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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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Elicitor-induced AOS increase in guard cells of the control and NbVPE-silenced plants. In all cases, AOS dye DHR was loaded into cells of epidermal peels, and fluorescence was detected after incubation in PBS (10 mM), boehmerin (50 nM), harpin (50 nM), and Nep1 (50 nM). For each treatment, fluorescence and bright-field images were shown. Results from several experiments were compiled in this figure. Experiments were repeated at least three times, and representative images were shown in (A). Green indicates AOS burst. (B) Quantitative analysis of in vivo AOS generation monitored using DHR fluorescence as shown in (A). Results were presented as mean (n ≥3) fluorescence intensity per pixel.
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fig7: Elicitor-induced AOS increase in guard cells of the control and NbVPE-silenced plants. In all cases, AOS dye DHR was loaded into cells of epidermal peels, and fluorescence was detected after incubation in PBS (10 mM), boehmerin (50 nM), harpin (50 nM), and Nep1 (50 nM). For each treatment, fluorescence and bright-field images were shown. Results from several experiments were compiled in this figure. Experiments were repeated at least three times, and representative images were shown in (A). Green indicates AOS burst. (B) Quantitative analysis of in vivo AOS generation monitored using DHR fluorescence as shown in (A). Results were presented as mean (n ≥3) fluorescence intensity per pixel.

Mentions: It was found that VPE silencing compromised elicitor-induced stomatal closure via the suppression of NO accumulation in guard cells. To evaluate further whether VPE silencing affected the generation of other AOS in elicitor-induced stomatal closure, peroxide and peroxynitrite levels were analysed via incubation with DHR that is oxidized to form the fluorochrome rhodamine 123 in the presence of AOS (Schulz et al., 1996). Neither control nor gene-silenced plants showed AOS fluorescence after PBS treatment. As shown in Fig. 7A, treating guard cells with one of the indicated elicitors resulted in obvious AOS fluorescence. Fluorescence in response to boehmerin in NbVPE1a- and NbVPE1b-silenced plants did not differ from the response in control plants. However, boehmerin-induced AOS fluorescence in NbVPE1a/1b-silenced plants increased markedly compared with that in control plants. Similar results were obtained with harpin and Nep1 treatment (Fig. 7B). These results suggest that NbVPE1a and NbVPE1b show an overlap in the negative regulation of elicitor-induced AOS production in guard cells.


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)

Elicitor-induced AOS increase in guard cells of the control and NbVPE-silenced plants. In all cases, AOS dye DHR was loaded into cells of epidermal peels, and fluorescence was detected after incubation in PBS (10 mM), boehmerin (50 nM), harpin (50 nM), and Nep1 (50 nM). For each treatment, fluorescence and bright-field images were shown. Results from several experiments were compiled in this figure. Experiments were repeated at least three times, and representative images were shown in (A). Green indicates AOS burst. (B) Quantitative analysis of in vivo AOS generation monitored using DHR fluorescence as shown in (A). Results were presented as mean (n ≥3) fluorescence intensity per pixel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Elicitor-induced AOS increase in guard cells of the control and NbVPE-silenced plants. In all cases, AOS dye DHR was loaded into cells of epidermal peels, and fluorescence was detected after incubation in PBS (10 mM), boehmerin (50 nM), harpin (50 nM), and Nep1 (50 nM). For each treatment, fluorescence and bright-field images were shown. Results from several experiments were compiled in this figure. Experiments were repeated at least three times, and representative images were shown in (A). Green indicates AOS burst. (B) Quantitative analysis of in vivo AOS generation monitored using DHR fluorescence as shown in (A). Results were presented as mean (n ≥3) fluorescence intensity per pixel.
Mentions: It was found that VPE silencing compromised elicitor-induced stomatal closure via the suppression of NO accumulation in guard cells. To evaluate further whether VPE silencing affected the generation of other AOS in elicitor-induced stomatal closure, peroxide and peroxynitrite levels were analysed via incubation with DHR that is oxidized to form the fluorochrome rhodamine 123 in the presence of AOS (Schulz et al., 1996). Neither control nor gene-silenced plants showed AOS fluorescence after PBS treatment. As shown in Fig. 7A, treating guard cells with one of the indicated elicitors resulted in obvious AOS fluorescence. Fluorescence in response to boehmerin in NbVPE1a- and NbVPE1b-silenced plants did not differ from the response in control plants. However, boehmerin-induced AOS fluorescence in NbVPE1a/1b-silenced plants increased markedly compared with that in control plants. Similar results were obtained with harpin and Nep1 treatment (Fig. 7B). These results suggest that NbVPE1a and NbVPE1b show an overlap in the negative regulation of elicitor-induced AOS production in guard cells.

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