Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Feb;85(6):1961-76.
doi: 10.1007/s00253-009-2269-0. Epub 2009 Oct 14.

Silencing of Vlaro2 for Chorismate Synthase Revealed That the Phytopathogen Verticillium Longisporum Induces the Cross-Pathway Control in the Xylem

Affiliations
Free PMC article

Silencing of Vlaro2 for Chorismate Synthase Revealed That the Phytopathogen Verticillium Longisporum Induces the Cross-Pathway Control in the Xylem

Seema Singh et al. Appl Microbiol Biotechnol. .
Free PMC article

Abstract

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.

Figures

Fig. 1
Fig. 1
Isolation of the gene for CS in V. longisporum, Vlaro2.a∆ARO2 yeast complementation assay on SC medium lacking the three aromatic amino acids. BY4751, wild-type yeast strain; Y04515 (∆ARO2), yeast CS mutant; Y04515 (∆ARO2) +Vlaro2-1, yeast CS mutant complemented with gene for CS in V. longisporum. b Illustration of the Vlaro2-1 locus consisting of two exons and one intron. c Determination of the isogene Vlaro2-2 of Vlaro2-1 by Southern hybridization analysis of V. dahliae and V. longisporum. The genomic DNA was digested with SalI and XhoI. A 416-bp sequence of Vlaro2-1 was used as a probe. Arrows indicate the signal generation by probe binding
Fig. 2
Fig. 2
Characterization of the Vlaro2 silenced mutants. a Southern hybridization analysis of Vlaro2 silenced mutants to detect integration of T-DNA after A. tumefaciens-mediated transformation. Five Vlaro2 silenced mutants (15) and wild-type (wt) gDNA was digested with HindIII and the hygromycin resistance gene was used as a probe. All mutants showed single integration of the gene. b RT-PCR analysis of Vlaro2 mRNA expression in the Vlaro2 silenced mutants. For RNA integrity, the actin gene was used as a control. 15, Vlaro2 silenced mutants; wt, wild type; NTC, no template control. c Western hybridization analysis of Vlaro2 expression in the Vlaro2 silenced mutants compared to the wild type. Proteins were extracted from the Vlaro2 silenced mutants and wild type, ran on SDS-polyacrylamide gel, blotted and probed with N. crassa CS antibody. The same blot was stripped and probed again with Rat IgG tubulin antibody as a control. 15, Vlaro2 silenced mutants; wt, wild type. In the graph, CS expression was quantified and normalized against the tubulin level for the different samples using Kodak Molecular Imaging 4.05 software. Data represent average ± standard deviations of three experimental replicates
Fig. 3
Fig. 3
Phenotypic analysis of the Vlaro2 silenced mutants. a Light microscopy images of V. longisporum wild type (wt) and Vlaro2 silenced mutant (Vlaro2sm) cultured on CDA as observed at 1 and 2 dpi. The colonies were examined microscopically for vegetative growth and spore formation. Black arrows indicate conidia. b Light microscopy images of V. longisporum wild type (wt) and Vlaro2sm cultured on CDA supplemented with 5 mM 5-MT as observed at 1 and 2 dpi. The colonies were examined microscopically for vegetative growth and spore formation. Black arrows indicate conidia. c The graph shows the growth rates of wild-type (wt) and Vlaro2sm on CDA determined by plate-based growth assays. Agar plates were inoculated with 5,000 spores, and colony diameter from three replicates was measured following 3, 6, 9, and 12 days of incubation at 25°C. Data represent average ± standard deviations of three experimental replicates. d The graph shows the growth rates of wild type (wt) and Vlaro2sm on CDA amended with 5-MT determined as described in c
Fig. 4
Fig. 4
Vlaro2 and VlcpcA expression. a Relative expression of Vlaro2 measured by quantitative real-time PCR. Vlaro2 cDNA was normalized to the ß-tubulin cDNA. wt, wild type, Vlaro2sm + 5-MT, V. longisporum strains grown in the presence of 5-MT. The error bars represent the standard deviations of three different measurements of the same cDNA. bVlcpcA expression measured relative to the ß-tubulin cDNA as described in a
Fig. 5
Fig. 5
Assessment of pathogenicity of the Vlaro2 silenced mutant. a Assessment of disease development by scoring for disease symptoms according to Eynck et al. (2007). Plants were scored for disease symptoms at 7, 14, 21, 28, and 35 dpi. Data represent average ± standard deviations of 20 experimental replicates. wt, wild type; Vlaro2sm, Vlaro2 silenced mutant; mock, mock inoculation with water. b Assessment of stunting of rapeseed due to V. longisporum infection. The height of 20 replicates each of rapeseed plants infected with wild type (wt) and Vlaro2 silenced mutant (Vlaro2sm) was measured at 7, 14, 21, 28, and 35 dpi. For comparison, the height of rapeseed plants mock-inoculated (mock) with tap water was also measured. The plants are heavily infected at 28 and 35 dpi. Data represent average ± standard deviations of 20 experimental replicates. c Rapeseed infection assay. Representative B. napus plants shown at 35 dpi. d Assessment of disease development in A. thaliana by measuring the leaf area. The area of the leaves of A. thaliana plants infected with wild type (wt), Vlaro2 silenced mutant (Vlaro2sm) or mock-inoculated with water was measured at 7, 14, 21, and 28 dpi. Data represent average ± standard deviations of 30 experimental replicates. eArabidopsis infection assay. A. thaliana plants are shown at 28 dpi
Fig. 6
Fig. 6
Infection assay and determination of the V. longisporum DNA concentration in the infected plant tissue. aV. longisporum DNA concentration in the plant stem. bV. longisporum DNA concentration in the hypocotyl. V. longisporum DNA was measured with real-time PCR in stem and hypocotyl of B. napus inoculated with Vlaro2 silenced mutant (Vlaro2sm) and wild type (wt) at 14, 21, 28, and 35 dpi. Data represent average ± standard deviations of three experimental replicates. The mock-inoculated plants as a control did not show presence of any V. longisporum DNA. N VL DNA/g FW = nanogram V. longisporum DNA/gram fresh weight of plant tissue. (In the inset, a representative rapeseed plant depicting the stem (5–6 cm from the top) and the hypocotyl harvested for the quantification of the V. longisporum DNA is shown). cV. longisporum DNA concentration in A. thaliana. DNA was measured with real-time PCR in A. thaliana inoculated with Vlaro2 silenced mutant (Vlaro2sm) and wild type (wt) at 28 dpi. Data represent average ± standard deviations of three experimental replicates. The mock-inoculated plants as a control did not show presence of any V. longisporum DNA. P Vl DNA/108molecules of actin, picogram V. longisporum DNA/108 molecules of actin of A. thaliana
Fig. 7
Fig. 7
Quantification of Vlaro2 and VlcpcA expression in B. napus and A. thaliana.a Relative expression of Vlaro2 in B. napus measured by quantitative real-time PCR. Vlaro2 cDNA was normalized to the ß-tubulin cDNA. cDNA from host plants mock-inoculated with water served as control. wt, wild type, Bn_wt, cDNA from B. napus infected with wild type, Bn_Vlaro2sm, cDNA from B. napus infected with Vlaro2 silenced mutant. The error bars represent the standard deviations of triplicates. b Expression of Vlaro2 relative to the ß-tubulin cDNA measured in A. thaliana by quantitative real-time PCR. cDNA from host plants mock-inoculated with water served as control. wt, wild type, At_wt, cDNA from A. thaliana infected with wild type, At_Vlaro2sm, cDNA from A. thaliana infected with Vlaro2 silenced mutant. The error bars represent the standard deviations of triplicates. c Relative expression of VlcpcA in B. napus measured by quantitative real-time PCR. VlcpcA cDNA was normalized to the ß-tubulin cDNA. cDNA from host plants mock-inoculated with water served as control. wt, wild type, Bn_wt, cDNA from B. napus infected with wild type, Bn_Vlaro2sm, cDNA from B. napus infected with Vlaro2 silenced mutant. The error bars represent the standard deviations of triplicates. d Relative expression of VlcpcA in A. thaliana measured by quantitative real-time PCR. VlcpcA cDNA was normalized to the ß-tubulin cDNA. cDNA from host plants mock-inoculated with water served as control. wt, wild type, At_wt, cDNA from A. thaliana infected with wild type, At_Vlaro2sm, cDNA from A. thaliana infected with Vlaro2 silenced mutant. The error bars represent the standard deviations of triplicates

Similar articles

See all similar articles

Cited by 11 articles

See all "Cited by" articles

References

    1. Albrecht G, Mösch HU, Hoffmann B, Reusser U, Braus GH. Monitoring the Gcn4 protein mediated response in the yeast Saccharomyces cerevisiae. J Biol Chem. 1998;273:12696–12702. doi: 10.1074/jbc.273.21.12696. - DOI - PubMed
    1. Arteca RN, Arteca JM. Effects of brassinosteroid, auxin, and cytokinin on ethylene production in Arabidopsis thaliana plants. J Exp Bot. 2008;59:3019–3026. doi: 10.1093/jxb/ern159. - DOI - PMC - PubMed
    1. Benavente LM, Alonso JM. Molecular mechanisms of ethylene signaling in Arabidopsis. Mol Biosyst. 2006;2:165–173. doi: 10.1039/b513874d. - DOI - PubMed
    1. Bentley R, Meganathan R. Biosynthesis of vitamin K (menaquinone) in bacteria. Microbiol Rev. 1982;46:241–280. - PMC - PubMed
    1. Bhaskaran R. In vitro production of indole-3-acetic acid by Verticillium dahliae. Sci Cult. 1972;38:523.

Publication types

Feedback