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Bolt-bearing strength of wood and modified wood : Bearing strength of commercial aircraft plywood under aircraft bolts
Forest Products Laboratory (U.S.); McLeod, A. M.
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FPL_1523-Cocr.pdf
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Information reviewed and reaffirmed March 1956. Original report issued Nov. 1946.
Plywood -- TestingDouglas fir -- TestingYellow birch -- TestingLiriodendron tulipifera -- Testing
Early reports of research findings from U. S. Forest Service Research Stations.A RanGAP protein physically interacts with the NB-LRR protein Rx, and is required for Rx-mediated viral resistance - Sacco - 2007 - The Plant Journal - Wiley Online Library
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Race-specific disease resistance in plants is mediated by the products of host disease resistance (R) genes. Plant genomes possess hundreds of R gene homologs encoding nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. NB-LRR proteins induce a disease resistance response following recognition of pathogen-encoded avirulence (Avr) proteins. However, little is known about the general mechanisms by which NB-LRR proteins recognize Avr proteins or how they subsequently induce defense responses. The Rx NB-LRR protein of potato confers resistance to potato virus&X (PVX). Using a co-purification strategy, we have identified a Ran GTPase-activating protein (RanGAP2) as an Rx-interacting protein. We show by co-immunoprecipitation that this interaction is mediated in&planta through the putative signaling domain at the Rx amino terminus. Overexpression of RanGAP2 results in activation of certain Rx derivatives. Likewise, knocking down RanGAP2 expression in Nicotiana benthamiana by virus-induced gene silencing compromises Rx-mediated resistance to PVX. Thus, we have demonstrated a novel role for a RanGAP in the function of a plant disease resistance response.IntroductionDisease resistance in plants operates through multiple mechanisms to protect the host from potential pathogens and parasites. If the pathogen is able to breach the physical barrier imposed by the plant waxy cuticle and cell wall, pathogens encounter a line of defense directed by membrane-spanning extracellular proteins that recognize conserved pathogen-associated molecular patterns (PAMPs). This PAMP-triggered immunity (PTI) is in some ways analogous to innate immune responses controlled by animal Toll-like receptors, and is directed towards a broad range of microbes (). Many host-adapted pathogens express effector proteins that are delivered to the host cytoplasm and promote virulence in part by suppressing PTI. A stronger and more specific form of resistance may be encountered by pathogens, the effectors of which are recognized by the plant rendering the pathogen avirulent. In this case the effectors are termed as avirulence (Avr) factors, and the resulting response is termed as effector-triggered immunity (ETI). ETI is mediated by the products of plant disease resistance genes (R proteins) that confer resistance specifically to pathogens expressing a corresponding Avr gene (). This interaction triggers a defense response that limits pathogen growth and may lead to local programmed cell death, referred to as the hypersensitive response (HR) ().Disease resistance research has progressed rapidly in recent years with the identification of a growing catalog of cloned R genes conferring resistance to bacterial, viral, fungal and oomycete pathogens, as well as insect and nematode parasites (; ). The most numerous classes of R genes that confer recognition of intracellular effectors encode proteins with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains. These NB-LRR proteins show similarities in domain structure to the NACHT-LRR proteins of animals, which have been demonstrated to play important roles in animal innate immunity (; ; ).The carboxy-terminal LRR domains of NB-LRR proteins are highly variable in primary structure and number of repeats, and appear to be under diversifying selection (). Molecular genetic studies have shown that the LRR region determines recognition specificity (; ; ; ; ). The NB domains of two plant R proteins have been shown to bind and hydrolyze ATP, and it is thought that this domain acts to regulate NB-LRR protein activity (). Between the NB and LRR domains lies a conserved domain known as the ARC (Apaf-1, R&proteins and CED-4) domain (; ; ). The combination of the NB and ARC domains is often referred to as the NBS or NB-ARC domain. Through its interaction with the NB and LRR domains, the ARC domain plays an important role in regulating the activity of NB-LRR proteins (). The NB-LRR proteins are grouped into two classes based on the domain present at their amino termini. The amino terminus of the TIR-NB-LRR class shows homology to the toll/interleukin-1 receptor (TIR) cytoplasmic domain, whereas the non-TIR class has a less conserved domain with a predicted coiled-coil (CC) structure in some members, and is often termed the CC-NB-LRR class (). The amino termini of NB-LRR proteins are thought to act as signal adaptor domains, although the molecules through which they signal have yet to be identified.Plant NB-LRR proteins undergo at least two intramolecular interactions (; ), and recognition of Avr determinants appears to trigger a switch mechanism by altering these interactions (). However, general mechanisms of how recognition occurs or how subsequent signal transduction events are initiated remain elusive. This is in part because very few proteins have been identified that interact with NB-LRR proteins. The proteins RAR1, SGT1 and HSP90 are required for the function of multiple NB-LRR proteins, and physically interact with some NB-LRRs; however, these proteins appear to be largely required for the proper folding and stability of NB-LRR proteins (; ; ; ). Three proteins, RIN4, PBS1 and Pto, have been shown to bind to the amino-terminal domains of the NB-LRR proteins RPM1, RPS5 and Prf, respectively. RIN4, PBS1 and Pto also interact with the cognate Avr determinants of their NB-LRR partners, suggesting that these proteins mediate indirect recognition by their associated R proteins (; ; ; ). In addition, the barley MLA proteins interact with WRKY transcription factors through their amino termini, although it is unclear whether this interaction is involved in recognition or in signaling ().The Rx and Rx2 genes of potato encode two highly similar CC-NB-LRR proteins that mediate resistance to potato virus&X (PVX), with the viral coat protein (CP) acting as Avr determinant (). We undertook a biochemical approach to identify cellular proteins that interact with Rx-like proteins, which may function in pathogen recognition and/or signal transduction. Sucrose gradient and native polyacrylamide-gel electrophoresis (PAGE) suggest that prior to elicitation Rx exists in a stable complex of relatively low molecular weight. We show that a Ran GTPase-activating protein (RanGAP2) constitutively interacts with the CC domain of Rx. Overexpression of potato RanGAP2 results in an Rx-dependent activation of defense responses, whereas silencing of RanGAP2 expression in Nicotiana benthamiana resulted in a compromise of Rx-mediated resistance to PVX. The involvement of a RanGAP in the function of an NB-LRR points to a novel function for this protein, and lends insight into the molecular mechanisms of disease resistance in plants.ResultsIdentification of a RanGAP as a component of an Rx-containing protein complexWe first sought evidence that Rx constitutively interacted with other proteins in the Nicotiana tabacum cell line NT-1 and in N.&benthamiana leaves. Using sucrose density gradient centrifugation, we found that Rx, expressed from its genomic promoter with four C-terminal HA tags (Rx:4HA), fractionated with proteins of a molecular weight &232&kDa. When these fractions were resolved by native PAGE, Rx:4HA migrated as two distinct bands at approximately 115 and 180&kDa, relative to protein standards (). Similar results were also observed for Rx:4HA from transgenic N.&benthamiana extracts (data not shown). Although accurate estimation of size using native gels requires more in-depth analysis, these results suggested that Rx (predicted size approximately 113&kDa) is present in a monomeric form, as well as forming a stable complex with at least one other protein. Notably, the size of the complex seen by both sucrose gradient fractionation and native PAGE was in contradiction to size exclusion chromatography (SEC) data, where Rx:4HA eluted in a peak centered around 300&kDa (Figure&S1). The appearance of a higher molecular weight complex for Rx:4HA by SEC is likely to be the result of aberrant mobility caused by the non-globular nature of the LRR domain (; ).Figure&1. &Biochemical demonstration of Rx protein complexes.(a) Rx exists in more than one native form. Extracts from a tobacco NT-1 cell line expressing Rx:4HA were separated through a 10&40% sucrose gradient, with gel filtration protein standards centrifuged in parallel. Fractions (0.8&ml) collected from the top (1&12) were subject to native polyacrylamide-gel electrophoresis (PAGE) (upper panel) or sodium dodecyl sulphate (SDS)&PAGE (lower panel). The same gel filtration protein standards used for centrifugation were run on the native PAGE gel for comparison. Following transfer of proteins to polyvinylidene difluoride membrane, Rx was detected using an anti-HA mAb. Rx was detected as two native protein bands (arrowheads), which migrated in sucrose gradients to fractions with proteins of &232&kDa.(b) Rx&CC:HA forms a higher molecular weight complex. Rx&CC:HA was expressed transiently in Nicotiana benthamiana, and the native forms of this single domain construct were resolved by size exclusion chromatography. Fractions were analyzed by anti-HA immunoblots of SDS&PAGE gels. Elution volumes (in ml) of fractions are indicated, with the elution peaks of the protein standards used to calibrate the column indicated with arrowheads.(c) Rx&CC:HA co-immunoprecipitates a protein of approximately 65&kDa. Immunoprecipitation with anti-HA agarose beads was performed on soluble extracts prepared from control wild-type N.&benthamiana leaves or leaves transiently expressing Rx&CC:HA. Extracts were analyzed by Coomassie Brilliant Blue G250 staining of SDS-PAGE resolved proteins (left panel) to ensure immunoprecipitation inputs were equal. Silver staining was performed to detect immunoprecipitated proteins eluted with HA peptide from the anti-HA agarose beads (right panel). Rx&CC:HA and the approximately 65-kDa protein (p65) are indicated.As the amino-terminal domains of NB-LRR proteins have been proposed to serve as signal adaptors, the Rx CC domain (Rx&CC:HA) was examined by SEC after transient expression in N&benthamiana. Rx&CC:HA eluted in a peak corresponding to the predicted molecular weight of 25&kDa, as well as in a peak corresponding to approximately 150&kDa (), suggesting that it may be responsible for the interaction with another protein(s).Both Rx:4HA and Rx&CC:HA were used to perform immunoaffinity purification. Several proteins were found to co-purify with the Rx&CC domain, however only an approximately 65-kDa protein was found to consistently co-purify with both Rx&CC:HA () and with full-length Rx (data not shown) in silver-stained gels. This protein was excised from gels stained with Coomassie G-250 and was subjected to tandem mass spectrometry (MS/MS) for identification. Two independent experiments resulted in acquisition of the same two peptide sequences: FLNLSDNALGEK and SPLLEDFR. Database searches with the two peptide sequences identified a potato (Solanum tuberosum) expressed sequence tag (EST596034) encoding a plant RanGAP. The Arabidopsis thaliana genome encodes two RanGAPs (). EST596034 is most similar to AtRanGAP2, and is hereafter designated StRanGAP2 (). We identified an additional clone (EST425553) encoding StRanGAP1 that shares 68% identity with StRanGAP2, but has amino acid substitutions in the peptide sequences obtained by MS/MS (Figure&S2). Using sequence data from the potato RanGAP genes, we also amplified portions of NbRanGAP1 and NbRanGAP2. The encoded proteins showed very strong similarity to the potato proteins (Figure&S2).Figure&2. &Phylogenetic relationships among RanGAP1 and RanGAP2 protein homologs. RanGAP homologs from Solanum tuberosum (potato) and Nicotiana benthamiana were aligned with a previously described Medicago truncatula homolog and Arabidopsis thaliana homologs used to designate clone names (Figure&S2). Bootstrap values show the percentage occurrence in 500 random samplings. The scale bar represents 0.1 expected amino acid residue replacements per site.StRanGAP2 interacts with the CC domains of Rx-like proteinsAs Rx originates from potato, we used the potato RanGAP proteins to confirm the interaction with Rx. Both StRanGAP1 and StRanGAP2 were tagged with a C-terminal FLAG plus six histidine (FH) tag and were transiently expressed in N.&benthamiana leaves with HA-tagged Rx (Rx:HA), or with several deletion constructs thereof. StRanGAP2:FH co-immunoprecipitated with all Rx derivatives that included the CC domain, but not with NB-ARC-LRR, LRR, EGFP:HA or an HA-tagged PVX CP (). StRanGAP1:FH did not interact with any Rx fragments, EGFP or CP (; data not shown), indicating that Rx stably and specifically binds to only StRanGAP2 through its CC domain.Figure&3. &StRanGAP2 specifically interacts with Rx-related coiled-coil (CC) domains.(a) HA-tagged Rx derivatives, PVX&CP and EGFP were tested for interaction with StRanGAP1:FH and StRanGAP2:FH. Soluble extracts from Nicotiana benthamiana transiently expressing tagged proteins were immunoprecipitated (IP) with anti-HA agarose beads. Input and immunoprecipitated proteins were detected by immunoblotting (IB), as indicated.(b) StRanGAP1:FH or StRanGAP2:FH were transiently co-expressed in N.&benthamiana with HA-tagged CC domain fragments of Rx, Rx2, Gpa2, Bs2 and HRT. Recombinant RanGAPs were immunoprecipitated with anti-FLAG agarose beads and detected by anti-FLAG immunoblot analysis (lower panel). Input and co-immunoprecipitating CC domains were detected with anti-HA blots.(c) Rx interacts with a native RanGAP. Protein extracts from Rx4HA-2 transgenic N.&benthamiana were immunoprecipitated using anti-HA agarose beads alongside wild-type N.&benthamiana extracts. Coomassie G-250 staining of the sodium dodecyl sulphate&polyacrylamide-gel electrophoresis gel shows equal protein input into immunoprecipitations. Single bands corresponding to Rx:4HA (top right) and to RanGAP (bottom right) were detected by immunoblot analyses of immunoprecipitates using anti-HA and anti-AtRanGAP1 antibodies, respectively.We predicted that StRanGAP2 would interact with the CC domains of the Rx-like proteins Rx2 and Gpa2 (; ) because these domains share 93 and 95% amino acid identity with Rx, respectively. As expected, both the Rx2 and Gpa2 CC:HA domains were co-immunoprecipitated with StRanGAP2:FH (). We also tested for interaction between the StRanGAPs and two other CC domains. Of characterized R proteins, the Bs2 protein from pepper () has a CC domain that is the most similar to the CC domains of the Rx-like proteins, whereas the HRT protein from A.&thaliana () is more diverged. Neither StRanGAP1 nor StRanGAP2 interacted with the CC domains of Bs2 or HRT, further demonstrating the specificity of the StRanGAP2 interaction with the Rx-like proteins (). Finally, we wished to determine whether Rx interacts with RanGAP proteins when both are expressed at physiological levels. Protein extracts from transgenic N.&benthamiana expressing Rx:4HA from the Rx promoter were subjected to anti-HA immunoprecipitation followed by immunoblotting with an antibody raised against AtRanGAP1 (). As shown in , a single band of the predicted size for RanGAP (approximately 61&kDa) was co-immunoprecipitated with Rx:4HA. Within the alignment shown in Figure&S2, NbRanGAP1 and NbRanGAP2 are 67 and 66% identical to AtRanGAP1, with multiple regions of identity shared among all three proteins for potential antiserum cross-reactivity. Furthermore, the anti-AtRanGAP1 serum showed reactivity to StRanGAP2:FH immunoprecipitated with anti-FLAG antibodies (data not shown). Thus, although this antibody does not distinguish between NbRanGAP1 and NbRanGAP2, this suggests that RanGAP2 is a bona fide Rx-interacting protein in vivo.RanGAP-induced activation of Rx and Rx2 fragmentsWe next assessed the role of RanGAP2 in Rx-like protein function by overexpressing StRanGAP2. We observed no change in the HR strength or timing of onset when Rx or Rx2 were co-expressed with CP (data not shown). However, overexpression of StRanGAP2:FH plus the CC-NB:HA fragments of Rx and Rx2 in tobacco leaves induced a visible HR within 36&h following agroinfiltration (). Overexpression of Rx has been observed to result in a relatively weak CP-independent HR that is augmented by the deletion of the LRR and ARC domains (); however, this effect was seen only in very young tobacco leaves, but neither in N.&benthamiana and nor with the CC-NB fragment of Rx2.Figure&4. &RanGAP interacts functionally with Rx proteins.(a) RanGAP2 activates coiled-coil nucleotide-binding (CC-NB) fragments of Rx-like proteins. Rx or Rx2 CC-NB:HA fragments were expressed in tobacco leaves by agroinfiltration together with StRanGAP2 or EGFP as indicated. Representative hypersensitive responses (HRs) induced within 2&days post-infiltration are shown, and photographed at 9&days post-infiltration. Overexpression of StRanGAP2 alone showed no HR induction (not shown).(b) RanGAP2 activation of a series of Rx and Rx2 derivatives. Various combinations of Rx and Rx2 derivatives were expressed via agroinfiltration in Nicotiana tabacum and Nicotiana benthamiana leaves, as indicated with EGFP:HA, StRanGAP2:FH or StRanGAP2WP/AA:FH. Elicitor-independent HRs induced by RanGAP2 overexpression developed within 2 (+++) or 3&days (++), except for the weakest reactions (+), which induced cell collapse by 5&days, whereas (&) indicates no visible response. All combinations were tested at least three times.In N.&benthamiana, only the Rx2 CC-NB-ARC gave a well-developed, confluent HR when expressed with StRanGAP2:FH (). The CC-NB fragments of Rx and Rx2 eventually induced visible cell collapse in N.&benthamiana on the underside of the infiltrated leaf patches in the presence of excess StRanGAP2, demonstrating a clear, albeit weaker, gain of function. No activation of CC-NB-ARC fragments of Bs2 or HRT by StRanGAP2:FH was observed. We also observed a weak activation of full-length Rx in tobacco by StRanGAP2 overexpression ().Plant RanGAP proteins show localization in the cytoplasm with concentration at the nuclear envelope (NE) (). Localization to the NE is mediated by an amino-terminal WPP domain, so named for the three highly conserved residues (WPP) present in this domain. The WPP domain is present in AtRanGAP1, AtRanGAP2 and three other proteins encoded in the Arabidopsis genome, all of which show localization to the NE (). Mutation of the conserved WPP residues to AAP disrupts NE localization of AtRanGAP1 and WPP1 (; ). We engineered a similar mutation in StRanGAP2 (StRanGAP2WP/AA). Disruption of this motif had no effect on the ability of StRanGAP2 to induce an HR in the presence of Rx(2) fragments (), suggesting that NE localization is unlikely to be required for this activity.NbRanGAP2 is required for Rx-mediated defense responsesTo assess the requirement for RanGAP proteins in Rx-mediated resistance we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) vectors. TV:StRanGAP2, TRV:NbRGAP1, TRV:NbRGAP2, TRV:NbRGAP1+2 and TV:Sgt1.2 contain inserts of StRanGAP2, NbRanGAP1, NbRanGAP2, NbRanGAP1 plus NbRanGAP2 and NbSgt1, respectively (). Silenced plants were agroinfiltrated with HR-inducing combinations of Rx plus a GFP-tagged version of CP (), Pto plus AvrPto or Bs2 plus AvrBs2. As Rx plus CP results in a very rapid HR response (HR after 1&day), we also included in this assay an autoactivating combination of the Rx LRR plus the Gpa2 CC-NB-ARC domains (), which gives a weaker HR (after 3&days), as well as a 35S promoter-driven Gpa2 construct that results in a very weak HR (after 5&7&days). With the exception of Sgt1-silenced plants, we neither observed any reduction in the Avr-induced HRs of Rx, Bs2 or Pto () nor observed a break of PVX resistance in Rx transgenic plants (data not shown). However, the slower, Avr-independent, HR induced by Rx LRR plus Gpa2 CC-NB-ARC or 35S::Gpa2 were consistently blocked in plants silenced with TV:StRanGAP2, TRV:NbRGAP2 and TRV:NbRGAP1+2 (). No effect on these HRs was observed in plants silenced with TRV:NbRGAP1, which contains no stretches of identity to NbRanGAP2 &11 nucleotides.Figure&5. &Virus-induced gene silencing of Nicotiana benthamiana RanGAPs. TRV vectors containing fragments of NbSgt1, StRanGAP2, NbRanGAP1, NbRanGAP2 or both NbRanGAP1 and NbRanGAP2 were used to silence expression of the corresponding genes in wild-type N.&bethamiana. Various hypersensitive response (HR)-inducing protein combinations were expressed via agroinfiltration alongside control plants infected with empty TRV vector (TV:00) to test for HR competence. Infiltration patches are circled in black where co-infiltration resulted in an HR or in white where there was no response, and correspond to the diagram on the left as follows: (1) pB1RxHA; (2) pBin61CP32GFP; (3) pB1RxHA&+&pBin61CP32GFP; (4) 35S::Pto&+&35S::AvrP (5) pBin61Gpa2; (6) pBin61Gpa2 CC-NB-ARC:HA&+&Rx LRR:HA; and (7) pB1Bs2 + 35S::AvrBs2.We did not observe any visible phenotype in VIGSed plants, even when both NbRanGAP gene inserts were targeted (TRV:NbRGAP1+2; data not shown), which is contrary to what we would expect from eliminating RanGAP activity. To assess the level of NbRanGAP silencing, we investigated NbRanGAP protein levels in silenced plants. As the anti-AtRanGAP1 protein does not detect NbRanGAP proteins in immunoblots of crude extract, we examined the level of RanGAP protein able to bind to the Rx CC domain. We found that in NbRanGAP2-silenced plants, we were able to co-immunoprecipitate NbRanGAP, at reduced levels, with Rx&CC:HA (Figure&S3), suggesting that downregulation of NbRanGAP by VIGS is incomplete.In potato and Nicotiana spp., the Rx gene confers extreme resistance to PVX, so named because viral accumulation is not detected and the HR lesions associated with many viral resistance genes do not develop (; ). This type of resistance may be more difficult to break using the gene &knockdown& techniques available for Solanaceous plants. Because point mutations in the ARC domain result in a quantitative decrease in Rx function (), we generated transgenic N.&benthamiana plants expressing Rx with a mutation (D399V) in the ARC domain. Unlike transgenic N.&benthamiana expressing wild-type Rx, rub-inoculation of Rx(D399V) plants with PVX resulted in a limited number of HR-type lesions on inoculated leaves (). Thus, like many viral resistance genes, Rx(D339V) plants appear to allow limited viral replication in infected tissue that results in an HR but still restricts the virus to within the inoculated area.Figure&6. &Silencing of NbRanGAP2 compromises Rx-mediated resistance to potato virus&X (PVX). Transgenic Nicotiana benthamiana plants expressing Rx(D399V) were inoculated with either empty TRV vector (TV:00) or TV:StRanGAP2. Plants were rub-inoculated with sap containing PVX-GFP virions 21&days later.(a) Local leaves demonstrated few and discrete hypersensitive response (HR) lesions on TV:00 plants, whereas RanGAP2-silenced plants had more and spreading lesions (top panel). The same leaves were photographed under UV illumination to visualize GFP fluorescence (lower panel). Lesions in RanGAP2-silenced plants also extended into veins (b) and to systemic leaves (c), indicating viral movement.(d) Typical symptoms seen in the uppermost leaves of plants silenced with TV:00 (left) or with TV:StRanGAP2 (right) followed by PVX-GFP infection.(e) Protein extracts from the uppermost leaves of PVX-GFP infected plants were immunoblotted with anti-CP Ab. The presence of PVX was confirmed in systemic tissues of RanGAP2- and Sgt1-silenced plants, but not in TV:00 vector controls. Numbers over lanes indicate individual plants. A duplicate gel stained with Coomassie G-250 is shown below to demonstrate equivalent protein loading.When Rx(D399V) transgenic plants were silenced with TV:StRanGAP2, rub-inoculation of leaves with PVX-GFP resulted in more lesions than in plants infected with empty TRV vector (TV:00) (). In TV:00-infected plants, GFP fluorescence was restricted to narrow bands surrounding a small number of lesions, whereas many non-necrotic fluorescent foci could be observed in plants silenced with TV:StRanGAP2 (). Moreover, RanGAP2-silenced plants were compromised in their ability to restrict viral replication and spread: PVX-induced necrotic lesions spread into the veins of inoculated leaves () and the virus was able move to upper leaves, as observed by a spreading systemic necrosis (). Evidence of systemic PVX spread could also be detected by anti-CP immunoblotting ().DiscussionIdentification of an Rx-interacting proteinMany NB-LRR proteins have been shown to require chaperone-like molecules such as RAR1, SGT1 and HSP90, and to associate with such proteins in co-immunoprecipitation or yeast two-hybrid assays (; ; ; ; ). It has been suggested that NB-LRR proteins might exist in large molecular weight complexes with chaperone-like molecules. Although Rx requires Hsp90 and Sgt1 for proper function (; ), we have not been able to detect interactions between Rx and either Sgt1 or Hsp90 in&planta (data not shown). Our analytical methods suggest rather, that when expressed under physiological conditions, Rx forms relatively small protein complexes that include RanGAP2 as a major component, and that a significant pool of Rx is associated with RanGAP2 in&vivo.RanGAP2 is required for Rx functionAlthough silencing of a number of genes by VIGS has been shown to compromise the function of other R genes, the loss of resistance is often only partial (; ; ). Extreme resistance conferred by the Rx gene is likely to occur as a result of a very strong and rapid response, rather than being qualitatively different from the responses induced by HR-type resistance genes (). A quantitative difference has also been proposed to explain the fact that Rx2 and HRT confer either extreme or HR-type resistance depending on R gene expression levels (; ). The strength of the Rx response may explain why gene downregulation breaks resistance in so few cases. By using a mutant version of Rx that induces a weaker response than in wild type, we have lowered the threshold of downregulation needed to demonstrate a requirement for NbRanGAP2 in Rx-mediated disease resistance. Thus, the combination of physical binding between Rx and RanGAP2, gain of function by overexpression, and the loss of function from VIGS demonstrates that RanGAP2 plays an important role in Rx-mediated disease resistance. Additionally, a similar report has shown that downregulation of NbRanGAP2 by VIGS compromises Rx-mediated resistance ().The Arabidopsis genome encodes two RanGAP proteins, AtRanGAP1 and AtRanGAP2, which have highly similar subcellular localization patterns, and are involved in the assembly of the mitotic spindle, nuclear membrane and the cell plate (; ; ). Given the importance of these processes, it would be expected that a complete loss of RanGAP function in plants would be lethal, as it is in yeast (; ). Although our binding assays showed that Rx interacts with StRanGAP2, but not with StRanGAP1 (), we cannot formally rule out the possibility of a role for NbRanGAP1 in Rx function. For example, RanGAP1 might bind transiently to Rx or might bind only in the presence of Avr protein, as has been suggested for the interaction between WRKY2 and MLA10 ().The role of RanGAP2 in Rx functionAlthough we have demonstrated a requirement for RanGAP2 in Rx-mediated resistance, the question remains as to what role RanGAP2 plays in this response. RanGAP proteins play essential roles in eukaryotic cells by acting as a GAP for Ran GTPase. Ran functions by cycling between GTP and GDP bound states, with the two states having different protein&protein interaction properties. In this manner, Ran activity is spatially regulated by the cellular location of RanGAP, as well as by the Ran guanidine exchange factor, RanGEF, which allows Ran to exchange GDP for GTP (; ; ). Recently, it has been reported that attachment of a nuclear export signal to the N and MLA10 NB-LRR proteins abrogates their function, suggesting that nuclear localization is important for their activity (; ). Mutations in two other NE-localized proteins, a nucleoporin 96 homolog and an importin & homolog, have been shown to partially rescue the phenotypic effects induced by a weakly autoactivating mutation in the TIR-NB-LRR protein, SNC1 (; ). The functional relationship between these proteins has not yet been elucidated. Although it is possible that RanGAP2 is necessary for nuclear localization of Rx, this does not seem likely as plant RanGAPs are localized on the cytoplasmic side of the NE in interphase cells, with a significant pool present in the cytoplasm (; ). Although RanGAP plays a critical role in nucleocytoplasmic transport by maintaining a gradient of RanGTP and RanGDP between the nucleus and the cytoplasm, it has not been reported to act as a receptor itself for nuclear-localized proteins. Moreover, mutation of the WPP motif in the StRanGAP2 amino-terminal domain that is essential for RanGAP NE localization had no effect on the HR induced by overexpression of StRanGAP2 and Rx(2) fragments (), suggesting that NE localization of RanGAP2 is not important for Rx function.StRanGAP2 binds to the CC domain of Rx-like proteins (). This interaction appears similar to the interactions between RIN4, Pto and PBS1, and the amino termini of RPM1, Prf and RPS5, respectively (; ; ). RIN4, Pto and PBS1 also bind the cognate Avr proteins of their NB-LRR interaction partners and are thought to mediate recognition (; ; ). Such examples have given rise to a model of indirect recognition often referred to as the guard hypothesis (; ; ). The basic premise of this model is that pathogen effectors promote virulence by modifying host proteins, and that these modifications are in turn sensed by NB-LRR proteins. Given the above examples, it seems plausible that RanGAP2 might play a role in Avr recognition by Rx, Rx2 and possibly by Gpa2. It seems less likely that RanGAP2 is generally involved in signaling as both the physical and functional interactions were specific for Rx-like proteins, and overexpression of RanGAP2 alone did not induce a response (; data not shown). We have been unable to demonstrate an interaction between RanGAP2 and PVX CP in&planta (), and a potential direct or indirect role for RanGAP2 in CP recognition will require further investigation.Overexpression of RanGAP2 also induces recognition-independent activation of Rx(2) derivatives (). This may occur because excess RanGAP2 induces a portion of the Rx(2) derivatives to adopt a signaling competent state normally induced by recognition, or vice versa. Alternatively, the complex of the two molecules might be required to interact with some as yet unidentified signal adaptor protein(s). In addition, RanGAP2 is required for the weak responses of auto-active Rx-like molecules (). These results do not, however, rule out a role for RanGAP2 in recognition, but may indicate a concomitant role for RanGAP2 in priming Rx to be competent for signaling. Similarly, the HR induced by overexpression of Prf requires concomitant overexpression of Pto, the latter playing a well-defined role in recognition, although Pto has not been shown to bind AvrPto in&planta ().Unlike RIN4, Pto and PBS1, the only known functions of which are in Avr recognition and regulation of defense, RanGAP2 represents an example of an NB-LRR co-factor with demonstrated roles in housekeeping functions (; ; ). This finding suggests that some NB-LRR proteins have co-opted proteins involved in fundamental cellular processes for the detection of pathogens.Experimental proceduresPlant material and transient expressionNicotiana benthamiana and N.&tabacum plants were germinated and grown in a glass house maintained at 21&C. All experiments were repeated at least three times. For transient expression of proteins, plants were infiltrated by syringe with Agrobacterium& tumefaciens strain C58C1 carrying the virulence plasmid pCH32 and an appropriate binary expression vector, as previously described (). A.&tumefaciens cultures were diluted to OD550&=&0.1 in individual or co-infiltrations. Plants were transferred to a Conviron growth cabinet (Conviron, ) maintained with 16-h light and 8-h darkness at 21&C for 38&42&h prior to collection of leaf material for protein extraction. For VIGS, plants were co-infiltrated with A.&tumefaciens carrying plasmid pBINTra6 (TRV RNAI cDNA) and constructs derived from either pTRV2 () or pTV:00 (). A tobacco cell line driving expression of Rx4HA from the Rx genomic promoter was generated by transformation of N.&tabacum cv. Bright Yellow&2 (BY-2) line NT-1 with the binary vector pB1Rx4HA (). NT-1 cells were grown in NT1 medium [Murashige and Skoog salts (Invitrogen, ), 3% (w/v) sucrose, 3&&m thiamine, 0.58&mm myoinositol, 1.3&mm KH2PO4, 1&&m 2,4-dichlorophenoxyacetic acid (Sigma, ), 2.5&mm 2-(N-morpholino)ethanesulfonic acid, pH&5.7]. Transformation was performed by co-cultivation of A.&tumefaciens strain LBA4404 carrying pB1Rx4HA with NT-1 cells for 3&days in the dark. Transformed cell lines were selected by plating NT-1 cells onto NT-1 agar medium containing 100&mg&l&1 kanamycin plus 300&mg&l&1 timentin. Kanamycin-resistant calli were transferred to KCMS liquid medium () with 100&mg&l&1 kanamycin, and suspension cultures were maintained at 21&C on a rotary shaker in the dark.Transgenic N.&benthamiana line Rx4HA-2 has been previously described (). Transgenic N.&benthamiana plants were also generated to stably express Rx(D399V). This mutation results in a protein that is weakly autoactivating in tobacco, but not in N.&benthamiana, and shows a slightly delayed CP-mediated HR in transient expression assays (). A binary vector was constructed wherein Rx(D399V) was placed under the transcriptional control of the geminiviral AV1 promoter that drives low levels of expression in plants (). These plants were found to constitutively express Rx(D399V), confer resistance to PVX and to undergo an HR when agroinfiltrated with PVX&CP, but showed no apparent morphological phenotype (data not shown).To generate PVX-GFP sap, pGr208 was agroinfiltrated into wild-type N.&benthamiana plants and viral sap was prepared 10&days later as described by . Plants were manually inoculated with sap using a light dusting of carborundum.Biochemical purification and identification an Rx-associated proteinNicotiana benthamiana leaves transiently expressing Rx&CC:HA were used to prepare protein extracts. Leaves were homogenized in a blender using 2.5&ml of ice cold extraction buffer per gram of leaf tissue {GTEN [10% (v/v) glycerol, 25&mm Tris&HCl, pH&7.5, 1&mm EDTA, 150&mm NaCl], 10&mm dithiothreitol (DTT), 2% (w/v) polyvinylpolypyrrolidone and protease inhibitors: 1&mm 1,10-phenathroline, 1&mm phenylmethylsulfonyl fluoride, 1&&g&ml&1 pepstatin&A, 1&&m bestatin, 5&&mN-[N-(l-3-trans-carboxyoxirane-2-carbonyl)-l-leucyl]-agmatine (E-64) and 5&&g&ml&1 leupeptin}. Protein extracts were kept chilled on ice or at 4&C for all subsequent steps. The crude extract was filtered through four layers of cheesecloth pre-wet with GTEN and cleared by centrifugation at 11&000&g for 15&min, using a GSA rotor (S Thermo Scientific, ). Proteins were precipitated from the extract by the addition of pulverized ammonium sulfate crystals to a final concentration of 50% (w/v). Precipitates were collected by centrifugation at 11&000&g for 15&min, at 4&C using a Sorvall GSA rotor, resuspended with 8&ml GTEN and dialyzed overnight against buffer TEN (25&mm Tris&HCl, pH&7.5, 1&mm EDTA and 150&mm NaCl). Insoluble material was cleared from the dialyzed extract by centrifugation for 20&min at 32&500&g using a Sorvall SS-34 rotor. The extract was prepared for affinity purification by desalting through a Bio-Gel P6 DG column pre-equilibrated with TEN buffer, then by adjusting the extract to 0.15% (v/v) Nonidet P-40 (NP-40). Antibody affinity purification (immunoprecipitation) was performed by first pre-clearing the extract by incubating end-over-end with 100&&l goat IgG-agarose beads (Rockland, ) for 30&min and spinning briefly to remove beads. Rx&CC:HA and associated proteins were immunoprecipitated from extracts by end-over-end incubation overnight with 50&&l anti-HA agarose beads. Beads were washed extensively with ice-cold IP buffer (TEN, 0.15% NP-40). Proteins were eluted from beads in three sequential fractions by incubation for 10&min at 37&C with HA peptide (Roche, ) dissolved in TEN to a concentration of 1&mg&ml&1. Eluted proteins were pooled and separated on a 7.5&10% SDS-PAGE gel and stained with colloidal Coomassie G-250 (Sigma). An approximately 65-kDa band was excised and sent for tandem mass spectrometric analysis at the Donald Danforth Plant Science Center Proteomics and Mass Spectrometry Facility ().Plasmid constructionAll constructs containing Rx or Gpa2 as well as expression vectors pBin61 (35S promoter and terminator) and pB1 (Rx genomic promoter and terminator), TV:Sgt1.2, pBin61CP32GFP and PVX-GFP have been previously described (; ; ; ). For generation of expression clones, all inserts were ligated into 5&XbaI and 3&BamHI sites of a pBin61 binary vector series, unless otherwise indicated. This vector series contains epitope tags, or the enhanced red-shifted variant of jelly fish green fluorescent protein (EGFP) with an HA epitope tag, positioned for carboxy-terminal tagging of inserts in frame with the BamHI site (; ). To prepare inserts, StRanGAP2 from S.&tuberosum (potato) cultivar Desir&e was PCR amplified from genomic DNA isolated using the REDExtract-N-Amp Plant PCR kit (Sigma). PCR was performed with Expand Hi Fidelity Taq DNA polymerase (Roche) and primers RanGAP2Xba (5&-CTCTAGACCATGGATGCCACAACAGCTAAC-3&) and RanGAP2Rev (5&-GGATTCATTGCTATCTGGTGTGTCAAG-3&). A cloned PCR product was re-amplified with StRanGAP2Xba plus RanGAP2Bam (5&-GATATCGGATCCATTGCTATCTGGTGTGTCAGG-3&). StRanGAP1 was PCR amplified from EST425553 plasmid clone cSTB16H12 (accession number ) with primers RGAP1ForXba (5&-CTCTAGACCAGCTATTGAAGATGGATTC-3&) and RGAP1RevBam (5&-GGATCCTTCTTCCTGCTTGATATCGAGATCCTTG-3&). The StRanGAP2WP/AA was generated by PCR mutagenesis as previously described for AtRanGAP1 (). Rx2 and Gpa2 CC (aa&1&144) and CC-NB (aa&1&293) constructs were generated using the same strategy as previously described for the analogous Rx derivatives (). The pB1Bs2 construct was generated by transferring Bs2HA from pBin61Bs2HA () as an XbaI/BamHI fragment into the same sites of pB1.For construction of TRV VIGS vectors, the StRanGAP2 insert was cut from pGEM-T using vector flanking EcoRI sites. Fragments of StRanGAP2 were generated by digestion of the isolated insert with Sau3A1, and a 926-bp fragment was subcloned into the tobacco rattle virus TV:00 vector BamHI sites (). The StRanGAP2 sequence demonstrated 91% nucleotide identity to the closest N.&benthamiana homolog sequence. RanGAP fragments from N.&benthamiana were cloned using primers designed from the potato RanGAP sequences by RT-PCR. Total RNA was isolated from N.&benthamiana using the RNeasy miniprep kit (Qiagen, ). First-strand DNA was synthesized using Superscript&III reverse transcriptase (Invitrogen) with an oligo-dT anchor primer (5&-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3&). The complete NbRanGAP1 coding sequence was amplified from cDNA with primers RGAP1ForXba and RGAP1RevBam. A fragment of NbRanGAP2 was amplified with primers DeltaMAF2 (5&-CTCTAGACATGGAATGCAGCAAGCTATCC-3&) and DeltaCRev (5&-GGATCCAAGCATTTCAGGAG-3&). Products cloned into pGEM-T were amplified with primers NbRGAP1XhoF (5&-CTCGAGTTTGCTCACAACAGCTTG-3&) and NbRGAP1XhoR (5&-CTCGAGGTTCTGGAGATGGATG-3&) or NbRGAP2EcoF (5&-GAATTCGGAGAGCCTTAGCAATCTG-3&) and NbRGAP2EcoR (5&-GAATTCCTCAGCAAACATGAAAAGC-3&) for subcloning into XhoI and EcoRI sites, respectively, of the TRV2 vector (). For construction of the NbRanGAP1+2 double insert, NbRanGAP1 and NbRanGAP2 PCR products were ligated and re-amplified with primers NbRGAP2EcoF and NbRGAP1XhoR.DNA and protein sequencesDNA sequences were translated to protein and aligned using the translator and clustalw-based aligner programs of the justbio suite (Pierre RJustBio, ). The plant RanGAP sequence alignment was processed through boxshade version&3.21 to mark conserved residues. Phylogenetic analysis was performed with the phylip phylogeny inference package () using the programs seqboot, protdist with the Dayhoff PAM matrix, neighbor employing the neighbor-joining method, and consense to generate a dendogram and to obtain bootstrap values for 500 data replicates. New sequences reported with this study have been deposited to GenBank/EMBL databases under the following accession numbers:
(StRanGAP1),
(StRanGAP2),
(NbRanGAP1) and
(NbRanGAP2). Additional sequences relevant for this report can be retrieved from the GenBank/EMBL databases under the following accession numbers:
(AtRanGAP1),
(AtRanGAP2),
(MtRanGAP),
(EST596034),
(EST425553).Co-immunoprecipitationProtein extracts were prepared from N.&benthamiana leaves transiently expressing epitope-tagged proteins by grinding in 2.5&ml GTEN extraction buffer (see above) per gram of leaf tissue. For co-immunoprecipitation of tagged proteins, 1&g of leaf tissue was used, whereas 4&g of leaf tissue was used to co-immunoprecipitate endogenous NbRanGAP proteins in TRV vector-silenced plants. Extracts were centrifuged twice at 12&000&g for 10&min at 4&C and desalted with GTEN buffer using Bio-Gel P6 DG. Extracts were pre-cleared by incubation for 30&min at 4&C with 25&&l non-specific IgG agarose in a 2-ml volume of extract adjusted to 0.15% (v/v) NP-40, followed by centrifugation at 12&000&g for 1&min. Supernatants were incubated end-over-end with EZview Red anti-HA affinity gel (Sigma) or anti-FLAG M2 affinity gel (Sigma) overnight at 4&C, with 20&&l of agarose beads used with 950&&l of clarified extract. Beads were washed three times with IP buffer, aspirated dry and eluted by boiling in 100&&l of 2&&&sodium dodecy sulphate (SDS)&PAGE loading dye. Samples separated by SDS&PAGE were electroblotted to polyvinylidene difluoride membranes (Bio-Rad, ). Blots were pre-blocked for 30&min with Tris-buffered saline (TBS) containing 5% (w/v) powdered skimmed milk, 0.1% (v/v) Tween&20 and probed for 1&h with horseradish peroxidase-conjugated anti-HA (3&F10; Roche) or anti-FLAG (M2; Sigma) antibodies diluted in TBS plus 0.1% (v/v) Tween&20. For RanGAP detection, blots were sequentially probed with anti-AtRanGAP1 serum () and horseradish peroxidase-conjugated goat anti-rabbit IgG. Epitope-tagged proteins were visualized using the ECL chemiluminescent system (Pierce, ), whereas NbRanGAPs were detected using ECL-Plus (Amersham, ).Size exclusion chromatography (SEC)Protein extracts prepared as described above were pre-cleared for SEC by ultracentrifugation at 100&000&g for 30&min at 4&C. Supernatant was loaded onto a Superdex&200 16/60 column in an AKTA FPLC (GE Healthcare, ) using a 2-ml loop and TEND buffer (TEN, 1&mm DTT). Fractions collected (2&ml) were mixed with loading buffer for PAGE analysis. For native PAGE of Rx4HA-2&N.&benthamiana plants, pooled fraction pairs were immunoprecipitated with anti-HA agarose beads overnight and eluted with 1&mg&ml&1 HA peptide in TBS (25&mm Tris&HCl, pH&7.5, 150&mm NaCl) prior to preparation of PAGE samples by addition of native PAGE loading buffer to 1& final concentration [25&mm Tris&HCl, pH&6.8, 10% (v/v) glycerol, 0.001% (w/v) bromophenol blue]. Gel filtration standards (catalog no. 15-1901, Bio-R catalog no. 17-0441-0, Amersham Biosciences) were used for calibration of the column. All experiments were performed at least three times.Sucrose density gradient centrifugationProtein extracts were prepared by grinding 1&g of NT-1 cells in 2.5&ml of cold sucrose gradient extraction buffer (purification extraction buffer plus 20&mm sodium molybdate) as described for SEC. Cleared extracts were desalted with GTEN plus 20&mm sodium molybdate. Protein extracts (1&ml) were layered over 10&40% continuous sucrose gradient cushions prepared in TENDM (TEN plus 1&mm DTT, 10&mm sodium molybdate) and centrifuged for 15&h at 4&C at 100&000&&&g using a Beckman SW41 Ti rotor (Beckman Coulter, ). Calibration standards thyroglobulin (669&kDa), ferritin (440&kDa) and catalase (223&kDa) were centrifuged in parallel (catalog no. 17-0441-0, Amersham). Fractions (0.8&ml) were removed by pipetting from the top of the tube, and proteins were mixed with loading dye for native or SDS&PAGE separation and immunoblot analysis to detect the Rx HA-tag. Experimental repetitions performed without molybdate and experiments using extracts from Rx4HA transgenic N.&benthamiana gave similar fractionation results (not shown).AcknowledgementsWe thank Joyce Van Eck for providing the tobacco NT-1 cell line and Iris Meier for sharing the anti-AtRanGAP1 serum. We acknowledge Dr Sixue Chen for advice on sample preparation and MS/MS identification of RanGAP. We are grateful to the Boyce Thompson Institute greenhouse staff for plant care and the BTI Lab Services for research support, to Daniel Klessig for critical review of the manuscript and to members of the Moffett lab for fruitful discussions. This work was supported by funds from the National Science Foundation (Grant IOB-0343327) and The Triad Foundation to PM.
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