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1 Departamento de Genetica Molecular y Microbiologia, Pontificia Universidad Catolica de Chile, Santiago, Chile
2 Centre for Bioinformatics (CBUC), Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile
Correspondence
Patricio Arce-Johnson
parce{at}bio.puc.cl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary figure is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The HR is initiated when a pathogen avirulence gene product (avr) is recognized – directly or indirectly – by a plant resistance gene (R) product (Nimchuk et al., 2003). Upon recognition, defence pathways are activated, leading to an increase in salicylic acid and the appearance of necrotic lesions exclusively in the infected plant tissue. As a result of HR induction, systemic acquired resistance is activated and plants become resistant to attack by a broad spectrum of other pathogens (Dempsey & Klessig, 1994).
Over 200 naturally occurring R genes that recognize virus elicitors have been identified and mapped in crop plants, their wild relatives and the model plant Arabidopsis thaliana (reviewed by Kang et al., 2005). In most cases, a viral gene product, which is essential for the viral life cycle, is the elicitor of the resistance response mediated by an R gene. In the genus Tobamovirus, three viral genome-encoded proteins can elicit the HR; the replicase enzyme, the movement protein (MP) and the coat protein (CP). The 126/183 kDa replicase enzymes of tobacco mosaic virus (TMV) can elicit the HR in Nicotiana tabacum plants carrying the N gene (Padgett & Beachy, 1993). In tomato, the MP of tomato mosaic virus is recognized by the Tm-2 or the allelic Tm-22 resistance genes (Lanfermeijer et al., 2004). CPs of several tobamoviruses induce resistance in pepper carrying L genes and in Nicotiana sylvestris plants carrying the N' gene (Culver & Dawson, 1989; Gilardi et al., 2004).
CP is essential for covering and protecting the RNA genome and also plays several roles during the viral life cycle, such as movement and the integration of the viral replication complex (Saito et al., 1990; Asurmendi et al., 2004). Besides, transgenic plants that produce self-assembling CP are resistant to TMV infection and it has been shown that specific structure based mutations in CP can enhance or reduce CP-mediated resistance in transgenic crops (Bendahmane et al., 1997, 2007; Asurmendi et al., 2007). Due to the close relationship between viral pathogens and their hosts, it is not surprising that CP also participates in the induction of host responses and disease development (reviewed by Culver, 2002). During the infection process, it has been proposed that intermediate aggregates of viral CPs elicit N'-mediated HR (Culver et al., 1994). Using a set of TMV strains encoding different mutant CPs, Culver and colleagues showed that structural characteristics of TMV-CP were responsible for the N'-mediated recognition by the host. They demonstrated that a P20L mutation within CP of the U1 strain of TMV, normally a non-elicitor, leads to the induction of HR in N. sylvestris (Taraporewala & Culver, 1996). Therefore, it has been suggested that alterations affecting the stability of tertiary and quaternary CP structures influence recognition by the host.
We have previously reported that, unlike all known tobamovirus, the crucifer and garlic-infecting strain of TMV (TMV-Cg) induces local and systemic necrotic lesions when inoculated onto N. tabacum cv. Xanthi nn sensitive plants lacking the N gene (Arce-Johnson et al., 2003). However, this response is not sufficient enough to restrict the virus and it therefore spreads from the inoculation site to the whole plant, developing systemic necrotic symptoms. Given that the defence pathways are activated, but the plant becomes infected, this response has been described as an HR-like response (Ehrenfeld et al., 2005). It has been suggested previously that during this response an N homologue gene named NH could be responsible for triggering this failed resistance (Stange et al., 2008). Using a set of hybrid viruses in which each of the TMV-U1 genes were replaced by the corresponding TMV-Cg genes, the CP of TMV-Cg (CPCg) was identified as the elicitor of this HR-like response (Ehrenfeld et al., 2005). The induction of the HR-like response by TMV-Cg occurs specifically in tobacco Xanthi nn and not in Nicotiana benthamiana where it induces severe chlorosis (Stange et al., 2004), neither in Arabidopsis plants where it spreads systemically without causing any resistance response (Arce-Johnson et al., 2003).
In the present study, we examined the influence of specific CPCg amino acid substitutions on the development of the host defence response. Insights into the induction of the HR-like response were gained from molecular modelling and molecular dynamic experiments using the three-dimensional structure of CPCg. Based on previous studies of TMV-U1 CP (Taraporewala & Culver, 1997; Toedt et al., 1999), P21L and R46G mutations were introduced into CPCg. A S54K substitution was also included to increase sequence identity of CPCg and CPU1 in the more divergent region (aa 51–60). In addition, the double CPCg mutant P21L-S54K was studied to determine whether these mutations were redundant, additive or synergistic. The effects of CPCg mutations on the electrostatic surface of the protein, and the formation of inter-subunit aggregates were also addressed. We propose that the stability of CP structures and lateral aggregates affects the induction of the HR-like response in sensitive tobacco Xanthi nn plants.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Virus purification.
For the generation of full-length viral RNA transcripts, 10 µg plasmid DNA (see below) was digested at the KpnI or BstEII sites flanking the viral genome. Transcription reactions were carried out using the mMessage mMachine T7 transcription system (Ambion). Once plants were inoculated and systemically infected, wild-type and hybrid viruses were purified following the method of Asseling & Zaitlin (1998) and resuspended in 20 mM phosphate buffer pH 7.2. Viability of purified viruses were checked in Xanthi NN resistant plants for the generation of local lesions and used for the experiments.
Samples of infected tissues were ground in 20 mM phosphate buffer containing 5 mM EDTA pH 8 and the extracts were centrifuged for 10 min at 20 000 g. Supernatants were collected and spotted onto carbon-coated grids, and stained with 1 % uranyl acetate. Grids were examined under a JEOL 100Cx transmission electron microscope to observe the ultrastructure of mutant viruses.
Construction of mutant hybrid viruses.
Wild-type full-length TMV-Cg (Cg-8) was cloned previously (Arce-Johnson et al., 2003) and U1CPCg hybrid was previously described (Ehrenfeld et al., 2005). Mutant hybrid viruses were constructed using site-directed mutagenesis in the CPCg gene. The sequence of forward and reverse 30 mer oligonucleotides perfectly matched the CPCg region except at the point in which a mutation was introduced. The 5' fragment of CPCg was amplified using a forward primer (CPCg short) and a reverse primer that included the mutation. The 3' region of CPCg and 3' UTR was amplified with a forward primer that carried the codon mutation and a reverse primer (KpnI 3Cg). Both PCR products were purified from agarose gels and the fragments obtained were added in equal amounts to a second amplification, using primers CPCg short and KpnI 3Cg. Fragments of the expected size were cloned into pGEM-T Easy (Promega) and sequenced to confirm the presence of the desired mutations. In all single amino acid mutations, clone Cg-8 was used as template, whereas to generate the double amino acid mutant, the clone containing CPCgP21L was used. The mutant CPs were introduced in the SnaBI and KpnI sites of the vector TMV-U1
CP, which does not contain a CP. The primers used for the site-directed mutagenesis were (introduced mutation underlined): CPCgP21L-F, 5'-CGATGTGGGCAGAGCTGACAGCGATGTTTAACC-3'; CPCgP21L-R, 5'-GGTTAAGCATCGCTGTCAGCTCTGCCCACATCG-3'; CPCgR46G-F, 5'-CAAGAGATACTGTTGGTCAACAGTTCTCAAACCTG-3'; CPCgR46G-R, 5'-CAGGTTTGAGAACTGTTGACCAACAGTATCTCTTG-3'; CPCgS54K-F, 5'-GTTCTCAAACCTGCTAAAGGCGATTGTGACACCA-3'; CPCgS54K-R, 5'-TGGTGTCACAATCGCCTTTAGCAGGTTTGAGAAC-3'; CPCg short, 5'-GGATATCATGTCTTACAACATCACG-3'; KpnI 3Cg, 5'-AGAGGATCCGGGTACCTGG-3'.
Comparative modelling of TMV-CPCg.
A comparative model of the CPCg was built using MODELLER v.8 (Sali & Blundell, 1993). Template coordinates were taken from the closely related ribgrass mosaic virus CP, PDBid: 1RMV (Wang et al., 1997, 1998) possessing 95.5 % sequence identity and 89.8 % of positive residues with CPCg. Subsequently, the four CP mutant variants: P21L, R46G, S54K and P21L-S54K were modelled using the build mutant module implemented in MODELLER. The 100 models generated (20 models per variant) were ranked based on their stereochemistry (Laskowski et al., 1993), the potential energy values computed by MODELLER and the Verify-3D score (Eisenberg et al., 1997). At the end, the five best-ranked models for the wild-type protein and four mutants were chosen for molecular dynamic (MD) experiments. Selected structures were energy minimized to reduce close contacts using a steepest-descents minimization protocol within GROMACS 3.1 (Van der Spoel et al., 2005) until the maximum force fell to 100 [kJ mol–1 nm–1] using the GROMOS-96 force field (van Gunsteren et al., 1998).
Lateral CPCg assemblies.
Lateral assemblies were constructed by arranging CP molecular models in a similar way to that which is found within the disc aggregates of TMV (Bhyravbhatla et al., 1998). Coordinate adjustment was accomplished by C
superimposition of the models onto two laterally adjacent protein subunits in the four-layer aggregate of the TMV CP determined at 2.4 Å (0.24 nm) resolution: PDBid: 1EI7. As a result, five lateral assemblies composed of two protein subunits were generated for the wild-type, P21L, R46G, S54K and P21L-S54K mutants, respectively. The final complexes were adjusted by energy minimization until convergence.
MD experiments.
MD simulations were performed using GROMOS-96 force field (van Gunsteren et al., 1998) within GROMACS 3.1 (Van der Spoel et al., 2005). Single and laterally assembled models were embedded in a solvent box with the simple point charge water model to obtain a periodic boundary condition (Berendsen et al., 1987). Long-range electrostatic interactions were calculated with the particle-mesh Ewald method. Lennard–Jones and short-range Coulombic interactions were restricted at 0.9 nm. The simulations were performed under normal pressure and temperature conditions. Thus, a constant pressure of 1 bar was applied independently in all three directions with a coupling constant of P=0.5 ps and compressibility of 4.5e–5 bar–1. Additionally, Na+ ions were added to compensate the net charge of the systems. Temperature was controlled by independently coupling the protein, solvent and counterions to a 300 K temperature bath, using a coupling constant of 0.1 ps. The structures were energy minimized as stated before. Subsequently, the systems were equilibrated to 300 K during 100 ps with 2 fs integration steps, using the LINCS algorithm to restrain all bond lengths. Finally, constraints over bonds were removed and a full 1 ns MD simulation at 300 K was performed with 1 fs integration steps. Trajectory frames were collected every picosecond.
Calculations of electrostatic potentials by DelPhi.
Calculations of electrostatic potentials were performed by solving the Poisson–Boltzmann equation, employing the finite difference method as implemented in DelPhi within Insight II (Accelrys Inc.). A standard protein formal atomic charge and internal and external dielectric constant values of 2 and 80, respectively, were employed. Solvent probe radii of 1.4 Å (0.14 nm) and a physiological ionic strength of 0.145 M were used. Figures were prepared using the solvent accessible molecular surface area of every model, and coloured according to their electrostatic potential grid values.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This is an incomplete resistant response associated with the appearance of necrotic lesions in the inoculated leaf followed by viral systemic movement and development of systemic necrotic lesions. Previous work allowed us to identify CPCg as the elicitor of the HR-like response in Xanthi nn sensitive plants (Ehrenfeld et al., 2005). The hybrid virus U1CPCg that contains the 5' UTR, the 126/183 kDa replicase enzymes and the MP of TMV-U1 but the CP and 3' UTR of TMV-Cg was the only hybrid that also induced this response in sensitive tobacco plants. Four hybrid mutant viruses were constructed in this work based on the U1CPCg hybrid virus. Therefore, all hybrid mutant viruses contain the complete sequence of TMV-U1 replicase enzymes and the MP, and each one has the sequence of TMV-Cg CP with different amino acid substitutions. These mutants were named as follows: U1CPCgP21L, U1CPCgR46G, U1CPCgS54K and the double mutant U1CPCgP21L-S54K. RNA of all virus mutants was synthesized by in vitro transcription and then inoculated onto tobacco plants. The hybrid virus U1CPCg and the wild-type TMV-U1 were used as controls of transcription and inoculation. All mutants induced the appearance of necrotic lesions and viral spread was restricted to the inoculated leaves in Xanthi NN plants, indicating that all were capable of normal replication and cell-to-cell movement.
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). It induced the formation of readily discernible large necrotic lesions on the inoculated leaves of sensitive Xanthi nn plants (Fig. 1e
), but no lesions appeared in systemic leaves even after 50 days p.i. Samples of systemic tissues and also of inoculated leaves of Xanthi nn infected plants with the U1CPCgP21L mutant were observed under electron microscopy and viral particles were not found (Fig. 2). Additionally, CP could not be detected by immunoblot analysis, using polyclonal antibodies, in systemic tissues of inoculated N. tabacum or N. benthamiana plants. However, the presence of U1CPCgP21L viral RNA in the infected leaves of sensitive tobacco was confirmed by sequencing of RT-PCR products, confirming that this mutant was restricted to the initial infected leaf.
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). Mutant hybrid virus U1CPCgS54K induced mosaic symptoms in the apical leaves around 7 days p.i. (Table 1) and there was no appearance of local or systemic necrotic lesions. Electron microscopy of U1CPCgS54K-inoculated leaf extracts showed abundant virions (Fig. 2). The presence of U1CPCgR46G and U1CPCgS54K in apical tissues was confirmed by immunoblot assays detecting CPCg (data not shown).
Since the CPCgP21L substitution caused an HR phenotype associated with the lack of systemic movement, we evaluated whether the S54K substitution could counteract this effect. The infection of sensitive tobacco with the double mutant hybrid virus U1CPCgP21L-S54K induced the appearance of necrotic lesions on the inoculated leaves (Fig. 1h) and also in the intermediate leaves of the plant after 25 days p.i. Thus, the double CPCg mutant P21L-S54K recovered the capacity to form virions and to induce an HR-like response. The systemic movement of this double mutant was lower than all the other mutant and wild-type viruses (Table 1). To corroborate that the mutations had not been lost during the infection process, specific amplifications and subsequent sequencing of the PCR products demonstrated that the virus found in apical leaves of these infected plants contained a CPCg mutated specifically at the position P21
L or S54K.
Structure and electrostatic surface properties of CPCg comparative models
As expected, the CPCg models fold into a right-handed -helical bundle, a common feature of tobamovirus CPs. The four core helices, named left and right slewed (LS and RS), and left and right radial (LR and RR), are denoted in Fig. 3. All amino acid substitutions generated in CPCg by in vitro mutagenesis are close to the LS and RS helices. Previous evidence points to the presence of a recognition surface within the right face of the CPU1 -helical bundle for the N' gene-mediated induction of HR in tobacco plants (Taraporewala & Culver, 1997). Moreover, various findings indicate that charged surface residues within the elicitor active site contribute to the N' gene-mediated recognition of all tobamoviruses (Taraporewala & Culver, 1996). Based on this evidence, a comparative analysis of the electrostatic surface potential map for all CPCg models was conducted as shown in Fig. 4. As depicted, mutations in the CP structure would produce changes of different intensities over the surface charge distribution. Comparative analysis highlights major differences between the CPCgP21L structure (Fig. 4) and the rest of the models. Local variations in charge distribution are also observed in the remaining mutant models when compared with CPCg wild-type. Those changes were consistent with the mutation introduced, being more positive for the inclusion of lysine in the CPCgS54K and CPCgP21L-S54K (green arrowheads in Fig. 4), and more negative for the loss of the positively charged arginine in the CPCgR46G mutant (yellow arrowheads).
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). According to these values, CPU1 and CPCgS54K were the most stable structures, whereas CPCgP21L showed higher fluctuations. Nonetheless, secondary structure elements and the overall architecture were maintained in all the assayed structures during the entire simulation.
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, the reference assembly structure corresponding to the CPU1 protein subunits is the most stable. The lateral assemblies of CPCgS54K, CPCgR46G, CPCgP21L-S54K and the wild-type CPCg, computed RMSD values around 0.3 nm, whereas the CPCgP21L lateral assembly computed the highest RMSD value around 0.5 nm. Distances calculated during the MD experiment between the centres of mass of the subunits in the assemblies are depicted in Fig. 5(c), where it can be observed that all distances decrease during the MD experiment. Under this analysis, the CPCgR46G lateral assembly exhibited the shortest monomer–monomer distance. On the other hand, the monomer distance became larger in the CPCgP21L and CPCgP21L-S54K lateral assemblies.
The inner surface long loop plays the most significant part in destabilizing CPCg
To examine the contribution of individual amino acids on the structural stability of CP, the C root-mean-square fluctuation (RMSF) of model structures were computed during the last 500 ps of the MD experiment (Fig. 6). The averaging time period was determined based on the observation of backbone RMSD drift. As observed in Fig. 6, the highest atomic fluctuations (>0.2 nm) occur in the inner surface loop from residues 90 to 110 in agreement with the high temperature factors observed for the analogous region in crystallographic structures of TMV-U1 (Namba & Stubbs 1986; Bhyravbhatla et al., 1998). Substantial fluctuations were also observed in the N- and C-terminal regions, again consistent with these crystallographic diffraction reports.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and in the present work we constructed and analysed several CP mutants to gain insight into the structural determinants that induce this host response. Molecular and histological studies have shown that even when sensitive tobacco plants do not carry the N and N' genes, a defence response that leads to cell death is mounted (Stange et al., 2004, Ehrenfeld et al., 2005). Therefore, in these cases, CPCg is acting as a symptom determinant. Since 1 % of the plant genome encodes resistance genes (Hammond-Kosack & Jones, 1997; Meyers et al., 2003), sensitive plants might have unknown resistance genes, which to different degrees can be capable of recognizing diverse pathogens, such as the N homologous gene in Xanthi nn tobacco (Stange et al., 2004).
The U1CPCgP21L mutant was constructed with the aim of inducing a complete HR in sensitive plants, since the equivalent mutation enhances the stability of U1CP and transforms the non-elicitor U1CP into an HR inducer in the N. sylvestris system (Culver et al., 1994). However, even though U1CPCgP21L virus induced larger necrotic lesions than U1CPCg virus in the inoculated leaves of sensitive tobacco, it failed to spread systemically (Table 1). The induction of large necrotic lesions in the inoculated leaves has been related to slow viral cell-to-cell movement (Deom et al., 1994). Viral RNA moves to neighbouring cells associated with the viral replication complex (VRC), of which the CP is a component (Asurmendi et al., 2004). The inability of U1CPCgP21L virus to systemically infect the Xanthi nn plants was possibly caused by its inability to form virus particles (Table 1, Fig. 2). Structural changes in the CPCgP21L may be affecting the formation of VRCs and therefore the efficiency of local movement, reflected in larger necrotic lesions. The data obtained by MD experiments of U1CPCgP21L are in agreement with the absence of virions of this hybrid mutant virus. As the RMSD and RMSF plots obtained from the MD experiments show the greatest atomic fluctuations of the single models and lateral assemblies correspond to CPCgP21L. These results indicate that the P21L mutation has disturbing effects over the CP tertiary structure (Fig. 5a), electrostatic surface potential (Fig. 4) and consequently on the quaternary arrangement (Fig. 5b and c). In the TMV-U1 strain, the P20L substitution contributes to a hydrophobic interaction between neighbouring CP molecules, since little effect on tertiary structure was detected (Culver et al., 1994). In contrast, for the CPCgP21L structure, the MD experiment reveals tertiary structure fluctuations with major changes observed within both loops and the contiguous residues in the RS–RR helices (Fig. 6), suggesting a possible interfering role in CP aggregation.
Two mutant viruses U1CPCgR46G and U1CPCgS54K overcome the HR-like response of sensitive tobacco plants. Both move systemically and cause mosaic symptoms like TMV-U1 (Table 1). The R46G mutation in CPU1 has been extensively studied by Culver et al. (1994) and Toedt et al. (1999). The mutation induces a moderate HR in N. sylvestris but virions move systemically. As in CPCg, the R46 residue in the CPU1 structure is located within the RS helix and participates in both lateral and axial interactions with neighbouring subunits (Culver et al., 1994). It was demonstrated that even though the mutant protein does not possess important three-dimensional structural variations compared with the wild-type CPU1, the equilibrium of intermediate assembling aggregates is affected in the mutant (Toedt et al., 1999). In the case of CPCgR46G, no major changes were observed in the tertiary structure. However, the absence of the bulky positive arginine side chain generates local changes in the surface charge distribution (Fig. 4, yellow arrows) that could modify the binding site involved in host recognition as well as the interactions with a neighbouring CP within the lateral assembly. Nevertheless, whilst this may affect the aggregation kinetics of multiple CPCgR46G subunits, virions were still observed in infected tissue (Table 1).
The mutant virus U1CPCgS54K was designed in an attempt to generate a CPCg similar to CPU1, changing the neutral serine residue present in CPCg to a positive lysine residue, as found in CPU1. The mutant virus was capable of infecting sensitive tobacco without generating an HR-like response. The CPCgS54K mutation made CPCg more similar to TMV-U1 in its infection capacity and probably its structure reduces the possibility of recognition by the host plant. By analysing the in silico results, we postulate that the stability gained by the CPCgS54K mutant with respect to the wild-type monomer could be explained by the presence of an intrasubunit salt-bridge between the exposed K54 mutated residue and the E20 residue (see Supplementary Fig. S1 available in JGV Online). In support of this finding, an analogous LS–RS ionic interaction is observed in the CPU1 protein between the E22 and K53 ionic groups (Caspar & Namba, 1990).
Considering that mutations P21L and S54K induced opposite effects on the virion assembly and movement, we evaluated the effect of the 2 aa substitutions using the U1CPCgP21L-S54K construct. The infection of sensitive tobacco with this double mutant virus induced the appearance of necrotic lesions on the inoculated leaves with systemic movement of the virus followed by the appearance of necrotic lesions in the apical leaves (Fig. 1). The induction of the HR-like response was recovered and the behaviour of the hybrid mutant virus was similar to the hybrid U1CPCg. The analysis performed on the MD experiment data showed that the double mutant retains the electrostatic surface potential properties of the CPCgS54K mutant (see Fig. 4). However, it has a reduced interaction with a neighbouring CP, to that of CPCgP21L when it was arranged laterally with an identical subunit (Fig. 5c). This suggests that the formation of lateral assemblies in the double mutant could have a different stoichiometry and/or kinetics of small-order CP aggregates due to the disturbing effect of the P21L substitution, explaining in this way the observed HR-like response.
The CP mutations analysed in this study affected the radial zone of CPCg, and the in silico experiments suggest that the amino acid substitutions could have an important influence in the association capacity of the CPs and consequently in the host recognition response. Collectively, our results suggest that the CPCg proteins or small intermediates (laterally assembled dimers) are important in the recognition and subsequent elicitation of the HR-like response in sensitive tobacco plants.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 August 2007;
accepted 21 November 2007.
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