- Open Access
Rice black-streaked dwarf virus P6 self-interacts to form punctate, viroplasm-like structures in the cytoplasm and recruits viroplasm-associated protein P9-1
© Wang et al; licensee BioMed Central Ltd. 2011
- Received: 26 October 2010
- Accepted: 18 January 2011
- Published: 18 January 2011
Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus within the family Reoviridae, can infect several graminaceous plant species including rice, maize and wheat, and is transmitted by planthoppers. Although several RBSDV proteins have been studied in detail, functions of the nonstructural protein P6 are still largely unknown.
In the current study, we employed yeast two-hybrid assays, bimolecular fluorescence complementation and subcellular localization experiments to show that P6 can self-interact to form punctate, cytoplasmic viroplasm-like structures (VLS) when expressed alone in plant cells. The region from residues 395 to 659 is necessary for P6 self-interaction, whereas two polypeptides (residues 580-620 and 615-655) are involved in the subcellular localization of P6. Furthermore, P6 strongly interacts with the viroplasm-associated protein P9-1 and recruits P9-1 to localize in VLS. The P6 395-659 region is also important for the P6-P9-1 interaction, and deleting any region of P9-1 abolishes this heterologous interaction.
RBSDV P6 protein has an intrinsic ability to self-interact and forms VLS without other RBSDV proteins or RNAs. P6 recruits P9-1 to VLS by direct protein-protein interaction. This is the first report on the functionality of RBSDV P6 protein. P6 may be involved in the process of viroplasm nucleation and virus morphogenesis.
- Green Fluorescent Protein
- Onion Epidermal Cell
- Onion Cell
- BiFC Assay
- Bimolecular Fluorescence Complementation Assay
Rice black-streaked dwarf virus (RBSDV), an important pathogen that belongs to the genus Fijivirus in the family Reoviridae, causes rice black-streaked dwarf and maize rough dwarf diseases, which lead to severe yield losses of crops in southeast Asian countries [1–4]. The virus is transmitted to graminaceous plant species via the planthopper Laodelphax striatellus in a persistent, circulative manner [4–6]. Typical symptoms caused by RBSDV include stunting, darkening of leaves and white tumours or black-streaked swellings along the veins on the back of the leaves, leaf blades and sheaths. Microscopy of ultrathin sections has shown that the virions are restricted to the phloem tissues in infected plants and that viroplasms, virus crystals and tubular structures are abundantly synthesized in both infected plants and insect cells [1, 4, 7, 8].
The RBSDV virion is an icosahedral, double-layered particle with a diameter of 75-80 nm and consists of ten genomic dsRNA segments [9–12]. Protein sequence analysis suggested that S1 encodes a putative 168.8-kDa RNA-dependent RNA polymerase. S2 and S4 encode a core protein and an outer-shell B-spike protein, respectively [8, 11, 12]. The protein encoded by S3 is assumed to have some guanylyltransferase activity . Proteins translated from S8 and S10 are the components of the major capsid and outer capsid, respectively [8, 14, 15]. Both S7 and S9 encode nonstructural proteins. S7 ORF1 P7-1 and S9 ORF1 P9-1 are components of the tubular structures and viroplasm produced in infected cells, respectively . Recent studies have demonstrated that P9-1, an α-helical protein with a molecular mass of 40 kDa, self-interacts to form dimers, and it is proposed to be the minimal viral component required for viroplasm formation . P6 is a large nonstructural protein containing 792 amino acids with a molecular mass of 89.6 kDa that is translated from S6, which is 2645 bp in length and contains a single long ORF. It is synthesized abundantly in RBSDV-infected plants and viruliferous planthoppers . However, further characterization and elucidation of the functions of P6 have not yet been reported.
In this study, we investigated the homologous interaction P6-P6 using a yeast two-hybrid (YTH) assay and bimolecular fluorescence complementation assay (BiFC) and determined the subcellular localization of P6 and P6 derivatives using two different fluorescent markers. P6 self-interacts and forms large discrete viroplasm-like structures (VLS) in plant cytoplasm. The minimal region of P6 necessary for P6 self-interaction in vivo is composed of amino acids residing between positions 395 and 659. The exact residues in this region that greatly affect the subcellular distribution of P6 were also determined. Furthermore, a strong interaction between P6 and the viroplasm-associated protein P9-1 was apparent from YTH analyses and co-expression experiments. These results might provide deeper understanding of the process of viroplasm formation of RBSDV.
P6 forms punctate, cytoplasmic viroplasm-like structures in vivo and self-interacts in YTH system
Subsequently, a YTH assay was performed to find out whether P6 had an intrinsic ability to self-interact in vivo. Combinations of plasmids expressing bait protein BD-P6 and prey protein AD-P6 were transformed into Y187 and AH109 strains, respectively. Making sure there was no transcriptional activation or toxicity of BD-P6 for yeast strains, western blot analysis was carried out to verify that both BD-P6 and AD-P6 were expressed in the yeast (data not shown). Cotransformation and yeast mating assays showed that independent yeast colonies containing pGADT7-P6 and pGBKT7-P6 grew well and turned blue in the β-galactosidase colony-lift filter assay (data not shown), indicating that there were strong interactions between P6 molecules. In contrast, no growth was observed for the negative controls (Figure 1B). This suggested that P6 has an inherent ability to self-interact and is able to form VLS when expressed alone in plant cells.
YTH assays indicate the centrally located region spanning residues 395 to 659 is necessary for P6 self-interaction
As there was not much information available from the literature about P6, protein sequence analysis was performed. BLAST searches indicated that the region approximately inclusive of residues 400 to 675 exhibited limited conservation of amino-acid sequence with the ATPase domain of structural maintenance of chromosomes proteins (SMCs), which play an essential role in chromosome segregation, condensation and organization .
The YTH analysis indicated that a centrally located domain between positions 395 and 659 was required for P6-P6 interaction. All truncations harbouring this region were able to interact with intact P6. However, as their N and C termini approached this region, the abilities of the P6 mutants to associate with intact P6 decreased. Varying interaction abilities were indicated by the rates of yeast growth on the selective medium. When the deletion comprised exactly the region from positions 395 to 659, the interaction with P6 was very weak, and the colonies transformed with pGADT7-P6395-659/pGBKT7-P6 or pGADT7-P6/pGBKT7-P6395-659 showed obvious growth inhibition and the streaks turned dark red. Mutant P61-449, in which most of the central and C-terminal region was deleted, showed complete inability to interact with P6 (Figure 2B). Binding capabilities between these deletions were also investigated, and the results demonstrated that, even when both the N and C termini were absent, the deletions had some ability to associate with each other (data not shown). The results suggested that the region from residues 395 to 659 is necessary to sustain the P6 self-interaction and that further truncation might abolish this interaction.
Transient expression experiments of P6 derivatives indicate residues 395 to 659 are important for P6 self-interaction
Recombinant plasmids that can express P6274-792, P6395-703 and P6395-659, fused in-frame to the N terminus of GFP (P6mutant-GFP) or the C terminus of DsRed2 (DsRed-P6mutant), were constructed and their subcellular localization was determined. Plasmids expressing P6mutant-GFP were delivered into onion epidermal cells via biolistic bombardment, whereas those expressing DsRed-P6mutant were introduced into epidermal cells of N. benthamiana leaves by agroinfiltration assay .
Generally, the fluorescence distribution patterns of the three mutants (P6274-792, P6395-703 and P6395-659) indicated that the 395-659 region is important for P6 localization and that self-assembly is possible outside of the P6 native environment. The results also suggested that residues on both sides of the 395-659 region might be engaged in the process, based on the numbers and the size of the fluorescent foci.
Bimolecular fluorescence complementation assay confirms that P6 molecules self-interact in planta
Polypeptides consisting of residues 580 to 620 and 615 to 655 are involved in VLS formation
To sum up, two polypeptide chains, comprising residues 580 to 620 and 615 to 655, are implicated in VLS formation, and loss of them alters the subcellular localization of P6.
YTH assays demonstrate P6 interacts with P9-1
P9-1 cannot form inclusion-like structures when expressed alone
Colocalization experiments indicate P6 relocalizes the distribution of P9-1 and recruits P9-1 to VLS
Co-expression experiments were developed to investigate potential P6-P9-1 interactions (Figure 8B). We introduced two plasmids expressing P6-GFP and DsRed-P9-1 into onion cells by cobombardment. Contrary to the case when DsRed-P9-1 was expressed alone, when P6-GFP and DsRed-P9-1 were co-expressed, a striking relocalization of red fluorescence emerged. DsRed-P9-1 displayed a nearly complete coincidence with the intracellular distribution of P6-GFP. The two proteins were colocalized and exclusively presented in discrete punctate VLS, identical to those formed by P6-GFP alone, and no diffuse green or red fluorescent signals were observed in the cytoplasm or the nuclei. Control combinations were also investigated to rule out the possibility that GFP or DsRed2 expression might have some aberrant effects on the DsRed-P9-1 or P6-GFP distribution. The colocalization of P6-GFP and DsRed-P9-1 confirmed that P6 has a dramatic effect on the distribution of P9-1 and that it is caused by the direct association between these two proteins.
YTH assays confirm residues 395 to 659 of P6 are necessary for P6-P9-1 heterologous interaction
Further YTH analyses were performed to examine the regions of P6 crucial for P6-P9-1 heterologous interaction. P6 AD-fused deletions, including AD-P61-449, AD-P6341-792, AD-P6274-703, AD-P6271-703, AD-P6395-703 and AD-P6395-659, were tested and all P6 deletions except AD-P61-449 were able to interact with P9-1. Transformants expressing BD-P9-1 and AD-P61-449 showed no growth on the selective medium, whereas those containing other combinations grew well (Figure 7B). The results indicated that the region located between amino acids 395 and 659 is indispensable for P6-P9-1 interaction.
YTH assays indicate deletion mutants of P9-1 do not interact with P6
We also investigated P9-1 regions crucial for P6-P9-1 interaction. A dozen P9-1 BD-fused deletions that express fusions BD-P9-11-197, BD-P9-11-207, BD-P9-11-248, BD-P9-176-347, BD-P9-1167-347, BD-P9-1198-347, BD-P9-1208-347 and BD-P9-176-207 were constructed. YTH results indicated that all deletions completely lost the ability to interact with P6 (Figure 7C). It is supposed that minor changes in the protein sequence might affect the properties and protein structure of P9-1 and thereby abrogate P6-P9-1 interaction.
Compared to animal reoviruses, most events in the Fijivirus life cycle, such as virus entry, replication, and packaging and particle assembly and systemic movement, are poorly understood, as are the functions of proteins encoded by the viral genome. In this study, we investigated the uncharacterized protein P6 of RBSDV, a member of the Fijivirus genus, by employing the related experiments in protein-protein interactions.
YTH analysis and/or subcellular localization experiments showed that P6 interact (Figure 2B) and establish punctate VLS when solely expressed in plant cells (Figure 1; Additional file 1, Figure S1), and BiFC assays also indicated that the truncated version P6274-703 (equivalent to one-third of the whole P6 protein) is able to interact intimately to form aggregate-like structures (Figure 4). These results, which clearly demonstrated that P6 has a strong ability to self-assemble, prompted us to question whether P6 is capable of forming multimeric structures. Multimerization of viral proteins always plays an essential role in the virus cycle [20–22]. In Reoviridae, the viroplasm determinants, such as NSP2 and NSP5 of rotaviruses, μNS and σNS of orthoreoviruses, NS2 of orbiviruses, and Pns12 of rice dwarf virus, all share this characteristic to assemble into higher-order complexes to recruit other viral proteins or RNAs [23–29]. The self-interaction of RBSDV P6 might be prerequisite for its multimerization and subsequently for its biological functions.
The coiled-coil region might be involved in P6-P6 interactions. Coiled-coil motifs are increasingly recognized as key determinants in both intra- and inter-molecular interactions. In our experiments, the P6 region spanning residues 365 to 659, which is predicted to harbour a coiled-coil structure and show some sequence homology with the ATPase domain of SMCs, is crucial for VLS formation. Deleting two peptide chains (aa 580-620 and aa 624-654) abolishes VLS formation (Figure 6), which suggests that loss of this region might have a pronounced effect in altering the context of the whole protein and perturb the correct folding of the coiled-coil domain and thereby inhibit molecular interactions. On the basis of the different rates of yeast growth in the YTH assay and the different numbers of fluorescent foci formed in transient expression experiments, we conclude that, whereas the central region spanning residues 365 to 659 is identified as important for P6-P6 or P6-P9-1 interactions, the amino acid sequences near to this region might also affect these interactions by changing the stability of the newly-built protein complexes.
A strong interaction between P6 and P9-1 was detected in our experiments. The two proteins are both expressed at high levels in infected plants and viruliferous insects, as detected by using antibodies against them [8, 17]. Previous experiments indicated that the viroplasm matrix was densely and evenly immunolabelled with antibodies against P9-1 . Although corresponding electron microscopy results have not been obtained for P6, the ability of P6 to form VLS and the heterologous interaction between P6 and P9-1, as well as the localization of P9-1 in hosts, hint that P6 might associate with viroplasm and play a role in the viroplasm nucleation. It is noteworthy that orthoreovirus μNS, which plays an essential role in the process of viroplasm formation, is able to assemble into globular VLS when expressed alone and recruit another viroplasm-associated protein σNS to the VLS [24, 30, 31]. This is quite similar to our results.
Despite the lack of detectable protein sequence homology with animal reovirus proteins, P6 possesses some common features with their viroplasm determinants. Being expressed at high level in hosts, possessing the ability to form VLS and recruiting the viroplasm-associated protein P9-1, P6 protein prediction showed that the P6 fragment located between amino acids 400 and 675 has a low homology with the SMC ATPase domain, whereas the region from positions 404 to 439 is likely to be a pumilio RNA-binding repeat profile, which indicates that P6 might be involved in ATP hydrolysis and binding of RNA. Generally, viroplasm determinants are often inferred to possess NTP-hydrolysis and RNA-binding activities to assist in the process of RNA replication, especially in Reoviridae[32, 33]. It is necessary to do further work to elucidate the biochemical and biophysical properties of P6 and to determine whether P6 functions according to a mechanism that is similar to other viroplasm determinants of reoviruses in the process of viroplasm formation.
RBSDV P9-1 was previously reported to form inclusion bodies when tagged with GFP at its C-terminus and expressed in Arabidopsis protoplasts , which is contrary to our findings. We investigated the P9-1 distribution by two different transient expression strategies. Whenever the protein is fused with GFP at its C terminus or DsRed2 at its N terminus and expressed in onion (Figure 8A) or tobacco epidermal cells or tobacco protoplasts (Additional file 3, Figure S3), P9-1 has a diffuse distribution pattern in the cytoplasm and cannot form aggregates. Consistent with the conclusion reached by Zhang et al , we confirmed that P9-1 self-interacts in the YTH system and forms stable dimers in vitro (data not shown). P9-1 itself might not be the nucleating factor in plant cells for it is located in the VLS only when coexpressed with P6.
Being highly homologous to RBSDV P9-1 (64.5% identity) , Mal de Rio Cuarto virus (MRCV) P9-1, which was detected in viroplams in both infected plants and planthoppers , was found to be sufficient for the formation of viral inclusion body (VIB)-like structures when expressed in Spodoptera frugiperda Sf9 cells . There might be distinct mechanisms involved in viroplasm formation in insect and plant hosts. These questions need to be addressed in future work.
This is the first report on the functionality of RBSDV nonstructural protein P6, which previously was completely uncharacterized. Our results showed RBSDV P6 self-interacts and forms punctate cytoplasmic VLS when expressed alone. Furthermore, P6 strongly interacts with the viroplasm-associated protein P9-1 and recruits P9-1 to localize in VLS. The P6 and P9-1 regions necessary for these homologous or heterologous interactions were also determined, as well as the exact residues essential for P6 VLS formation. Results presented here might provide clues for understanding the viroplasm nucleation of RBSDV and allow us to gain further insight into the relationship between P6 and P9-1 in the virus life cycle.
Healthy N. benthamiana plants were grown at 23 °C under 1,000 lumens with a 16-hour daylight regimen. Agrobacterium tumefaciens strain EHA105 was grown on LB agar containing 50 g/ml rifampin. The yeast strains, Saccharomyces cerevisiae AH109 and Y187, and the yeast vectors, pGBKT7 and pGADT7, as well as the positive control plasmids, pGBKT7-T and pGADT7-p53, were used for YTH analyses (Clontech). The binary expression vectors pGDR and pGDp19 used to express Tomato bushy stunt virus (TBSV) p19 for suppressing gene silencing were obtained as generous gifts from Professor Andrew O. Jackson of the University of California at Berkeley, USA, while another plasmid, pEGFP (Clontech) which harboured EGFP segment, was kindly provided by Professor Zaifeng Fan, China Agricultural University, PR China. BiFC vectors, pSPYNE-35S and pSPYCE-35S, were kindly provided by Professor Jörg Kudla, Universität Müneter, Germany. Both RBSDV S6 (GenBank: AY144570) and S9 (GenBank: AF536564) full-length cDNA clones were maintained in our lab .
Construction of recombinant plasmids
Primers used for PCR amplification.
Sequence (5′→3′) a
Locations b and modifications
full-length reverse primer with stop codon; Sac I
CG ggatcc ATGTCTGCCC
1aa; Bam HI
CTAG ccatgg GA ATGTCTGCCCACCTGACCAATTTAG
1aa; Nco I
CG ggatcc TTACTCAGAGCTTAGTTGCCAGAGG
full-length reverse primer with stop codon; Bam HI
CCG ctcgag CTCAGAGCTTAGTTGCC
full-length reverse primer without stop codon; Xho I
CCG ctcgag ATCAGCTACTTCGTCAG
449aa; Xho I
CG ggatcc AC ATGTCTGCCCACCTG
1aa; Bam HI
CCG ctcgag ccatgg AAGCTTCTGATGTCCAG
274aa; Xho I, Nco I
CCG ctcgag ccatgg ACTTGATTAATCATGCC
395aa; Xho I, Nco I
CG ggatcc ggtacc ATCTCCAAAGTTAGCATCTAC
703aa; Bam HI, Kpn I
CG ggatcc ggtacc CGTTTCATTAGCAGATGTTTTG
659aa; Bam HI, Kpn I
TCC cccggg GAACAGATCGGCATGATTAATC
403aa; Sma I
TCC cccggg GTGAATGATTTAACTGACGAAG
440aa; Sma I
CATG gggccc GTCTTTCTCTTTTAGTAAAGAACAG
615aa; Apa I
CATG gggccc TCTGCTAATGAAACGAATGATG
655aa; Apa I
CATG gggccc GGCAATCTGTTCTTTAGCTTGTC
580aa; Apa I
CATG gggccc GAGAACGAAATGTTGAAGGAACAG
620aa; Apa I
GC tctaga ccatgg ACGTACTCAACCTGTCCAA
98aa; Xba I, Nco I
GC tctaga ccatgg AAGCTTCTGATGTCCAGTC
274aa; Xba I, Nco I
CCG ctcgag ggtacc CTCAGAGCTTAGTTGCCAGAG
full-length reverse primer without stop codon; Xho I Kpn I
CTAG ccatgg GA ATGGCAGACCAAGAGCG
1aa; Nco I
CG ggatcc AACGTCCAATTTCAAGG
full-length reverse primer without stop codon; Bam HI
CG ggatcc ATGGCAGACC AAGAGCG
1aa; Bam HI
CCG ctcgag AACGTCCAATTTCAAGG
full-length reverse primer without stop codon; Xho I
CCG gaattc TCTCATCTCCCTAACC
76aa; Eco RI
CG ggatcc CAAATACATTAAAAAGCC
207aa; Bam HI
CCG gaattc GGTGAAAATCCAAACTC
208aa; Eco RI
CG ggatcc GTGATTAACTTCTTTATTTG
248aa; Bam HI
CCG ctcgag CT ATGGCAGACCAAGAGCG
1aa; Xho I
ggtacc ggatcc TCAAACGT CCAATTTCAAG
full-length reverse primer, Kpn I, Bam HI
gaattc gtcgac ATGGCAGACCAAGAGC
1aa, Eco RI, Sal I
gaattc gtcgac ATGTCGTTGTTGCCAAT
167aa, Eco RI, Sal I
gaattc gtcgac ATGTATATAAAAGGCTT
198aa, Eco RI, Sal I
To generate yeast plasmids for the two-hybrid assay, P6 ORF was amplified using primers PS6-4/PS6-5. PCR products were digested with Nco I/Bam HI, and then ligated into the same sites of pGADT7 to generate pGADT7-S6. Vectors pGADT7-S9-1 and pGBKT7-S9-1 were created using similar strategies. P6 ORF was amplified using primers PS6-6/PS6-9 and the PCR products were ligated to pMD19-T. The clone was digested with Bam HI/Sal I, and P6 Bam HI/Sal I-fragments were ligated into the corresponding sites of pGBKT7 to obtain pGBKT7-S6. P6 Nco I/Bam HI-fragments excised from the pMD19-T intermediates (containing P6274-703, P6395-703, P6395-659) were ligated into Nco I/Bam HI sites of pGADT7 and pGBKT7 to generate vectors expressing truncations fused with AD or BD. pGADT7-S6 was digested with Eco RI/Bam HI and Eco RV, and the liberated fragments were ligated into pGADT7 Eco RI/Bam HI and Sma I to obtain constructs expressing AD-P6341-792 and AD-P6271-703. P6 Bam HI/Xho I-fragment from the intermediate harbouring P61-449 was inserted into the corresponding sites of pET30a, and then the clone was digested with Nco I/Xho I. The liberated fragment was cloned into the Nco I/Xho I-digested pGADT7 to obtain constructs expressing AD-P61-449. P6 Nco I/Xho I-fragments excised from the pMD19-T intermediates (containing P698-792 and P6274-792) were ligated into the same sites of pGBKT7 to generate vectors expressing truncations BD-P698-792 and BD-P6274-792. Similar strategies were used to generate the constructs expressing P9-1 mutants fused with BD.
To obtain construction binary vectors for BiFC, the intermediate which contained P6274-703 was digested with Xba I/Bam HI, and the P6274-703 fragment was then inserted into the same sites of pSPYNE-35S and pSPYCE-35S to generate vectors expressing P6274-703-NE and P6274-703-CE.
The primers used in the experiments are shown in Table 1. All clones derived from the PCR products were verified by sequencing, and the recombinant plasmids were confirmed by restriction analyses.
YTH and β-galactosidase assays
Yeast transformations were conducted using the small-scale lithium acetate method. Two-hybrid assays were performed using the Matchmaker GAL4 Two-Hybrid System3 (Clontech), according to the manufacturer's protocols. Cotransformants were plated on synthetic defined (SD) minimal medium minus adenine, histidine, leucine, and tryptophan (SD/-Ade/-His/-Leu/-Trp), and positive yeast colonies that could grow on the auxotrophic medium were lysed in liquid nitrogen and then tested for β-galactosidase activity as mentioned in the β-galactosidase colony-lift filter assay.
Transient expression of protein in onion cells
To introduce plasmid DNA into onion epidermal cells, particle bombardment was conducted using a helium-driven particle accelerator PDS-1000/He (Bio-Rad). 2-5 μg plasmid DNA in 5 μL distilled water were mixed with 8 μL of a 60 mg/mL 1.0-μm-diameter gold particle solution, 20 μL of 2.5 M CaCl2, and 8 μL of 0.1 M fresh prepared spermidine. The resultant suspension was incubated for 10 min with intermittent mixing every 1 to 2 min at room temperature. The golden particles coated with plasmid DNA were collected by 5-s pulse centrifugation. After the supernatant was removed, the pellet was washed with 100 μL of 70% cold ethanol followed by the same volume of 100% cold ethanol, and then suspended in 10 μL 100% ethanol. After being dried on the center of an aluminum foil rupture disk, the gold particles were bombarded into onion cells under a vacuum of 28 mm Hg with 6-cm target distances. The bombarded onion epidermal cells were cultured on 0.6% agar with 2,4-D-free MS medium at 25 °C in darkness. Fluorescence signals were detected at 16 to 24 h after bombardment [37, 38].
Subcellular localization of RBSDV P6 derivatives and BiFC assay in N. benthamiana leaves
Different binary plasmids were transformed into A. tumefaciens EHA105 by a freeze-thaw method. Cultures of EHA105 harbouring a relevant binary plasmid were grown in LB medium containing rifampicin (50 g/ml) and kanamycin (100 g/ml) at 28 °C for 16 h. For expression of different fusions, EHA105 strains containing the pGDR derivatives and pGDp19 plasmid were resuspended and adjusted to an OD600 of 0.5:0.3 with infiltration medium (10 mM MES, pH 5.6, 10 mM MgCl2, 150 mM acetosyringone). For the BiFC assay, Agrobacterium cultures containing the BiFC plasmids and the pGDp19 plasmid were resuspended at a final OD600 of 0.5:0.5:0.3. The cells were incubated at room temperature for 2 to 4 h, and then infiltrated into 5-6-week-old N. benthamiana leaves. Underside epidermal cells of tobacco infiltrated leaves were assayed for fluorescence 48-96 h after infiltration .
Laser-scanning confocal microscopy
Fluorescence analysis was performed using a Nikon ECLIPSE TE2000-E inverted fluorescence microscope equipped with a Nikon D-ECLIPSE C1 spectral confocal laser scanning system. GFP and YFP were both detected with an excitation at 488 nm and emission capture at 550-590 nm. DsRed2 was excited at 543 nm using a 543-nm helium neon laser, and the emission was captured at 570 to 600 nm . For analysis of coexpression assays, multi-tracking was used to prevent emission cross-talk between the channels.
We are grateful to Professor Andrew O. Jackson (Department of Plant and Microbial Biology, University of California, Berkeley) and Sek-Man Wong (National University of Singapore, Singapore) for providing valuable suggestions. We also thank Professors Jörg Kudla (Universität Münster, Germany) for providing BiFC vectors. This research was supported by the National Basic Research Program (2006CB101903) and National Department Public Benefit Research Funds (nyhyzx07-051, 2008ZX08003-001 and 2009ZX08003-010B).
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