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Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

Li D, Zhang H, Song Q, Wang L, Liu S, Hong Y, Huang L, Song F - BMC Plant Biol. (2015)

Bottom Line: Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes.The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells.VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China. dyli@zju.edu.cn.

ABSTRACT

Background: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive.

Results: A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells.

Conclusion: VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

No MeSH data available.


Related in: MedlinePlus

Transient expression of Sl3-MMP in N. benthamiana conferred an increased resistance to B. cinerea. a Expression of Sl3-MMP. Agrobacteria carrying pFGC-Sl3-MMP or pFGC-eGFP were infiltrated into leaves of N. benthamiana and expression of Sl3-MMP was analyzed by qRT-PCR. Relative expression levels were calculated by comparing with the corresponding values at 0 h (as a control) after infiltration. b Immunoblot analysis of Sl3-MMP-GFP fusion proteins in N. benthamiana leaves at 48 h after agroinfiltration. A GFP-specific antibody was used for detection of GFP-fusion protein. Equal loading of total proteins was examined by Ponceau staining. c and d Disease symptom and lesion size. e Expression of defense-related genes. Opposite part of the leaves infiltrated with Sl3-MMP-GFP or pFGC-eGFP was inoculated by dropping spore suspension (2 × 105 spores/mL) of B. cinerea and lesion sizes were measured at 5 days after inoculation. Data presented in d are the means ± SD from a minimum of 60 lesions. Data presented in a and e are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level
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Fig9: Transient expression of Sl3-MMP in N. benthamiana conferred an increased resistance to B. cinerea. a Expression of Sl3-MMP. Agrobacteria carrying pFGC-Sl3-MMP or pFGC-eGFP were infiltrated into leaves of N. benthamiana and expression of Sl3-MMP was analyzed by qRT-PCR. Relative expression levels were calculated by comparing with the corresponding values at 0 h (as a control) after infiltration. b Immunoblot analysis of Sl3-MMP-GFP fusion proteins in N. benthamiana leaves at 48 h after agroinfiltration. A GFP-specific antibody was used for detection of GFP-fusion protein. Equal loading of total proteins was examined by Ponceau staining. c and d Disease symptom and lesion size. e Expression of defense-related genes. Opposite part of the leaves infiltrated with Sl3-MMP-GFP or pFGC-eGFP was inoculated by dropping spore suspension (2 × 105 spores/mL) of B. cinerea and lesion sizes were measured at 5 days after inoculation. Data presented in d are the means ± SD from a minimum of 60 lesions. Data presented in a and e are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level

Mentions: To further confirm the function of Sl3-MMP in disease resistance, we examined whether overexpression of Sl3-MMP could confer an increased resistance to B. cinerea. In our qRT-PCR experiments, transcripts of putative N. benthamiana homolog(s) of Sl3-MMP was detected using Sl3-MMP-specific primers in GFP-infiltrated plants, probably due to high level of sequence similarity/identity among Sl3-MMP and the homologous MMP genes in N. benthamiana. However, agroinfiltration did not significantly affect the transcript levels of endogenous N. benthamiana homologous genes in GFP-infiltrated plants (Fig. 9a). When transiently expressed in N. benthamiana leaves, high levels of Sl3-MMP expression, as estimated by the significant increases in the transcript levels of Sl3-MMP and the endogenous homologous genes in Sl3-MMP-infiltrated plants over the levels of the endogenous homologous genes in GFP-infiltrated plants, and the Sl3-MMP-GFP (a fusion of Sl3-MMP with GFP) fusion protein were detected during a period of 48 h after infiltration (Fig. 9a and b). In disease assays, the lesions on leaves from Sl3-MMP-infiltrated N. benthamiana plants were significantly smaller than that in GFP-infiltrated control plants (Fig. 9c), leading to approximately 40 % of reduction in lesion size at 5 days after inoculation (Fig. 9d). To examine whether an increased defense response was linked to the enhanced resistance resulted from the transient expression of Sl3-MMP, we analyzed and compared the expression of some selected defense-related genes in leaves of the GFP- and Sl3-MMP-infiltrated N. benthamiana plants. As shown in Fig. 9e, the expression of PR1, PR2, PR3 and PR4 in Sl3-MMP-infiltrated plants were significantly upregulated at 24 h after infiltration, showing 5-24 folds of increases over those in GFP-infiltrated plants, whereas no significant difference in the levels of these defense-related genes was observed between GFP-infiltrated plants at 0 h and 24 h and between GFP- and Sl3-MMP-infiltrated plants at 0 h after infiltration (Fig. 9e). These data demonstrate that transient expression of Sl3-MMP in N. benthamiana plants conferred an increased resistance against B. cinerea through an activated defense response resulted from the upregulated expression of defense-related genes.Fig. 9


Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

Li D, Zhang H, Song Q, Wang L, Liu S, Hong Y, Huang L, Song F - BMC Plant Biol. (2015)

Transient expression of Sl3-MMP in N. benthamiana conferred an increased resistance to B. cinerea. a Expression of Sl3-MMP. Agrobacteria carrying pFGC-Sl3-MMP or pFGC-eGFP were infiltrated into leaves of N. benthamiana and expression of Sl3-MMP was analyzed by qRT-PCR. Relative expression levels were calculated by comparing with the corresponding values at 0 h (as a control) after infiltration. b Immunoblot analysis of Sl3-MMP-GFP fusion proteins in N. benthamiana leaves at 48 h after agroinfiltration. A GFP-specific antibody was used for detection of GFP-fusion protein. Equal loading of total proteins was examined by Ponceau staining. c and d Disease symptom and lesion size. e Expression of defense-related genes. Opposite part of the leaves infiltrated with Sl3-MMP-GFP or pFGC-eGFP was inoculated by dropping spore suspension (2 × 105 spores/mL) of B. cinerea and lesion sizes were measured at 5 days after inoculation. Data presented in d are the means ± SD from a minimum of 60 lesions. Data presented in a and e are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4465618&req=5

Fig9: Transient expression of Sl3-MMP in N. benthamiana conferred an increased resistance to B. cinerea. a Expression of Sl3-MMP. Agrobacteria carrying pFGC-Sl3-MMP or pFGC-eGFP were infiltrated into leaves of N. benthamiana and expression of Sl3-MMP was analyzed by qRT-PCR. Relative expression levels were calculated by comparing with the corresponding values at 0 h (as a control) after infiltration. b Immunoblot analysis of Sl3-MMP-GFP fusion proteins in N. benthamiana leaves at 48 h after agroinfiltration. A GFP-specific antibody was used for detection of GFP-fusion protein. Equal loading of total proteins was examined by Ponceau staining. c and d Disease symptom and lesion size. e Expression of defense-related genes. Opposite part of the leaves infiltrated with Sl3-MMP-GFP or pFGC-eGFP was inoculated by dropping spore suspension (2 × 105 spores/mL) of B. cinerea and lesion sizes were measured at 5 days after inoculation. Data presented in d are the means ± SD from a minimum of 60 lesions. Data presented in a and e are the means ± SD from three independent experiments and different letters above the columns indicate significant differences at p < 0.05 level
Mentions: To further confirm the function of Sl3-MMP in disease resistance, we examined whether overexpression of Sl3-MMP could confer an increased resistance to B. cinerea. In our qRT-PCR experiments, transcripts of putative N. benthamiana homolog(s) of Sl3-MMP was detected using Sl3-MMP-specific primers in GFP-infiltrated plants, probably due to high level of sequence similarity/identity among Sl3-MMP and the homologous MMP genes in N. benthamiana. However, agroinfiltration did not significantly affect the transcript levels of endogenous N. benthamiana homologous genes in GFP-infiltrated plants (Fig. 9a). When transiently expressed in N. benthamiana leaves, high levels of Sl3-MMP expression, as estimated by the significant increases in the transcript levels of Sl3-MMP and the endogenous homologous genes in Sl3-MMP-infiltrated plants over the levels of the endogenous homologous genes in GFP-infiltrated plants, and the Sl3-MMP-GFP (a fusion of Sl3-MMP with GFP) fusion protein were detected during a period of 48 h after infiltration (Fig. 9a and b). In disease assays, the lesions on leaves from Sl3-MMP-infiltrated N. benthamiana plants were significantly smaller than that in GFP-infiltrated control plants (Fig. 9c), leading to approximately 40 % of reduction in lesion size at 5 days after inoculation (Fig. 9d). To examine whether an increased defense response was linked to the enhanced resistance resulted from the transient expression of Sl3-MMP, we analyzed and compared the expression of some selected defense-related genes in leaves of the GFP- and Sl3-MMP-infiltrated N. benthamiana plants. As shown in Fig. 9e, the expression of PR1, PR2, PR3 and PR4 in Sl3-MMP-infiltrated plants were significantly upregulated at 24 h after infiltration, showing 5-24 folds of increases over those in GFP-infiltrated plants, whereas no significant difference in the levels of these defense-related genes was observed between GFP-infiltrated plants at 0 h and 24 h and between GFP- and Sl3-MMP-infiltrated plants at 0 h after infiltration (Fig. 9e). These data demonstrate that transient expression of Sl3-MMP in N. benthamiana plants conferred an increased resistance against B. cinerea through an activated defense response resulted from the upregulated expression of defense-related genes.Fig. 9

Bottom Line: Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes.The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells.VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China. dyli@zju.edu.cn.

ABSTRACT

Background: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive.

Results: A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells.

Conclusion: VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

No MeSH data available.


Related in: MedlinePlus