Induction of Necrosis Via Mitochondrial Targeting of Melon Necrotic Spot Virus Replication Protein P29 by Its Second Transmembrane Domain

Virology 390 (2009) 239–249

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Virology

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Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain

Tomofumi Mochizuki a,1, Katsuyuki Hirai a, Ayami Kanda a, Jun Ohnishi b, Takehiro Ohki a, Shinya Tsuda a,⁎ a National Agricultural Research Center, Tsukuba, Ibaraki 305-8666, Japan b National Institute of Vegetable and Tea Science, Tsu, Mie 514-2392, Japan article info abstract

Article history: The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease Received 19 February 2009 on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using Returned to author for revision a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy 21 March 2009 revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and Accepted 10 May 2009 confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an Available online 7 June 2009 integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic Keywords: transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to MNSV deﬁciencies in both the mitochondrial localization and virulence of p29. Taken together, these results Mitochondria indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor Necrosis causing the observed necrosis. RNA replication protein © 2009 Elsevier Inc. All rights reserved. Pathogenesis

Introduction the recessive “nsv” gene (Díaz et al., 2004). The MNSV isolate that can overcome the resistance conferred by “nsv” is able to infect non- Melon necrotic spot virus (MNSV) causes a necrotic spot disease on cucurbit plants, such as Nicotiana benthamiana, and induce systemic melon plants (Cucumis melo)(Kishi, 1966). Although different types of necrotic symptoms similar to those in melons (Díaz et al., 2004). necrotic symptoms are observed at each growth stage of the melon Thus far, several pathogenic factors of plant viruses that are plants, the most common symptom of MNSV infection is necrotic responsible for necrotic symptoms have been discovered (Carette et spots on the large leaves (Hibi and Furuki, 1985; Yoshida et al., 1980). al., 2002; Divéki et al., 2004; Fernandez et al., 1999; Király et al., 1999; MNSV belongs to the Carmovirus genus in the Tombusviridae family. Ozeki et al., 2006; Tribodet et al., 2005; van Wezel et al., 2001). In the The virus is 30 nm in diameter with an icosahedral virion (Hibi and Tombusviridae family, the p19 protein of Tomato bushy stunt virus Furuki, 1985) and contains a single-stranded positive-sense RNA (TBSV), which is a suppressor of RNA silencing, induces systemic genome of 4.2 kb (Riviere and Rochon, 1990). The MNSV genome necrosis in N. benthamiana and Nicotiana clevelandii and local necrotic encodes at least ﬁve proteins: the viral RNA replication proteins p29 spots on inoculated leaves of Nicotiana tabacum. TBSV CP induces the and p89, which are translated from the viral genome (Genovés et al., production of necrotic spots on inoculated leaves of Nicotiana 2006); the cell-to-cell movement proteins p7A and p7B, known as the glutinosa and Nicotiana edwardsonii (Scholthof et al., 1995). Hybrid double gene block (DGB), which are translated from a large sub- viruses were used to show that combinations of the replication genomic RNA (Genovés et al., 2006; Navarro et al., 2006); and the coat protein open reading frame 1 (ORF1) and p19 derived from Carnation protein (CP) p42, which is translated from a small sub-genomic RNA Italian ringspot virus (CIRV) and Cymbidium ringspot virus (CymRSV) and is involved in fungal transmission (Mochizuki et al., 2008). The inﬂuenced the induction of systemic lethal necrosis on N. benthamiana p29 ORF terminates with an amber stop codon, and the read-through (Burgyán et al., 2000). In addition, the CymRSV ORF1 product p33 was product can be translated to give p89 (Riviere and Rochon, 1990). A shown to induce systemic lethal necrosis using an individual particular variant of the 3′-untranslated region of the viral genome expression system with a Potato virus X (PVX) vector (Burgyán et al., allows the virus to overcome a MNSV resistance gene in melon plants, 2000). However, it has not yet been elucidated at the molecular level how viral pathogenicity factors induce necrosis in infected plant tissues, although in some virus–host interactions, the development of ⁎ Corresponding author. Fax: +81 29 838 8101. necrotic symptoms upon viral systemic infection has been associated E-mail address: [email protected] (S. Tsuda). 1 Present address: Graduate School of Life and Environmental Sciences, Osaka with the hypersensitive response (HR) (Ehrenfeld et al., 2005; Kim et Prefecture University, Sakai, Osaka 599-8531, Japan. al., 2008; Xu and Roossinck, 2000).

Previously, it was reported that the MNSV CP indirectly contributes to the appearance of necrotic symptoms on infected melon plants by affecting viral movement at the tissue level (Genovés et al., 2006). However, the viral virulence factors and the mechanism of necrosis induction upon MNSV infection are not yet fully understood. Here, we investigated the mechanism of necrosis induction in N. benthamiana expressing MNSV genome-encoded proteins using a foreign gene- expression system based on the Cucumber mosaic virus (CMV). Our results indicated that the MNSV RNA replication protein p29 causes necrotic spots in N. benthamiana by association with the mitochondria. The second transmembrane domain (TMD) in p29 plays a role in mitochondrial targeting. Possible induction mechanisms for necrosis caused by MNSV p29 are discussed.

Results

Determination of an MNSV virulence factor for necrosis

The proteins encoded by the MNSV genome were individually expressed in N. benthamiana plants using a CMV vector system (Figs. 1A and B). For expression of p89, the p89 gene lacking the p29 ORF (named as p89Δ) was expressed because cloning of the full- length p89 ORF for expression from a CMV-based vector was unsuccessful. Necrotic spots appeared on the inoculated leaves only when the replication protein p29 gene was expressed by the vector (CMV-p29; Fig. 1C). In contrast, inoculation with vectors carrying other viral genes (p89Δ,p7A,p7B,andCP)ortheendoplasmic reticulum (ER)-localized highly fluorescent green fluorescent protein (GFP) variant erG3GFP did not induce any necrotic spots on the leaves. Western blot analysis with an antibody against the p29 protein detected a signal with the expected size in the tissues surrounding the necrotic spots on N. benthamiana leaves inoculated with CMV-p29 (Fig. 1D). Also, bands of the expected sizes for the other MNSV proteins and erG3GFP were detected in the tissues of N. benthamiana inoculated with CMV-p89Δ, CMV-p7A, CMV-p7B, CMV-MNCP, and CMV-erG3GFP even though there were no necrotic spots on the leaves (Fig. 1D). When N. benthamiana was inoculated with the CMV-Δp29 which had double stop codons just down stream of the initiation codon of the full-length p29 ORF gene being anontranslatablep29gene(Fig. 1D), any necrotic spots did not appear on the leaves (Fig. 1C), resulting showing that the p29 RNA itself was not the virulence factor for the necrosis induction. These results showed that the MNSV-encoded replication protein p29 could act as a viral virulence factor for the induction of necrotic Fig. 1. (A) Genomic structure of MNSV. The five ORFs are denoted by boxes. For p89 spots. In this experiment, we could not rule out the involvement of expression, the region of the p89 ORF lacking the p29 ORF, with an initiation codon introduced just downstream of the termination codon of the p29 ORF, was used (named intact p89 in the induction of necrotic spots, although p89Δ,which p89Δ). (B) Diagram of a CMV-based expression-vector system. A foreign gene was does not include the p29 sequence, was not involved in necrotic spot introduced into an EcoR V cloning site of CMV RNA3. (C) N. benthamiana leaves development. inoculated with a CMV vector carrying each MNSV gene or the erG3GFP gene. Each transcript from the pCP3-MN series mixed with transcripts from pCP1II and pCP2II was Ultra-structural analysis of CMV-p29-infected cells mechanically inoculated. D: Western blot analysis was used to detect each viral protein expressed in N. benthamiana leaves. Photographs were taken at 4 days post-inoculation for p29 and at 6 days post-inoculation for the other proteins. To understand histologically the mechanisms of induction of necrotic spots by p29 expression, the cytological changes in N. benthamiana cells inoculated with CMV-p29 were observed using transmission electron microscopy. In the cells around the necrotic 2D–F) and in the mock-inoculated cells (data not shown). This spots of CMV-p29-inoculated N. benthamiana, non-uniform aggrega- confirmed that the CMV-p29-induced cytological changes were not tions of mitochondria, spherical and swollen chloroplasts, and caused by CMV vector inoculation. disrupted tonoplast were observed (Fig. 2A). In the mitochondria, the cristae structures were characteristically disrupted (Fig. 2B), Subcellular localization of p29 and influence on mitochondrial function whereas the mitochondria in the cells of the CMV-erG3GFP- inoculated plants appeared normal (Fig. 2E). Although chromatin Previously, it was reported that multivesicular bodies (MVBs), condensation and plasmolysis are characteristic apoptotic features which are considered to be replication-complex structures, were (Greenberg and Yao, 2004; Hatsugai et al., 2004; Vianello et al., 2007; observed on the mitochondrial membrane in MNSV-infected melon Yao et al., 2004), these phenomena were not observed in the cells cells (Di Franco and Martelli, 1987; Yoshida et al., 1980). In addition, (Figs. 2A and C). These cytological features in the CMV-p29-inoculated presence of a mitochondrial targeting signal (MTS) in the amino (N)- cells were not observed in the CMV-erG3GFP-inoculated cells (Figs. terminus of p29 was predicted by a computer program (Ciuffreda T. Mochizuki et al. / Virology 390 (2009) 239–249 241

Fig. 2. Electron micrographs of N. benthamiana tissues infecting the CMV-p29 showing necrotic spots (A–C) or CMV-erG3GFP-inoculated N. benthamiana tissues (D–F). N. benthamiana leaves were mechanically inoculated with RNAs transcribed from pCP1II, pCP2II, and pCP3-MN29dstp or pCP3S2-erG3GFP. B and E show high-magniﬁcation views of mitochondria in dotted line boxes of A and D, respectively. C and F show the structure of the nucleus in a CMV-p29- or CMV-erG3GFP-infected cell, respectively. Note that the cristae structure in the mitochondria of the CMV-p29-infected leaf was disrupted (B), and mitochondrial aggregation was visible in the cell (A, in a rectangle with a dotted line), whereas mitochondria having normal cristae structure were observed in the CMV-erG3GFP-infected leaf (E). In addition, an integral vacuolar membrane was not observed in the CMV-p29- infected cell (A). N: nucleus, Chl: chloroplast, Mito: mitochondria, VAC: vacuole. Bars=2 μm (A, C, D and F) or 500 nm (B and E).

et al., 1998). Thus, we analyzed the subcellular localization of p29 by N. benthamiana leaves were further fractionated by discontinuous cell fractionation analysis. percoll density gradient centrifugation (Gualberto et al., 1995). Two Cell organelles extracted from CMV-p29-inoculated N. benthamiana distinct organelle bands were observed at 0–13% (F1 fraction) and leaves were separated into the following four fractions by centrifuga- 21–45% (F2 fraction) on the percoll gradients. These bands were tion: the precipitates present after centrifugation at 1000, 3000, and collected separately, and the accumulation of p29, erG3GFP, and COX II 10,000 ×g (P1, P3, and P10, respectively), and the supernatant present as a marker protein for the mitochondrial membrane fraction was after centrifugation at 10,000 ×g (S). p29 was detected in fractions P3 analyzed in each fraction. p29 was detected mainly in the F2 fraction and P10, but not in fraction S, which contained a large amount of from CMV-p29-inoculated tissues (Fig. 3C), whereas erG3GFP was cytosolic Hsp70 (Fig. 3A). This indicated that p29 was a membrane- concentrated in the F1 fraction (Fig. 3C), indicating that p29 was not associated protein. Furthermore, when the P10 membrane fraction was localized to the ER. Also, major anti-COX II IgG immuno-reactive bands treated on ice with either an extraction solution (detergent) or an were detected in the F2 fraction from both CMV-p29- and CMV- elution solution (urea, high pH, high salt), only treatment with 2% erG3GFP-inoculated tissues, even though a minor anti-COX II IgG Triton X-100 led to a solubilization of p29, suggesting that is an integral immuno-reactive band appeared in each F1 fraction (Fig. 3C). membrane protein. Some p29 remained in a membrane (P) fraction Moreover, ultra-structural analysis showed that the normal or after this treatment (Fig. 3B). abnormal mitochondria mostly observed in the F2 fraction (Figs. 3D To investigate the mitochondrial association of p29, the P10 and E), whereas the F1 fraction included abundant pieces of thylakoid fractions from CMV-p29-inoculated or CMV-erG3GFP-inoculated from chloroplasts (Fig. 3F arrowheads) and a few mitochondria. 242 T. Mochizuki et al. / Virology 390 (2009) 239–249

Fig. 3. (A) Western blot analysis of the accumulation of MNSV p29 and cytosolic HSP70 in each fraction of N. benthamiana leaf tissues inoculated with CMV-p29 or mock inoculation. The fractions were obtained by continuous differential centrifugation. P1: precipitate after 1000 ×g centrifugation. P3: precipitate after 3000 ×g centrifugation. P10: precipitate after 10,000 ×g centrifugation. S: supernatant after 10,000 ×g centrifugation. The size of the marker protein is indicated on the left. (B) Extraction of p29 from the membrane fraction. The

P10 precipitate of p29-expressing leaf homogenate was treated with either an extraction solution (2% Triton X-100) or elution solutions (4 M urea, 0.1% NaCO3 pH 11.0 or 1 M KCl), following by western blotting with anti-p29 for the membrane (P) and the solubilized (S) fractions. (C) Western blot analysis of the accumulation of MNSV p29, erG3GFP, and COX II in fractions from the P10 precipitate that were re-fractionated by discontinuous percoll density gradient centrifugation. F1: 0–13% percoll interface fraction. F2: 21–45% percoll interface fraction. The size of the marker proteins is indicated on the left. (D) Electron micrograph of mitochondria having cristae structure in the 21–45% percoll interface fraction (F2 in C) of CMV-p29-inoculated N. benthamiana. (E) Electron micrograph of abnormal mitochondria having bilayer membrane that the cristae structure was disrupted in the 21–45% percoll interface fraction (F2 in C) of CMV-p29-inoculated N. benthamiana. (F) Electron micrograph of pieces of thylakoid (arrowheads) in the 0–13% percoll interface fraction (F1 in C) of CMV-p29-inoculated N. benthamiana. F1 fraction mostly contained the pieces of thylakoid. Bars=500 nm (D-F).

Further analysis of p29 in mitochondria was carried out using a Determination of a virulence region of p29 and TMD prediction CMV vector expressing GFP-tagged p29 (CMV-p29GFP)—GFP- tagged p29 was used because immunohistochemical detection To further understand the mechanism by which p29 expression with an anti-p29 antibody was unsuccessful. CMV-p29GFP could induces necrosis, we determined the region of p29 associated with successfully induce necrotic spots on N. benthamiana leaves similar virulence. The formation of necrotic spots on N. benthamiana leaves to those caused by CMV-p29 inoculation (data not shown). N. caused by inoculationwith CMV vectors carrying p29 deletion fragments benthamiana protoplasts inoculated with CMV-p29GFP were treated was examined (Fig. 5A). The deletion of 46 amino acid residues (aa) from with the mitochondria membrane potential (ΔΨm)-sensitive the N-terminus or 202 aa from the carboxy (C)-terminus of p29 mitochondrial marker dye, Mito-Tracker Orange, to visualize prevented the formation of necrotic spots (fragments 29-11 versus 29- mitochondria with a normal membrane potential. Fluorescence of 12 and 29-1 versus 29-14, respectively; see Fig. 5A). This suggested that GFP and the Mito-Tracker dye were simultaneously observed using the minimum region of the protein required for virulence was from 36 to confocal laser-scanning microscopy at 3, 6, 9, 12, and 18 h post- 76 aa. However, the p29 fragment containing only aa 36 to 76 (fragment inoculation (hpi). At 6 and 9 hpi, p29GFP co-localized with Mito- 29-20) did not induce necrotic spots, while necrotic spots appeared Tracker fluorescence in the cell (Fig. 4A upper panel and B), when extra aa were added to the C-terminus (fragments 29-21–29-23). indicating that p29 was associated with the mitochondria. In a Using four computer programs, we further analyzed the potential protoplast expressing the erG3GFP as negative control, Mito-Tracker hydrophobic α-helices, which can act as TMDs, in p29. Three regions fluorescence was able to separate from the erG3GFP fluorescence were identified as putative TMDs; their probabilities are shown in (Fig. 4A lower panels), indicating that spectral “bleed-through” Fig. 5B. Two of the programs (TMpred and TopPred2) predicted a first between GFP and Mito-Tracker Orange fluorescence did not occur. TMD (TMD1) from aa 25 to 50. All four programs—DAS, HMMTOP, From the data shown in Figs. 3 and 4B, we concluded that p29 is a TMpred, and TopPred2—predicted a second TMD (TMD2) from aa 55 mitochondrial membrane-associated protein and that it is an inte- to 75. Two of the programs (DAS and TMpred) also predicted a third gral membrane protein. TMD (TMD3) from aa 191 to 200. The minimum virulence region of In most CMV-p29GFP-inoculated protoplasts, however, Mito- p29 (fragment 29-21), which was necessary for the induction of Tracker fluorescence disappeared by 12–18 hpi (Fig. 4Aupper necrotic spots, included TMD2. In fact, fragment 29-30, in which panel). In the CMV-erG3GFP-inoculated and mock-inoculated proto- TMD2 was deleted, did not produce necrotic spots (Fig. 5A). plasts, Mito-Tracker fluorescence was continuously detected through- Accumulation of the 29-10, 29-11, 29-13, and 29-30 protein fragments out the experimental period (Fig. 4B lower panels and data not in N. benthamiana leaves was confirmed by western blotting with an shown). This indicates that the disappearance of Mito-Tracker anti-p29 antibody (Fig. 5C), showing that the loss of virulence of fluorescence in CMV-p29GFP-inoculated cells was not due to infection 29-10, 29-11, and 29-30 was not due to a failure to express the protein. with the empty vector. These results demonstrated that TMD2 is necessary, but not sufficient, T. Mochizuki et al. / Virology 390 (2009) 239–249 243

Fig. 4. Time-course confocal images of the distribution of GFP-tagged p29 and the detection of active mitochondria in N. benthamiana protoplasts. (A) Protoplasts were inoculated with CMV-p29GFP (upper panel) or CMV-erG3GFP (lower panel) and were treated with a ΔΨm sensitive dye (Mito-Tracker orange CM-H2TMRos) 45 min before observation. The distribution of GFP and the Mito-Tracker dye was observed at 3, 6, 9, 12, and 18 hpi with confocal laser-scanning microscopy. To discern between fluorescence of the Mito-Tracer Orange dye and chloroplast auto-fluorescence, the Mito-Tracker dye (upper lane), GFP (middle lane), and chloroplast auto-fluorescence (lower lane) signals are represented in light- blue, green, and red, respectively. (B) Mitochondrial localization of p29 in the CMV-p29GFP-infected protoplasts at 9 hpi. The cells on the left, center, and right show the distribution of the Mito-Tracker dye, p29GFP, and an overlay image of the two, respectively. The superimposition of the red and green signals is shown in yellow in the panel on the right.

for the virulence of p29. Because the anti-p29 antibody was raised Subcellular localization of p29 deletion fragments against artificially synthesized polypeptide cocktails (CVKVDINE- DAEEESDTGE; 98–114 aa, CTNVNPEVRAKRRHRSRP; 127–143 aa, and To analyze the role of the TMDs in the mitochondrial localization CGLPDWKAFKLVN; 256-268 aa), the antibody failed to detect some of of p29, the subcellular localization of the p29 deletion fragments the p29 fragments (29-1, 29-14–29-21). tagged with GFP was observed in N. benthamiana protoplasts treated In all gene-expression experiments using a CMV vector in this with Mito-Tracker dye (Fig. 6). The distribution of each p29 deletion study, the Kozak consensus-like sequence (AACATG) was added just fragment was observed at 6 hpi. The GFP fluorescence of 29-11GFP upstream of the initiation codon of each ORF for effective translation and 29-13GFP, which contained the TMD2 and TMD3 domains, co- initiation. When the Kozak consensus-like sequence was not added, localized with the Mito-Tracker dye fluorescence, similar to that of the N-terminal deletion fragments 29-12 and 29-13 did not induce an intact form of p29. Fragments 29-1GFP, 29-7GFP, and 29-30GFP, necrotic spots on N. benthamiana leaves after inoculation (data not in which TMD2 was missing, showed a diffuse pattern. The shown). This result also ruled out a role for the p29 RNA itself as the fluorescence of 29-17GFP, which lacked TMD3, was partially co- factor responsible for the necrosis induction. localized with the Mito-tracker dye. These observations revealed 244 T. Mochizuki et al. / Virology 390 (2009) 239–249

ance was detected over time. Fig. 7B shows the average of the three time points from two or three experiments. In intact p29-expressing cells, fluorescence of the Mito-Tracker dye was not observed in N80% of the infected cells. Similarly, in cells expressing the 29-13, 29-17, and 29-21 fragments, Mito-Tracker fluorescence was not observed in ∼70%, 80%, and 80% of cells, respectively. With fragment 29-11, the frequency of the disappearance of Mito-Tracker fluorescence was moderate (b40%). However, in cells expressing fragments 29-1, 29-20, and 29-30, Mito-Tracker fluorescence was detectable in almost cells (b20%). The absence of Mito- Tracker fluorescence in the cells expressing p29 fragments that induced necrotic spots (29, 29-13, 29-17, and 29-21) significantly differed from the absence of Mito-Tracker fluorescence in the cells expressing fragments that did not induce necrotic spots (29-1, 29-11, 29-20, and 29-30) (Tukey's multiple comparison test, p b0.05; different letters on the bars indicate significantly different values). Thus, the induction of necrotic spots by p29 fragments containing TMD2 correlated with the localization of the fragments to the mitochondria, which lead to an alteration in mitochondrial bioactivity. In these experiments, each necrotic spot-inducing p29 fragment consistently induced the disappearance of Mito-Tracker fluorescence at 9 hpi. In contrast, Mito-Tracker fluorescence in cells expressing GFP-tagged p29 was still detectable even at 9 hpi, but not at 12 hpi (Fig. 4A). Thus, the timing of the disappearance of Mito-Tracker dye fluorescence was altered by GFP tagging of p29. We considered that the tagging GFP to the p29 protein might affect the conformation or function of p29, thereby altering the timing of the disappearance of Mito-Tracker dye fluorescence.

Discussion

Here, we showed that expression of the MNSV replication protein p29 triggered necrotic spots on N. benthamiana leaves. Our results also showed that p29 is found on the mitochondria, a localization that is dependent on an internal hydrophobic region, TMD2, putatively consisting of α-helices. Deletion of TMD2 from p29 resulted in a lack of mitochondrial association of p29 and a loss of MNSV virulence. These findings indicated that the mitochondrial association of p29 via TMD2 was necessary for the development of necrotic symptoms on Fig. 5. Diagram of the series of p29 deletion fragments and the induction of necrotic MNSV-infected plants. spots by CMV vector inoculation. The CMV-p29 deletion series was mechanically The deletion fragment analysis of p29 and the subcellular inoculated into N. benthamiana leaves, and the development of necrotic spots was observed. +: positive for the induction of necrotic spots on inoculated N. benthamiana distribution of each deletion fragment, as shown in Figs. 5 and 6, leaves. −: negative for the induction of necrotic spots on inoculated N. benthamiana indicated that TMD2 was involved in mitochondrial targeting of p29 leaves. (B) Potential hydrophobic regions and putative TMDs in p29. The line above the during the process of necrotic spots expression. On the other hand, hydrophobicity graph indicates the positions of the TMDs predicted by the four TMD2 alone was unable to induce the necrotic spots. In fact, the different structural programs. (C) Western blot analysis for detection of p29 fragments in 29-10, 29-11, 29-13 or 29-30 fragment-expressing N. benthamiana leaves. Arrow- fragment 29-11 contained an intact TMD2 but did not induce any heads indicate p29 fragments. The size of the marker proteins is indicated on the left. necrotic spots even though 29-11GFP localized to the mitochondria. The disappearance of Mito-Tracker fluorescence due to expression of 29-11 occurred in 40% of cells, which was higher than the level of the that TMD2 was necessary for the complete mitochondrial localiza- disappearance of Mito-Tracker fluorescence for the other fragments tion of p29. that did not induce necrotic spots (Fig. 7). These results suggest that mitochondrial bioactivity is weakly affected by the mitochondrial Influence of the expression of p29 deletion fragments on targeting of fragment 29-11. Fragment 29-11 does not induce high mitochondrial bioactivity levels of Mito-Tracker fluorescence disappearance, probably because of a lack of amino acid sequences around TMD2. A functional protein We further confirmed the relationship among the disappearance of conformation that includes an extra amino acid region just before the Mito-Tracker dye in cells, the mitochondrial localization, and the TMD2 (between aa 36 and 46) might be important in the induction of virulence of each of the representative p29 deletion fragments mitochondrial damage and in the virulence of p29. There is also a mentioned above. To control for any unexpected effects caused by possibility that the stability of p29 was decreased by the deletion of its expression of the GFP tag, GFP was separately expressed from the CMV N-terminus. For instance, the N-terminal deletion fragments 29-12 RNA2-based vector in inoculated cells (pCP2a-erG3; Fig. 7A). and 29-13 without the Kozak consensus-like sequence did not induce Fluorescence of the Mito-Tracker dye was observed at 9, 12, and 18 the necrotic spots on N. benthamiana, suggesting that the induction of hpi in ∼30 protoplasts in which GFP fluorescence was also detectable virulence by p29 is dose-dependent. (Fig. 7C). The percentage of GFP-expressing cells in which the Mito- ORF1 of the Tombusvirus genus in the family Tombusviridae also Tracker dye was not observed was scored at 9, 12, and 18 hpi, and no encodes the RNA replication protein, likely to the p29 protein of difference of the percentage of Mito-Tracker fluorescence disappear- MNSV. It has previously been described that the p33 of TBSV, T. Mochizuki et al. / Virology 390 (2009) 239–249 245

Fig. 6. Subcellular localization of the p29 deletion fragments tagged with GFP. (A) Upper closed boxes indicate the locations of the predicted TMDs (TMD1–TMD3) in p29. The letters on the right indicate the subcellular localization of each p29 fragment. mito: mitochondria. diffuse: cytoplasm. (B) Confocal images of the distribution of p29 deletion fragments tagged with GFP in each CMV vector-inoculated N. benthamiana protoplast sample at 6 hpi. N. benthamiana protoplasts treated with Mito-Tracker dye were treated 45 min before observation. Mito-Tracker ﬂuorescence is represented in red. The superimposition of the red (Mito-Tracker) and green (GFP) signals is shown in yellow in the overlay image (lower panel).

Cucumber necrosis virus and CymRSV localize the peroxisome that TMD3 might play a role in anchoring p29 to the mitochondrial (McCartney et al., 2005; Navarro et al., 2004; Panavas et al., 2005) membrane. while p36 of CIRV targeting the mitochondria (Rubino et al., 2000, In the current study, we did not investigate how MNSV induces 2001). Interestingly, the chimeric viruses having a hybrid ORF1 necrotic symptoms during the infection cycle in melon plants. between CymRSV and CIRV did not elicit wild type symptoms whereas However, it was previously reported that viral replication proteins of both CymRSV and CIRV induced systemic lethal necrosis on infected N. positive-sense RNA viruses caused a rearrangement of the host benthamiana (Burgyán et al., 2000). Authors speculated that necrotic organelle membrane morphology in order to establish RNA replication symptom was attenuated because of chimeras having the hybrid sites (Schwartz et al., 2004). In fact, MVBs, which are considered to be between the RNA replication proteins that localized in different replication-complex structures, were observed on the mitochondrial organelle. Our results proved this speculation; the causality between membranes of MNSV-infected melon cells (Di Franco and Martelli, subcellular localization of RNA replication protein and the virulence of 1987; Yoshida et al., 1980). The localization of p29 in our studies Tombusviruses. suggested that targeting of p29 and p89 to the mitochondria could Although the presence of an MTS on the N-terminus of MNSV p29 enable the formation of MVBs as viral replication-complex structures was previously predicted by computer analysis (Ciuffreda et al., 1998), by altering the mitochondrial membranes in melon cells. Further- this region was not necessary for the mitochondrial localization of p29 more, the disappearance of Mito-Tracker fluorescence was also in this study (Fig. 6). Similar to the mitochondrial targeting caused by observed in MNSV-inoculated melon cells (Mochizuki et al. unpub- TMD2 of MNSV p29, the mitochondrial localization of a viral RNA lished data). Similar results were seen in N. benthamiana cells (Figs. 4 replication protein with internal α-helices that function as TMDs was and 7). Based on these findings, we hypothesize that p29 modifies shown for the plant virus CIRV (Hwang et al., 2008; Rubino et al., mitochondrial membrane structures to allow the formation of MVBs 2000, 2001; Weber-Lotfi et al., 2002) and for the animal virus Greasy in order to replicate viral RNAs, which causes mitochondrial damage grouper nervous necrosis virus (Guo et al., 2004). Recently, it was and leads to necrosis. shown that p36 of CIRV interacts with a variety of import receptors in The observations made using Mito-Tracker dye indicated that the certain components of the mitochondrial outer membrane complex expression of necrotic spot-inducing p29 fragments affected the (Hwang et al., 2008). However, it is not yet clear how these replication mitochondrial bioactivity in cells (Fig. 7). This suggests that proteins access mitochondria using regions of the TMD. In addition to mitochondrial bioactivity might be involved in the induction of the TMD2, MNSV p29 has two additional putative TMDs: TMD1 and necrotic symptoms caused by p29. In plant cells, some physiological TMD3. Our data showed that TMD1 was not required for the changes in the mitochondria have been reported to be involved in mitochondrial localization of p29 (Fig. 6). In contrast, fragment 29- programmed cell death (PCD) (Curtis and Wolpert, 2004; Krause and 17, which lacked TMD3 but contained TMD1 and TMD2, partially Durner, 2004; Lam et al., 2001; Takabatake et al., 2007; Tiwari et al., localized to the mitochondria and was partially diffuse, suggesting 2002; Xie and Chen, 2000). In our study, however, chromatin 246 T. Mochizuki et al. / Virology 390 (2009) 239–249

Fig. 7. Effect of the expression of p29 deletion fragments on mitochondrial bioactivity in cells. (A) Diagrams of the CMV RNA2-based vector for GFP expression and the CMV RNA3- based vector for expression of p29 deletion fragments. (B) Percentage (average and standard deviation) of cells in which the Mito-Tracker dye disappeared. N. benthamiana protoplasts were co-inoculated with transcripts of RNA1, GFP-expressing RNA2, and each RNA3 carrying a p29 deletion fragment. Mito-Tracker dye was added 45 min before observation. At 9, 12, and 18 hpi, Mito-Tracker fluorescence was observed in the GFP-expressing protoplasts. GFP fluorescence was used to confirm expression of the desired proteins. Approximately 30 cells were observed in each experimental period, and the number of cells in which Mito-Tracker fluorescence was not observed was counted. The experiments were repeated two or three times, and statistical analysis was performed with Tukey's multiple comparison test (pb0.05). Different letters indicate significantly different values. (C) Representative confocal images of mitochondrial bioactivity in N. benthamiana protoplasts (GFP) infected with each CMV expression vector at 12 hpi. White line arrowheads indicate the protoplasts with Mito-Tracker fluorescence in which GFP was detectable. White arrowheads indicate the protoplasts without Mito-Tracker fluorescence in which GFP was detectable. Note that fluorescence of the Mito-Tracker dye was observed in all healthy protoplasts in which the vector infection failed. condensation and plasmolysis, which are characteristic apoptotic Construction of a CMV-based expression vector features of these cells (Greenberg and Yao, 2004; Hatsugai et al., 2004; Vianello et al., 2007; Yao et al., 2004), were not observed in the In order to create a CMV vector from the three plasmids described necrotic tissues of CMV-p29-inoculated N. benthamiana (Fig. 2). above, pCP1II and pCP2II were prepared as modified clones in which a Moreover, resistance genes against MNSV that induce hypersensitive BamH I restriction site located upstream of the T7 promoter sequence responses in melon plants have not previously been reported. In order on pCP1TP1 and pCP2TP1 was removed. pCP3TP2 was modified to to understand the mechanisms of necrosis induction caused by the enable insertion of the erG3GFP (Tamai and Meshi, 2001) by replacing expression of p29, further analysis of the physiological responses of the CP gene to create pCP3S2-erG3. This was done according to the mitochondrial bioactivity to MNSV infection and of the replication protocol previously described (Kim et al., 2004) using the following mechanisms of MNSV are needed. primers: pepodCP-fw (5′-AAGGGAGCTCTTCCGTGTTTTCCAGAACCC- 3′), pepodCP-rv (5′-ATGGGAGCTCGACTCGACTCAATTCTACGA-3′), Materials and methods pepo3ad33-rv (5′-ATAGCACCAAAGTGCTAACTGCGCGCATTCTGAT-3′), and P3R-123-bam (see ref. Saitoh et al., 1999). To make it easy to insert Virus genes, plants, and inoculation a foreign gene into the CMV RNA3 vector in place of erG3GFP, the erG3GFP sequence was replaced by an EcoR V restriction site in The genes encoding the five proteins (p29, p89, p7A, p7B, and CP) pCP3S2-erG3 using the following primers: CMVvec-fw (5′- expressed by the MNSV chiba strain (MNSV-chi) were amplified from GCGGATATCGCTCGAGCTCTTCCGTGTTTTCCAG-3′) and P3R-123-bam the full-length cDNA clone pTMNW1 (GenBank/European Molecular (see ref. Saitoh et al., 1999). The resulting vector was referred to as Biology Laboratory Nucleotide Sequence Database (EMBL)/DNA Data pCP3S2-vec. Bank of Japan (DDBJ) accession no. AB250684, Mochizuki et al., Each MNSV gene was inserted into pCP3S2-vec. For p29 expression, 2008). To develop a CMV vector system, the plasmids pCP1TP1, an additional termination codon was added just downstream of the pCP2TP1, and pCP3TP2, each containing one of the three segments of original leaky termination codon of the p29 gene, resulting in the the genomic RNA (RNA1, RNA2, and RNA3, respectively) of the CMV formation of a double termination codon. For p89, the sequence of p89 pepo strain, were used (Saitoh et al., 1999) (GenBank accession nos. lacking the p29 ORF and with an initiation codon just after the AB124834, AB124835, and AF103991, respectively). N. benthamiana termination codon of the p29 ORF was employed (named p89Δ). For was mechanically inoculated with RNAs transcribed in vitro from the effective translation initiation, the Kozak consensus-like sequence CMV vector plasmids after linearization with appropriate restriction (AACATG) was added just before the initiation codon of each gene. endonucleases. In vitro transcription was performed using the T7 Each MNSV ORF was amplified using the following primers: p29 (fw: RiboMAX™ large-scale RNA-production system (Promega, Madison, 5′-CGCGGATCCAACATGGATACTGGTTTGAAATTTCTTGTTTCC-3′,rv WI). The inoculated N. benthamiana plants were grown in an 5′-CTACTAGTTGACCAACTTGAAGGCCTTCC-3′), p89 (fw 5′-GCTCTA- artificially controlled chamber kept at 25 °C with a 16 h light/8 h GAAACATGGGGTGCCTAGAGGAACTCGCTGGGTTCTGT-3′,rv dark cycle. 5′-CCGCTCGAGTTACTACAGTTCGTTGAGGGTCCATGAGATTGG-3′), p7A T. Mochizuki et al. / Virology 390 (2009) 239–249 247

(fw 5′-CGCGGATCCTCATACCATAGAGTGGGGTTTCGTGCCCC-3′, Electron microscopy rv 5′-CCGCTCGAGTTAACCATCGCCATTTGTAGAGATGCCAAC-3′), p7B (fw 5′ -AACATGGCTTGTTACCGTTGTGA-3′ ,rv5′ - The leaf tissue samples were ﬁxed with 4% glutaraldehyde in 50 mM

CCGCTCGAGTTAACCATCGCCATTTGTAGAGATGCCAAC-3′), CP (fw 5′- KPO4, pH 7.2, overnight at 4 °C, and then post-fixation was performed CGCGGATCCAACATGGCGATGGTTAAACGCATTAATAATTTA-3′,rv5′- with 1% osmium tetroxide in 50 mM KPO4 overnight at 4 °C. After CGCGAGCTCTTAGGCGAGGTAGGCAGTTTCAGGAGAGCA-3′), and Δp29 dehydration in a graded series of ethanol/water mixtures, the tissues (fw: 5′-AACATGGATTGATGATTGAAATTTCTTGTTTCC-3′, underline indi- were embedded in Spur resin (Nisshin EM, Tokyo, Japan). Ultra-thin cates the additional double stop codons). Each MNSV gene fragment was sections (90–100 nm) were prepared, mounted on grids, and stained cloned into the EcoR V site of pCP3S2-vec to create pCP3-MN29dstp, with 2% uranyl acetate and Reynolds lead citrate for 120 min and 30 min, pCP3-MN89Δ, pCP3-MNDGB1 (p7A), pCP3-MNDGB2 (p7B), pCP3- respectively. The sections were observed at 80 kV under a JE1230 MNCP, and pCP3-Δp29, respectively. transmission electron microscope (JEOL, Tokyo, Japan). To prepare the p29 deletion mutants, several primers were designed, and the p29 deletion fragments were amplified by PCR Fractionation of cellular material from N. benthamiana leaves using a combination of the appropriate primers. The Kozak consensus-like sequence was also added to each fragment for the Fractionation of cellular material was performed according to the effective initiation of translation in the manner described above. The methods described by Gualberto et al. (1995) with some modifications. deletion fragments were expressed after cloning into the EcoR V site N. benthamiana leaves that had been inoculated with three transcripts of pCP3S2-vec. The resulting vectors were named pCP3-29-1 through from pCP1II, pCP2II, and pCP3-MN29dstp or with buffer as a mock pCP3-29-30. inoculation were ground in 10 ml of mitochondria extraction buffer To create a p29-GFP fusion protein, the G3GFP gene was added just (0.4 M sucrose, 50 mM Tris–HCl, pH 7.5, 3 mM EDTA, 0.1% bovine serum after the 3′-terminus of the p29 sequence by a two-step PCR albumin (BSA), and 4 mM 2-mercaptoethanol) and homogenized with procedure, and then the p29-G3GFP sequence was inserted into a blender. After incubation for 15 min on ice, the homogenates were pCP3S2-vec to create pCP3-29GFP. p29 deletion fragments tagged passed through two layers of miracloth (Calbiochem, Darmstadt, with G3GFP were also generated by PCR using pCP3-29GFP as a Germany). The extracts were precipitated step by step by a series of template, follow by cloning into the EcoR V site of pCP3-vec to create centrifugations: 1000 ×g for 10 min for fraction P1, 3000 ×g for 15 min pCP3-29-1GFP through pCP3-29-30GFP. for fraction P3, and 10,000 ×g for 15 min for fraction P10. The final The plasmid pCP2II was modified to express a foreign gene from supernatant was added to 10 volumes of acetone and then centrifuged the second segment of the CMV vector system by replacing the 2b at 9000 ×g for 15 min. After washing with 80% acetone, the precipitate gene of CMV with an EcoR V restriction site, according to the was used as the cytosolic fraction (fraction S). These fractions were previously described method (Matsuo et al., 2007) using the following subjected to western blotting with anti-p29 antibody and anti-heat primers: CP2-1168-fw (5′-CGTAATGCTGACGTTCCAGA-3′), CP2aStop- shock protein 70 (Hsp70) monoclonal antibody to identify the cytosolic fw (5′-GTGGACGCGTCCTACGTTAAATTCAATATTTCTTT-3′), CP2a-Stop- fraction (GeneTex Inc., San Antonio, TX). rv (5′-ATGGGAGCTCGACTCGACTCAATTCTACGA-3′), P3R-123-bam To verify that p29 is an integral membrane protein, the P10 fraction

(see ref. Saitoh et al., 1999). The resulting construct was referred to was incubated with either 2% Triton X-100, 4 M urea, 0.1 M NaCO3,pH as pCP2astop. The erG3GFP tag was then introduced into the EcoR V 11.0, or 1 M KCl in 50 mM Tris–HCl, pH 7.5, 3 mM EDTA, and 4 mM site of pCP2astop to create pCP2a-erG3. phenylmethylsulfonyl fluoride for 30 min on ice and then centrifuged at 30,000 ×g for 30 min to separate membrane (P) and solubilized (S) Western blot analysis fractions. Separation of the fractions was followed by western blot analysis with anti-p29 antibody. Polyclonal rabbit antibodies that recognize each MNSV-encoded Further fractionation was performed by discontinuous percoll protein (p29, p89, p7A, and p7B) were raised against polypeptide gradient centrifugation. Extracts from N. benthamiana leaves that were cocktails, which were artificially synthesized based on the respective inoculated with the transcripts from pCP1II, pCP2II, and pCP3-MN28dstp amino acid sequences. A polyclonal antibody to MNSV CP was newly or with the transcript from pCP3S2-erG3 were centrifuged at 1000 ×g for prepared using denatured MNSV virions because a previously 10 min to remove the cellulardebris, starch, and nuclei. The supernatants produced antibody to the intact virion was not able to detect CP in were centrifuged at 10,000 ×g for 15 min, and the precipitates were the western blot analysis. The immunoglobulin G (IgG) fractions of fractionated by discontinuous percoll gradient centrifugation (Gualberto each antiserum were isolated as antibody by the MabTrap Kit (GE et al., 1995). Cytochrome Oxidase Subunit II (COX II) was detected as a Healthcare UK Ltd.; Buckinghamshire, UK). marker protein for the mitochondrial membrane fraction using an anti- Leaf samples were ground in a protein extraction buffer composed COX II antibody (AgriSera, Vännäs, Sweden). of 7 M urea, 2 M thio-urea, 4% Triton X-100, 2% 2-mercaptoethanol, Electron microscope observations of the intercellular organelles 100 mM phosphate buffer pH 7.5, and protease inhibitor cocktail from the percoll fractions embedded in 1% low-melting agarose were (complete mini EDTA-free; Roche Diagnostics, Penzberg, Germany) performed according to the methods described above. and incubated for 30 min at room temperature. Crude homogenates were centrifuged at 4 °C (9000 ×g for 30 min) to remove cellular Protoplast inoculation and confocal laser-scanning microscopy debris. The supernatant was then centrifuged again (20,000 ×g for 30 min). The supernatant (8 μL) was mixed with 2 μl LiDS solution The N. benthamiana leaves peeled lower epidermal layer were (10% lithium dodecyl sulfate and 0.02% bromophenol blue), separated incubated for 3 h in a solution of 1.0% cellulase Onozuka R-10 (Yakult by SDS polyacrylamide gel electrophoresis, and transferred onto a Pharmaceutical, Tokyo, Japan), 0.1% macerozyme (Yakult Pharmaceu- polyvinylidene difluoride membrane or a nitrocellulose membrane. tical), and 0.5 M mannitol, pH 5.8. The protoplasts were then collected After blocking with 3% skim milk in Tris-buffered saline (TBS) and washed three times with a mannitol solution (0.5 M mannitol and including 0.05% Tween 20, the membranes were treated with each 10 mM CaCl2, pH 5.8). For transcript inoculation, the protoplasts were antibody to the MNSV-encoded proteins, followed by treatment with prepared at a density of 5×105 cells per 700 μl electroporation buffer an alkaline-phosphatase-conjugated anti-rabbit antibody. Immuno- (0.5 M mannitol, 10 mM MES, and 70 mM KCl, pH 5.8) containing 20 μl detection was visualized by the bromo-chloro-indolyl phosphate/ RNA in vitro-transcribed from the CMV vector system, as described nitroblue tetrazolium (BCIP/NBT) liquid substrate system (Sigma- above. Electroporation was carried out by a Gene Pulser II and Aldrich, St. Louis, MO). Capacitance Extender (Bio-Rad Laboratories, Hercules, CA) at 190 V, 248 T. Mochizuki et al. / Virology 390 (2009) 239–249

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