Stepwise artificial evolution of a plant diseaseresistance geneC. Jake Harrisa, Erik J. Slootwegb, Aska Goverseb, and David C. Baulcombea,1

aDepartment of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom; and bLaboratory of Nematology, Department of PlantSciences, Wageningen University, 6708 PB, Wageningen, The Netherlands

Edited by Brian J. Staskawicz, University of California, Berkeley, CA, and approved November 13, 2013 (received for review June 12, 2013)

Genes encoding plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins confer dominant resistance to diverse pathogens.The wild-type potato NB-LRR protein Rx confers resistance againsta single strain of potato virus X (PVX), whereas LRR mutantsprotect against both a second PVX strain and the distantly relatedpoplar mosaic virus (PopMV). In one of the Rx mutants there wasa cost to the broad-spectrum resistance because the response toPopMV was transformed from a mild disease on plants carryingwild-type Rx to a trailing necrosis that killed the plant. To explorethe use of secondary mutagenesis to eliminate this cost of broad-spectrum resistance, we performed random mutagenesis of the N-terminal domains of this broad-recognition version of Rx andisolated four mutants with a stronger response against the PopMVcoat protein due to enhanced activation sensitivity. These muta-tions are located close to the nucleotide-binding pocket, a highlyconserved structure that likely controls the switch between ac-tive and inactive NB-LRR conformations. Stable transgenic plantsexpressing one of these versions of Rx are resistant to the strainsof PVX and the PopMV that previously caused trailing necrosis. Weconclude from this work that artificial evolution of NB-LRR diseaseresistance genes in crops can be enhanced by modification of bothactivation and recognition phases, to both accentuate the positiveand eliminate the negative aspects of disease resistance.

plant immunity | genetically modified | arms race | NLR | plant defense

Plant resistance (R) genes confer dominant resistance againstdiverse pests and pathogens including viruses, fungi, andinvertebrates (1). The vast majority of R genes encode NB-LRR(nucleotide-binding leucine-rich repeat) proteins (2) that forma subgroup within the STAND ATPases (signal transductionATPases with numerous domains) (3). NB-LRRs are thought tofunction as a molecular switch, existing in ATP-bound activeor ADP-bound inactive states (47). Activated NB-LRRs ini-tiate a downstream signaling cascade triggering defense responses,which often culminate in a form of programmed cell death knowas the hypersensitive response (HR) (8).Activation of NB-LRRs occurs following molecular recogni-

tion in which a pathogen-derived elicitor interacts, either directlyor indirectly, with the LRR and/or other domains of the protein(915). In different R proteins the molecular mechanisms mayvary but, in Rx from potato (16), which confers resistance topotato virus X (PVX), the domains required to initiate down-stream signaling (17, 18) are likely exposed by conformationalchanges (19, 20) following recognition.NB-LRR genes represent a useful target for generating disease-

resistant crops, and have long been selected unknowingly by cropbreeders. More recently, transgenic approaches have been usedto transfer R genes between plant species (2123). However, theusefulness of NB-LRR genes in both conventional breeding andtransgenic approaches is limited by the availability of R proteingenes with useful recognition specificities. In addition, there maybe a cost to carrying NB-LRR R genes. The disease-resistantplants may have reduced fitness in competition with susceptibleplants (24), there may be a tradeoff with plant growth (25),the plants may exhibit hybrid incompatibility (26), or they may

exhibit a partial resistance against some strains of pathogen sothat there is a spreading necrosis that kills the plant (2730).Artificial evolution through random mutagenesis has been

explored previously as a strategy to expand the number of usefulvariants of Rx (27). The evolved products were forms of Rx withmutant LRR domains that recognized more strains of PVX thanthe wild-type protein. The elicitor of Rx-mediated resistance isthe viral coat protein (CP), and the wild-type protein responds tostrains in which there is a T and a K at positions 121 and 127,respectively (CPTK), but not those with a K and an R at thesepositions (CPKR) (31). The artificial evolution focused on theLRR domain (27), and the selected mutants were elicited byboth CPKR and the original CPTK. However, this broad recog-nition had a cost in that one of the mutants, RxM1 (N846D),displayed systemic necrosis when the plants were challenged withpoplar mosaic virus (PopMV). This virus is a distant relative ofPVX, and on plants with wild-type Rx, or without Rx, it onlyinduces a mild mosaic. To explain the lethal symptom associatedwith PopMV on RxM1 plants we proposed that the mutationmediated weak recognition of a PopMV structure that was in-visible to the wild-type Rx. The systemic necrosis would havearisen because the weak recognition by Rx triggers a delayed HRresponse that is too late to prevent spread of the virus from thesite of initial infection.This side effect of the M1 mutation illustrates the balance

between the costs and benefits of disease resistance, and it cre-ated an opportunity to find out whether artificial evolution couldbe used to reduce the costs associated with disease resistance ofNB-LRR proteins. Our approach was random mutagenesis ofRxM1 combined with selection for response to the PopMV coatprotein rather than CPKR. In addition, rather than the LRR, wemutagenized the amino-terminal coiled coil (CC), NB, and

Significance

Plant nucleotide-binding leucine-rich repeat (NB-LRR) proteinsare responsible for detecting potential pathogens and trig-gering a defense response. However, disease resistance canalso involve a cost. Here we show that the trailing necrosis costassociated with a broad-recognition version of the NB-LRRprotein, Rx, can be overcome by selecting for mutations thatincrease the sensitivity of the NB-LRR protein. Using homologymodeling, we predict that these mutations are colocalized tothe ATP/ADP nucleotide-binding pocket, which is thought tocontrol the switch between active and inactive states.These results suggest that a stepwise approach to coordinatingrecognition and response could be used to design improvedNB-LRRs for use in agriculture.

Author contributions: C.J.H., A.G., and D.C.B. designed research; C.J.H. performed re-search; E.J.S. contributed new reagents/analytic tools; C.J.H. and E.J.S. analyzed data;and C.J.H. and D.C.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: dcb40@cam.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1311134110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1311134110 PNAS | December 24, 2013 | vol. 110 | no. 52 | 2118921194

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ARC (shared by Apaf-1, resistance genes, and CED-4) domains.By selecting mutants with enhanced recognition of PopMV, wepredicted to isolate mutants that give a strong and rapid responseto this virus and eliminate the spreading HR. By focusing ondomains other than the LRR, we reasoned that we would avoidcompromising the beneficial aspects of the RxM1 phenotype.Here we describe five amino acid changes that gave the se-

lected response to the PopMV-CP. These mutants increase theactivation sensitivity rather than the recognition phase of the Rxresistance mechanism and, from protein modeling, we concludethat they are localized around the conserved ATPase nucleotide-binding pocket. Transgenic plants containing these versions ofRx retained broad-spectrum resistance against PVX strains andPopMV without systemic necrosis: The cost of RxM1 resistancehad been eliminated. The results show that Rx can be enhancedby a stepwise process that targets both recognition and activationmechanisms. Based on these results, we suggest that artificialevolution could be a useful enhancement of all disease resistancethat is to be transferred by genetic manipulation of NB-LRRs.

ResultsArtificial Evolution Screen Across RxM1 N-Terminal Domains.R genescan be coexpressed with cognate pathogen proteins in leaf seg-ments of Nicotiana tabacum or N. benthamiana using an Agro-bacterium-mediated transient expression assay (32). If the Rgene recognizes the pathogen protein, a hypersensitive responseis induced that manifests as necrosis in the infiltrated leaf seg-ment. RxM1 does not induce HR when transiently coexpressedwith the coat protein of PopMV (PopMV-CP), but RxM1transgenic plants display trailing necrosis when infected withPopMV, and electrolyte leakage assays suggest that RxM1 mountsa delayed response to PopMV (27).To find versions of RxM1 with an accelerated PopMV re-

sponse, we generated a library of 1,500 RxM1 variants by randomlymutagenizing the N-terminal CC-NB-ARC1-ARC2 domains (Fig.1A) at an error rate of 5.1 bp changes per 1.5-kb molecule, en-suring an average coverage of 5 bp changes per position. Theselection was by transient coexpression of these mutants withPopMV-CP. The weak recognition of PopMV-CP by RxM1 doesnot lead to visible necrosis but, of the 1,500 RxM1 variants, weidentified 22 RxM1 mutants that produce a necrotic response(Table S1). Of these, there were 18 candidates that also producea necrotic response when coexpressed with GFP, indicating thatthese mutations cause constitutive activation of the protein. Theremaining four clones, denoted RxS1*M1, RxS2*M1, RxS3*M1,and RxS4M1, induced necrosis when coexpressed with PopMV-CP but no necrosis when coexpressed with GFP (Fig. 1 B and Cand Fig. S1A) and were selected for further analysis. The basis forthis nomenclature is explained in later sections of this text: S isfor sensitized, and the asterisk indicates that the mutants containmore than one amino acid change.

We used a chlorophyll content assay to quantify the level ofnecrosis induced by different Rx variant/elicitor combinationsafter transient expression in N. tabacum (33). The four candi-dates, RxS1*M1, RxS2*M1, RxS3*M1, and RxS4M1, retainedbroad recognition of PVX-CP, producing a strong necrotic re-sponse when coexpressed with both versions of the PVX coatprotein, PVX-CPTK and PVX-CPKR (Fig. 1C). The RxM123construct, which contains three previously identified broad rec-ognition-conferring mutations combined in the LRR domain(N846D, N796D, and L607P, respectively) was used as a positivecontrol for recognition of PopMV-CP (27). Western blots ofinfiltrated leaf tissue indicated that the mutations in RxS1*M1,RxS2*M1, RxS3*M1, and RxS4M1 do not increase Rx proteinstability (Fig. S1B). This control observation rules out that thephenotypes are due to enhanced accumulation of the mutantNB-LRR proteins.

Sensitized Mutations Are Close to the ATP/ADP Binding Site. TheRxS1*M1, RxS2*M1, and RxS3*M1 proteins each contain morethan one amino acid change in addition to M1 (N846D). RxS1*M1contains four mutations (L179M, R291C, M293L, K482N);RxS2*M1 contains three (E55K, T178A, P187Q); RxS3*M1 con-tains four (A88T, N147D, V337A, V362I); and RxS4M1 containsonly one (G340R) (Fig. 2A, Fig. S2A, and Table S1). To determinewhich amino acid changes are responsible for the PopMV-CPresponse in the RxS1*M1, RxS2*M1, and RxS3*M1 clones, weused site-directed mutagenesis to generate single-amino acidchange constructs corresponding to each mutation.All single-amino acid change constructs produced functional

proteins that respond to PVX coat proteins in transient expres-sion assays (Fig. S2B). The M293L (referred to as S1) and T178A(referred to as S2) single mutations were necessary and sufficientto recapitulate the phenotypes of RxS1*M1 and RxS2*M1, re-spectively, whereas for RxS3*M1, both N147D and V337A (to-gether referred to as S3) were required (Figs. S2C and S3). Asthere was only one newly introduced amino acid change in RxS4M1(G340R), this mutation is referred to as S4.The five amino acid changes in these four mutants (S14) are

located in the NB and ARC1 domains of Rx and are dispersed onthe primary structure (Fig. 2A and Fig. S4). However, when theseresidues are mapped onto a 3D homology model of the Rx NB-ARC1-ARC2 domains, these positions colocalize around the nu-cleotide-binding pocket (Fig. 2B) (20). The N147D and V337Amutations (derived from RxS3*M1) are positioned on oppositesides of the nucleotide, in motifs that appear together in theATPase family phylogeny (34). N147D is located in the charac-teristic STAND ATPase hhGRExE motif of the NB domain (34)and V337A is located in the GxP motif, which provides the maincontribution of the ARC1 domain to the nucleotide-bindingpocket. The G340R mutation (S4) in RxS4M1 is also located inthe GxP motif. A direct contact of this residue with the nucle-otide is not likely, but the change from a small glycine to a large

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Fig. 1. N-terminal random mutagenesis of RxM1and identification of candidates. (A) Schematic ofRxM1. Red, N-terminal domains; green, C-terminalLRR domain. Arrows depict error-prone PCR targetregion. (B) Transient expression in leaf segments ofN. tabacum. PopMV-CP is coexpressed with RxM1and the RxM1 variants identified in the screen,RxS1*M1, RxS2*M1, RxS3*M1, and RxS4M1. Photoswere taken 5 d postinoculation (dpi). (Scale bar,1 cm.) (C) Quantification of necrosis after transientcoexpression of Rx variants with viral CP elicitors inN. tabacum (5 dpi) by chlorophyll content assay. Alow level of chlorophyll indicates high levels of ne-crosis. GFP and RxM123 are used as negative andpositive controls, respectively. Each bar represents anaverage of five biological replicates. Error bars rep-resent 95% confidence intervals.

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positively charged arginine may affect the structure of ARC1, theinteraction of the GxP motif with the nucleotide, and/or the in-teraction with ARC2. The M293L mutation (S1) in RxS1*M1lies just outside the nucleotide-binding pocket, in a linker be-tween the fifth -strand of NB and the first -helix of ARC1, aspart of the RNBS-C motif (35). The T178A mutation identifiedin RxS2*M1 (S2) is located in the P-loop motif of the NB do-main, immediately C-terminal to the GKT sequence. The P loopis highly conserved and plays an important role in nucleotidebinding (6, 34, 36). Although most mutations in the P loop causeloss of function, a mutation in the T178A homologous posi-tion in the tomato NB-LRR, Mi-1.2 (T557S), causes autoacti-vation (37).

Mutations Increase Activation Sensitivity. The increased response toPopMV-CP in the RxM1 mutants could be due either to improvedrecognition of the elicitor or to increased activation sensitivity. Totest these hypotheses, we exploited the weak HR induced wheneither Rx or RxM1 is overexpressed in the absence of CP elicitor(27, 38). If the mutations affect activation, then overexpression inthe absence of elicitor should produce more robust HR thanRxM1 when overexpressed. Alternatively, if the mutations affectrecognition of the elicitor, then overexpression-induced HR shouldremain unchanged: the phenotype would be dependent on thepresence of elicitor. In each instance, the overexpression ofRxS1M1, RxS2M1, RxS3M1, and RxS4M1 from the strong CaMV35S promoter produced a more rapid and robust HR in N.tabacum leaf segments than 35S::RxM1 (Fig. S5A).In an additional test of elicitor-independent HR, we coex-

pressed the original Rx promoter constructs with RanGAP2, an Rxcytoplasmic retention factor causing overaccumulation of Rx in thecytoplasm and mild HR with Rx (39, 40). As in the overexpressionassay, each of the S14 mutants produced a stronger HR thanRxM1 (Fig. S5B). These findings suggest that the S14 mutationsincrease the sensitivity for activation of the NB-LRR proteinrather than via improved specific recognition of PopMV-CP.In a third test, we replaced the M1 LRR mutation (N846D)

with the wild-type LRR sequence (N846) in S14 mutants andcoexpressed them with PopMV-CP. If these mutations had en-hanced the recognition of PopMV-CP, there would have been anHR in the absence of the M1 mutation. However, none of theseconstructs without M1 produced a robust HR on coexpressionwith PopMV-CP (Fig. 3A). However, with the coat protein fromthe resistance-breaking PVX isolate PVX-CPKR, HR was en-hanced in the absence of the M1 mutation (Fig. 3B). Presumably,the wild-type Rx has weak recognition of CPKR that is sensitizedby the S14 mutants.In a fourth test of sensitization, we assayed the phenotype of

RxS1234M1 and RxS1234 mutants that carry all five S mutations(Fig. 4A). We predicted that the combined effects of thesemutants on sensitization would result in a necrotic response inthe absence of an elicitor, whereas recognition-mediated phe-notypes would be elicitor-dependent. The results were in linewith the sensitization model (Fig. 4B). Thus, all four tests re-inforce our conclusion that the M1 and S14 mutations affectdifferent stages of the Rx resistance mechanism: M1 mutationaffects recognition, and S14 sensitize the responsiveness.

Sensitization Requires an Intact P Loop. The nucleotide-binding Ploop, also known as the Walker A motif, is essential for thefunction of most (6, 34, 41) but not all NB-LRRs (42, 43). To findout whether the S14 mutants are P loop-dependent, we introducedthree inactivating mutations (G175A, K176R, T177A) (6, 42) intothe invariant nucleotide-binding GKT motif of the P loop in theautoactive RxS1234 background.All three P-loop mutations suppressed autoactivity, but only

the K176R mutation completely abolished RxS1234-inducednecrosis (Fig. 4C). From Western blots, we rule out that this lossof function is due to protein stability (Fig. 4D). The results areconsistent with nucleotide binding as an essential step in RxS1234autoactivity. Because the S1, S2, S3, and S4 mutations are in close

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Fig. 2. Amino acid changes cluster around the nucleotide-binding pocket.(A) Schematic of RxM1 variants identified from the screen, with error-prone PCRinduced amino acid changes. Responsible amino acid changesare encapsulated in red boxes. (B) Homology model of NB-ARC1-ARC2domains of Rx (20). Red, positions of the responsible amino acid changesidentified; blue, previously reported residues involved in nucleotidebinding. K176 in the P loop (NB); P332 in the GxP motif (ARC1); H549 inthe MHD motif (ARC2). Yellow, ADP nucleotide. The image was generatedby PyMOL software.

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) Fig. 3. S mutations sensitize the response. (A) Re-sponsible amino acid change constructs S1, S2, S3,and S4, with or without the M1 mutation in the LRR,coexpressed with PopMV-CP or (B) S1, S2, S3, and S4without the M1 mutation coexpressed with PVX-CPKRand quantified by chlorophyll content assay 5 dpi inN. tabacum. Rx (light gray bar) and Rx coexpressedwith PVX-CPTK (dark gray bar) are used as negativeand positive controls for necrosis, respectively. Eachbar represents an average of five biological repli-cates. Error bars represent 95% confidence intervals.

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proximity to the nucleotide-binding pocket (Fig. 2B), they couldincrease activation sensitivity by affecting the affinity or catalysisof the ATP/ADP nucleotide (4).

Transgenic Plants Are Resistant to PopMV. To determine whetherthe sensitized versions of RxM1 provide broad-spectrum re-sistance to PVX and PopMV strains without the systemic ne-crosis, we generated transgenic lines of N. benthamiana expressingRxS1*M1, RxS2*M1, RxS3*M1, and RxS4M1 and comparedthem with Rx and RxM1. When challenged with PopMV, thewild-type and Rx plants developed mosaic symptoms and mildstunting whereas the RxM1 plants exhibited trailing necrosis, aspreviously reported (27).In contrast, the RxS1*M1, RxS2*M1, RxS3*M1, and RxS4M1

transgenic lines showed resistance against PopMV infection. Theresistance in the RxS2*M1, RxS3*M1, and RxS4M1 plants wasvariable, and these plants exhibited complete resistance or trailingnecrosis in the T1 generation (progeny of primary transformants)when challenged with PopMV. RxS1*M1, however, displayed fullresistance to PopMV (Fig. 5A). RT-PCR of systemic leaf tissuefrom RxS1*M1 plants inoculated with PopMV revealed a perfectanticorrelation between expression of the transgene and accu-mulation of PopMV, demonstrating cosegregation of transgeneand resistance to PopMV (Fig. 5B). The RxS1*M1-expressingplants were also fully resistant to both strains of PVX (CPTK andCPKR), like RxM1 (Fig. S6).

DiscussionAn initial interpretation of Rx and other NB-LRR proteins was thatthe LRR domain would carry out the elicitor recognition functionin disease resistance and that the amino-terminal domains, in-cluding NB, would mediate the response phase leading to in-tracellular signaling and disease resistance. In this and a previousstudy (27), we provide strong evidence for these two separatephases of Rx-mediated resistance. The S14 mutations identifiedhere, for example, could activate an HR in our transient assay inthe absence of elicitor (Fig. 4 and Fig. S5), and they have thepredicted properties of response-phase mutants. Conversely, themodified HR responses of the previously identified M13 mu-tations were absolutely dependent on the presence of elicitor andhave the properties of recognition-phase mutants.With Rx and other NB-LRRs, there is evidence that the LRR

plays some role in recognition, as predicted by the originalmodel, because sequence variants in that domain influence thespecificity of the disease resistance (911, 27, 44). Similarly, it islikely that the amino-terminal domains, including NB, have some

role in the response phase, as predicted by the original model(17, 45, 46). Supporting this idea, we describe five mutationsaffecting the structure of the conserved nucleotide-binding pocket(formed by the NB-ARC domains; Fig. 2B) that increased acti-vation sensitivity of Rx-mediated responses. However, other anal-yses have blurred the relationship between structure and functionof NB-LRR proteins (12, 47, 48), and it no longer seems likely thatthere is a clean separation of the activation and recognition func-tions between the amino- and carboxyl-terminal domains. Our datado not rule out that recognition and response functions are dis-persed throughout the structure of Rx.

The Effect of the N-Terminal Mutations on Activation of Rx in theResponse Phase. That individual mutants S1, S2, S3, and S4 in-crease activation sensitivity of Rx was only evident when theywere overexpressed or expressed together with RanGAP2 (Fig.S5). However, when the five mutations were combined in RxS1234,the Rx HR was activated even when the protein was expressedfrom the native Rx promoter and without RanGAP2 coexpression(Fig. 4 A and B). This effect could be explained in terms of anATPase that mediates the switch between ATP-bound on andADP-bound off NB-LRR activation states (4). These two formsof Rx would be in dynamic equilibrium and, in the absence of coatprotein elicitor with wild-type Rx, the pool would be predomi-nantly in the inactive state. The S1, S2, S3, and S4 mutations couldeach shift the balance toward the activated form so that, inRxS1234, it is likely that the full activation was achieved withoutany further stimulus from overexpression of Rx or RanGAP2. Thesame mechanism is indicated by the increased preference forbinding ATP (49) in an autoactive version of the Flax M NB-LRR. Similarly, an autoactive version of the tomato NB-LRRprotein, I-2, is impaired in nucleotide hydrolysis (50). Ultimately,it will be necessary to perform biochemical tests on these ver-sions of Rx to test these hypotheses.Several other observations are consistent with this mechanistic

explanation of the S14 mutants. For example, the requirementfor an intact P loop (Fig. 4 C and D) indicates an essential role ofnucleotide binding for the S14 mutations to have their effect. Anintact P loop has also been shown to be required for otherautoactive NB-LRR variants, including Rx and I-2 (38, 50).Similarly, the proximity of the S14 mutations to the nucleotide-binding pocket (Fig. 2B) is also consistent with an effect on ATPturnover. However, we cannot exclude the possibility that themutations affect the interaction with the LRR domain ordownstream signaling components (18, 19, 40).

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Fig. 4. Combining the S mutations causes autoac-tivity and requires the P loop. (A) Schematic showingthe combined amino acid change constructs (S14) inthe M1 (Upper) and wild-type (Lower) LRR back-grounds. (B) Transient expression of constructsencoding S1234 induces elicitor-independent necrosis inthe M1 and wild-type LRR backgrounds. Correspondingconstructs (RxM1 and Rx) with no S mutations in theN-terminal region are not autoactive. (Scale bars,0.5 cm.) Pictures were taken 5 dpi. (C) Chlorophyllcontent assay to quantify necrosis after transientexpression of the autoactive RxS1234 constructs con-taining mutations in the GKT motif 5 dpi in N. tabacum.Rx and RxS1234 are used as negative and positivecontrols for autoactivity, respectively. Each bar repre-sents an average of five biological replicates. Error barsrepresent 95% confidence intervals. (D) Western blotfor accumulation of the RxS1234 GKT mutants aftertransient expression in N. tabacum. RxS1234 does notshow a clear band, as the tissue was extensively necroticwhen collected at 3 dpi. RuBisCo is used as a load-ing control.

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Artificial and Natural R Gene Evolution in Crop Protection. In thispaper, we have used the RxM1 mutant to illustrate the tradeoffof costs and benefits in disease resistance. We show that a costspreading necrosis following PopMV infectioncan be miti-gated by secondary mutation and, if PVX were a problem incrops, it may well be that one or more of the RxS1-4M1 geneswould be more useful than the natural alleles of Rx: they wouldconfer resistance against both the CPTK and CPKR strains ofPVX. However, the crops with evolved RxM1 alleles would needto be tested against a range of pathogens to rule out that newcosts had been introduced.The idea that the LRR and N-terminal domains must coevolve

has been indicated by recombination experiments showing in-compatible interactions between domains of closely related NB-LRRs (20, 47), and it may well be that other NB-LRR genescould be enhanced by stepwise artificial evolution as with RxM1(51). The NB-LRRs in crops would have originated in wild spe-cies where the tradeoff of costs and benefits selected in thenatural environment would have influenced the resistance phe-notype. However, the optimal tradeoff in a natural environmentmay not be the same as in a managed agricultural setting, and itcould be that the range of useful R genes for crop protectioncould be expanded by stepwise artificial evolution. In the firststage, a gene could be evolved in one domain to derive mutantsin which the degree or specificity of resistance is modified. The

mutants could then be further refined by mutation of a seconddomain to ensure that costs have been minimized. This approachmight be particularly useful if resistance-breaking strains ofa pathogen have emerged or if the resistance of the natural Rgene is too weak to contain the pathogen. Alternatively, broadrecognition could be paired with a reduced level of activationsensitivity to minimize the resistance tradeoff with plant growth(25) and ensuring that only very strongly recognized pathogensignals initiate a defense response. As we have shown that theseS mutations can act independently of the broad recognition-conferring mutation in the LRR (Figs. 3A and 4B), we can beginto fine-tune the balance between NB-LRR recognition andresponse functions.How does our sequential in vitro selection approach compare

with evolution in natural populations? According to Ossowskiet al. (52), the spontaneous mutation rate of Arabidopsis is es-timated at 7 109 nt changes per site per generation, 1 105changes per 1.5-kb region per generation. Here we surveyedaround 7,500 changes (1,500 clones at an average mutationalfrequency of 5 changes per molecule, 5 1,500 = 7,500) across a1.5-kb region. Therefore, this study surveys the mutational spec-trum equivalent to 750 million Arabidopsis plants (7,500/1 105) in each round of mutation: The combined effect of a step-wise strategy is therefore equivalent to much larger populations.An additional consideration is that the selection in our artifi-

cial evolution approach is much more stringent and targeted tothe NB-LRR of interest than selection on individual NB-LRRsin a natural plant population (53). Stepwise artificial evolutiontherefore represents a powerful approach to modulate NB-LRRcharacteristics that is more efficient in time and space thanevolution in the field. This study demonstrates that a heuristicapproach, combining in vitro directed evolution and structuralmodeling, can be used to design improved NB-LRRs for use incrop protection.

Materials and MethodsPlasmids. The constructs used are described in SI Materials and Methods.

Homology Modeling. Homology modeling has been described previously (20).Images were generated using PyMOL software, http://www.pymol.org/.

Transient Expression. Agrobacterium tumefaciens GV3101 or C58C1 was infil-trated in 3- to 4-wk-old N. tabacum or N. benthamiana plants using similarmethods described previously (32).

Chlorophyll Content Assay. Based on ref. 33, the protocol is described in detailin SI Materials and Methods.

Western Blots. Total protein was extracted from infiltrated N. tabacum tissueusing methods described by ref. 54. HA-tagged Rx was detected using ananti-HA rat monoclonal antibody (clone 3F10; Roche 1867423) and goat anti-rat (LI-COR; IRDye 800CW, 926-32219) secondary antibody and visualized ona LI-COR Odyssey CLx.

Transgenic Plants. Previously described N. benthamiana transgenic lines usedin this study were wild type, Rx (RxH3), RxM1 (2/227 2B10), and RxM123 (GR3F11) (27). RxS14*M1 transgenic N. benthamiana were generated by leafdisc transformation (55).

RT-PCRs. Total RNA was extracted from noninoculated leaf tissue using TRIzolreagent, and cDNA was generated using random hexamer-primed RTsynthesis (Invitrogen; 18080-051). RT-PCR was performed using gene-specificprimers (Table S2).

ACKNOWLEDGMENTS. We thank our colleagues Attila Molnar andNatasha Elina for helpful discussions and their critical review of the manu-script, and Wladimir Tameling for facilitating a fruitful collaboration.This work was supported by the Biotechnology and Biological SciencesResearch Council (C.J.H.), the Gatsby Charitable Foundation, and theRoyal Society Edward Penley Abraham Research Professorship (to D.C.B.).E.J.S. and A.G. are supported by the European Commission IntegratedBIOEXPLOIT project.

PopMV inoculated

wt Rx RxM1 RxM1

RxM123

A

PopMV

Rx

GAPDH

B

S1*

RxS1*M1 + PopMV

Fig. 5. Transgenic plants carrying RxS1*M1 are resistant to PopMV. (A)Representative plants 6 wk after inoculation with PopMV. Wild-type and Rxplants show mild stunting and mosaic symptoms; RxM1 plants display thelethal trailing necrosis phenotype; and RxS1*M1 plants do not present viralsymptoms. The RxM123 transgenic line is used as a positive control forPopMV resistance (27). (B) RT-PCR of cDNA from noninoculated leaf tissueharvested 3 wk postinoculation from eight different RxS1*M1 plants. Foreach plant, the same cDNA was used in three RT-PCR reactions to amplifyPopMV coat protein, RxS1*M1 transgene, or the housekeeping geneGAPDH. Because RxS1*M1 plants are in the T1 generation, the transgene issegregating, providing an additional blinding control in the experiment(uninfected RxS1*M1 T1 plants are phenotypically identical). These resultswere repeated on at least three separate occasions.

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