HopAS1 recognition significantly contributes to Arabidopsis nonhost resistance to Pseudomonas syringae pathogens

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HopAS1 recognition significantly contributes to Arabidopsis nonhostresistance to Pseudomonas syringae pathogens

Author for correspondence:Jonathan D. G. Jones

Tel: +44 01603 450400

Email: [email protected]

Received: 7 September 2011

Accepted: 30 September 2011

Kee Hoon Sohn1, Simon B. Saucet1, Christopher R. Clarke2, Boris A.

Vinatzer2, Heath E. O’Brien3, David S. Guttman3 and Jonathan D. G. Jones1

1The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK; 2Department of Plant Pathology,

Physiology and Weed Science, Virginia Tech, Latham Hall, Blacksburg VA 24061, USA; 3Centre for the Analysis of Genome

Evolution and Function, University of Toronto, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada

New Phytologist (2012) 193: 58–66doi: 10.1111/j.1469-8137.2011.03950.x

Key words: Arabidopsis, effector-triggeredimmunity, nonhost resistance, Pseudomonas,type III effector.

Summary

• Plant immunity is activated by sensing either conserved microbial signatures, called patho-

gen ⁄ microbe-associated molecular patterns (P ⁄ MAMPs), or specific effectors secreted by

pathogens. However, it is not known why most microbes are nonpathogenic in most plant

species.

• Nonhost resistance (NHR) consists of multiple layers of innate immunity and protects plants

from the vast majority of potentially pathogenic microbes. Effector-triggered immunity (ETI) has

been implicated in race-specific disease resistance. However, the role of ETI in NHR is unclear.

• Pseudomonas syringae pv. tomato (Pto) T1 is pathogenic in tomato (Solanum lycopersicum)

yet nonpathogenic in Arabidopsis. Here, we show that, in addition to the type III secretion

system (T3SS)-dependent effector (T3SE) avrRpt2, a second T3SE of Pto T1, hopAS1, triggers

ETI in nonhost Arabidopsis.

• hopAS1 is broadly present in P. syringae strains, contributes to virulence in tomato, and is

quantitatively required for Arabidopsis NHR to Pto T1. Strikingly, all tested P. syringae strains

that are pathogenic in Arabidopsis carry truncated hopAS1 variants of forms, demonstrating

that HopAS1-triggered immunity plays an important role in Arabidopsis NHR to a broad-

range of P. syringae strains.

Introduction

The plant innate immune system provides resistance to mostmicrobes. Effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI)are major components of plant innate immunity (Jones & Dangl,2006). PTI is activated via recognition of conserved microbial pat-terns by cell surface-localized pattern recognition receptors (PRRs)(Zipfel et al., 2004; Jones & Dangl, 2006; Segonzac & Zipfel,2011). Successful pathogens secrete virulence factors and suppressPTI (Jones & Dangl, 2006). Pathogenic microbes differ from eachother in the range of plant species on which they can grow andreproduce. Several studies have suggested that PTI is involved innonhost resistance (NHR) and that pathogen effector-mediatedsuppression of PTI partially compromises NHR (Ham et al.,2007; Ferrante et al., 2009; Lacombe et al., 2010; Zhang et al.,2010). NHR requires multiple defense layers and therefore is

considered to be more durable than race-specific resistance, whichis usually dependent on the recognition of a single effector (Holub& Cooper, 2004; Nurnberger & Lipka, 2005; Schulze-Lefert &Panstruga, 2011).

Although ETI is a major component of host ⁄ pathogen racespecificity, little is known of the extent to which ETI plays a rolein NHR. In some cases, avirulence (Avr) effectors trigger ETI innonhost plants, suggesting a role for ETI in determiningpathogen host range (Staskawicz et al., 1987; Kobayashi et al.,1989; Wei et al., 2007; Wroblewski et al., 2009). However,because Avr effectors contribute to pathogen virulence on hostplants, they can be maintained in pathogen populations eventhough they trigger ETI on some plants (Kearney & Staskawicz,1990).

The Gram-negative plant pathogenic bacterium Pseudomonassyringae infects and causes disease in a wide range of plant hosts(Hirano & Upper, 2000). Pseudomonas syringae pv. tomato

Research

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(Pto) T1 causes bacterial speck disease in tomato (Solanumlycopersicum) but is nonpathogenic in Arabidopsis, whereas adifferent strain, Pto DC3000, is pathogenic in tomato andArabidopsis. The genomes of Pto DC3000 and T1 are fullysequenced and provide a useful tool to dissect the roles of ETIin NHR in the model plant Arabidopsis (Buell et al., 2003;Almeida et al., 2009).

Materials and Methods

Plasmid constructions and mobilizations to Pseudomonasand Agrobacterium tumefaciens strains

All Pto T1 T3SS-dependent effectors (T3SEs) and hopAS1 werecloned in the pENTR-SD-D-TOPO vector (Invitrogen),sequenced and maintained in Escherichia coli DH5a. Subse-quently, an LR reaction was performed using pBS46 (Swingleet al., 2008) and pER8:N-HS (a binary vector to express estra-diol-induced N-terminally HA (haemagglutinin)-tagged proteinin plant cells) as destination vectors for expression in Pseudomonasand Arabidopsis thaliana (L.) Heynh., respectively. Plasmidmobilizations from E. coli DH5a to Pseudomonas strains wereperformed using standard triparental mating as described previ-ously (Sohn et al., 2007). Agrobacterium tumefaciens AGL1 wastransformed with pER8:N-HS:hopAS1 by electroporation. Primersequences are available upon request.

Generation of Pto T1 DhopAS1 and DavrRpt2DhopAS1mutant strains

To generate Pto T1 DhopAS1, a central 660-bp-long fragment ofthe hopAS1PtoT1 gene was amplified. Stop codons were addedin-frame to the 5¢ end of the primers and an ApaI site was addedto the 5¢ end of the forward primer and a ClaI site to the 5¢ endof the reverse primer. The fragment was cloned into pBAV208and the resulting plasmid was introduced into PtoT1 as previ-ously described (Mohr et al., 2008), giving Pto T1 DhopAS1,which was confirmed by sequencing to have two fragments of thehopAS1 gene, one 5¢ fragment with a stop codon after bp 174and one 3¢ fragment starting with a stop codon at bp 838. Pto T1DavrRpt2DhopAS1 was generated as follows: a 1-kb upstreamfragment of hopAS1PtoT1 was PCR-amplified, digested withBamHI and XbaI, and cloned in pRK415 (Keen et al., 1988) tocreate pRK415AS1A. A 1.5-kb downstream fragment ofhopAS1PtoT1 was PCR-amplified, digested with XbaI andHindIII, and cloned in pRK415AS1A to create pRK415AS1AB.Subsequently, a spectinomycin resistance gene was amplified,digested with HindIII and cloned in pRK415AS1AB to createpRK415AS1ASPB. pRK415AS1ASPB was mobilized into a PtoT1 strain that already had an insertion of plasmid pBA208between the avrRpt2 hypersensitive response and pathogenicity(hrp) box and the avrRpt2 start codon and that does not triggerresistance to P. syringae 2 (RPS2)-dependent hypersensitiveresponse (HR) (data not shown). Transformants were selected on

King’s B agar media containing kanamycin (50 lg ml)1), tetra-cycline (10 lg ml)1) and spectinomycin (100 lg ml)1). A singlecolony was used to inoculate 10 ml of NYG (nutrient broth,yeast extract and glucose) broth and cultured for 2 d withoutantibiotics. After three subcultures, cells were spread on King’s Bagar media containing kanamycin (50 lg ml)1) and spectino-mycin (100 lg ml)1). After 3 d of incubation in 28�C, selectedcolonies were tested for tetracycline resistance. The colonies thatwere resistant to kanamycin (50 lg ml)1) and spectinomycin(100 lg ml)1) but sensitive to tetracycline (10 lg ml)1) wereselected and DNA was isolated for PCR analysis. Replacement ofthe hopAS1 open reading frame (ORF) by the spectinomycinresistance gene was confirmed by sequencing the PCR product.

Generation of transgenic Arabidopsis plants

A transgenic Arabidopsis line was generated by following the pro-tocol described previously (Clough & Bent, 1998).

In planta bacterial growth and HR assays

Leaves of Arabidopsis, turnip (Brassica rapa) or tomato were infil-trated with bacterial suspensions using a 1-mL needleless syringefor the HR assay or the bacterial growth assay. For spraying infec-tion, Arabidopsis plants were sprayed with bacterial suspensions(0.05% silwet L-77) and covered with a transparent plastic coverfor 48 h. The infected leaf samples were collected at 3 or 4 d postinfection (dpi), ground in sterilized 10 mM MgCl2, seriallydiluted and spotted on NYG or low-salt LB (Luria-Bertani) agarmedium containing appropriate antibiotics. Numbers of colonieswere counted after 2 d of incubation at 28�C. The infectedplants were kept in a growth chamber up to 7 dpi to observe dis-ease symptoms. For the HR assay, the infected leaves wereobserved up to 48 h post infection (hpi).

Immunoblot analysis

Protein extraction and immunoblot analyses were performed aspreviously described (Sohn et al., 2009). In short, PFO1-T3SSstrains carrying pBS46:T3SE constructs were harvested in lysisbuffer (140 mM NaCl, 2.7 mM KCL, 10 mM Na2HPO4 and1.8 mM KH2PO4, pH 7.3) after growing on King’s B agar med-ium (30 ug ml)1 chloramphenicol, 5 ug ml)1 tetracycline and20 lg ml)1 gentamycin) for 2 d at 28�C. After sonication(3 · 10 s) and brief centrifugation, the supernatant was mixedwith SDS-loading buffer and boiled for 4 min before beingloaded in a sodium dodecyl sulphate–polyacrylamide gel electro-phoresis (SDS-PAGE) gel for immunoblot analysis. TransgenicArabidopsis T2 lines expressing HA-HopAS1PtoT1 were grownon Murashige and Skoog (MS) agar medium (hygromycin 40 lgml)1) for 10–14 d and then transferred to the same medium con-taining 50 lM estradiol. After 24 h of incubation, two to threeseedlings were collected and frozen in liquid nitrogen for proteinextraction. Immunoblot analysis was performed using anti-HAantibody (Roche; 3F10).

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Results and Discussion

The Pto T1 T3SE hopAS1 triggers ETI in Arabidopsis

To better understand the role of ETI in host range determina-tion, we compared the T3SE repertoires of Pto DC3000 and T1(Almeida et al., 2009). To proliferate and cause disease intomato, both strains require a functional type III secretion system(T3SS) (Fig. 1a). However, on Arabidopsis, wild-type Pto T1

shows as little growth as Pto T1 or Pto DC3000 DhrcC (hrp-conserved) mutant lacking a functional T3SS, whereas wild-typePto DC3000 is highly virulent (Fig. 1b). We set out to investi-gate why Pto T1 is nonpathogenic in Arabidopsis whereas PtoDC3000 is highly pathogenic.

Pto T1 carries the T3SE avrRpt2, which triggers RPS2-dependent resistance (Bent et al., 1994; Mindrinos et al., 1994;Almeida et al., 2009). Strain T1 also shows slightly enhancedvirulence in an Arabidopsis mutant lacking functional RPS2

Fig. 1 The Pseudomonas syringae pv. tomato (Pto) T1 T3SE hopAS1 triggers effector-triggered immunity (ETI) in nonhost Arabidopsis plants. (a, b) Pto T1is a nonhost pathogen in Arabidopsis. In planta growth was measured for Pto DC3000 and T1 wild type or DhrcC strains in tomato (cv Moneymaker) (a) orArabidopsis (b) leaves. Five-wk-old leaves were hand-infiltrated using a 1-ml needless syringe with bacterial suspensions (optical density (OD)600 = 0.0001for tomato and OD600 = 0.001 for Arabidopsis) and samples were taken at 3 d post infection (dpi) to measure bacterial number in infected leaves. Resultsare the mean ± SE of bacterial colonies recovered from nine leaf samples each containing four leaf discs (1 cm2). Means labeled with the same letter arenot statistically different at the 5% confidence level based on Tukey’s test (a, b). This experiment was repeated twice with similar results. (c) hopAS1

triggers P. syringae 2 (RPS2)-independent hypersensitive response (HR) in Arabidopsis. Arabidopsis leaves were infiltrated with PFO1-T3SS carrying emptyvector (pBBR 1MCS-5), pBS46:avrRpt2PtoT1-HA or pBS46:hopAS1PtoT1-HA (OD600 = 0.4). Photographs were taken at 36 h post infection (hpi). Thisexperiment was repeated 4 times with similar results. (d) hopAS1-triggered immunity does not require RPS2. Wild-type or mutant Arabidopsis accessionColumbia (Col-0) leaves were infected with Pto DC3000 carrying empty vector (pBBR 1MCS-5) or pBS46:hopAS1PtoT1-HA as explained in (a). Four-wk-oldplants were infected by spraying with bacterial suspensions (OD600 = 0.1) and kept in a humidity chamber for 2 d, and samples were taken at 4 d postinfection (dpi) to measure bacterial number in infected leaves. Results are the mean ± SE of bacterial colonies recovered from eight leaf samples eachcontaining four leaf discs (1 cm2). Means labeled with the same letter are not statistically different at the 5% confidence level based on Tukey’s test. Thisexperiment was repeated twice with similar results. (e) Transient expression of HA-HopAS1 in Arabidopsis cells causes cell death. Photographs were takenat 3 d after estradiol treatment. (f) hopAS1-triggered immunity does not require EDS1, NDR1, SID2 and RAR1. Experimental conditions and statisticalanalysis were same as in (d).





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(rps2-101c) (Mindrinos et al., 1994), yet still does not cause dis-ease (Fig. 1b). Based on these results, we hypothesized that theinability of Pto T1 to cause disease in rps2-101c is attributable toinefficient suppression of PTI by Pto T1 T3SEs and ⁄ or addi-tional ETI triggered by a Pto T1 T3SE other than avrRpt2. Thus,we decided to search for additional Pto T1 T3SEs that are recog-nized in an RPS2-independent manner.

To identify an additional Avr T3SE from Pto T1, we clonedall 13 predicted T3SEs of Pto T1 that are absent or significantlydifferent (< 90% amino acid identity) from those of PtoDC3000 (Supporting Information Table S1) (Almeida et al.,2009). Each of the 13 Pto T1-specific T3SEs was expressed underthe constitutive nptII (neomycin phosphotransferase II) promoterin the nonpathogenic Pseudomonas fluorescens PFO1 strain carry-ing a functional T3SS (PFO1-T3SS) (Thomas et al., 2009). AllPto T1-specific T3SEs were expressed well in PFO1-T3SS exceptHopS1-HA, which we could not detect (Supporting InformationFig. S1). PFO1-T3SS expressing AvrRpt2PtoT1 triggered anRPS2-dependent HR within 20 hpi (Fig. 1c), while the Pto T1T3SE HopAS1PtoT1 triggered a weak HR in Arabidopsis Colum-bia (Col-0) and rps2-101c at 36–48 hpi (Fig. 1c). No other PtoT1-specific T3SE triggered an HR in Arabidopsis Col-0 within48 hpi (data not shown).

As HR is often, but not always, associated with ETI, we testedif any Pto T1 T3SE can restrict virulent bacterial growth inArabidopsis Col-0, by constructing Pto DC3000 strains carryingindividual Pto T1-specific T3SEs and measuring bacterial popu-lations in infected leaves at 3 dpi. Only Pto DC3000 carryingavrRpt2PtoT1 or hopAS1PtoT1 reproducibly showed significantlyreduced growth compared with Pto DC3000 (empty vector) (Fig.S2). Moreover, Pto DC3000 carrying hopAS1PtoT1 triggered HRand resistance in rps2-101c, verifying that hopAS1PtoT1-triggeredimmunity is independent of RPS2 (Fig. 1c,d).

hopAS1PtoT1 is preceded by an hrp box in the 5¢ regulatoryregion and HopAS1PtoT1 protein is delivered to plant cells in aT3SS-dependent manner (Almeida et al., 2009). A conserveddomain search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) using the HopAS1PtoT1 protein (1362 amino acids)sequence predicted that the N-terminal region of HopAS1PtoT1

shares 10% similarity over 17% of the sequence with the bacterialchromosome segregation protein SMC (structural maintenanceof chromosomes); no other homologies were detected.

Transient expression of Avr proteins in resistant plant cellsoften causes cell death responses (McNellis et al., 1998). Toinvestigate whether HopAS1PtoT1 also triggers cell death whentransiently expressed in plant cells, we generated a stable Arabid-opsis Col-0 line (T2 generation) conditionally expressing HA-HopAS1PtoT1 (hence, At-EST:HA:HopAS1PtoT1) upon estradioltreatment. The HA-HopAS1PtoT1 protein was well expressed inArabidopsis cells after 24 h of estradiol treatment of 2-wk oldAt-EST:HA:HopAS1PtoT1 seedlings grown on MS agar plates(Fig. 1e). Unlike several P. syringae T3SEs (e.g. AvrRpt2 andAvrRps4) (Mudgett & Staskawicz, 1999; Sohn et al., 2009),HA-HopAS1PtoT1 protein expressed in At-EST:HA:HopAS1PtoT1

seedlings migrated similarly to HopAS1PtoT1-HA protein fromPFO1-T3SS, indicating the post-translational processing of

HopAS1PtoT1 into a smaller form does not occur in plant cells(Fig. 1e). At-EST:HA:HopAS1PtoT1 seedlings showed severe celldeath after 72–96 h of estradiol treatment (Fig. 1e). This resultis in accordance with the previous HR (Fig. 1c) and T3SS-dependent secretion (Almeida et al., 2009) data when Ho-pAS1PtoT1 was secreted from PFO1-T3SS or Pto DC3000, anddemonstrates that HopAS1PtoT1 triggers ETI from inside plantcells.

Previously, it was shown that Pto T1 virulence was not signifi-cantly enhanced in an Arabidopsis mutant lacking RAR1(required for Mla12 resistance), which is required for some ETI(Muskett et al., 2002; Tornero et al., 2002; Almeida et al.,2009). In many cases, signaling components downstream of Rgenes are well conserved (i.e. EDS1 (enhanced disease suscep-tibility 1)- or NDR1 (non race-specific disease resistance 1)-dependent ETI). To investigate the genetic requirements ofhopAS1PtoT1-triggered immunity in more detail, we comparedthe growth of Pto DC3000 carrying empty vector (pBBR 1MCS-5) or hopAS1PtoT1 in several mutants impaired in ETI signaling.Pto DC3000 carrying empty vector or hopAS1PtoT1 showedenhanced virulence in eds1, ndr1, sid2 (salicylic acid inductiondeficient 2) and rar1 mutants compared with Col-0 wild-typeplants (with the exception of Pto DC3000 carrying hopAS1PtoT1

in the rar1 mutant) (Fig. 1f). Nonetheless, Pto DC3000 carryinghopAS1PtoT1 showed significantly reduced virulence and diseasesymptom development compared with Pto DC3000 (empty vec-tor) in all four tested mutants, demonstrating that hopAS1PtoT1-triggered immunity is, at least partially, independent of thesesignaling components (Figs 1f, S3). Several Arabidopsis R genes(e.g. RPP7 (resistance to peronospora parasitica 7), RPP8 andRPP13) conferring resistance to downy mildew (Hyaloperonosporaarabidopsidis) function mostly independently of EDS1, NDR1and salicylic acid (SA) pathways (McDowell et al., 2000; Bittner-Eddy & Beynon, 2001). Recently, it was shown that P. syringaeeffector hopZ1a-triggered immunity also does not require knownETI-signaling components (Lewis et al., 2010). hopAS1PtoT1-and hopZ1a-triggered Arabidopsis immunity thus uses EDS1-and NDR1-independent signaling.

A mutation in hopAS1 confers enhanced and reducedvirulence of Pto T1 in Arabidopsis and tomato, respectively

As hopAS1 triggers immunity in Arabidopsis mutants lackingRPS2, NDR1, SID2 or RAR1, which are required for avrRpt2-triggered immunity, we hypothesized that Pto T1 DhopAS1might confer enhanced virulence compared with wild-type PtoT1 in rps2-101c. To test this hypothesis, Pto T1 DhopAS1 wasgenerated and in planta growth was measured. Pto T1 DhopAS1showed slightly but significantly enhanced virulence comparedwith Pto T1 wild-type or the DhrcC mutant in wild-type Col-0plants, indicating that hopAS1-triggered immunity plays a rolein Arabidopsis NHR to Pto T1 (Fig. 2a). Moreover, Pto T1 Dho-pAS1 showed further enhanced virulence compared with Pto T1wild-type in rps2-101c, indicating that hopAS1- and avrRpt2-triggered immunity quantitatively contribute to ArabidopsisNHR to Pto T1 (Fig. 2a).






Why does Pto T1 carry functional hopAS1PtoT1 which canrestrict its host range? One plausible scenario is that hopAS1 is animportant virulence factor for growth of Pto T1 on its naturalhost plants. Therefore, we investigated whether hopAS1 isinvolved in Pto T1 virulence on tomato. Pto T1 DhopAS1 showedslightly, but significantly, less growth compared with Pto T1 wildtype or DhopAS1 complemented with a plasmid-bornehopAS1PtoT1-HA in tomato (cv Moneymaker) leaves (Fig. 2b).This suggests that hopAS1 is required for full virulence of Pto T1during colonization of tomato leaves. We conclude that Pto T1maintains a functional hopAS1 because of its function invirulence during colonization of host plants despite the resultingreduction in host range.

The phytotoxin coronatine is one of the major virulence deter-minants of Pto DC3000 (Brooks et al., 2004). Comparativegenomic studies have shown that Pto T1 lacks biosynthetic genesfor coronatine (Almeida et al., 2009). Thus, we investigatedwhether exogenous application of coronatine (purified fromP. syringae pv. glycinea; Sigma) can suppress Arabidopsis NHR,which would result in enhanced Pto T1 virulence. We also gener-ated a Pto T1 DavrRpt2DhopAS1 double mutant strain andmeasured its virulence with or without coronatine application.Infiltration of Arabidopsis Col-0 leaves with coronatine enhancedthe growth of Pto DC3000 Cor- (a Pto DC3000 AK87 mutantwhich carries mutations in cmaA (coronamic acid A) and cfa6(coronafacic acid 6)) (Brooks et al., 2004) compared with wild-type Pto DC3000 in Arabidopsis Col-0 (Fig. 2c). In addition,coronatine treatment enabled the Pto T1 wild type and theDavrRpt2DhopAS1 mutant to show slightly enhanced growth inArabidopsis Col-0 plants, indicating that coronatine may play arole in suppressing NHR (Fig. 2c). By contrast, we could notdetect any enhanced growth on Arabidopsis of the Pto T1 DhrcCmutant or wild-type Pto DC3000 when treated with coronatine(Fig. 2c). As coronatine treatment enhanced the growth of PtoT1 wild type, T1 DavrRpt2DhopAS1 and DC3000 Cor- strainsbut had no effect on Pto T1 DhrcC growth, coronatine is not suf-ficient for suppression of NHR and may require the action ofone or more T3SEs. Alternatively, coronatine may suppress ETItriggered by another T3SE from Pto T1. It is interesting to notethat, even though avrRpt2-triggered ETI, hopAS1-triggered ETIand lack of coronatine additively contribute to NHR (10–50times reduced bacterial growth), Pto T1 DavrRpt2DhopAS1(+ coronatine) still showed up to 100 times less growth comparedwith Pto DC3000 wild type (Fig. 2a,c). However, we cannotexclude the possibility that exogenous application of coronatinemay not fully compensate for the coronatine deficiency of PtoDC3000 Cor- strains. Recently, it was shown that P. syringae sax(survival in Arabidopsis extracts) genes play an essential role inovercoming isothiocyanate-based defenses, resulting in enhancedvirulence of nonhost Pseudomonas strains, including Pto T1, inArabidopsis (Fan et al., 2011). However, introduction of saxCABgenes in Pto T1 DhopAS1 or Pto T1 DavrRpt2DhopAS1 did notresult in enhanced bacterial growth in the Col-0 wild type or therps2-101c mutant (Fig. S4). These results suggest the involve-ment of other factors in Arabidopsis NHR to Pto T1. Conceiv-ably, Pto T1 T3SEs are not as efficient at suppressing PTI in

Fig. 2 Pseudomonas syringae pv. tomato (Pto) T1 DhopAS1 showsenhanced virulence in Arabidopsis and reduced virulence in tomato plants.(a) Pto T1 DhopAS1 shows enhanced growth in Arabidopsis. Leaves of5-wk-old Arabidopsis accession Columbia (Col-0) wild type or rps2-101cmutant plants were infected with Pto T1 wild type or its derivatives asdescribed in Fig. 1(b). Results were obtained from eight leaf samples each.Means labeled with the same letter are not statistically different at the 5%confidence level based on Tukey’s test. This experiment was repeated threetimes with similar results. (b) hopAS1 is required for full virulence of Pto T1 intomato leaves. The bacterial growth assay was performed as described inFig. 1(a), except that results were obtained from four leaf samples. Meanslabeled with the same letter are not statistically different at the 5% confi-dence level based on Tukey’s test. This experiment was repeated twice withsimilar results. (c) Application of coronatine enhances survival of nonhostbacteria in Arabidopsis. Bacterial infection and the growth assay were per-formed as described in (a) with bacterial suspensions (OD600 = 0.0002) with(+Cor) or without ()Cor) coronatine. Samples were taken at 4 d post infec-tion (dpi). Results were obtained from four leaf samples. Means labeled withthe same letter are not statistically different at the 5% confidence levelbased on Tukey’s test. Error bars (a–c) represent standard error (±SE). Thisexperiment was repeated twice with similar results.






Arabidopsis as those of Pto DC3000. In this case, Pto DC3000T3SEs that are not present in Pto T1 would be good candidatesfor testing whether they enhance the virulence of Pto T1DhopAS1 in rps2-101c.

Arabidopsis-infecting P. syringae strains carry truncatedhopAS1 alleles

We investigated hopAS1 alleles from a diverse array of P. syringaestrains to assess the diversity in this T3SE family and determineits impact on plant immunity. In particular, we analyzed hopAS1nucleotide (NT) sequences from 28 P. syringae strains belongingto 11 pathovars and isolated from many different plant species(Table 1). Among the 28 strains, all strains pathogenic onArabidopsis (Debener et al., 1991; Whalen et al., 1991; Rohmeret al., 2003; Yan et al., 2008) carry truncated hopAS1 alleles(Table 1), including Pto DC3000, which has a nonsense

mutation (CAT to TAG) at nucleotide position 838–840, andP. syringae pv. maculicola (Pma) ES4326, which has a prematurestop brought about by a 2-bp deletion at nucleotide position3803–3804 (Fig. 3a). All hopAS1 alleles from the 19 strainsnonpathogenic on Arabidopsis carry full-length hopAS1 alleles,indicating that hopAS1-triggered immunity is strongly associatedwith Arabidopsis NHR to these strains (Table 1).

To test the avirulence activity of hopAS1 alleles from strainsthat do not cause disease in Arabidopsis Col-0, we cloned hopAS1from five nonpathogenic and two pathogenic P. syringae strains(Fig. 3a). When expressed in PFO1-T3SS, the truncatedhopAS1PtoDC3000 and hopAS1PmaES4326 alleles did not trigger anyvisible HR, whereas the full-length hopAS1 from the five non-pathogenic strains triggered macroscopic HR at 36–48 hpi inArabidopsis Col-0 (Fig. 3b). In addition, Pto DC3000 carryinghopAS1PmaES4326 or empty vector (pBBR 1MCS-5) showedsimilar levels of growth, while Pto DC3000 carrying hopAS1

Table 1 Pseudomonas syringae strains virulent in Arabidopsis carry a truncated hopAS1 allele

P. syringae straina Virulence in ArabidopsisHopAS1 polymorphism(aa length ⁄ mutationb)

HopAS1-triggeredHR in Arabidopsis

Pto DC3000 (hopAS1)virulence in Arabidopsisc

Pto T1 Nonpathogenic Full-length (1362) HRd DecreasedPto JL1065 Nonpathogenic Full-length (1362) HR DecreasedPph 1448A Nonpathogenic Full-length (1361) HR DecreasedPan 126 Nonpathogenic Full-length (1371) HR DecreasedPca CFBP2341 Nonpathogenic Full-length (1369) HR DecreasedPta 11528 Nonpathogenic Full-length (1361) NAe NAPav BPIC631 Nonpathogenic Full-length (1361) NA NAPph HB10Y1 Nonpathogenic Full-length (1361) NA NAPph 1302A Nonpathogenic Full-length (1361) NA NAPta 6606 Nonpathogenic Full-length (1361) NA NAPse HC_1 Nonpathogenic Full-length (1361) NA NAPph NPS3121 Nonpathogenic Full-length (1361) NA NAPph Y5_2 Nonpathogenic Full-length (1361) NA NAPla 107 Nonpathogenic Full-length (1361) NA NAPla YM7902 Nonpathogenic Full-length (1361) NA NAPgy KN44 Nonpathogenic Full-length (1361) NA NAPgy LN10 Nonpathogenic Full-length (1361) NA NAPgy UnB647 Nonpathogenic Full-length (1361) NA NAPgy BR1 Nonpathogenic Full-length (1361) NA NAPma M6 Pathogenicf Truncated (402 ⁄ frame shift) NA NAPto DC3000 Pathogenic Truncated (279 ⁄ frame shift) No HR SimilarPto ICMP3443 Pathogenic Truncated (400 ⁄ frame shift) NA NAPma ES4326g Pathogenic Truncated (1329 ⁄ frame shift) No HR SimilarPma F1 Pathogenic Truncated (216 ⁄ frame shift) NA NAPma F9 Pathogenic Truncated (279 ⁄ frame shift) NA NAPma M3 Pathogenic Truncated (279 ⁄ frame shift) NA NAPal CFBP6866 Pathogenic Truncated (1329 ⁄ frame shift) NA NAPal T3C Pathogenic Truncated (1329 ⁄ frame shift) NA NA

aPto, Pseudomonas syringae pv. tomato; Pph, Pseudomonas syringae pv. phaseolicola; Pan, Pseudomonas syringae pv. antirrhini; Pca, Pseudomonas

cannabina; Pta, Pseudomonas syringae pv. tabaci; Pav, Pseudomonas syringae pv. avellanae; Pse, Pseudomonas syringae pv. sesami; Pla, Pseudomonassyringae pv. lachrymans; Pgy, Pseudomonas syringae pv. glycinea; Pma, Pseudomonas syringae pv. maculicola; Pal, Pseudomonas cannabina pv. alisalensis.bMutations were caused by nucleotide change, insertion or deletion of one or two nucleotides.chopAS1-triggered immunity was determined by comparing in planta growth of Pto DC3000 (pBBR 1MCS-5:hopAS1) with that of Pto DC3000 (pBBR1MCS-5) in Arabidopsis accession Columbia (Col-0).dHR, hypersensitive response. HR was triggered by infiltrating 5- or 6-wk-old Arabidopsis Col-0 leaves with PFO1-T3SS carrying hopAS1 (OD600 = 0.4).HR was scored at 36–48 h post infection (hpi).eNA, not available.fAll pathogenic strains analyzed cause disease in at least one Arabidopsis accession (data not shown).gPma ES4326 belongs to P. cannabina pv. alisalensis based on multilocus sequence typing (Bull et al., 2010).






alleles from the nonpathogenic strains showed significantlyreduced growth as compared with Pto DC3000 (empty vector) inArabidopsis at 4 dpi (Fig. 3c).

To assess the role of hopAS1 alleles in alternative hosts, we usedPFO1-T3SS to deliver Pto T1, Pto JL1065 and Pto DC3000 ho-pAS1 alleles into turnip (cv Just Right) leaves. Consistent withthe Arabidopsis results, the full-length hopAS1PtoT1 and ho-pAS1PtoJL1065 alleles, but not the truncated hopAS1PtoDC3000

allele, triggered HR-like symptoms at 8–10 dpi, suggesting thathopAS1-triggered immunity is conserved in brassicaceous hosts(Fig. 3d). These results indicate that hopAS1-triggered immunityplays an important role in the NHR of Arabidopsis and turnip todiverse P. syringae strains and emphasize the importance of ETI

not only in pathogen race determination but also in host rangeand NHR. Interestingly, several closely related strains, for exam-ple Pto DC3000 ⁄ Pto T1 or Pca CFBP2341 ⁄ Pal CFBP6866,show different pathogenicity levels in Arabidopsis. Because full-length hopAS1 alleles must necessarily have evolved beforedisrupted hopAS1 alleles, one could infer that the most recentcommon ancestor of each of these groups of strains was not path-ogenic in Brassicaceae. Alternatively, the most recent commonancestor was pathogenic on ancestors of extant Brassicaceae spe-cies that had not yet evolved hopAS1-triggered immunity. Identi-fying the genetic basis of hopAS1-triggered immunity inArabidopsis and other Brassicaceae will enable further insights tobe obtained into the dynamics of the evolutionary arms racebetween the Brassicaceae family and P. syringae.

However, hopAS1 is clearly not the only factor determininghost specificity in Arabidopsis, as some P. syringae strains that arenot pathogenic in Arabidopsis do not carry any hopAS1 allele (forexample Psy B728a) (Feil et al., 2005). In addition, the Pto T1DavrRpt2DhopAS1 mutant grows significantly less well than PtoDC3000 in Arabidopsis Col-0 (Fig. 2c). Consequently, theabsence of hopAS1 and avrRpt2 is necessary but not sufficient forP. syringae compatibility with Arabidopsis. Other virulencefactors (e.g. coronatine and virulence-promoting T3SEs) absentfrom Pto T1 are needed to overcome Arabidopsis NHR, andthese await identification.

By definition, we would expect negligible genetic variationwithin a species for nonhost resistance. As the absence of full-length and functional hopAS1 in the P. syringae strains we testedis completely associated with the ability to colonize Arabidopsisaccession Col-0, we wondered how well hopAS1-triggered immu-nity is conserved among Arabidopsis accessions. Therefore, wetested the responses of 24 diverse Arabidopsis accessions toPFO1-T3SS-delivered HopAS1. We also tested avrRpm1,avrRpt2 or avrPphB-triggered HR as controls to see if other Avrgenes isolated from nonpathogenic P. syringae strains of Arabid-opsis are broadly recognized or not. All 24 accessions showed HRto PFO1-delivered HopAS1, whereas several accessions did notshow HR in response to avrRpm1, avrRpt2 or avrPphB (TableS2). This suggests that hopAS1-triggered immunity more signifi-cantly contributes to Arabidopsis NHR to P. syringae pathogensthan other more well-known ETIs.

Acknowledgements

We are grateful to Jeff Dangl (University of North Carolina,USA) and Marc Nishimura (University of North Carolina) forsharing unpublished sequences with us and providing helpfulcomments on our manuscript. We also thank Jeff Chang and BillThomas (Oregon State University) for Pf. PFO1, Bryan Swingle(USDA) for pBS46 plasmid, Jane Parker (MPIZ, Cologne,Germany) for pER8:N-HS plasmid, Alan Collmer (Cornell Uni-versity, USA) for pRK415 plasmid, Barbara Kunkel (WashingtonUniversity, USA) for the Pto DC3000 Cor- mutant, Jun Fan(John Innes Centre, UK) for pME6012:saxCAB plasmid, andMatthew Smoker, Jodie Pike and JIC horticultural staff for gen-erating and maintaining Arabidopsis transgenic lines. This work

Fig. 3 Virulent Pseudomonas syringae strains, Pseudomonas syringae pv.tomato (Pto) DC3000 and Pseudomonas syringae pv. maculicola (Pma)ES4326, carry nonfunctional hopAS1. (a) Comparison of amino acidsequences of HopAS1 from seven Pseudomonas strains. Full names forthe strains are listed in Table 1. Numbers indicate the amino acid identity(%) compared with HopAS1PtoT1. For HopAS1PtoDC3000, 279 aa ofHopAS1PtoT1 was used to determine identity level. Asterisks indicate thepositions of mutation. (b) hopAS1PtoDC3000 and hopAS1PmaES4326 do nottrigger HR. Five-wk-old Arabidopsis leaves were infiltrated with PFO1-T3SS carrying hopAS1 (OD600 = 0.4). Photographs were taken at 36 hpost infection (hpi). This experiment was repeated three times with similarresults. (c) hopAS1PtoDC3000 and hopAS1PmaES4326 do not trigger ETI.Arabidopsis Col-0 leaves were hand-infiltrated with Pto DC3000 strainscarrying hopAS1-HA (OD600 = 0.001). Results were obtained from nineleaf samples. Means labeled with the same letter are not statistically differ-ent at the 5% confidence level based on Tukey’s test. This experiment wasrepeated twice with similar results. Error bars (c) represent standard error(±SE). (d) hopAS1PtoT1 and hopAS1PtoJL1065 trigger cell death responsein turnip. Leaves of 4-wk-old turnip (cv Just Right) plants were hand-inoculated with PFO1-T3SS carrying hopAS1-HA (OD600 = 0.01).Photographs were taken at 10 d post infection (dpi).






was carried out with the support of the Gatsby Foundation (UK)and the ‘Cooperative Research Program for Agriculture Science& Technology Development (Project No. PJ007850201006)’Rural Development Administration (Republic of Korea).Research in the Vinatzer lab was funded by the National ScienceFoundation (Award IOS 0746501).

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Immunoblot analysis of Pseudomonas syringae pv. tomato(Pto) T1 T3SE-HA proteins and avirulence D (AvrD)-HAexpressed in PFO1-T3SS.

Fig. S2 Pseudomonas syringae pv. tomato (Pto) T1 T3SEs, avrRpt2and hopAS1, confer reduced virulence when delivered from PtoDC3000 in Arabidopsis accession Columbia (Col-0).

Fig. S3 HopAS1 confers reduced disease symptom developmentwhen delivered from Pseudomonas syringae pv. tomato (Pto)DC3000 in wild-type or mutant Arabidopsis accession Columbia(Col-0).

Fig. S4 Expression of SAX does not confer enhance growth ofPseudomonas syringae pv. tomato (Pto) T1 DhopAS1 or Dho-pAS1DavrRpt2 in Arabidopsis.

Table S1 List of Pseudomonas syringae pv. tomato (Pto) T1 typeIII effectors analyzed in this study

Table S2 List of Arabidopsis accessions tested for hypersensitiveresponse (HR) in responses to PFO1-T3SS carrying hopAS1PtoT1,avrRpm1, avrRpt2 or avrPphB

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HopAS1 recognition significantly contributes to Arabidopsis nonhost resistance to Pseudomonas syringae pathogens