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Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular Stimulations and Its Role in Verticillium Resistance.

Duan X, Zhang Z, Wang J, Zuo K - PLoS ONE (2016)

Bottom Line: Verticillium pathogens secrete various disease-causing effectors in cotton.Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments.In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology Research Center, School of Agriculture and Life Sciences, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Verticillium wilt is a disastrous vascular disease in plants caused by Verticillium dahliae. Verticillium pathogens secrete various disease-causing effectors in cotton. This study identified a subtilase gene GbSBT1 from Gossypium babardense and investigated the roles against V. dahliae infection. GbSBT1 gene expression is responsive to V. dahliae defense signals, jasmonic acid, and ethylene treatments. Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments. Silencing GbSBT1 gene expression through virus-induced GbSBT1 gene silencing reduced the tolerance of Pima-90 (resistant genotype), but not facilitated the infection process of V. dahliae in Coker-312 (sensitive genotype). Moreover, the ectopically expressed GbSBT1 gene enhanced the resistance of Arabidopsis to Fusarium oxysporum and V. dahliae infection and activated the expression levels of defense-related genes. Furthermore, pull-down, yeast two-hybrid assay, and BiFC analysis revealed that GbSBT1 interacts with a prohibitin (PHB)-like protein expressed in V. dahliae pathogens during infection. In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton.

No MeSH data available.


Related in: MedlinePlus

Reduced resistance of cotton to Verticillium dahliae after GbSBT1 gene silencing.(A–C) Disease symptoms in Gossypium barbadense Pima-90 and the VIGS plants inoculated with V. dahliae strain V991; (D) Enlarged region of disease symptoms on leaf of Pima-90 inoculated with V. dahliae strain V991. Left panels: Pima-90; right panels: Pima-90 containing silenced GbSBT1 gene; dpi: days post-inoculation. (E–G) Disease symptoms in Gossypium hirsutum Coker-312 and the VIGS plants inoculated with V. dahliae strain V991; (H) Enlarged region of disease symptoms on leaf of Coker-312 inoculated with V. dahliae strain V991. Left panels: Coker-312; right panels: Coker-312 containing silenced GhSBT1 gene; dpi: days post-inoculation. (I–I/II) DAB staining in the control and VIGS cotton leaves of G. barbadense after V. dahliae V991 inoculation. (J) Disease index measurement in Pima-90 and Coker-312 cotton plants after inoculation with V991. VIGS experiments were repeated at least three times with more than 10 cotton plants for each construct. Double asterisks represent significant difference between VIGS plants and wild-type plants (P < 0.01) in t-test.
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pone.0153988.g005: Reduced resistance of cotton to Verticillium dahliae after GbSBT1 gene silencing.(A–C) Disease symptoms in Gossypium barbadense Pima-90 and the VIGS plants inoculated with V. dahliae strain V991; (D) Enlarged region of disease symptoms on leaf of Pima-90 inoculated with V. dahliae strain V991. Left panels: Pima-90; right panels: Pima-90 containing silenced GbSBT1 gene; dpi: days post-inoculation. (E–G) Disease symptoms in Gossypium hirsutum Coker-312 and the VIGS plants inoculated with V. dahliae strain V991; (H) Enlarged region of disease symptoms on leaf of Coker-312 inoculated with V. dahliae strain V991. Left panels: Coker-312; right panels: Coker-312 containing silenced GhSBT1 gene; dpi: days post-inoculation. (I–I/II) DAB staining in the control and VIGS cotton leaves of G. barbadense after V. dahliae V991 inoculation. (J) Disease index measurement in Pima-90 and Coker-312 cotton plants after inoculation with V991. VIGS experiments were repeated at least three times with more than 10 cotton plants for each construct. Double asterisks represent significant difference between VIGS plants and wild-type plants (P < 0.01) in t-test.

Mentions: We generated GbSBT1 gene-silenced cotton plants through VIGS to study the function of the GbSBT1 gene during V. dahliae infection. After growing in a greenhouse for 7 days, the V. dahliae resistance of the control (Pima-90) and VIGS plant lines was subsequently examined and quantified. qPCR results showed that GbSBT1 was successfully repressed in VIGS plants. Results indicated that GbSBT1 expression in the VIGS plants was repressed significantly (about 76% lower than control) compared with control plants (S1 Fig). The GbSBT1 gene-silenced and control plants were inoculated with V. dahliae strain V991. As shown in Fig 5A, GbSBT1-silenced plants grew as normal as the control plants before fungal inoculation. Ten days after inoculation, the silenced plants began to display disease symptoms in the form of margin-wilted leaves; by contrast, the control cotton plants grew normally (Fig 5B). Thirteen days after inoculation, the GbSBT1-silenced plants were severely affected and the majority of leaves turned yellow and tended to wilt (Fig 5C, right panel). Moreover, the rate of diseased plants and the disease index were significantly higher in the GbSBT1-silenced plants than in the control plants (Fig 5J). This result suggests that GbSBT1 knockdown in cotton plants increases plant susceptibility to V. dahliae infection.


Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular Stimulations and Its Role in Verticillium Resistance.

Duan X, Zhang Z, Wang J, Zuo K - PLoS ONE (2016)

Reduced resistance of cotton to Verticillium dahliae after GbSBT1 gene silencing.(A–C) Disease symptoms in Gossypium barbadense Pima-90 and the VIGS plants inoculated with V. dahliae strain V991; (D) Enlarged region of disease symptoms on leaf of Pima-90 inoculated with V. dahliae strain V991. Left panels: Pima-90; right panels: Pima-90 containing silenced GbSBT1 gene; dpi: days post-inoculation. (E–G) Disease symptoms in Gossypium hirsutum Coker-312 and the VIGS plants inoculated with V. dahliae strain V991; (H) Enlarged region of disease symptoms on leaf of Coker-312 inoculated with V. dahliae strain V991. Left panels: Coker-312; right panels: Coker-312 containing silenced GhSBT1 gene; dpi: days post-inoculation. (I–I/II) DAB staining in the control and VIGS cotton leaves of G. barbadense after V. dahliae V991 inoculation. (J) Disease index measurement in Pima-90 and Coker-312 cotton plants after inoculation with V991. VIGS experiments were repeated at least three times with more than 10 cotton plants for each construct. Double asterisks represent significant difference between VIGS plants and wild-type plants (P < 0.01) in t-test.
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Related In: Results  -  Collection

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pone.0153988.g005: Reduced resistance of cotton to Verticillium dahliae after GbSBT1 gene silencing.(A–C) Disease symptoms in Gossypium barbadense Pima-90 and the VIGS plants inoculated with V. dahliae strain V991; (D) Enlarged region of disease symptoms on leaf of Pima-90 inoculated with V. dahliae strain V991. Left panels: Pima-90; right panels: Pima-90 containing silenced GbSBT1 gene; dpi: days post-inoculation. (E–G) Disease symptoms in Gossypium hirsutum Coker-312 and the VIGS plants inoculated with V. dahliae strain V991; (H) Enlarged region of disease symptoms on leaf of Coker-312 inoculated with V. dahliae strain V991. Left panels: Coker-312; right panels: Coker-312 containing silenced GhSBT1 gene; dpi: days post-inoculation. (I–I/II) DAB staining in the control and VIGS cotton leaves of G. barbadense after V. dahliae V991 inoculation. (J) Disease index measurement in Pima-90 and Coker-312 cotton plants after inoculation with V991. VIGS experiments were repeated at least three times with more than 10 cotton plants for each construct. Double asterisks represent significant difference between VIGS plants and wild-type plants (P < 0.01) in t-test.
Mentions: We generated GbSBT1 gene-silenced cotton plants through VIGS to study the function of the GbSBT1 gene during V. dahliae infection. After growing in a greenhouse for 7 days, the V. dahliae resistance of the control (Pima-90) and VIGS plant lines was subsequently examined and quantified. qPCR results showed that GbSBT1 was successfully repressed in VIGS plants. Results indicated that GbSBT1 expression in the VIGS plants was repressed significantly (about 76% lower than control) compared with control plants (S1 Fig). The GbSBT1 gene-silenced and control plants were inoculated with V. dahliae strain V991. As shown in Fig 5A, GbSBT1-silenced plants grew as normal as the control plants before fungal inoculation. Ten days after inoculation, the silenced plants began to display disease symptoms in the form of margin-wilted leaves; by contrast, the control cotton plants grew normally (Fig 5B). Thirteen days after inoculation, the GbSBT1-silenced plants were severely affected and the majority of leaves turned yellow and tended to wilt (Fig 5C, right panel). Moreover, the rate of diseased plants and the disease index were significantly higher in the GbSBT1-silenced plants than in the control plants (Fig 5J). This result suggests that GbSBT1 knockdown in cotton plants increases plant susceptibility to V. dahliae infection.

Bottom Line: Verticillium pathogens secrete various disease-causing effectors in cotton.Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments.In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology Research Center, School of Agriculture and Life Sciences, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Verticillium wilt is a disastrous vascular disease in plants caused by Verticillium dahliae. Verticillium pathogens secrete various disease-causing effectors in cotton. This study identified a subtilase gene GbSBT1 from Gossypium babardense and investigated the roles against V. dahliae infection. GbSBT1 gene expression is responsive to V. dahliae defense signals, jasmonic acid, and ethylene treatments. Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments. Silencing GbSBT1 gene expression through virus-induced GbSBT1 gene silencing reduced the tolerance of Pima-90 (resistant genotype), but not facilitated the infection process of V. dahliae in Coker-312 (sensitive genotype). Moreover, the ectopically expressed GbSBT1 gene enhanced the resistance of Arabidopsis to Fusarium oxysporum and V. dahliae infection and activated the expression levels of defense-related genes. Furthermore, pull-down, yeast two-hybrid assay, and BiFC analysis revealed that GbSBT1 interacts with a prohibitin (PHB)-like protein expressed in V. dahliae pathogens during infection. In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton.

No MeSH data available.


Related in: MedlinePlus