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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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Local induction of hypersensitivity responses with boehmerin (50 nM), harpin (50 nM), and Nep1 (50 nM). (A) Leaves (representative of three replicate treatments) from control PVX, NbVPE1a-, NbVPE1b-, and NbVPE1a/1b-silenced N. benthamiana were infiltrated with the elicitors simultaneously. The red and black circles indicate cell death and no cell death, respectively. Leaves were removed from plants after 3 d of treatment (left panels) and bleached in ethanol (right panels). (B) Trypan blue staining of leaves from control and NbVPE1a-, NbVPE1b-, and NbVPE1a/1b-silenced plants in response to boehmerin, harpin, and Nep1. Responses in the inoculated leaf are shown. Pictures were taken from plants after 24 h of treatment. The experiments were performed in triplicate. Bars, 1 cm.
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fig2: Local induction of hypersensitivity responses with boehmerin (50 nM), harpin (50 nM), and Nep1 (50 nM). (A) Leaves (representative of three replicate treatments) from control PVX, NbVPE1a-, NbVPE1b-, and NbVPE1a/1b-silenced N. benthamiana were infiltrated with the elicitors simultaneously. The red and black circles indicate cell death and no cell death, respectively. Leaves were removed from plants after 3 d of treatment (left panels) and bleached in ethanol (right panels). (B) Trypan blue staining of leaves from control and NbVPE1a-, NbVPE1b-, and NbVPE1a/1b-silenced plants in response to boehmerin, harpin, and Nep1. Responses in the inoculated leaf are shown. Pictures were taken from plants after 24 h of treatment. The experiments were performed in triplicate. Bars, 1 cm.

Mentions: Typical hypersensitive cell death occurred in the leaves 24 h after the elicitor (i.e. boehmerin, harpin, or Nep1) was infiltrated into control PVX-infected N. benthamiana leaves. As shown in Fig. 2A, typical hypersensitive cell death was also observed in the gene-silenced plants after boehmerin or Nep1 infiltration. However, typical hypersensitive cell death did not occur in NbVPE1a- or NbVPE1a/1b-silenced leaves after harpin infiltration. Although ethanol bleaching can be used to enhance the visualization of HR in leaves (Schornack et al., 2004; Weber et al., 2005; Gan et al., 2009), HR remained undetectable in NbVPE1a- and NbVPE1a/1b-silenced leaves 72 h after harpin treatment (Fig. 2A). Cell death was further investigated in situ using trypan blue, which accumulated in dead cells. The application of boehmerin, harpin, and Nep1 induced blue staining that was localized to treated tissues, whereas leaves of NbVPE1a- and NbVPE1a/1b-silenced plants infiltrated with harpin remained unstained, with a negligible number of blue spots (Fig. 2B). These results indicate that NbVPE1a, but not NbVPE1b, is required for harpin-mediated cell death, but not for the boehmerin- or Nep1-triggered cell death response. The results also suggest that the molecular basis of hypersensitive cell death triggered by harpin may differ from that after boehmerin or Nep1 treatment.


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)

Local induction of hypersensitivity responses with boehmerin (50 nM), harpin (50 nM), and Nep1 (50 nM). (A) Leaves (representative of three replicate treatments) from control PVX, NbVPE1a-, NbVPE1b-, and NbVPE1a/1b-silenced N. benthamiana were infiltrated with the elicitors simultaneously. The red and black circles indicate cell death and no cell death, respectively. Leaves were removed from plants after 3 d of treatment (left panels) and bleached in ethanol (right panels). (B) Trypan blue staining of leaves from control and NbVPE1a-, NbVPE1b-, and NbVPE1a/1b-silenced plants in response to boehmerin, harpin, and Nep1. Responses in the inoculated leaf are shown. Pictures were taken from plants after 24 h of treatment. The experiments were performed in triplicate. Bars, 1 cm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Local induction of hypersensitivity responses with boehmerin (50 nM), harpin (50 nM), and Nep1 (50 nM). (A) Leaves (representative of three replicate treatments) from control PVX, NbVPE1a-, NbVPE1b-, and NbVPE1a/1b-silenced N. benthamiana were infiltrated with the elicitors simultaneously. The red and black circles indicate cell death and no cell death, respectively. Leaves were removed from plants after 3 d of treatment (left panels) and bleached in ethanol (right panels). (B) Trypan blue staining of leaves from control and NbVPE1a-, NbVPE1b-, and NbVPE1a/1b-silenced plants in response to boehmerin, harpin, and Nep1. Responses in the inoculated leaf are shown. Pictures were taken from plants after 24 h of treatment. The experiments were performed in triplicate. Bars, 1 cm.
Mentions: Typical hypersensitive cell death occurred in the leaves 24 h after the elicitor (i.e. boehmerin, harpin, or Nep1) was infiltrated into control PVX-infected N. benthamiana leaves. As shown in Fig. 2A, typical hypersensitive cell death was also observed in the gene-silenced plants after boehmerin or Nep1 infiltration. However, typical hypersensitive cell death did not occur in NbVPE1a- or NbVPE1a/1b-silenced leaves after harpin infiltration. Although ethanol bleaching can be used to enhance the visualization of HR in leaves (Schornack et al., 2004; Weber et al., 2005; Gan et al., 2009), HR remained undetectable in NbVPE1a- and NbVPE1a/1b-silenced leaves 72 h after harpin treatment (Fig. 2A). Cell death was further investigated in situ using trypan blue, which accumulated in dead cells. The application of boehmerin, harpin, and Nep1 induced blue staining that was localized to treated tissues, whereas leaves of NbVPE1a- and NbVPE1a/1b-silenced plants infiltrated with harpin remained unstained, with a negligible number of blue spots (Fig. 2B). These results indicate that NbVPE1a, but not NbVPE1b, is required for harpin-mediated cell death, but not for the boehmerin- or Nep1-triggered cell death response. The results also suggest that the molecular basis of hypersensitive cell death triggered by harpin may differ from that after boehmerin or Nep1 treatment.

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