Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 12;16(9):1629-1643.
doi: 10.1111/pbi.12900. Online ahead of print.

Host-induced gene silencing of a regulator of G protein signalling gene (VdRGS1) confers resistance to Verticillium wilt in cotton

Jun Xu  1 , Xinyu Wang  2 , Yongqing Li  1 , Jianguo Zeng  2 , Guilin Wang  1 , Chaoyang Deng  1 , Wangzhen Guo  1
Affiliations

Host-induced gene silencing of a regulator of G protein signalling gene (VdRGS1) confers resistance to Verticillium wilt in cotton

Jun Xu et al. Plant Biotechnol J. .

Abstract

Verticillium wilt (VW), caused by soil-borne fungi of the genus Verticillium, is a serious disease affecting a wide range of plants and leading to a constant and major challenge to agriculture worldwide. Cotton (Gossypium hirsutum) is the world's most important natural textile fibre and oil crop. VW of cotton is a highly devastating vascular disease; however, few resistant germplasms have been reported in cotton. An increasing number of studies have shown that RNA interference (RNAi)-based host-induced gene silencing (HIGS) is an effective strategy for improving plant resistance to pathogens by silencing genes essential for the pathogenicity of these pathogens. Here, we have identified and characterized multifunctional regulators of G protein signalling (RGS) in the Verticillium dahliae virulence strain, Vd8. Of eight VdRGS genes, VdRGS1 showed the most significant increase in expression in V. dahliae after treating with the roots of cotton seedlings. Based on the phenotype detection of VdRGS1 deletion and complementation mutants, we found that VdRGS1 played crucial roles in spore production, hyphal development, microsclerotia formation and pathogenicity. Tobacco rattle virus-mediated HIGS in cotton plants silenced VdRGS1 transcripts in invaded V. dahliae strains and enhanced broad-spectrum resistance to cotton VW. Our data demonstrate that VdRGS1 is a conserved and essential gene for V. dahliae virulence. HIGS of VdRGS1 provides effective control against V. dahliae infection and could obtain the durable disease resistance in cotton and in other VW-susceptible host crops by developing the stable transformants.

Keywords: Verticillium dahliae; cotton (Gossypium hirsutum); host-induced gene silencing; pathogenicity; regulator of G protein signalling.

PubMed Disclaimer

Conflict of interest statement

The authors declared that they had no competing interests.

Figures

Figure 1
Figure 1
Identification and expression of RGS genes in Verticillium dahlia. (a) Phylogenetic relationship of the eight RGS genes in V. dahliae. (b) Prediction of the domains of the eight RGS proteins. The eight RGS proteins not only have the RGS domain but also contain other functional domains. (c) Expression of eight RGS genes in V. dahliae induced by the roots of cotton seedlings at different time points. The roots of 2‐week‐old cotton seedlings are used to induce the V. dahliae. Verticillium dahliae spore suspensions induced for 0, 2, 4, 6, 12 and 24 hours (h) were collected separately and used for RNA isolation. RGS gene expressions were examined by qRT–PCR. The data represent the mean ± SD of three samples from three independent tests at each time point. ‘*’: significant difference at P < 0.05; ‘**’: significant difference at P < 0.01.
Figure 2
Figure 2
Strategies and confirmation of VdRGS1 deletion and complementation. (a) Schematic diagram of VdRGS1 replacement by HPH via homologous recombination. (b) Confirmation of VdRGS1 deletion and complementation mutants by RT‐PCR using VdRGS1 gene‐specific primers. The β‐tubulin gene was used as the internal control. (c) Confirmation of VdRGS1 deletion by DNA sequencing of the VdRGS1 locus. (d) Confirmation of VdRGS1 deletion by Southern blot analysis. Total genomic DNA wild‐type strain Vd8 (WT), ΔVdRGS1 and VdRGS1‐com were digested with Sac I and subjected to Southern blot analysis. The probe location is indicated in Figure 2a.
Figure 3
Figure 3
Effects of VdRGS1 deletion on spore germination, hyphal development, spore production and microsclerotia formation. (a) Microscopy of spores of ΔVdRGS1 and VdRGS1‐com compared to Vd8. Bars = 100 μm. (b) Microscopy of hyphal growth from single spore of Vd8, ΔVdRGS1 and VdRGS1‐com strains cultured on CM agar plates. Photographs were taken at 24 and 36 h postincubation, respectively. (c) The strains of Vd8, ΔVdRGS1 and VdRGS1‐com from single spore cultured in the CM medium for 5 days at 24 °C. Further, observation of microsclerotia produced in these three strains on BM medium. The spore suspensions of the Vd8, ΔVdRGS 1 and VdRGS 1‐com were diluted to 1 × 105 spores/mL, and sprayed on cellulose membrane placed onto BM plates and incubated at 24 °C for 5 days. (d) Microscopy of microsclerotia produced by Vd8, ΔVdRGS1 and VdRGS1‐com 5 days postinoculation under 4× and 10× mean objective magnification of microscope. (e) Expression of genes involved in melanin biosynthesis in each of Vd8, ΔVdRGS1 and VdRGS1‐com strains. The data represent the mean ± SD of three samples from three independent tests. ‘**’: significant difference at P < 0.01.
Figure 4
Figure 4
Virulence tests of ΔVdRGS 1. (a) Disease symptoms of cotton plants (Junmian 1) infected with ΔVdRGS 1 compared with wild‐type Vd8 and VdRGS1‐com strains. Photographs were taken at 11, 15 and 20 days postinoculation. (b) Vascular discoloration of cotton plants inoculated with indicated strains. Plants were inoculated with Verticillium dahliae spore suspensions (107 conidia/mL) for 15 days, and then, the stems were cut and photographed by stereoscope (Olympus MVX10, Tokyo, Japan). (c) The percentage of diseased leaves inoculated with ΔVdRGS1 compared with wild‐type Vd8 and VdRGS1‐com strains. (d) Disease index (DI) of cotton plants inoculated the wild‐type Vd8, ΔVdRGS1 and VdRGS1‐com at 20 dpi. These experiments were repeated for three times using at least 40 seedlings per treatment. Error bars show the standard deviation of three biological replicates. Asterisks indicate statistically significant differences in the percentage of diseased leaves and the DI of plants treated with ΔVdRGS1 and VdRGS1‐com mutants and the wild‐type Vd8 strain, as determined by Student's t‐tests (*P < 0.05, **P < 0.01).
Figure 5
Figure 5
Effects of TRV‐based HIGS on GFP expression in spores of V991‐GFP strain. GFP expression in spores of V991‐GFP strain was observed after its challenge of cotton plant infiltrated with TRV: GFP construct. For spore recovery, stems of cotton plants infiltrated with TRV: 00, TRV: GFP constructs, respectively, were sampled at 15 days after V991‐GFP inoculation, then incubated on the CM medium at 24 °C for 3 days. The GFP fluorescence in recovered spores from different treatments was observed under the confocal microscopy. Bar = 10 μm.
Figure 6
Figure 6
Effects of VdRGS1 HIGS on cotton resistance to Verticillium dahliae Vd8 infection. (a) Expression levels of VdRGS1 in Vd8‐infected cotton plants infiltrated with various TRV: VdRGS1 constructs. VdRGS1‐1, VdRGS1‐2, VdRGS1‐3 and VdRGS1‐4 represent the different cDNA fragments within the VdRGS1 coding region that were inserted in TRV vector. The data represent the mean ± SD of three samples from three independent tests. ‘*’: significant difference at P < 0.05; ‘**’: significant difference at P < 0.01. (b) Disease symptoms of the VdRGS1 HIGS cotton plants infiltrated with TRV: VdRGS1‐1, VdRGS1‐2, VdRGS1‐3, VdRGS1‐4 constructs compared with ΔVdRGS 1 infected and the controls. (c) The percentage of diseased leaves in TRV: VdRGS1‐1, VdRGS1‐2, VdRGS1‐3, VdRGS1‐4 infiltrated cotton plants and the controls after Vd8 inoculation. These experiments were repeated for three times using at least 40 seedlings per treatment. Error bars show the standard deviation of three biological replicates. Asterisks indicate statistically significant differences determined by Student's t‐tests (*P < 0.05, **P < 0.01). (d) Vascular discoloration in stems of different HIGS and control plants inoculated with V991‐GFP strain. Plants were inoculated with V. dahliae spore suspensions (107 conidia/mL) and photographed by stereoscope (Olympus MVX10, Japan) at 15 dpi.
Figure 7
Figure 7
Effects of VdRGS1 HIGS on cotton resistance to Verticillium dahliae V991 infection. (a) Expression levels of the VdRGS1 in the invaded V991 of different VIGS plants and compared with the TRV: 00 seedlings. Fifteen days after V991 inoculation, the stems of the different treatments were sampled and incubated on the CM medium at 24 °C for 3 days, and the strains were used for RNA extraction and qRT‐PCR analysis. The data represent the mean ± SD of three samples from three independent tests at each time point. ‘*’: significant difference at P < 0.05; ‘**’: significant difference at P < 0.01. (b) Disease symptoms of the VdRGS1‐1, VdRGS1‐2, VdRGS1‐3, VdRGS1‐4‐silenced, TRV: 00 and CK plants at 20 and 25 days after V. dahliae V991 inoculation. (c) The percentage of diseased leaves of the VdRGS1‐1, VdRGS1‐2, VdRGS1‐3, VdRGS1‐4‐silenced plants and controls after V991 inoculation. These experiments were repeated using at least 40 seedlings per treatment. Error bars show the standard deviation of three biological replicates. Asterisks indicate statistically significant differences in the percentage of diseased leaves between treated plants and TRV: 00 controls, as determined by Student's t‐tests (*P < 0.05, **P < 0.01).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ballon, D.R. , Flanary, P.L. , Gladue, D.P. , Konopka, J.B. , Dohlman, H.G. and Thorner, J. (2006) DEP‐domain‐mediated regulation of GPCR signaling responses. Cell, 126, 1079–1093. - PubMed
    1. Baum, J.A. , Bogaert, T. , Clinton, W. , Heck, G.R. , Feldmann, P. , Ilagan, O. , Johnson, S. et al. (2007) Control of coleopteran insect pests through RNA interference. Nat. Biotechnol. 25, 1322–1326. - PubMed
    1. Bolek, Y. , El‐Zik, K.M. , Pepper, A.E. , Bell, A.A. , Magill, C.W. , Thaxton, P.M. and Reddy, O.U.K. (2005) Mapping of Verticillium wilt resistance genes in cotton. Plant Sci. 168, 1581–1590.
    1. Burchett, S.A. (2000) Regulators of G protein signaling: a bestiary of modular protein binding domains. J. Neurochem. 75, 1335–1351. - PubMed
    1. Burchett, S.A. , Flanary, P. , Aston, C. , Jiang, L. , Young, K.H. , Uetz, P. , Fields, S. et al. (2002) Regulation of stress response signaling by the N‐terminal dishevelled/EGL‐10/pleckstrin domain of Sst2, a regulator of G protein signaling in Saccharomyces cerevisiae . J. Biol. Chem. 277, 22156–22167. - PubMed

LinkOut - more resources