1 School of Biological Sciences, University of Nebraska, Lincoln, NE 68588-0118, USA
2 Department of Biology and Medicinal Science, Pai Chai University, Daejeon 302-735, Korea
Correspondence
T. Jack Morris
jmorris{at}unlnotes.unl.edu
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ABSTRACT |
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INTRODUCTION |
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A second well-studied active plant defence mechanism is the resistance response, which is mediated by a specific resistance gene (R gene) in the plant host. This form of defence is conditioned by the presence of a corresponding avirulence or effector gene in the pathogen. This gene-for-gene interaction triggers a hypersensitive response at the site of invasion that limits the spread of the pathogen (Dangl & Jones, 2001). The specificity of this innate defence system has long been speculated to be due to direct recognition of the effector protein by the host R protein (Gabriel & Rolfe, 1990
). However, molecular characterization of several R proteins and corresponding effector proteins, followed by attempts to demonstrate their physical interactions, has verified such direct recognition events in only three systems (Tang et al., 1996
; Jia et al., 2000
; Deslandes et al., 2003
). Instead, several reports have identified other novel host proteins [e.g. TCV-interacting protein (TIP), RIN4 and PBS1] that play an essential role in mediating the R gene response (Ren et al., 2000
; Mackey et al., 2002
, 2003
; Shao et al., 2003
). In these cases, the pathogen effectors interact with these novel host proteins and modify them biochemically, leading to R gene-dependent resistance. These findings lend fresh support to the guard hypothesis' (Dangl & Jones, 2001
) as an explanation of how R genes function. This hypothesis postulates that R proteins in the nucleotide-binding sites/leucine-rich repeat domains class, to which most of the characterized R proteins belong, function by guarding key components (such as TIP, RIN4 or PBS1) of host basal defence pathways. It is proposed that pathogens would have evolved the ability to target key components of the basal defence system in order to invade successfully. To counteract the pathogen invasion, plants have evolved a surveillance system composed of R genes that effectively detect changes in components of the basal defence system that are brought about by pathogen attack. Although the plant basal defence system has not been well-defined, our recent results from studies on Turnip crinkle virus (TCV) and its interaction with the host plant Arabidopsis thaliana raised the speculation that the RNA-silencing pathway could well represent a basal defence system that is targeted by viruses, components of which could then be surveyed by R genes.
The coat protein (CP) of TCV is a potent effector protein because it is both a suppressor of RNA silencing (Qu et al., 2003) and an elicitor of R gene-mediated resistance that is conditioned by the HRT gene, identified in ecotype Di-17 of A. thaliana (Cooley et al., 2000
). We have shown that TCV CP also interacts specifically with the host protein TIP and established that this interaction correlates with the ability of ecotype Di-17 to mount an HRT-mediated resistance response (Ren et al., 2000
). These results were cited by Dangl & Jones (2001)
as evidence in support of extending the guard hypothesis' to include virus pathogens. Since then, we have also established that the TIP protein is a member of the NAC family of transcription factors and that interaction between TCV CP and TIP protein in plant cells prevents the transcription factor from localizing to the nucleus (T. Ren, F. Qu & T. J. Morris, unpublished results). Based on these data, we find it tempting to suggest that RNA silencing might function as a basal resistance pathway that is suppressed by TCV CP through CPTIP interaction, and that HRT might operate by detecting this interaction as the trigger for mounting the resistance response. This model was supported by Thomas et al. (2003)
in a paper on the characterization of TCV CP as a suppressor of silencing. In that study, they deleted the N-terminal 25 aa in TCV CP that we had implicated in CPTIP interaction and found that the resulting deletion mutant had lost the ability to suppress RNA silencing. We felt that it was very important to test our resistance-breaking TCV CP mutants rigorously for their ability to suppress RNA silencing, as these results have important implications with respect to connecting CPTIP interaction and, hence, the R gene-mediated resistance response, to the RNA-silencing pathway. In the current report, we show that TCV CPTIP interaction is unnecessary for the suppression of RNA silencing by TCV CP. Our data suggest, instead, that the suppressor and resistance-elicitor functions of TCV CP may be distinct, supporting the conclusion that TCV CP interferes with multiple host basal defence pathways.
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METHODS |
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Analysis of RNA and proteins extracted from infiltrated leaves.
RNA extraction and Northern blot analysis were carried out as reported previously (Qu et al., 2003). Protein extracts were prepared from infiltrated leaves by grinding the leaves in liquid nitrogen and suspending the ground tissue in PBS containing 0·1 % mercaptoethanol. Extracts were then centrifuged at 8000 g for 10 min and the supernatant fractions were subjected to SDS-PAGE and Western blot analysis following standard procedures (Sambrook et al., 1989
).
Particle bombardment of detached Arabidopsis leaves.
The protocol of Després et al. (2003) was used with minor modifications. A. thaliana (ecotype Col-0) was maintained in a growth chamber at 21 °C (day) and 18 °C (night) with a 10 h photoperiod. Leaves from 4- to 5-week-old plants were placed on plates containing medium with MS salts, micronutrients, B5 vitamins and 1 % sucrose at pH 5·8, and solidified with 0·8 % agar (A-1296; Sigma). The leaves were bombarded at a pressure of 1100 p.s.i. (7500 kPa) and a flight distance of 4 cm by using a Bio-Rad PDS-1000/He biolistic particle delivery system with M10 tungsten particles coated with 1 µg pRTL2GFP plasmid mixed with 1 µg pRTL2TCVCP, pRTL2CP
or pRTL2R6A plasmids. Five leaves were bombarded for each combination of plasmids. The plates containing the bombarded leaves were then kept in the growth chamber for 48 h. The leaves treated with the same plasmids were pooled and total RNA was extracted and subjected to Northern blot analysis.
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RESULTS AND DISCUSSION |
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Suppression of RNA silencing by TCV CP is compromised by in-frame deletions within all structural domains of TCV CP
In view of our results, we felt that it was important to examine more directly the possible connection between the TIP-binding function of the TCV CP R domain and its potential silencing-suppression activity, which was inferred in the report of Thomas et al. (2003). These authors showed that removal of the first 25 aa abolished the silencing-suppression activity of TCV CP and suggested that this might be related to the inability of TCV CP to bind to TIP. Our data showed that point mutations in the R domain that abolish TIP interaction do not abolish suppressor activity, indicating that the two phenomena are not connected. However, their results raise the need to examine whether suppression of RNA silencing might require several different domains or an intact CP. To test this possibility, we made a series of deletion mutants by removing specific regions of TCV CP and examined each mutant for its ability to suppress RNA silencing. The structural domains of TCV CP are shown in Fig. 1
(Qu & Morris, 1999
). The R domain is located at the N-terminus and is composed of the first 52 aa that interacts with the viral RNA inside the virus shell. The R domain is connected by a 29 aa arm to the surface (S) domain, which makes up the shell of the virion. The S domain connects through a 5 aa hinge to the protruding (P) domain, which forms the spike on the virion surface. The ability of each major structural domain to suppress RNA silencing was tested by transiently expressing each of the six deletion mutants that are shown in Fig. 1
in N. benthamiana leaves. The two R-domain mutants contained smaller deletions than the mutant that was tested by Thomas et al. (2003)
.
ST had 11 aa deleted (aa 212) and
STa had only 4 aa deleted (aa 69). The entire P domain was removed in
P; mutants S and P each contained the entire respective domain, together with the 5 aa hinge. Mutant SP lacked the entire R domain and arm. Agrobacterium cultures harbouring binary vectors that were engineered to express each of the mutant proteins were infiltrated into GFP 16c plant leaves, together with the GFP-expressing Agrobacterium suspension. GFP mRNA accumulation was monitored by Northern blot analysis as described above (Fig. 3
, top panel). It is clear that none of the deletion mutants was able to suppress the silencing of GFP mRNA to the level of full-length TCV CP. Only the
STa mutant, with a deletion of 4 aa in the region of the R domain that encompassed both the R6A and R8A mutations, retained some silencing-suppression activity. It is also noteworthy that the
P mutant with an intact N-terminal R domain was completely ineffective at suppressing RNA silencing. When the blot was reprobed with a TCV CP-specific probe (middle panel), only
STa mRNA was detected in appreciable quantities, confirming that this was the only mutant to retain some suppressor activity. These experiments support the conclusion that retention of CPTIP interaction ability is not necessary for silencing suppression. Moreover, these results show that large deletions in any part of the protein debilitate the silencing-suppression function, suggesting that the activity probably requires intact TCV CP.
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Our inability to connect the silencing-suppressor activity of TCV CP directly with its ability to bind to the TIP factor, which is associated with the R gene-mediated resistance response in Arabidopsis, is disappointing. It is clear, however, that the hypothesis emerging from studies on bacterial plant pathogens is that the signalling cascade leading to the resistance response initiates from a multiprotein complex of R proteins, effectors and as yet unidentified components of the basal resistance system (Ellis & Dodds, 2003). Our most recent results have confirmed the interaction of TCV CP with the TIP transcription factor in vivo. We have also shown that wild-type TCP CP prevents accumulation of TIP in the nucleus, suggesting that this interaction regulates host gene expression. The fact that TCV CP accumulates to such high concentrations in infected plants may explain why it has been selected for multiple functions associated with hostpathogen interactions. Although we have not yet been able to link the two resistance pathways directly, our results do not detract from a role for TCV CP as the effector protein consistent with the guard hypothesis, but rather suggest that TCV CP may interfere with multiple basal defence pathways.
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ACKNOWLEDGEMENTS |
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Received 27 May 2004;
accepted 21 July 2004.