- Email: [email protected]
Heat shock protein 70 is required for tabtoxinine-β-lactam-induced cell death in Nicotiana benthamiana
Journal of Plant Physiology 171 (2014) 173–178
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short communication
Heat shock protein 70 is required for tabtoxinine--lactam-induced cell death in Nicotiana benthamiana Makoto Ito a , Yu Yamamoto b , Chul-Sa Kim b , Kouhei Ohnishi c , Yasufumi Hikichi a , Akinori Kiba a,∗ a
Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kochi University, Nankoku 783-8502, Japan Laboratory of Bioactive Substance Chemistry, Faculty of Agriculture, Kochi University, Nankoku 783-8502, Japan c Research Institute of Molecular Genetics, Kochi University, Nankoku 783-8502, Japan b
a r t i c l e
i n f o
Article history: Received 1 July 2013 Received in revised form 15 October 2013 Accepted 22 October 2013 Available online 28 November 2013 Keywords: Cell death Heat shock protein 70 Nicotiana benthamiana Tabtoxinine--lactam Virus-induced gene silencing
a b s t r a c t Tabtoxinine--lactam (TL), a non-specific bacterial toxin, is produced by Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease. TL causes death of plant cells through the inhibition of glutamine synthetase, which leads to an abnormal accumulation of ammonium ions and the characteristic necrotic wildfire lesions. To better understand the mechanisms involved in TL-induced cell death, we studied its regulation in Nicotiana benthamiana. TL-induced lesions, similar to those in controls, could be observed in SGT1-, RAR1- and Hsp90-silenced plants. In contrast, Hsp70-silenced plants showed suppression of lesion formation. Expression of hin1, a marker gene for the hypersensitive response (HR), which is a characteristic of programmed cell death in plants, was strongly induced in controls by TL treatment but only slightly in Hsp70-silenced plants. However, in these TL-treated Hsp70-silenced plants, the amount of ammonium ions was considerably increased. Furthermore, the silencing of Hsp70 also suppressed l-methionine sulfoximine-induced cell death and hin1 expression and caused the over-accumulation of ammonium ions. When inoculated directly with P. syringae pv. tabaci, Hsp70-silenced plants showed only reduced symptoms. Our results suggest that the TL-induced pathway to cell death in N. benthamiana is at least partially similar to HR response, and that Hsp70 might play an essential role in these events. © 2013 Elsevier GmbH. All rights reserved.
Introduction Programmed cell death (PCD) has important roles not only in the process of plant development but also in incompatible plant–microbe interaction (Jones and Dangl, 2006). Plants may use apoptotic machinery similar to those of animals and yeast, because similar morphological and biochemical features are shared for PCD in these organisms (Gilchrist, 1998; Beers and McDowell, 2001; Greenberg and Yao, 2004). Furthermore, cell death in plants is suppressed by expression of an animal anti-apoptosis gene CED9/Bcl-2 (Mitsuhara et al., 1999; Dickman et al., 2001), and cell death is induced by the expression of animal pro-apoptotic Bax (Lacomme and Santa-Cruz, 1999; Mitsuhara et al., 1999; Xu
Abbreviations: HR, hypersensitive response; Hsp, heat shock protein; MSX, lmethionine sulfoximine; PCR, polymerase chain reaction; PCD, programmed cell death; RT-PCR, reverse transcription-polymerase chain reaction; qRT-PCR, quantitative real time polymerase chain reaction; TL, tabtoxinine--lactam; VIGS, virus-induced gene silencing. ∗ Corresponding author. Tel.: +81 88 864 5196; fax: +81 88 864 5196. E-mail address: [email protected] (A. Kiba). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.10.012
et al., 2004). Hypersensitive response (HR) is the most characterized PCD in plants and is defined as rapid, localized cell death triggered by an incompatible pathogen (Benoit and Dangl, 1997). Recently, significant progress has been made in understanding the signal transduction pathways leading to HR. It has been shown that molecular chaperones act as a critical component of HR-mediated defense responses. Hsp90 is reportedly required by HR mediated by the resistant genes, Pto and Rx (Lu et al., 2003). Another Hsp, Hsp70, is required for induction of HR in response to INF1 elicitin and Pseudomonas cichorii (Kanzaki et al., 2003). The RAR1 gene is reportedly required for N-mediated HR (Liu et al., 2002). SGT1 is also a critical signaling component required for R gene-mediated HR in several plant species against various plant pathogens, including fungi, bacteria and viruses (Austin et al., 2002; Azevedo et al., 2002; Tör et al., 2002; Liu et al., 2004). SGT1 has also been shown to be involved in cell death that promotes the pathogenesis of the necrotrophic fungal pathogen, Botrytis cinerea (EI Oirdi and Bouarab, 2007), and the hemibiotrophic fungal pathogen, Fusarium culmorum (Cuzick et al., 2009). Pseudomonas syringae pv. tabaci is the causal agent of wildfire disease in tobacco plants. A recent report showed that development of necrotic wildfire lesions caused by P. syringae pv. tabaci was
174
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
accompanied by oxidative burst and caspase-like proteases, suggesting the involvement of PCD during lesion formation of wildfire disease (Iakimova et al., 2004). This disease symptom is promoted by tabtoxinine--lactam (TL), a host non-specific bacterial toxin produced by P. syringae pv. tabaci. TL causes death of plant cells through the inhibition of glutamine synthetase, which leads to an abnormal accumulation of ammonium ions and the characteristic necrotic wildfire lesions (Turner and Debbage, 1982). However, the mechanism of TL-induced cell death is not clear. In this study, we used virus-induced gene silencing (VIGS) in Nicotiana benthamiana to investigate the involvement of molecular chaperones and cochaperones in induction of cell death and necrotic wildfire lesions by TL, and further characterized the involvement of Hsp70 in these events. We demonstrated that induction of cell death and necrotic wildfire lesions by TL was compromised in Hsp70-silenced plants and ammonium accumulation (inhibition of glutamine synthetase) was not inhibited by TL. Furthermore, the silencing of Hsp70 also suppressed cell death induced by treatment with l-methionine sulfoximine (MSX), which inhibits glutamine synthetase similarly to TL with over-accumulation of ammonia. These results suggest that Hsp70 might play an essential role in the TL-mediated cell death induction pathway downstream of glutamine synthetaseinhibition by TL.
Sequencing Sequencing analysis was carried out using M4 and RV primers with the reagents for the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems Foster, CA) and Applied Biosystems 3100 Avant Automated Sequencer (Applied Biosystems, Warrington, UK) according to the manufacturer’s instructions. The sequence analysis was carried out using DNASIS (version 3.6; Hitachi, Yokohama, Japan) and the BLAST network service from the National Center for Biotechnology Information (Altschul et al., 1990). Vector constructs and seedling infection for VIGS An Hsp70 cDNA fragment for a VIGS vector was described previously (Kanzaki et al., 2003). The cDNA fragments of Hsp90, RAR1 and SGT1 were described previously (Liu et al., 2004). The VIGS experiment was carried out with Agrobacterium tumefaciens strain GV3101 (Baulcombe et al., 1995) and inoculated into N. benthamiana leaves as described previously (Maimbo et al., 2007). We observed characteristic dwarf phenotypes of SGT1-, Hsp70- and Hsp90-silenced plants as shown previously (Supplemental Figure 1, Kanzaki et al., 2003; Liu et al., 2004). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2013.10.012.
Materials and methods Quantitative real time PCR Plant materials, bacterial isolates and culture conditions Nicotiana benthamiana was grown in a growth room (Maimbo et al., 2007). Pseudomonas syringae pv. tabaci 6605 was cultured in PY medium containing 20 g/mL rifampicin (Maimbo et al., 2010). Bacterial suspension was inoculated into N. benthamiana leaves as described previously (Kiba et al., 2007).
Chemicals MSX was obtained from Funakoshi Chemical (Tokyo, Japan). Primers used in this study are listed in Supplemental Table 1. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2013.10.012.
Purification of TˇL P. syringae pv. tabaci was grown with agitation in Woolley’s medium at 24 ◦ C. Purification of TL was carried out according to the method described by Thomas et al. (1983).
Quantitative real time PCR was carried out according to the method of Maimbo et al. (2007). Reverse transcription was carried out with 1 g total RNA using the PrimeScript RT reagent Kit (Takara). Quantitative reverse-transcription PCR (qRT-PCR) was carried out with a 20 L reaction mixture containing 1 L of the cDNA stock and 10 pM of the respective primers (Supplemental Table 1), using the SYBR GreenER qPCR Reagent System (Invitrogen, Tokyo, Japan), with an Applied Biosystems 7300 real time PCR system. Standard deviations and differences between expression ratios of non-treated controls and other samples were tested for statistical significance using the t-test. Cell death measurement Cell death was quantified by measuring ion leakage, as described by Asai et al. (2010) with slight modifications. For conductivity tests, a leaf disk (78.5 mm2 ) was obtained from TL- or MSX-treated areas of each leaf, and was floated in 1 mL of distilled water for 4 h at 25 ◦ C with gentle shaking. Conductivity was measured using a Twin Cond B-173 (HORIBA, Kyoto, Japan). We carried out the experiment with at least five biological replicates. Estimation of ammonium ion content
Chemical treatments MSX and TL were also treated by leaf infiltration as described in Maimbo et al. (2007).
Isolation of RNA and cDNA synthesis Total RNA was isolated from N. benthamiana leaves with RNAiso (Takara Shuzo, Shiga, Japan) according to the manufacturer’s instructions. RNA samples were treated with DNase I (RNase-free; Takara Shuzo) to degrade contaminating genomic DNA as described previously (Maimbo et al., 2007). Complementary DNA (cDNA) was synthesized by MMLV-reverse transcriptase (Takara) with oligodT according to the manufacturer’s instructions.
The amount of ammonium was determined according to the method described by Alkan et al. (2009). Leaf disks (78.5 mm2 ) were homogenized in 1 mL of deionized water and then centrifuged at 10,000 rpm for 5 min; 100 L of the supernatant was diluted to 900 L and ammonium was measured using a photometric ammonium test kit (Ammonium Test; Merck, Tokyo, Japan) to determine the final concentration in the supernatant. Results and discussion P. syringae pv. tabaci induces apoptosis-like PCD while the typical lesions of the wildfire disease are formed (Iakimova et al., 2004). Although this symptom is enhanced by TL (Fig. 1A), which inhibits glutamine synthetase (Thomas et al., 1983), it is not known whether
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
175
Fig. 1. Induction of hin1 gene expression in TL-treated Nicotiana benthamiana. (A) Purified TL (TL; 1.5 mM) and deionized water (W) was infiltrated into N. benthamiana leaves, and incubated at 25 ◦ C. Photograph was taken 4 days after TL treatment. (B) Total RNA was isolated from water (W; white box) and TL-treated leaves (TL; black box). Expression values of hin1 are expressed as [Qty] after normalization with actin. Values represent the means and SD from triplicate experiments. Asterisks denote values significantly different from empty water-treated controls (*; P < 0.05).
PCD is induced directly by the action of TL. To answer this question, we first checked if TL-induced cell death is similar to PCD-like cell death, and we estimated the expression of the hin1 gene, which is used as a molecular marker for the HR, a characteristic feature of plant PCD (Pontier et al., 1999). As shown in Fig. 1B, expression of hin1 was strongly induced in TL-treated N. benthamiana leaves, suggesting the possibility that TL-induced plant cell death resembles PCD mediated by intracellular signaling. Plant cellular components, including Hsp90, Hsp70, RAR1 and SGT1, play a critical role in HR. Recent reports show that SGT1 is involved in cell death that promotes disease development (EI Oirdi and Bouarab, 2007; Cuzick et al., 2009). We first focused on whether these cellular components participate in TL-induced cell death. Then, we created Hsp90-, Hsp70-, RAR1- and SGT1-silenced plants, and observed the effect of silencing these genes on TL-induced necrotic lesions (Supplementary Figure 2). Typical necrotic lesions were observed in control plants 1 day after infiltration of TL. We
could also observe clear necrotic wildfire lesions in Hsp90-, RAR1and SGT1-silenced plants, similar to those in the control plants. In contrast, significant suppression of necrotic wildfire lesions was observed in Hsp70-silenced plants (Supplementary Figure 2). These results suggest that Hsp70 might play a role in TL-induced necrotic wildfire lesions. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2013.10.012. To further analyze the effect of Hsp70 silencing on TL-induced cell death, we estimated cell death induction by monitoring ion leakage. In the control plants, cell death induced by TL infiltration was observed 24 and 48 h after infiltration, whereas significant suppression of TL-induced cell death was observed in Hsp70silenced plants (Fig. 2A). Expression of hin1 showed a peak in control plants 12 h after infiltration of TL but decreased significantly in Hsp70-silenced plants (Fig. 2B). These results suggest that
Fig. 2. Effect of Hsp70 silencing on TL-induced cell death, hin1 gene expression and ammonium ion accumulation. Control (PVX) and Hsp70-silenced (PVX:hsp70) leaves were infiltrated with TL (1.5 mM). (A) Cell death estimation was carried out by detection of ion leakages as described in Materials and Methods. (B) Total RNA was isolated from control and Hsp70-silenced leaves infiltrated with TL. Expression values of hin1 are expressed as [Qty] after normalization with actin. Relative expression of hin1 transcripts (relative to the absolute non-treated control, and normalized with actin) in control or Hsp70-silenced plants. Values represent the means and SD from triplicate experiments. (C) Ammonium ion content was determined as described in Materials and Methods. Asterisks denote values significantly different from empty PVX controls (*; P < 0.05, t = 5).
176
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
Fig. 3. Effect of Hsp70 silencing on MSX-induced cell death, ammonium ion accumulation and hin1 gene expression. Control (PVX) and Hsp70-silenced leaves were infiltrated with MSX. (A) Photographs were taken 4 days after infiltration of MSX. (B) MSX (150 M) was infiltrated into control and Hsp70-silenced plants. Cell death estimation was carried out by detection of ion leakages as described in Materials and Methods. (C) MSX (150 M) was infiltrated into control and Hsp70-silenced plants. Total RNA was isolated from control and Hsp70-silenced leaves infiltrated with MSX. Expression values of hin1 are expressed as [Qty] after normalization with actin. Relative expression of hin1 transcripts (relative to the absolute non-treated control, and normalized with actin) in control or Hsp70-silenced plants. Values represent the means and SD from triplicate experiments. (D) MSX (150 M) was infiltrated into control and Hsp70-silenced plants. Ammonium ion content was determined as described in Materials and Methods. Asterisks denote values significantly different from empty PVX controls (*; P < 0.05, t = 5).
TL-induced cell death was mediated by Hsp70-related mechanisms at least partially similar to HR induction. TL causes death of plant cells through the inhibition of glutamine synthetase, which leads to an abnormal accumulation of ammonium ions and the characteristic necrotic wildfire lesions (Frantz et al., 1982). To further understand the relationship between Hsp70 and induction of cell death and necrotic wildfire lesions by TL, we analyzed changes in ammonium ion concentration in TL-infiltrated control and Hsp70-silenced plants. As shown in Fig. 2C, accumulation of ammonium ions was observed in both control and Hsp70-silenced plants. Moreover, hyper-accumulation of ammonium ions was observed in Hsp70-silenced plants, and over twice the amount of ammonium ion was detected in the silenced plants 24 h after infiltration compared with control plants. These results suggest the glutamine synthetase-inhibiting action of TL is not inhibited but sensitivity to ammonium ions decreased in Hsp70-silenced plants. Hyper-accumulation of ammonium ions might be due to continuous production of ammonium resulting from glutamine synthetase inhibition by TL in living Hsp70-silenced leaves. MSX is known to be a potent and highly specific inhibitor of glutamine synthetase (Manning et al., 1969). Because MSX appears to mimic the effects of TL and is commercially available, whereas
TL is not, MSX has been used as a tool for studying wildfire disease in tobacco (Marek and Dickson, 1987). To further confirm the role of Hsp70 in induction of cell death by TL, we analyzed the effect of Hsp70 silencing on cell death induction by MSX. Cell death and necrotic lesions were induced by MSX infiltration, and cell death was observed at 24 and 48 h after infiltration in control plants. On the other hand, MSX-induced cell death and necrotic lesions were compromised in Hsp70-silenced plants (Fig. 3A and B). Expression of the hin1 gene was also compromised in Hsp70-silenced plants compared with control plants (Fig. 3C). Accumulation of ammonium ions was observed in both control and Hsp70-silenced plants. Hyper-accumulation of ammonium ions was observed in Hsp70silenced plants, and about twice the amount of ammonium ions was observed in the silenced plants 48 h after infiltration compared with control plants (Fig. 3D). These results suggest that Hsp70 might act downstream of glutamate synthase inhibition by TL and MSX (accumulation of ammonium ions) during cell death induction and necrotic lesion formation. Our present results show that TL-induced cell death and necrotic wildfire lesions were suppressed in Hsp70-silenced plants. These results led us to test the effect of Hsp70 silencing on the development of wildfire disease by inoculation with P. syringae pv.
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
177
Fig. 4. Effect of Hsp70 silencing on lesion formation, cell death and hin1 gene expression by inoculation with P. syringae pv. tabaci. Control (PVX) and Hsp70-silenced (PVX:hsp70) leaves were infiltrated with P. syringae pv. tabaci. (A) Cell death estimation was carried out by detection of ion leakages as described in Materials and Methods. (B) Total RNA was isolated from control and Hsp70-silenced leaves infiltrated with P. syringae pv. tabaci. Expression values of hin1 are expressed as [Qty] after normalization with actin. Relative expression of hin1 transcripts (relative to the absolute non-treated control, and normalized with actin) in control or Hsp70-silenced plants. Values represent the means and SD from triplicate experiments. (C) Lesion extension of wildfire was measured daily in control and Hsp70-silenced plants. (D) Photographs were taken 10 and 20 days after infiltration with P. syringae pv. tabaci. Asterisks denote values significantly different from empty PVX controls (*; P < 0.05, t = 5).
tabaci. Induction of cell death was observed 5–7 days after inoculation with P. syringae pv. tabaci in control plants, whereas cell death induction was not observed in Hsp70-silenced plants (Fig. 4A). Expression of hin1 showed a peak in control plants 2 days after inoculation with P. syringae pv. tabaci but decreased significantly in Hsp70-silenced plants (Fig. 4B). Inoculation with P. syringae pv. tabaci induced necrotic wildfire lesions within 4 days after inoculation in the control plants, and the leaves completely died within 10 days. In contrast, we did not observe any lesions in Hsp70-silenced plants 10 days after inoculation, and Hsp70-silenced leaves were still alive 20 days after inoculation with P. syringae pv. tabaci (Fig. 4C and D). Hsp70 exists widely within eukaryotic organisms (Hartl and Hayer-Hartl, 2002). Under various environmental and physiological stresses, Hsp70 is involved in protein assembly, folding (Bukau et al., 2006), localization (Ryan and Pfanner, 2002), degradation (Wickner et al., 1999), and tumor immunity (Nicchitta, 2003). High temperature stress causes extensive denaturation and aggregation of cellular proteins, leading to cell death. Through Hsp70’s chaperoning activity, Hsp70 is an anti-apoptotic chaperone protein highly expressed in human breast tumors and tumor cell lines (Nylandsted et al., 2000). In plant cells, Hsp70d prevents heatinduced damage of cellular proteins, and also protects plant cells from multiple environmental stresses (Krishna, 2004). OsHsp70 with MAPKs may contribute to cellular protection in rice roots from excessive Cu2+ toxicity (Chen et al., 2008). Over-expression of Hsp70 suppresses heat- and H2 O2 -induced PCD in rice (Qi et al.,
2011). Therefore, Hsp70 may function to prevent cell death by cell death-inducible stresses. In contrast, a recent study showed that Hsp70 is required for induction of cell death, since HR induction by INF1 elicitin and P. cichorii was compromised in Hsp70-silenced plants, suggesting the requirement of Hsp70 for PCD induction during HR (Kanzaki et al., 2003). In addition, our present data shows that Hsp70-silencing reduced cell death and necrotic lesions caused by the toxic effect of TL and MSX (Figs. 1–3). Moreover, hin1 gene expression, the marker gene for HR induction, was induced by TL and MSX treatment, whereas Hsp70 silencing compromised hin1 gene expression (Figs. 2B and 3C). Therefore, TL- and MSXinduced cell death may require PCD machinery at least partially similar to HR induction, and Hsp70 may contribute to the TLand MSX-induced PCD induction pathway(s), leading to necrotic lesions. Based on our experimental results and previous findings, Hsp70 appears to positively regulate the process of cell death during HR and disease-induced (TL-induced) plant cell death in N. benthamiana. Therefore, we propose that Hsp70 plays a dual role as a signaling component of cell death during both immunity and susceptibility. Our identifying a role for Hsp70 in disease-associated cell death will facilitate future studies aimed at determining shared and unique molecular events during cell death associated with plant immunity and disease development. In addition, identifying host factors associated with Hsp70-mediated cell death by TL may provide new insights into controlling the disease development processes and creating wildfire disease-resistant plants.
178
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
Acknowledgements The authors thank H. Kanzaki and R. Terauchi for the cDNA fragment of Hsp70 for the VIGS experiment, and D. Baulcombe for the pPVX201 vector. This work was supported by Grants in Aid for Scientific Research to AK (16780031 and 18780029) and to YH (15028214 and 16380037) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References Alkan N, Davydov O, Sagi M, Fluhr R, Prusky D. Mol Plant–Microbe Interact 2009;22:1484–91. Altschul SF, Gish W, Miller W, Liptman DJJ. Mol Biol 1990;215:403–10. Asai S, Mase K, Yoshioka H. Plant J 2010;62:911–24. Austin MJ, Muskett P, Kahn K, Feys BJ, Jones JDG, Paker JE. Science 2002;296: 2077–80. Azevedo C, Sadanandom A, Kitagawa K, Freialdenhoven A, Shirasu K, Schulze-Lefert P. Science 2002;295:2073–6. Baulcombe DC, Chapman S, Cruz SS. Plant J 1995;7:1045–53. Beers EP, McDowell JM. Curr Opin Plant Biol 2001;4:561–7. Benoit JB, Dangl JL. Cell Death Differ 1997;4:671–83. Bukau B, Weissman J, Horwich A. Cell 2006;125:443–51. Chen PY, Lee KT, Chi WC, Hirt H, Chang CC, Huang HJ. Planta 2008;228:499–509. Cuzick A, Maguire K, Hammond-Kosack KE. New Phytol 2009;181:901–12. Dickman MB, Park YK, Oltersdorf T, Li W, Clemente T, French R. Proc Natl Acad Sci U S A 2001;98:6957–62. EI Oirdi M, Bouarab K. New Phytol 2007;175:131–9. Frantz TA, Peterson DM, Durbin RD. Plant Physiol 1982;69:345–8. Gilchrist DG. Annu Rev Phytopathol 1998;36:393–414. Greenberg JT, Yao N. Cell Microbiol 2004;6:201–11.
Hartl FU, Hayer-Hartl M. Science 2002;195:1852–8. Iakimova E, Batchvarova R, Kapchina-Toteva V, Popov T, Atanassov A, Woltering E. Biotechnol Biotechnol Equip 2004;18:34–46. Jones JD, Dangl JL. Nature 2006;444:323–9. Kanzaki H, Saitoh H, Ito A, Fujisawa S, Kamoun S, Katou S, et al. Mol Plant Pathol 2003;4:383–91. Kiba A, Maimbo M, Kanda A, Tomiyama H, Ohnishi K, Hikichi Y. Plant Biotechnol 2007;24:409–16. Krishna P. Top Curr Genet 2004;4:73–101. Lacomme C, Santa-Cruz S. Proc Natl Acad Sci U S A 1999;96:7956–61. Liu Y, Schiff M, Marthe R, Kummar SPD. Plant J 2002;4:415–29. Liu Y, Burch-Smith T, Schiff M, Feng S, Dinesh-Kumar SP. J Biol Chem 2004;279:2101–8. Lu R, Malcuit I, Moffett P, Ruiz MT, Peart J, Wu AJ, et al. EMBO J 2003;22:5690–9. Maimbo M, Ohnishi K, Hikichi Y, Yoshioka H, Kiba A. Plant Physiol 2007;145:1588–99. Maimbo M, Ohnishi K, Hikichi Y, Yoshioka H, Kiba A. Plant Physiol 2010;152:2023–35. Manning JM, Moore S, Rowe WB, Meister A. Biochemistry 1969;8:2681–5. Marek ET, Dickson R. J Bacteriol 1987;169:2440–8. Mitsuhara I, Malik KA, Miura M, Ohashi Y. Curr Biol 1999;9:775–8. Nicchitta CV. Nat Rev Immunol 2003;3:427–32. Nylandsted J, Rohde M, Brand K, Bastholm L, Elling E, Jaattela M. Proc Natl Acad Sci U S A 2000;94:7871–6. Pontier D, Gan S, Amasino RM, Roby D, Lam E. Plant Mol Biol 1999;39:1243–55. Qi Y, Wang H, Zou Y, Liu C, Liu Y, Wang Y, et al. FEBS Lett 2011;585:231–9. Ryan MT, Pfanner N. Adv Protein Chem 2002;59:223–42. Thomas MD, Langston-Unkefer PJ, Uchytil TF, Durbin RD. Plant Physiol 1983;71:912–5. Tör M, Gordon P, Cuzick A, Eulgem T, Sinapidou E, Mert-Turk F, et al. Plant Cell 2002;14:993–1003. Turner JG, Debbage M. Physiol Plant Pathol 1982;20:223–33. Wickner S, Mauizi MR, Gottesman S. Science 1999;286:1888–93. Xu P, Rogers SJ, Roossinck MJ. Proc Natl Acad Sci U S A 2004;101:15805–10.
Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Short communication
Heat shock protein 70 is required for tabtoxinine--lactam-induced cell death in Nicotiana benthamiana Makoto Ito a , Yu Yamamoto b , Chul-Sa Kim b , Kouhei Ohnishi c , Yasufumi Hikichi a , Akinori Kiba a,∗ a
Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kochi University, Nankoku 783-8502, Japan Laboratory of Bioactive Substance Chemistry, Faculty of Agriculture, Kochi University, Nankoku 783-8502, Japan c Research Institute of Molecular Genetics, Kochi University, Nankoku 783-8502, Japan b
a r t i c l e
i n f o
Article history: Received 1 July 2013 Received in revised form 15 October 2013 Accepted 22 October 2013 Available online 28 November 2013 Keywords: Cell death Heat shock protein 70 Nicotiana benthamiana Tabtoxinine--lactam Virus-induced gene silencing
a b s t r a c t Tabtoxinine--lactam (TL), a non-specific bacterial toxin, is produced by Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease. TL causes death of plant cells through the inhibition of glutamine synthetase, which leads to an abnormal accumulation of ammonium ions and the characteristic necrotic wildfire lesions. To better understand the mechanisms involved in TL-induced cell death, we studied its regulation in Nicotiana benthamiana. TL-induced lesions, similar to those in controls, could be observed in SGT1-, RAR1- and Hsp90-silenced plants. In contrast, Hsp70-silenced plants showed suppression of lesion formation. Expression of hin1, a marker gene for the hypersensitive response (HR), which is a characteristic of programmed cell death in plants, was strongly induced in controls by TL treatment but only slightly in Hsp70-silenced plants. However, in these TL-treated Hsp70-silenced plants, the amount of ammonium ions was considerably increased. Furthermore, the silencing of Hsp70 also suppressed l-methionine sulfoximine-induced cell death and hin1 expression and caused the over-accumulation of ammonium ions. When inoculated directly with P. syringae pv. tabaci, Hsp70-silenced plants showed only reduced symptoms. Our results suggest that the TL-induced pathway to cell death in N. benthamiana is at least partially similar to HR response, and that Hsp70 might play an essential role in these events. © 2013 Elsevier GmbH. All rights reserved.
Introduction Programmed cell death (PCD) has important roles not only in the process of plant development but also in incompatible plant–microbe interaction (Jones and Dangl, 2006). Plants may use apoptotic machinery similar to those of animals and yeast, because similar morphological and biochemical features are shared for PCD in these organisms (Gilchrist, 1998; Beers and McDowell, 2001; Greenberg and Yao, 2004). Furthermore, cell death in plants is suppressed by expression of an animal anti-apoptosis gene CED9/Bcl-2 (Mitsuhara et al., 1999; Dickman et al., 2001), and cell death is induced by the expression of animal pro-apoptotic Bax (Lacomme and Santa-Cruz, 1999; Mitsuhara et al., 1999; Xu
Abbreviations: HR, hypersensitive response; Hsp, heat shock protein; MSX, lmethionine sulfoximine; PCR, polymerase chain reaction; PCD, programmed cell death; RT-PCR, reverse transcription-polymerase chain reaction; qRT-PCR, quantitative real time polymerase chain reaction; TL, tabtoxinine--lactam; VIGS, virus-induced gene silencing. ∗ Corresponding author. Tel.: +81 88 864 5196; fax: +81 88 864 5196. E-mail address: [email protected] (A. Kiba). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.10.012
et al., 2004). Hypersensitive response (HR) is the most characterized PCD in plants and is defined as rapid, localized cell death triggered by an incompatible pathogen (Benoit and Dangl, 1997). Recently, significant progress has been made in understanding the signal transduction pathways leading to HR. It has been shown that molecular chaperones act as a critical component of HR-mediated defense responses. Hsp90 is reportedly required by HR mediated by the resistant genes, Pto and Rx (Lu et al., 2003). Another Hsp, Hsp70, is required for induction of HR in response to INF1 elicitin and Pseudomonas cichorii (Kanzaki et al., 2003). The RAR1 gene is reportedly required for N-mediated HR (Liu et al., 2002). SGT1 is also a critical signaling component required for R gene-mediated HR in several plant species against various plant pathogens, including fungi, bacteria and viruses (Austin et al., 2002; Azevedo et al., 2002; Tör et al., 2002; Liu et al., 2004). SGT1 has also been shown to be involved in cell death that promotes the pathogenesis of the necrotrophic fungal pathogen, Botrytis cinerea (EI Oirdi and Bouarab, 2007), and the hemibiotrophic fungal pathogen, Fusarium culmorum (Cuzick et al., 2009). Pseudomonas syringae pv. tabaci is the causal agent of wildfire disease in tobacco plants. A recent report showed that development of necrotic wildfire lesions caused by P. syringae pv. tabaci was
174
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
accompanied by oxidative burst and caspase-like proteases, suggesting the involvement of PCD during lesion formation of wildfire disease (Iakimova et al., 2004). This disease symptom is promoted by tabtoxinine--lactam (TL), a host non-specific bacterial toxin produced by P. syringae pv. tabaci. TL causes death of plant cells through the inhibition of glutamine synthetase, which leads to an abnormal accumulation of ammonium ions and the characteristic necrotic wildfire lesions (Turner and Debbage, 1982). However, the mechanism of TL-induced cell death is not clear. In this study, we used virus-induced gene silencing (VIGS) in Nicotiana benthamiana to investigate the involvement of molecular chaperones and cochaperones in induction of cell death and necrotic wildfire lesions by TL, and further characterized the involvement of Hsp70 in these events. We demonstrated that induction of cell death and necrotic wildfire lesions by TL was compromised in Hsp70-silenced plants and ammonium accumulation (inhibition of glutamine synthetase) was not inhibited by TL. Furthermore, the silencing of Hsp70 also suppressed cell death induced by treatment with l-methionine sulfoximine (MSX), which inhibits glutamine synthetase similarly to TL with over-accumulation of ammonia. These results suggest that Hsp70 might play an essential role in the TL-mediated cell death induction pathway downstream of glutamine synthetaseinhibition by TL.
Sequencing Sequencing analysis was carried out using M4 and RV primers with the reagents for the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems Foster, CA) and Applied Biosystems 3100 Avant Automated Sequencer (Applied Biosystems, Warrington, UK) according to the manufacturer’s instructions. The sequence analysis was carried out using DNASIS (version 3.6; Hitachi, Yokohama, Japan) and the BLAST network service from the National Center for Biotechnology Information (Altschul et al., 1990). Vector constructs and seedling infection for VIGS An Hsp70 cDNA fragment for a VIGS vector was described previously (Kanzaki et al., 2003). The cDNA fragments of Hsp90, RAR1 and SGT1 were described previously (Liu et al., 2004). The VIGS experiment was carried out with Agrobacterium tumefaciens strain GV3101 (Baulcombe et al., 1995) and inoculated into N. benthamiana leaves as described previously (Maimbo et al., 2007). We observed characteristic dwarf phenotypes of SGT1-, Hsp70- and Hsp90-silenced plants as shown previously (Supplemental Figure 1, Kanzaki et al., 2003; Liu et al., 2004). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2013.10.012.
Materials and methods Quantitative real time PCR Plant materials, bacterial isolates and culture conditions Nicotiana benthamiana was grown in a growth room (Maimbo et al., 2007). Pseudomonas syringae pv. tabaci 6605 was cultured in PY medium containing 20 g/mL rifampicin (Maimbo et al., 2010). Bacterial suspension was inoculated into N. benthamiana leaves as described previously (Kiba et al., 2007).
Chemicals MSX was obtained from Funakoshi Chemical (Tokyo, Japan). Primers used in this study are listed in Supplemental Table 1. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2013.10.012.
Purification of TˇL P. syringae pv. tabaci was grown with agitation in Woolley’s medium at 24 ◦ C. Purification of TL was carried out according to the method described by Thomas et al. (1983).
Quantitative real time PCR was carried out according to the method of Maimbo et al. (2007). Reverse transcription was carried out with 1 g total RNA using the PrimeScript RT reagent Kit (Takara). Quantitative reverse-transcription PCR (qRT-PCR) was carried out with a 20 L reaction mixture containing 1 L of the cDNA stock and 10 pM of the respective primers (Supplemental Table 1), using the SYBR GreenER qPCR Reagent System (Invitrogen, Tokyo, Japan), with an Applied Biosystems 7300 real time PCR system. Standard deviations and differences between expression ratios of non-treated controls and other samples were tested for statistical significance using the t-test. Cell death measurement Cell death was quantified by measuring ion leakage, as described by Asai et al. (2010) with slight modifications. For conductivity tests, a leaf disk (78.5 mm2 ) was obtained from TL- or MSX-treated areas of each leaf, and was floated in 1 mL of distilled water for 4 h at 25 ◦ C with gentle shaking. Conductivity was measured using a Twin Cond B-173 (HORIBA, Kyoto, Japan). We carried out the experiment with at least five biological replicates. Estimation of ammonium ion content
Chemical treatments MSX and TL were also treated by leaf infiltration as described in Maimbo et al. (2007).
Isolation of RNA and cDNA synthesis Total RNA was isolated from N. benthamiana leaves with RNAiso (Takara Shuzo, Shiga, Japan) according to the manufacturer’s instructions. RNA samples were treated with DNase I (RNase-free; Takara Shuzo) to degrade contaminating genomic DNA as described previously (Maimbo et al., 2007). Complementary DNA (cDNA) was synthesized by MMLV-reverse transcriptase (Takara) with oligodT according to the manufacturer’s instructions.
The amount of ammonium was determined according to the method described by Alkan et al. (2009). Leaf disks (78.5 mm2 ) were homogenized in 1 mL of deionized water and then centrifuged at 10,000 rpm for 5 min; 100 L of the supernatant was diluted to 900 L and ammonium was measured using a photometric ammonium test kit (Ammonium Test; Merck, Tokyo, Japan) to determine the final concentration in the supernatant. Results and discussion P. syringae pv. tabaci induces apoptosis-like PCD while the typical lesions of the wildfire disease are formed (Iakimova et al., 2004). Although this symptom is enhanced by TL (Fig. 1A), which inhibits glutamine synthetase (Thomas et al., 1983), it is not known whether
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
175
Fig. 1. Induction of hin1 gene expression in TL-treated Nicotiana benthamiana. (A) Purified TL (TL; 1.5 mM) and deionized water (W) was infiltrated into N. benthamiana leaves, and incubated at 25 ◦ C. Photograph was taken 4 days after TL treatment. (B) Total RNA was isolated from water (W; white box) and TL-treated leaves (TL; black box). Expression values of hin1 are expressed as [Qty] after normalization with actin. Values represent the means and SD from triplicate experiments. Asterisks denote values significantly different from empty water-treated controls (*; P < 0.05).
PCD is induced directly by the action of TL. To answer this question, we first checked if TL-induced cell death is similar to PCD-like cell death, and we estimated the expression of the hin1 gene, which is used as a molecular marker for the HR, a characteristic feature of plant PCD (Pontier et al., 1999). As shown in Fig. 1B, expression of hin1 was strongly induced in TL-treated N. benthamiana leaves, suggesting the possibility that TL-induced plant cell death resembles PCD mediated by intracellular signaling. Plant cellular components, including Hsp90, Hsp70, RAR1 and SGT1, play a critical role in HR. Recent reports show that SGT1 is involved in cell death that promotes disease development (EI Oirdi and Bouarab, 2007; Cuzick et al., 2009). We first focused on whether these cellular components participate in TL-induced cell death. Then, we created Hsp90-, Hsp70-, RAR1- and SGT1-silenced plants, and observed the effect of silencing these genes on TL-induced necrotic lesions (Supplementary Figure 2). Typical necrotic lesions were observed in control plants 1 day after infiltration of TL. We
could also observe clear necrotic wildfire lesions in Hsp90-, RAR1and SGT1-silenced plants, similar to those in the control plants. In contrast, significant suppression of necrotic wildfire lesions was observed in Hsp70-silenced plants (Supplementary Figure 2). These results suggest that Hsp70 might play a role in TL-induced necrotic wildfire lesions. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jplph. 2013.10.012. To further analyze the effect of Hsp70 silencing on TL-induced cell death, we estimated cell death induction by monitoring ion leakage. In the control plants, cell death induced by TL infiltration was observed 24 and 48 h after infiltration, whereas significant suppression of TL-induced cell death was observed in Hsp70silenced plants (Fig. 2A). Expression of hin1 showed a peak in control plants 12 h after infiltration of TL but decreased significantly in Hsp70-silenced plants (Fig. 2B). These results suggest that
Fig. 2. Effect of Hsp70 silencing on TL-induced cell death, hin1 gene expression and ammonium ion accumulation. Control (PVX) and Hsp70-silenced (PVX:hsp70) leaves were infiltrated with TL (1.5 mM). (A) Cell death estimation was carried out by detection of ion leakages as described in Materials and Methods. (B) Total RNA was isolated from control and Hsp70-silenced leaves infiltrated with TL. Expression values of hin1 are expressed as [Qty] after normalization with actin. Relative expression of hin1 transcripts (relative to the absolute non-treated control, and normalized with actin) in control or Hsp70-silenced plants. Values represent the means and SD from triplicate experiments. (C) Ammonium ion content was determined as described in Materials and Methods. Asterisks denote values significantly different from empty PVX controls (*; P < 0.05, t = 5).
176
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
Fig. 3. Effect of Hsp70 silencing on MSX-induced cell death, ammonium ion accumulation and hin1 gene expression. Control (PVX) and Hsp70-silenced leaves were infiltrated with MSX. (A) Photographs were taken 4 days after infiltration of MSX. (B) MSX (150 M) was infiltrated into control and Hsp70-silenced plants. Cell death estimation was carried out by detection of ion leakages as described in Materials and Methods. (C) MSX (150 M) was infiltrated into control and Hsp70-silenced plants. Total RNA was isolated from control and Hsp70-silenced leaves infiltrated with MSX. Expression values of hin1 are expressed as [Qty] after normalization with actin. Relative expression of hin1 transcripts (relative to the absolute non-treated control, and normalized with actin) in control or Hsp70-silenced plants. Values represent the means and SD from triplicate experiments. (D) MSX (150 M) was infiltrated into control and Hsp70-silenced plants. Ammonium ion content was determined as described in Materials and Methods. Asterisks denote values significantly different from empty PVX controls (*; P < 0.05, t = 5).
TL-induced cell death was mediated by Hsp70-related mechanisms at least partially similar to HR induction. TL causes death of plant cells through the inhibition of glutamine synthetase, which leads to an abnormal accumulation of ammonium ions and the characteristic necrotic wildfire lesions (Frantz et al., 1982). To further understand the relationship between Hsp70 and induction of cell death and necrotic wildfire lesions by TL, we analyzed changes in ammonium ion concentration in TL-infiltrated control and Hsp70-silenced plants. As shown in Fig. 2C, accumulation of ammonium ions was observed in both control and Hsp70-silenced plants. Moreover, hyper-accumulation of ammonium ions was observed in Hsp70-silenced plants, and over twice the amount of ammonium ion was detected in the silenced plants 24 h after infiltration compared with control plants. These results suggest the glutamine synthetase-inhibiting action of TL is not inhibited but sensitivity to ammonium ions decreased in Hsp70-silenced plants. Hyper-accumulation of ammonium ions might be due to continuous production of ammonium resulting from glutamine synthetase inhibition by TL in living Hsp70-silenced leaves. MSX is known to be a potent and highly specific inhibitor of glutamine synthetase (Manning et al., 1969). Because MSX appears to mimic the effects of TL and is commercially available, whereas
TL is not, MSX has been used as a tool for studying wildfire disease in tobacco (Marek and Dickson, 1987). To further confirm the role of Hsp70 in induction of cell death by TL, we analyzed the effect of Hsp70 silencing on cell death induction by MSX. Cell death and necrotic lesions were induced by MSX infiltration, and cell death was observed at 24 and 48 h after infiltration in control plants. On the other hand, MSX-induced cell death and necrotic lesions were compromised in Hsp70-silenced plants (Fig. 3A and B). Expression of the hin1 gene was also compromised in Hsp70-silenced plants compared with control plants (Fig. 3C). Accumulation of ammonium ions was observed in both control and Hsp70-silenced plants. Hyper-accumulation of ammonium ions was observed in Hsp70silenced plants, and about twice the amount of ammonium ions was observed in the silenced plants 48 h after infiltration compared with control plants (Fig. 3D). These results suggest that Hsp70 might act downstream of glutamate synthase inhibition by TL and MSX (accumulation of ammonium ions) during cell death induction and necrotic lesion formation. Our present results show that TL-induced cell death and necrotic wildfire lesions were suppressed in Hsp70-silenced plants. These results led us to test the effect of Hsp70 silencing on the development of wildfire disease by inoculation with P. syringae pv.
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
177
Fig. 4. Effect of Hsp70 silencing on lesion formation, cell death and hin1 gene expression by inoculation with P. syringae pv. tabaci. Control (PVX) and Hsp70-silenced (PVX:hsp70) leaves were infiltrated with P. syringae pv. tabaci. (A) Cell death estimation was carried out by detection of ion leakages as described in Materials and Methods. (B) Total RNA was isolated from control and Hsp70-silenced leaves infiltrated with P. syringae pv. tabaci. Expression values of hin1 are expressed as [Qty] after normalization with actin. Relative expression of hin1 transcripts (relative to the absolute non-treated control, and normalized with actin) in control or Hsp70-silenced plants. Values represent the means and SD from triplicate experiments. (C) Lesion extension of wildfire was measured daily in control and Hsp70-silenced plants. (D) Photographs were taken 10 and 20 days after infiltration with P. syringae pv. tabaci. Asterisks denote values significantly different from empty PVX controls (*; P < 0.05, t = 5).
tabaci. Induction of cell death was observed 5–7 days after inoculation with P. syringae pv. tabaci in control plants, whereas cell death induction was not observed in Hsp70-silenced plants (Fig. 4A). Expression of hin1 showed a peak in control plants 2 days after inoculation with P. syringae pv. tabaci but decreased significantly in Hsp70-silenced plants (Fig. 4B). Inoculation with P. syringae pv. tabaci induced necrotic wildfire lesions within 4 days after inoculation in the control plants, and the leaves completely died within 10 days. In contrast, we did not observe any lesions in Hsp70-silenced plants 10 days after inoculation, and Hsp70-silenced leaves were still alive 20 days after inoculation with P. syringae pv. tabaci (Fig. 4C and D). Hsp70 exists widely within eukaryotic organisms (Hartl and Hayer-Hartl, 2002). Under various environmental and physiological stresses, Hsp70 is involved in protein assembly, folding (Bukau et al., 2006), localization (Ryan and Pfanner, 2002), degradation (Wickner et al., 1999), and tumor immunity (Nicchitta, 2003). High temperature stress causes extensive denaturation and aggregation of cellular proteins, leading to cell death. Through Hsp70’s chaperoning activity, Hsp70 is an anti-apoptotic chaperone protein highly expressed in human breast tumors and tumor cell lines (Nylandsted et al., 2000). In plant cells, Hsp70d prevents heatinduced damage of cellular proteins, and also protects plant cells from multiple environmental stresses (Krishna, 2004). OsHsp70 with MAPKs may contribute to cellular protection in rice roots from excessive Cu2+ toxicity (Chen et al., 2008). Over-expression of Hsp70 suppresses heat- and H2 O2 -induced PCD in rice (Qi et al.,
2011). Therefore, Hsp70 may function to prevent cell death by cell death-inducible stresses. In contrast, a recent study showed that Hsp70 is required for induction of cell death, since HR induction by INF1 elicitin and P. cichorii was compromised in Hsp70-silenced plants, suggesting the requirement of Hsp70 for PCD induction during HR (Kanzaki et al., 2003). In addition, our present data shows that Hsp70-silencing reduced cell death and necrotic lesions caused by the toxic effect of TL and MSX (Figs. 1–3). Moreover, hin1 gene expression, the marker gene for HR induction, was induced by TL and MSX treatment, whereas Hsp70 silencing compromised hin1 gene expression (Figs. 2B and 3C). Therefore, TL- and MSXinduced cell death may require PCD machinery at least partially similar to HR induction, and Hsp70 may contribute to the TLand MSX-induced PCD induction pathway(s), leading to necrotic lesions. Based on our experimental results and previous findings, Hsp70 appears to positively regulate the process of cell death during HR and disease-induced (TL-induced) plant cell death in N. benthamiana. Therefore, we propose that Hsp70 plays a dual role as a signaling component of cell death during both immunity and susceptibility. Our identifying a role for Hsp70 in disease-associated cell death will facilitate future studies aimed at determining shared and unique molecular events during cell death associated with plant immunity and disease development. In addition, identifying host factors associated with Hsp70-mediated cell death by TL may provide new insights into controlling the disease development processes and creating wildfire disease-resistant plants.
178
M. Ito et al. / Journal of Plant Physiology 171 (2014) 173–178
Acknowledgements The authors thank H. Kanzaki and R. Terauchi for the cDNA fragment of Hsp70 for the VIGS experiment, and D. Baulcombe for the pPVX201 vector. This work was supported by Grants in Aid for Scientific Research to AK (16780031 and 18780029) and to YH (15028214 and 16380037) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References Alkan N, Davydov O, Sagi M, Fluhr R, Prusky D. Mol Plant–Microbe Interact 2009;22:1484–91. Altschul SF, Gish W, Miller W, Liptman DJJ. Mol Biol 1990;215:403–10. Asai S, Mase K, Yoshioka H. Plant J 2010;62:911–24. Austin MJ, Muskett P, Kahn K, Feys BJ, Jones JDG, Paker JE. Science 2002;296: 2077–80. Azevedo C, Sadanandom A, Kitagawa K, Freialdenhoven A, Shirasu K, Schulze-Lefert P. Science 2002;295:2073–6. Baulcombe DC, Chapman S, Cruz SS. Plant J 1995;7:1045–53. Beers EP, McDowell JM. Curr Opin Plant Biol 2001;4:561–7. Benoit JB, Dangl JL. Cell Death Differ 1997;4:671–83. Bukau B, Weissman J, Horwich A. Cell 2006;125:443–51. Chen PY, Lee KT, Chi WC, Hirt H, Chang CC, Huang HJ. Planta 2008;228:499–509. Cuzick A, Maguire K, Hammond-Kosack KE. New Phytol 2009;181:901–12. Dickman MB, Park YK, Oltersdorf T, Li W, Clemente T, French R. Proc Natl Acad Sci U S A 2001;98:6957–62. EI Oirdi M, Bouarab K. New Phytol 2007;175:131–9. Frantz TA, Peterson DM, Durbin RD. Plant Physiol 1982;69:345–8. Gilchrist DG. Annu Rev Phytopathol 1998;36:393–414. Greenberg JT, Yao N. Cell Microbiol 2004;6:201–11.
Hartl FU, Hayer-Hartl M. Science 2002;195:1852–8. Iakimova E, Batchvarova R, Kapchina-Toteva V, Popov T, Atanassov A, Woltering E. Biotechnol Biotechnol Equip 2004;18:34–46. Jones JD, Dangl JL. Nature 2006;444:323–9. Kanzaki H, Saitoh H, Ito A, Fujisawa S, Kamoun S, Katou S, et al. Mol Plant Pathol 2003;4:383–91. Kiba A, Maimbo M, Kanda A, Tomiyama H, Ohnishi K, Hikichi Y. Plant Biotechnol 2007;24:409–16. Krishna P. Top Curr Genet 2004;4:73–101. Lacomme C, Santa-Cruz S. Proc Natl Acad Sci U S A 1999;96:7956–61. Liu Y, Schiff M, Marthe R, Kummar SPD. Plant J 2002;4:415–29. Liu Y, Burch-Smith T, Schiff M, Feng S, Dinesh-Kumar SP. J Biol Chem 2004;279:2101–8. Lu R, Malcuit I, Moffett P, Ruiz MT, Peart J, Wu AJ, et al. EMBO J 2003;22:5690–9. Maimbo M, Ohnishi K, Hikichi Y, Yoshioka H, Kiba A. Plant Physiol 2007;145:1588–99. Maimbo M, Ohnishi K, Hikichi Y, Yoshioka H, Kiba A. Plant Physiol 2010;152:2023–35. Manning JM, Moore S, Rowe WB, Meister A. Biochemistry 1969;8:2681–5. Marek ET, Dickson R. J Bacteriol 1987;169:2440–8. Mitsuhara I, Malik KA, Miura M, Ohashi Y. Curr Biol 1999;9:775–8. Nicchitta CV. Nat Rev Immunol 2003;3:427–32. Nylandsted J, Rohde M, Brand K, Bastholm L, Elling E, Jaattela M. Proc Natl Acad Sci U S A 2000;94:7871–6. Pontier D, Gan S, Amasino RM, Roby D, Lam E. Plant Mol Biol 1999;39:1243–55. Qi Y, Wang H, Zou Y, Liu C, Liu Y, Wang Y, et al. FEBS Lett 2011;585:231–9. Ryan MT, Pfanner N. Adv Protein Chem 2002;59:223–42. Thomas MD, Langston-Unkefer PJ, Uchytil TF, Durbin RD. Plant Physiol 1983;71:912–5. Tör M, Gordon P, Cuzick A, Eulgem T, Sinapidou E, Mert-Turk F, et al. Plant Cell 2002;14:993–1003. Turner JG, Debbage M. Physiol Plant Pathol 1982;20:223–33. Wickner S, Mauizi MR, Gottesman S. Science 1999;286:1888–93. Xu P, Rogers SJ, Roossinck MJ. Proc Natl Acad Sci U S A 2004;101:15805–10.