd-GFP (V. dahliae GFP strain obtained in this study) by delivering one of four different siRNAs—19-nt duplex with 2-nt 3′ overhangs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4)—into the Vd-GFP protoplasts using PEG-mediated transformation. Up to 100 % silencing of GFP was obtained with siRNA-gfp4; the other siRNAs were less effective (up to 10 % silencing). Verticillium transcription activator of adhesion (Vta2) gene of V. dahliae was also silenced with four siRNAs (siRNA-vta1, siRNA-vta2, siRNA-vta3 and siRNA-vta4) independently and together using the same approach; siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR (qRT-PCR) analysis. Conclusion Our quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0287-4) contains supplementary material, which is available to authorized users.