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The role of vacuolar processing enzyme (VPE) from Nicotiana benthamiana in the elicitor-triggered hypersensitive response and stomatal closure.

Zhang H, Dong S, Wang M, Wang W, Song W, Dou X, Zheng X, Zhang Z - J. Exp. Bot. (2010)

Bottom Line: Although NbVPE silencing does not affect H(2)O(2) accumulation triggered by boehmerin, harpin, or Nep1, the HR is absent in NbVPE1a- and NbVPE1a/1b-silenced plants treated with harpin alone.These results suggest that harpin-triggered HR is VPE-dependent.The accumulation of transcripts associated with defence and cell redox is modified by VPE silencing in elicitor signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, 210095, China.

ABSTRACT
Elicitors/pathogen-associated molecular patterns (PAMPs) trigger the plant immune system, leading to rapid programmed cell death (hypersensitive response, HR) and stomatal closure. Previous reports have shown that the vacuolar processing enzyme (VPE), a cysteine proteinase responsible for the maturation of vacuolar proteins, has caspase-1-like activity and mediates TMV- and mycotoxin-induced cell death. The role of VPE from Nicotiana benthamiana in the response to three elicitors: bacterial harpin, fungal Nep1, and oomycete boehmerin, is described here. Single-silenced (NbVPE1a or NbVPE1b) and dual-silenced (NbVPE1a/1b) N. benthamiana plants were produced by virus-induced gene silencing. Although NbVPE silencing does not affect H(2)O(2) accumulation triggered by boehmerin, harpin, or Nep1, the HR is absent in NbVPE1a- and NbVPE1a/1b-silenced plants treated with harpin alone. However, NbVPE-silenced plants develop a normal HR after boehmerin and Nep1 treatment. These results suggest that harpin-triggered HR is VPE-dependent. Surprisingly, all gene-silenced plants show significantly impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production in guard cells. Dual-silenced plants show increased elicitor-triggered AOS production in guard cells. The accumulation of transcripts associated with defence and cell redox is modified by VPE silencing in elicitor signalling. Overall, these results indicate that VPE from N. benthamiana functions not only in elicitor-induced HR, but also in elicitor-induced stomatal closure, suggesting that VPE may be involved in elicitor-triggered immunity.

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In situ detection of hydrogen peroxide using DAB staining on control, PVX-NbVPE1a, PVX-NbVPE1b-, and PVX-NbVPE1a/1b-infected N. benthamiana leaves in response to elicitors. (A) Photographs of representative ethanol-bleached leaves from control and NbVPE-silenced plants 6 h after PBS (10 mM), boehmerin (50 nM), harpin (50 nM), or Nep1 (50 nM) treatment; elicitation with the elicitor was conducted on plants by infiltrating an equivalent elicitor solution of 25 μl. (B) Quantitative scoring of staining in leaves of the control and NbVPE-silenced plants with elicitor treatment. The analysis was repeated for three sets of independently silenced plants in each experiment; the values shown were the means ±SD of duplicate assays. The experiment was repeated twice with similar results.
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fig3: In situ detection of hydrogen peroxide using DAB staining on control, PVX-NbVPE1a, PVX-NbVPE1b-, and PVX-NbVPE1a/1b-infected N. benthamiana leaves in response to elicitors. (A) Photographs of representative ethanol-bleached leaves from control and NbVPE-silenced plants 6 h after PBS (10 mM), boehmerin (50 nM), harpin (50 nM), or Nep1 (50 nM) treatment; elicitation with the elicitor was conducted on plants by infiltrating an equivalent elicitor solution of 25 μl. (B) Quantitative scoring of staining in leaves of the control and NbVPE-silenced plants with elicitor treatment. The analysis was repeated for three sets of independently silenced plants in each experiment; the values shown were the means ±SD of duplicate assays. The experiment was repeated twice with similar results.

Mentions: Although H2O2 production triggered by an elicitor is strongly correlated with the early defence response preceding HR (Garcia-Brugger et al., 2006; Pitzschke and Hirt, 2006), it is unclear whether elicitor-triggered H2O2 accumulation is dependent on VPE. The contribution of NbVPE1a and NbVPE1b to H2O2 production in response to elicitors was examined. DAB polymerizes on contact with H2O2 in a reaction requiring peroxidase; thus, H2O2 can be visualized in situ as a reddish-brown precipitate (Thordal-Christensen et al., 1997). A heavy staining was observed in control plants 6 h after boehmerin, harpin, or Nep1 treatment (Fig. 3A), which was consistent with the late peak of oxidative production during incompatible plant–pathogen interactions (Baker and Orlandi, 1995; Allan and Fluhr, 1997; Lamb and Dixon, 1997). Light staining was observed after PBS injection. Similar analyses were conducted with NbVPE-silenced plants. No change in DAB staining intensity was observed in NbVPE-silenced plants compared with control plants after boehmerin, harpin, and Nep1 infiltration (Fig. 3B). These data suggest that NbVPE1a and NbVPE1b contribute little to elicitor-induced H2O2 accumulation.


The role of vacuolar processing enzyme (VPE) from Nicotiana benthamiana in the elicitor-triggered hypersensitive response and stomatal closure.

Zhang H, Dong S, Wang M, Wang W, Song W, Dou X, Zheng X, Zhang Z - J. Exp. Bot. (2010)

In situ detection of hydrogen peroxide using DAB staining on control, PVX-NbVPE1a, PVX-NbVPE1b-, and PVX-NbVPE1a/1b-infected N. benthamiana leaves in response to elicitors. (A) Photographs of representative ethanol-bleached leaves from control and NbVPE-silenced plants 6 h after PBS (10 mM), boehmerin (50 nM), harpin (50 nM), or Nep1 (50 nM) treatment; elicitation with the elicitor was conducted on plants by infiltrating an equivalent elicitor solution of 25 μl. (B) Quantitative scoring of staining in leaves of the control and NbVPE-silenced plants with elicitor treatment. The analysis was repeated for three sets of independently silenced plants in each experiment; the values shown were the means ±SD of duplicate assays. The experiment was repeated twice with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2921209&req=5

fig3: In situ detection of hydrogen peroxide using DAB staining on control, PVX-NbVPE1a, PVX-NbVPE1b-, and PVX-NbVPE1a/1b-infected N. benthamiana leaves in response to elicitors. (A) Photographs of representative ethanol-bleached leaves from control and NbVPE-silenced plants 6 h after PBS (10 mM), boehmerin (50 nM), harpin (50 nM), or Nep1 (50 nM) treatment; elicitation with the elicitor was conducted on plants by infiltrating an equivalent elicitor solution of 25 μl. (B) Quantitative scoring of staining in leaves of the control and NbVPE-silenced plants with elicitor treatment. The analysis was repeated for three sets of independently silenced plants in each experiment; the values shown were the means ±SD of duplicate assays. The experiment was repeated twice with similar results.
Mentions: Although H2O2 production triggered by an elicitor is strongly correlated with the early defence response preceding HR (Garcia-Brugger et al., 2006; Pitzschke and Hirt, 2006), it is unclear whether elicitor-triggered H2O2 accumulation is dependent on VPE. The contribution of NbVPE1a and NbVPE1b to H2O2 production in response to elicitors was examined. DAB polymerizes on contact with H2O2 in a reaction requiring peroxidase; thus, H2O2 can be visualized in situ as a reddish-brown precipitate (Thordal-Christensen et al., 1997). A heavy staining was observed in control plants 6 h after boehmerin, harpin, or Nep1 treatment (Fig. 3A), which was consistent with the late peak of oxidative production during incompatible plant–pathogen interactions (Baker and Orlandi, 1995; Allan and Fluhr, 1997; Lamb and Dixon, 1997). Light staining was observed after PBS injection. Similar analyses were conducted with NbVPE-silenced plants. No change in DAB staining intensity was observed in NbVPE-silenced plants compared with control plants after boehmerin, harpin, and Nep1 infiltration (Fig. 3B). These data suggest that NbVPE1a and NbVPE1b contribute little to elicitor-induced H2O2 accumulation.

Bottom Line: Although NbVPE silencing does not affect H(2)O(2) accumulation triggered by boehmerin, harpin, or Nep1, the HR is absent in NbVPE1a- and NbVPE1a/1b-silenced plants treated with harpin alone.These results suggest that harpin-triggered HR is VPE-dependent.The accumulation of transcripts associated with defence and cell redox is modified by VPE silencing in elicitor signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, 210095, China.

ABSTRACT
Elicitors/pathogen-associated molecular patterns (PAMPs) trigger the plant immune system, leading to rapid programmed cell death (hypersensitive response, HR) and stomatal closure. Previous reports have shown that the vacuolar processing enzyme (VPE), a cysteine proteinase responsible for the maturation of vacuolar proteins, has caspase-1-like activity and mediates TMV- and mycotoxin-induced cell death. The role of VPE from Nicotiana benthamiana in the response to three elicitors: bacterial harpin, fungal Nep1, and oomycete boehmerin, is described here. Single-silenced (NbVPE1a or NbVPE1b) and dual-silenced (NbVPE1a/1b) N. benthamiana plants were produced by virus-induced gene silencing. Although NbVPE silencing does not affect H(2)O(2) accumulation triggered by boehmerin, harpin, or Nep1, the HR is absent in NbVPE1a- and NbVPE1a/1b-silenced plants treated with harpin alone. However, NbVPE-silenced plants develop a normal HR after boehmerin and Nep1 treatment. These results suggest that harpin-triggered HR is VPE-dependent. Surprisingly, all gene-silenced plants show significantly impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production in guard cells. Dual-silenced plants show increased elicitor-triggered AOS production in guard cells. The accumulation of transcripts associated with defence and cell redox is modified by VPE silencing in elicitor signalling. Overall, these results indicate that VPE from N. benthamiana functions not only in elicitor-induced HR, but also in elicitor-induced stomatal closure, suggesting that VPE may be involved in elicitor-triggered immunity.

Show MeSH
Related in: MedlinePlus