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. 2018 May 14;7:e34902. doi: 10.7554/eLife.34902

Figure 2. VdSCP41 functions to inhibit immunity in plants.

(A) VdSCP41 localizes to the base of the hyphopodia and forms ring signals surrounding the hyphal neck. The Vd∆scp41/VdSCP41-GFP strain and Vd∆scp41/ΔspVdSCP41-GFP was cultured on a cellophane membrane for 5 days to induce the formation of hyphopodia. Localization of VdSCP41-GFP was visualized using a Leica SP8 microscope. (B) Transiently expressed VdSCP41 preferentially localizes to the nucleus of Arabidopsis cells. Arabidopsis protoplasts were transfected with either a VdSCP41-mCherry or a ΔspVdSCP41-mCherry plasmid as indicated. mCherry fluorescence was visualized 16 hr post transfection. DAPI staining of the nucleus was visualized under UV light. (C–D) Expression of ΔspVdSCP41 in Arabidopsis inhibits nlp20Vd2-induced ICS1 (C) and FMO1 (D) expression. Wildtype (WT) and transgenic plants expressing ΔspVdSCP41 or ΔspVdSCP41-nls were treated with H2O or nlp20Vd2 as indicated. RNA was extracted for real-time PCR analyses. The experiments were repeated three times with similar results. Error bars indicate standard deviations. Student’s t-test was carried out to determine the significance of difference. * Indicates significant difference at a P-value of < 0.05, whereas ** indicates significant difference at a P-value of < 0.01.

Figure 2—source data 1. Source data for Figure 2.
elife-34902-fig2-data1.xlsx (13.8KB, xlsx)
DOI: 10.7554/eLife.34902.009

Figure 2.

Figure 2—figure supplement 1. VdSCP41 delivered by V. dahliae translocates into plant cells and inhibits immunity.

Figure 2—figure supplement 1.

(A) VdSCP41 contains a predicted signal peptide (SP) and a predicted nuclear localization signal (NLS) sequence. ASP predicted by SignalP is shown in red. A predicted NLS is shown in blue. (B) VdSCP41-GFP translocates into the nucleus of onion epidermal cells. Conidia of the V592-GFP, VdSCP41-GFP or VdSCP41-nls-GFP strain were inoculated onto onion epidermal cells. GFP and DAPI staining of nucleus (UV) fluorescence was visualized 3 days post inoculation. The experiments were repeated three times with similar results. (C) Expression of mcherry-tagged VdSCP41 and ΔspVdSCP41 in Arabidopsis protoplasts. Arabidopsis protoplasts were transfected with either VdSCP41-mCherry or ΔspVdSCP41-mCherry plasmid as indicated. Total protein was extracted for anti-mcherry immunoblot. (D) Transiently expressed VdSCP41 localizes to the nucleus of Nicotiana benthamiana (N. b.) cells. N. b. leaf cells were Agro-infiltrated with Agrobacterium strains carrying either VdSCP41-mCherry or ΔspVdSCP41-mCherry. mCherry fluorescence was visualized 48 hr post infiltration. (E) Expression of ΔspVdSCP41 in Arabidopsis inhibits flg22-induced gene expression. Wildtype (WT) and transgenic plants expressing VdSCP41 were treated with H2Oor flg22. RNA was extracted for real-time PCR analyses. The experiments were repeated three times with similar results. Error bars indicate standard deviations. Student’s t-test was carried out to determine the significance of difference between WT and transgenic plants in the same treatment. * Indicates significant difference at a P-value of < 0.05, whereas ** indicates significant difference at a P-value of < 0.01.
Figure 2—figure supplement 2. VdSCP41 inhibits pathogen-induced SA accumulation and gene expression.

Figure 2—figure supplement 2.

(A) ΔspVdSCP41 transgenic lines accumulated less free SA than did WT in response to Pst hrcC-. The plants were treated with or without Pst hrcC– and subjected to free SA measurement. (B–C) The VdΔscp41 mutant has greater expression of ICS1 and FMO1 than the V592 WT strain. Arabidopsis plants were inoculated without (mock) or with V592, or with the VdΔscp41 mutant. RNA was extracted for real-time PCR analyses of ICS1 (B) and FMO1 (C). Different letters indicate significant difference at a P-value of < 0.05. The experiments were repeated twice with similar results.