The Pseudomonas syringae effector protein, AvrRPS4, requires in planta processing and the KRVY domain to function

Authors


(fax +44 1603 450011; e-mail jonathan.jones@tsl.ac.uk).

Summary

A Pseudomonas syringae pv. pisi effector protein, AvrRPS4, triggers RPS4-dependent immunity in Arabidopsis. We characterized biochemical and genetic aspects of AvrRPS4 function. Secretion of AvrRPS4 from Pst DC3000 is type III secretion-dependent, and AvrRPS4 is processed into a smaller form in plant cells but not in bacteria or yeast. Agrobacterium-mediated transient expression analysis of N-terminally truncated AvrRPS4 mutants revealed that the C-terminal 88 amino acids are sufficient to trigger the hypersensitive response in turnip. N-terminal sequencing of the processed AvrRPS4 showed that processing occurs between G133 and G134. The processing-deficient mutant, R112L, still triggers RPS4-dependent immunity, suggesting that the processing is not required for the AvrRPS4 avirulence function. AvrRPS4 enhances bacterial growth when delivered by Pta 6606 into Nicotiana benthamiana in which AvrRPS4 is not recognized. Transgenic expression of AvrRPS4 in the Arabidopsis rps4 mutant enhances the growth of Pst DC3000 and suppresses PTI (PAMP-triggered immunity), showing that AvrRPS4 promotes virulence in two distinct host plants. Furthermore, full virulence activity of AvrRPS4 requires both proteolytic processing and the KRVY motif at the N-terminus of processed AvrRPS4. XopO, an Xcv effector, shares the amino acids required for AvrRPS4 processing and the KRVY motif. XopO is also processed into a smaller form in N. benthamiana, similar to AvrRPS4, suggesting that a common mechanism is involved in activation of the virulence activities of both AvrRPS4 and XopO.

Introduction

Plants sense pathogen-associated molecular patterns (PAMPs) of invading microbes through pattern-recognition receptors (PRRs). Several receptor-like kinases (RLKs) specifically recognize PAMPs from bacteria or fungi (Zipfel et al., 2004, 2006; Miya et al., 2007; Wan et al., 2008). Recognition of PAMPs by the corresponding PRRs generally initiates PAMP-triggered immunity (PTI), which includes activation of the mitogen-activated protein kinase (MAPK) pathway, callose deposition at the cell wall, production of reactive oxygen species (ROS), and defence gene expression (Felix et al., 1999; Asai et al., 2002; Chisholm et al., 2006; Jones and Dangl, 2006). Plants also activate strong defence responses by specifically recognizing one or more pathogen effectors (avirulence proteins); this is termed effector-triggered immunity (ETI) (Chisholm et al., 2006; Jones and Dangl, 2006). Direct or indirect recognition of pathogen effectors by extracellular or intracellular resistance (R) genes usually results in localized cell death at the site of infection (the hypersensitive response, HR) and restriction of pathogen growth (Chisholm et al., 2006; Jones and Dangl, 2006).

Plant pathogenic bacteria, such as Pseudomonas syringae, use a specialized type III secretion system (T3SS) to inject effector proteins into the host cell cytoplasm (Jin et al., 2003; Grant et al., 2006). Type III secreted effectors (T3SE) are the major virulence determinants of bacterial pathogens, as failure to assemble a functional T3SS or secrete effectors results in complete loss of pathogenicity on host plants (Lindgren et al., 1986). It is poorly understood how most of these T3SEs suppress plant innate immunity and enable the bacteria to proliferate in their host plants. P. syringae pv. tomato (Pst) DC3000 is a bacterial pathogen that causes leaf speck disease both on tomato and Arabidopsis. Genome sequencing of several plant pathogenic bacteria including Pst DC3000 has accelerated identification of the functions of many T3SEs during bacterial pathogenesis in plants (Fouts et al., 2002; Feil et al., 2005; Joardar et al., 2005). Bioinformatics-assisted prediction has identified dozens of T3SEs from each of several sequenced P. syringae strains (Petnicki-Ocwieja et al., 2002; Vinatzer et al., 2005). In addition, truncated forms of avirulence proteins, such as AvrRpt2 or AvrBs2, that lack domains required for T3SS-dependent secretion, have been introduced to bacterial chromosomes by engineered transposons to identify T3S effectors whose N-termini enable secretion of the reporter proteins (Guttman et al., 2002; Roden et al., 2004; Chang et al., 2005). Recently, it was shown that oomycete pathogen effectors can also be delivered by a T3SS when translationally fused to an N-terminal T3S signal domain of bacterial effectors (Sohn et al., 2007; Whisson et al., 2007; Rentel et al., 2008).

Bacterial plant pathogens, such as P. syringae, generally deliver approximately 30 T3SEs into host cells. It is widely believed that the primary role of many of these effectors is to suppress both PTI and ETI. The Pst DC3000 effectors HopM1, AvrE and AvrPto suppress PAMP-triggered callose deposition at the cell wall (Hauck et al., 2003; DebRoy et al., 2004), and HopM1 and AvrPto were further shown to target AtMIN7 and the PAMP receptors FLS2 and EFR, respectively, to suppress PTI in Arabidopsis (Nomura et al., 2006; Xiang et al., 2008). HopAU1 has been shown to be an ADP-ribosyltransferase, and is required for its ability to suppress PTI and enhance the virulence of the bacterial pathogen Pst DC3000 by binding to an RNA-recognition motif that is present in glycine-rich RNA-binding protein 7 (AtGRP7) (Fu et al., 2007). The P. syringae T3SE HopAI1 inactivates MAPKs through its phosphothreonine lyase activity, and thus suppresses PTI (Li et al., 2005; Zhang et al., 2007). The P. syringae pv. phaseolicola effector protein AvrPphB, a member of the YopT class of cysteine proteases, undergoes autoproteolytic processing in bacterial cells (Puri et al., 1997). Agrobacterium-mediated transient expression (agroinfiltration) of the P. syringae effector AvrRpt2 revealed that the C-terminal 136 amino acids are sufficient to trigger the HR in N. tabacum (RPS2) (Mudgett and Staskawicz, 1999). It was further shown that AvrRpt2 is a cysteine protease that is activated by eukaryotic cyclophilin in plant cells (Axtell et al., 2003; Coaker et al., 2005, 2006).

AvrRPS4 was identified from P. syringae pv. pisi based on its ability to trigger an HR on some Arabidopsis accessions (Hinsch and Staskawicz, 1996). DNA hybridization results showed that AvrRPS4 is present in P. syringae pv. pisi, pv. glycinea and pv. phaseolicola (Hinsch and Staskawicz, 1996). AvrRPS4 was identified in the genome sequence of P. syringae pv. phaseolicola 1448A and shown to be secreted by the T3SS (Chang et al., 2005; Joardar et al., 2005). The corresponding R gene, RPS4, which belongs to the class of Toll/interleukin 1 receptor nucleotide-binding site leucine-rich repeat (TIR-NB-LRR) type receptors, was shown to confer resistance in Arabidopsis Col-0 and Ws-0 to P. syringae carrying avrRPS4 (Gassmann et al., 1999). The Arabidopsis accession RLD is susceptible to Pst DC3000(avrRPS4) due to the lack of a functional RPS4 allele (Gassmann et al., 1999). Agroinfiltration of the full-length RPS4 protein both in tobacco and Nicotiana benthamiana triggers an AvrRPS4-independent but EDS1- and SGT1-dependent HR (Zhang et al., 2004). Recently, it was shown that nuclear localization of RPS4 is required for AvrRPS4-triggered immunity (Wirthmueller et al., 2007). However, there has been no detailed analysis of the effector functions of AvrRPS4.

Here we show that translocation of AvrRPS4 to the plant cell requires a functional T3SS. AvrRPS4 then undergoes processing into a smaller form in planta. We also show that the C-terminal 88 amino acids of AvrRPS4 are sufficient to trigger an HR. Expression of AvrRPS4 in plants lacking RPS4 promotes susceptibility to bacterial pathogens and suppresses PTI. The in planta processing occurs at G133/G134 and is required for the virulence but not the avirulence function of AvrRPS4. Moreover, the KRVY motif at the N-terminus of the processed AvrRPS4 is required for both virulence and avirulence functions. Furthermore, we show that the processing sites and the amino acids required for AvrRPS4 processing are conserved in a homologous Xanthomonas effector of AvrRPS4, XopO. XopO is also processed into a smaller form in plant cells.

Results

AvrRPS4 is secreted by the Pst DC3000 type III secretion system

An Hrp (hypersensitive response and pathogenicity) sequence motif (Hrp box) that is recognized by the HrpL transcriptional activator has been found upstream of many avirulence (avr) and hrp genes from P. syringae. A nucleotide sequence corresponding to an Hrp box is located approximately 100 bp upstream of the ORF in the promoter sequence of avrRPS4 (Hinsch and Staskawicz, 1996). We therefore tested whether secretion of AvrRPS4 is dependent on an intact T3SS by using a plasmid-borne avrRPS4 gene whose transcriptional expression is driven by its own promoter (498 bp upstream of ATG) with or without a Flag-epitope tag at the C-terminus (pMH10 and pV316-1A, respectively). Bacterial cells cultured in Hrp induction media were sedimented by low-speed centrifugation, and an aliquot of the supernatant was plated on agar medium to ensure that there was no bacterial contamination. Proteins from these two fractions were then immuno-analysed using SDS–PAGE. In wild-type Pst DC3000 transformed with pV316-1A or pMH10, full-length AvrRPS4 protein (approximately 28 kDa) was detected in both the cellular lysate and the culture fluid (Figure 1a). In contrast, NPTII (encoded by neomycin phosphotransferase II, which is present in the vector carrying avrRPS4) was detected only in the cellular lysate. In the T3SS-deficient Pst DC3000 ΔhrcU (hypersensitive response and pathogenicity conserved U) mutant, AvrRPS4 was detected only in the cellular lysate, indicating that secretion of AvrRPS4 from Pst DC3000 into culture fluids requires a functional T3SS (Figure 1b).

Figure 1.

 HrcU-dependent secretion of AvrRPS4 by Pst DC3000.
(a) Immunoblot analysis of protein extracts of wild-type Pst DC3000 expressing AvrRPS4 (pV316-1A) or AvrRPS4-Flag (pMH10) grown in hrp-inducing minimal medium. Expression of neomycin phosphotransferase II (NPTII) was determined as a control.
(b) The Pst DC3000 ΔhrcU mutant expressing AvrRPS4 (pV316-1A) or AvrRPS4-Flag (pMH10) grown in hrp-inducing minimal media.

The C-terminal 88 amino acids of AvrRPS4 are sufficient to trigger an HR in turnip

As agroinfiltration of AvrRPS4 did not reliably trigger an HR in Arabidopsis plants carrying RPS4 (data not shown), we used turnip plants (cv. Just Right) for our transient protein expression experiments. Agroinfiltration of wild-type AvrRPS4–HA protein triggered an HR in turnip at 4 days post-inoculation (dpi) (Figure S1a). Previously, several P. syringae strains including Pst DC3000 have been shown to cause disease on turnip cv. Just Right (Dong et al., 1991). We tested whether AvrRPS4 is recognized in turnip leaves when delivered by Pst DC3000. Wild-type Pst DC3000(pVSP61) multiplied to 5 × 106 cfu cm−2 at 4 dpi, and caused disease symptoms when inoculated at a lower density (1 × 105 cfu ml−1) (Figure S1b). In contrast, growth of Pst DC3000(pV316-1A) (harbouring avrRPS4) was restricted by approximately 100-fold compared to Pst DC3000(pVSP61) (empty vector) and the plants showed no visible symptoms, suggesting that the HR induced by transient expression of AvrRPS4–HA is due to AvrRPS4-triggered immunity in turnip plants. To define which part of AvrRPS4 triggers HR, we generated N-terminally truncated mutant AvrRPS4–HA and tested the avirulence (HR-inducing) activities in turnip leaves by agroinfiltration. Deletion of the first 52, 79, 119, 131, 132 or 133 amino acids (i.e. M53, M80, M120, M132, M133 or M134, respectively) of AvrRPS4–HA retained the HR-inducing activity in turnip leaves (Table 1). Expression of AvrRPS4–HA with deletions of 134 (M135) or more amino acids from the N-terminus failed to cause HR. These results indicate that 88 C-terminal amino acids are sufficient for the avirulence function of AvrRPS4 in turnip.

Table 1.   The C-terminal 88 amino acids of AvrRPS4 are sufficient to trigger HR after Agrobacterium-mediated transient expression in turnip
AvrRPS4–HA proteinaPhenotypebProtein expressionc
N. benthamianaTurnip
  1. aWild type and N-terminally truncated AvrRPS4 proteins were expressed under the 35S promoter with C-terminal HA epitope tag.

  2. bPhenotypes were observed 5 days after transient expression on turnip leaves. HR, hypersensitive response; NS, no symptoms.

  3. cProtein samples were taken from N. benthamiana and turnip leaves.

Wild-type
 1–221HRYesYes
N-terminal deletions
 53–221 (M53)HRYesYes
 80–221 (M80)HRYesYes
 120–221 (M120)HRYesYes
 132–221 (M132)HRYesYes
 133–221 (M133)HRYesYes
 134–221 (M134)HRYesYes
 135–221 (M135)NSNoNo
 136–221 (M136)NSNoNo
 137–221 (M137)NSNoNo
 138–221 (M138)NSNoNo
 139–221 (M139)NSYesYes
 140–221 (M140)NSYesYes
 141–221 (M141)NSYesYes
 153–221 (M153)NSYesYes

AvrRPS4 protein is processed to a smaller form when transiently expressed in plant cells

Interestingly, when we transiently expressed wild-type AvrRPS4–HA in turnip and N. benthamiana, we were able to detect the processed (11 kDa) protein using an anti-HA antibody (Figure 2). However, the AvrRPS4–HA protein is not processed when expressed either in bacteria (Figure 1) or yeast cells (Figure S2). Some of the N-terminally truncated AvrRPS4–HA (M135, M136, M137 and M138) were not detected using an anti-HA antibody when transiently expressed in N. benthamiana and turnip leaves (Figure 2 and Table 1). This suggests that these amino acids could be important for AvrRPS4 protein stability in plant cells. In addition, we detected much more unprocessed than processed AvrRPS4–HA protein when we transiently expressed M53. Moreover, M120 triggered an HR in turnip but the protein was not processed in our transient expression experiments, indicating that processing is not necessary for the AvrRPS4-triggered HR in turnip (Table 1 and Figure 2). Based on these results, we conclude that the N-terminal portion of AvrRPS4 is required for in planta processing of AvrRPS4.

Figure 2.

 Expression of N-terminally truncated AvrRPS4–HA proteins by Agrobacterium-mediated transient transformation in N. benthamiana.
The numbers refer to amino acids of AvrRPS4. The lower panel shows Ponceau staining of the same gel to demonstrate equal loading of the proteins.

As the size of the processed wild-type AvrRPS4 protein is approximately 11 kDa, which is similar to the size of the truncated AvrRPS4–HA proteins M132, M133 and M134, we predicted that the processing site is within or close to G132–G134. We next tested the in planta processing of the wild-type AvrRPS4–HA protein in resistant and susceptible Arabidopsis accessions, turnip, Nicotiana tabacum and N. benthamiana using an agroinfiltration method. The processed AvrRPS4–HA protein was detected in all Arabidopsis accessions tested, indicating that in planta processing of AvrRPS4–HA is independent of the presence of the corresponding R gene, RPS4, as the accession RLD does not carry a functional RPS4 allele (Figure 3a). In addition, processed AvrRPS4–HA was also detected from leaf extracts of turnip, N. tabacum and N. benthamiana plants (Figure 3a).

Figure 3.

 AvrRPS4 is processed to a smaller form after transient or inducible expression in plant cells.
(a) Immunoblot analysis of AvrRPS4–HA protein using anti-HA antibody and protein extracts of Arabidopsis, turnip, Nicotiana tabacum (N.t) and Nicotiana benthamiana (N.b) transiently transformed with Agrobacterium carrying the pBIN19 empty vector (1) or pBIN19:avrRPS4:HA (2).
(b) Immunoblot analysis of AvrRPS4–HA protein using protein extracts of N. benthamiana leaves transiently expressing AvrRPS4–HA.
(c) 12% SDS–PAGE gel stained with Coomassie brilliant blue, containing processed AvrRPS4–HA protein purified from N. benthamiana (N.b) leaves transiently expressing AvrRPS4–HA and from Arabidopsis (A.t) accession Col-0 (Dex-avrRPS4–HA) after 30 μm dexamethasone treatment.
(d) Amino acid sequences of the processed AvrRPS4–HA proteins identified by N-terminal Edman degradation. The amino acid sequences were identified using the processed AvrRPS4–HA proteins purified from N. benthamiana (N.b) and A. thaliana (A.t) shown in (c).
(e) Diagram showing the processing site and amino acid residue required for processing of AvrRPS4.

In planta processing of AvrRPS4 occurs at G133–G134 and requires R112

In order to determine the processing site of AvrRPS4, we transiently expressed AvrRPS4–HA under the control of the CaMV 35S promoter in N. benthamiana leaves by agroinfiltration and also by spraying Arabidopsis plants that conditionally express AvrRPS4–HA under the control of the glucocorticoid-inducible promoter with 30 μm dexamethasone. The C-terminal fragment of processed AvrRPS4–HA protein (11 kDa) was immunoprecipitated from leaf extracts of N. benthamiana and Arabidopsis Col-0 (Dex-avrRPS4-HA) and separated on 12% SDS–PAGE before transfer to PVDF membrane. The processed AvrRPS4 (11 kDa) was visualized by staining the PVDF membrane with Coomassie Brilliant Blue, and the correct size band was excised and subjected to N-terminal sequencing (Figure 3c). N-terminal sequencing of both samples revealed that the processing site of AvrRPS4–HA was at G133/G134 (Figure 3d). However, we have previously shown that M120 was not processed (Figure 2). To identify the amino acid(s) required for processing of AvrRPS4, we performed error-prone PCR to generate an AvrRPS4 mutant that is not processed when expressed in plant cells. Among 94 independent clones tested, one mutant was found to be unprocessed when transiently expressed in N. benthamiana leaves (Figure 3b). The unprocessed AvrRPS4 mutant had two nucleotide substitutions (CG to TT) that resulted in an amino acid change of Arg112 to Leu (R112L) (Figure 3e). These data, which are consistent with the M120 result (Figure 2), indicate that the N-terminal part of AvrRPS4 is required for in planta processing.

The avirulence function of AvrRPS4 in Arabidopsis and turnip requires the KRVY motif but is independent of processing

Based on the data obtained from transient expression analysis of the N-terminally truncated AvrRPS4–HA proteins and the N-terminal sequencing of processed AvrRPS4, we predicted that the N-terminal residues of the processed AvrRPS4 protein are important for protein stability, processing and/or effector function when expressed in planta. Site-directed mutagenesis was performed on the nucleotides encoding one or more amino acids between K135 and I140 of AvrRPS4 (Figure 4a), and the mutants were tested for protein expression, processing and avirulence activities, both in turnip plants by agroinfiltration and in Arabidopsis by delivery by Pst DC3000 (Table 2). The wild-type AvrRPS4–HA protein triggered a strong HR in turnip leaves when transiently expressed under the control of the CaMV 35S promoter using agroinfiltration, and was also recognized in Arabidopsis Col-0 plants when delivered by Pst DC3000, resulting in restricted bacterial growth (Table 2, Figure 4b and Figure S3). Mutation of two or more amino acids between K135 and Y138 to an alanine completely abolished the avirulence activity of AvrRPS4–HA when transiently expressed in turnip. In addition, bacterial proliferation of Pst DC3000 expressing the KR135–136AA, RV136–137AA, VY137–138AA or KRVY135–138AAAA mutants of AvrRPS4–HA in Arabidopsis Col-0 plants was equivalent to that of Pst DC3000 with the empty vector, and the host plants developed disease symptoms (Table 2 and Figure 4b). This suggests that these mutant proteins had lost their avirulence activities. In contrast, agroinfiltration of the processing-deficient mutant R112L of AvrRPS4 triggered an HR in turnip leaves (Table 2 and Figure S3). The growth of Pst DC3000 expressing the R112L mutant was similar to that of Pst DC3000 expressing wild-type AvrRPS4–HA, indicating that the R112L mutant retained its avirulence activity in Arabidopsis (Figure 4b). Pst DC3000 expressing K135R, R136K, YQ138–139AA, Q139A or I140A mutants of AvrRPS4–HA were avirulent in Arabidopsis Col-0 plants and produced an HR when expressed in turnip. (Table 2 and Figure 4b). In pathogen tests on Col-0 rps4-2 (Wirthmueller et al., 2007) plants, Pst DC3000 expressing the empty vector or wild-type or mutants of AvrRPS4 grew to similar levels (Figure 4b).

Figure 4.

 AvrRPS4-triggered immunity in Arabidopsis requires the KRVY motif but not in planta processing.
(a) Diagram showing in capital letters the amino acids of AvrRPS4 targeted for site-directed mutagenesis.
(b) Growth of Pst DC3000 carrying the empty vector, wild-type or mutants of avrRPS4 in Arabidopsis Col-0 or rps4-2. Leaves of 5-week old Arabidopsis plants were hand-inoculated with 5 × 105 cfu ml−1 suspensions of Pst DC3000 strains. Samples were taken at 0 and 4 dpi to determine the bacterial population in the leaves. Error bars represent the standard error of six samples for each strain. The experiments were repeated three times with similar results.

Table 2.   Mutational analysis of AvrRPS4 protein expressed in Pst DC3000
AvrRPS4–HA proteinaPhenotypebProteindProcessingd
Col-0brps4-2bTurnipcN. benthamianaTurnip
  1. aSite-directed mutagenesis was used to generate all mutations except R112L which was created by error-prone PCR.

  2. bArabidopsis wild-type or rps4-2 mutant Col-0 plants were hand-inoculated with 105 cfu ml−1 suspensions of Pst DC3000 carrying wild-type or mutant avrRPS4. The bacterial growth or disease symptoms of infected leaves were scored at 4 dpi (as shown in Figure 4) or 6 dpi, respectively. R, resistant; S, susceptible.

  3. cAll AvrRPS4 mutant proteins were expressed in turnip leaves by Agrobacterium-mediated transient transformation, and HR symptoms were scored at 4 dpi.

  4. dProteins were analysed by transiently expressing them in turnip and N. benthamiana leaves, and detected using anti-HA antibody.

Wild-typeRSHRYesYes
Mutations
 R112LRSHRYesNo
 K135RRSHRYesYes
 R136KRSHRYesYes
 KR135–136AASSNSYesYes
 RV136–137AASSNSYesYes
 VY137–138AASSNSYesYes
 YQ138–139AAR (±)SHR (±)YesYes
 KRVY135–138AAAASSNSYesYes
 Q139ARSHRYesYes
 I140ARSHRYesYes

Both in planta processing and the KRVY motif are required for the full virulence function of AvrRPS4 in N. benthamiana

As AvrRPS4 does not trigger an HR when transiently expressed in N. benthamiana, we investigated the virulence function of AvrRPS4 by exploiting the Pseudomonas syringae pv. tabaci (Pta) strain 6606–N. benthamiana pathosystem. N. benthamiana plants were infected by dipping them in bacterial suspensions. Pta 6606 grew about tenfold more and caused more severe symptoms if it carried avrRPS4–HA (Figure 5a). We also studied the virulence function of AvrRPS4 by infecting N. benthamiana leaves by hand infiltration of Pta strains using a 1 ml syringe (Figure S5). Pta 6606 expressing AvrRPS4(R112L)–HA showed reduced growth with milder disease symptoms in N. benthamiana plants compared to the Pta 6606 expressing wild-type AvrRPS4–HA (Figure 5 and Figure S5). However, Pta 6606 expressing AvrRPS4(R112L)–HA grew more than the strain carrying the empty vector (Figure 5b and Figure S5). We therefore conclude that AvrRPS4 requires in planta processing for full virulence activity when expressed and delivered by Pta 6606 in N. benthamiana. We also tested whether the KRVY motif is required for the virulence function. Pta 6606 carrying avrRPS4(KRVY135–138AAAA)–HA showed the same level of bacterial growth as the strain carrying the empty vector, suggesting that the KRVY motif is indispensable for the AvrRPS4 virulence function (Figure 5b and Figure S5).

Figure 5.

 The virulence function of AvrRPS4 requires both processing and the KRVY motif.
(a) N. benthamiana leaves were dipped in 107 cfu ml−1 suspensions (in 0.02% Silwet-77) of P. syringae pv. tabaci (Pta) 6606 expressing the empty vector, AvrRPS4(WT)–HA, AvrRPS4(R112L)–HA or AvrRPS4(KRVY135-138AAAA)–HA. The photographs were taken at 7 dpi.
(b) Infection was performed as in (a) and samples were taken at 4 dpi to determine the bacterial population in the leaves. Error bars represent the standard error of 12 samples taken from four infected plants for each strain. The experiments were repeated four times with similar results.

Expression of AvrRPS4 in Arabidopsis leaf cells promotes susceptibility to Pst DC3000 and suppresses PTI phenotypes

We generated transgenic Arabidopsis plants expressing wild-type AvrRPS4 or AvrRPS4(R112L)–HA under the control of either the dexamethasone-inducible promoter or the RPS2 promoter in Col-0 (rps4-2) or RLD, respectively. The Arabidopsis accession RLD carries a non-functional RPS4 allele that is unable to recognize AvrRPS4 (Gassmann et al., 1999). Expression of the AvrRpt2 effector under the control of the RPS2 promoter in a susceptible host plant has been reported previously (Chen et al., 2000). To investigate whether AvrRPS4 could promote virulence of P. syringae by expression in plant cell, we generated transgenic RLD plants stably expressing AvrRPS4–HA under the control of the RPS2 promoter. Transgenic RLD (RPS2p-avrRPS4–HA) plants showed slightly delayed growth and early flowering phenotypes compared to wild-type RLD plants under standard short-day conditions (Figure 6b, c). Infiltration of rps4-2 [Dex-avrRPS4–HA] leaves with dexamethasone (10 μm) caused disease-like symptoms at 5 days after infiltration, whereas rps4-2 or rps4-2 [Dex-avrRPS4(R112L)–HA] showed no or delayed symptom development, respectively (Figure 6c). We tested whether inducible or stable expression of AvrRPS4–HA in a susceptible Arabidopsis genotype confers enhanced susceptibility of host plants to the bacterial pathogen. Both transgenic rps4-2 (Dex-avrRPS4–HA) plants sprayed with 30 μm dexamethasone and RLD (RPS2pro-avrRPS4–HA) plants showed approximately tenfold enhanced growth of wild-type or ΔhrcC mutant Pst DC3000 compared to untreated or wild-type RLD plants (Figure 7a). However, transgenic rps4-2 plants conditionally expressing AvrRPS4(R112L)–HA did not show enhanced growth of Pst DC3000 after spraying with dexamethasone, confirming that processing of AvrRPS4 is required for its virulence function (Figure 7a).

Figure 6.

 Arabidopsis transgenic lines stably or transiently expressing AvrRPS4 show aberrant phenotypes.
(a) Total RNA samples were extracted from wild-type and transgenic RLD (RPS2p-avrRPS4–HA) plants, and first-strand cDNAs were generated for RT-PCR analysis. Expression of AvrRPS4 in transgenic rps4-2 [Dex-avrRPS4(WT)–HA] or [Dex-avrRPS4(R112L)–HA] plants was tested by infiltrating the leaves of 4-week-old transgenic plants with dexamethasone (10 μm). Samples were taken at 0 and 48 h post-infiltration (hpi). Wild-type or mutant AvrRPS4–HA protein was detected using anti-HA antibody (Sigma).
(b) Seven-week-old Arabidopsis accession RLD wild-type or transgenic plants expressing AvrRPS4 under the control of the RPS2 promoter. Plants were grown under short-day conditions (10 h light/14 h dark).
(c) Five-week-old rps4-2 or rps4-2 [Dex-avrRPS4(WT)–HA] or [Dex-avrRPS4(R112L)–HA] plants were infiltrated with 10 μm dexamethasone. Black dots indicate infiltrated leaves. Pictures were taken at 5 dpi.

Figure 7.

 Expression of AvrRPS4 in Arabidopsis cells suppresses PTI phenotypes.
(a) Growth of wild-type Pst DC3000 on wild-type or transgenic Arabidopsis plants as indicated. For rps4-2 [Dex-avrRPS4(WT)–HA] or [Dex-avrRPS4(R112L)–HA] plants, 20 μm dexamethasone (in 0.02% Silwet-77) was sprayed onto the plants 24 h prior to bacterial infection. Samples were taken at 0 and 4 dpi to determine the bacterial population in the leaves. Error bars represent the standard error of six samples taken for each strain. The experiments were repeated three times with similar results.
(b) Leaf discs from 5-week-old rps4-2 [Dex-avrRPS4(WT)–HA] or [Dex-avrRPS4(R112L)–HA] plants were taken and floated on water or 20 μm dexamethasone solution for 15 h prior to flg22 treatment. Luminescence was measured every 2 min after treatment with flg22 using a Photek camera system. Points and error bars represent the means and standard errors, respectively, of 48 independent samples tested at the same time. The experiments were perfomed twice with similar results.
(c) Five-week-old rps4-2 [Dex-avrRPS4(WT)–HA] or [Dex-avrRPS4(R112L)–HA] plants were sprayed with water or 20 μm dexamethasone (in 0.02% Silwet-77). The leaves were infiltrated 24 h later with water or 1 μm flg22. Leaf samples were taken at 8 h after flg22 infiltration and stained with aniline blue for visualization of callose (white dots). The experiment was repeated twice with similar results.

We next analysed whether the enhanced susceptibility of rps4-2 (Dex-avrRPS4–HA) plants to Pst DC3000 is correlated with the suppression of PTI phenotypes. Transient expression of wild-type AvrRPS4–HA in transgenic rps4-2 plants suppressed flg22 (a peptide corresponding to the most conserved domain of flagellin)-triggered production of ROS and callose deposition at the leaf cell walls, but expression of AvrRPS4(R112L)–HA failed to suppress these two PTI phenotypes to the same extent as wild-type AvrRPS4–HA. These results suggest that AvrRPS4 functions by suppressing PTI in susceptible Arabidopsis plants (Figure 7b, c) and that processing is required for full effector function.

In planta processing of AvrRPS4 is conserved in an Xcv effector protein, XopO

A Xanthomonas campestris pv. vesicatoria (Xcv) effector protein, XopO (Xanthomonas outer protein O), has been identified as a homologue of AvrRPS4 (Roden et al., 2004). In addition, XopO was the only T3SE carrying the KRVY motif in the Pseudomonas and Xanthomonas strains for which genome sequences are available (data not shown). Alignment of AvrRPS4 and XopO showed that approximately 41% of the amino acid residues are identical (data not shown). We found that R112, which is required for the processing of AvrRPS4, and the amino acids around the processing site (G133/G134) are also conserved in the XopO amino acid sequence (Figure 8a). We therefore tested whether XopO has similar avirulence activity and in planta processing property in turnip plants as AvrRPS4. Agroinfiltration of the full-length XopO–HA protein did not trigger HR, whereas the full-length AvrRPS4–HA triggered strong HR in turnip (Figure 8c). GUS–HA was also expressed as a negative control. We found that XopO–HA is also processed in turnip and N. benthamiana (Figure 8b).

Figure 8.

 An Xcv effector protein, XopO, is processed in plant cells similarly to AvrRPS4.
(a) Partial amino acid alignment of AvrRPS4 and XopO. The amino acid required for AvrRPS4 processing is labelled in blue and conserved amino acids around the processing site are labelled in red. Black capital letters represent amino acids that are conserved between AvrRPS4 and XopO.
(b) Immunoblot analysis of the full-length AvrRPS4–HA and XopO–HA proteins. Both proteins were transiently expressed in N. benthamiana leaves by agroinfiltration. Leaf discs were removed at 48 h post-infiltration for protein extraction. The bands represent processed AvrRPS4–HA and XopO–HA proteins of approximately 11 kDa.
(c) Agrobacterium-mediated transient expression of GUS–HA, AvrRPS4–HA and XopO–HA in turnip leaves. The red arrow indicates HR symptoms. Photographs were taken at 4 dpi.

Discussion

We demonstrate that the Pseudomonas syringae effector, AvrRPS4, is secreted by the DC3000 T3SS and requires the C-terminal 88 amino acids for its avirulence function. We found that AvrRPS4 is processed into a smaller form in plant cells, and that it contributes to enhanced bacterial pathogen growth and suppression of PAMP-triggered ROS production and callose deposition in Arabidopsis plants lacking a functional RPS4 gene. We also show that a conserved KRVY motif at the N-terminus of the processed AvrRPS4 is necessary for both avirulence and virulence functions.

Several P. syringae effectors require processing in order to function in plant cells. Auto-proteolytic activity of AvrPphB and AvrRpt2 is required for recognition by RPS5 and RPS2, respectively (Axtell and Staskawicz, 2003; Mackey et al., 2003; Shao et al., 2003). Therefore, we expected that processing of AvrRPS4 would be necessary to trigger RPS4-dependent defence activation. However, one processing-deficient mutant, R112L, retained full avirulence activity and triggered RPS4-dependent resistance, including an HR. This indicated that processing of AvrRPS4 is not required for its avirulence function. Based on its amino acid sequence, we could not find any evidence that AvrRPS4 has auto-proteolytic activity. Also, AvrRPS4 is processed only in plant cells and not in bacteria or yeast cells (Figures 1 and 3 and Figure S2). These results support the hypothesis that the processing of AvrRPS4 involves a mechanism distinct from that of either AvrPphB or AvrRpt2.

Our finding that processing is required only for full virulence but not for the avirulence function of AvrRPS4 suggests that there could be at least two distinct mechanisms by which AvrRPS4 triggers resistance or susceptibility in plant cells. Alternatively, different thresholds may be required for avirulence and virulence activities of AvrRPS4. Lim and Kunkel (2004) showed that several randomly generated AvrRpt2 mutants lost both their avirulence and any RIN4-degrading activities but still enhanced Pst DC3000 virulence on susceptible Arabidopsis plants lacking functional RPS2 (Lim and Kunkel, 2004). Surprisingly, AvrRpt2 enhances pathogen virulence in the absence of its target protein, RIN4, indicating the existence of at least one more host protein that is targeted by AvrRpt2 (Lim and Kunkel, 2004). The C-terminal domain of AvrRPS4 certainly confers avirulence activity independently of in planta processing. The virulence function of AvrRPS4, however, requires processing for full activity, as well as the KRVY motif, which is also required for avirulence. It was shown that expression of AvrRPS4 (1–136aa) in wild-type or ΔCEL mutant Pst DC3000 does not enhance bacterial virulence (Sohn et al., 2007). Therefore, the C-terminal domain of AvrRPS4 probably carries virulence activity. In addition, mutation of the KRVY motif abolished the virulence activity of AvrRPS4 in N. benthamiana (Figure 5), indicating that the C-terminal domain is necessary for virulence function. It would be very interesting to further investigate how the processed AvrRPS4 promotes the susceptibility of host plants by identifying the target host proteins.

The mechanism by which AvrRPS4 is processed may be conserved for other effectors that act inside in plant cells. The Xcv effector XopO shares approximately 41% amino acid identity with AvrRPS4. Our data show that XopO does not exhibit the same avirulence activity in Arabidopsis as AvrRPS4 but is nonetheless processed in a similar way, so Xcv pathogenesis could also involve the processing of XopO for virulence. This is consistent with the hypothesis that AvrRPS4 and XopO share a common ancestral origin that has evolved to acquire an in planta processing requirement for provision of a virulence function.

It is apparent that bacterial T3SEs suppress PTI in order to promote bacterial survival in host plants. One phenotype characterized by PTI is deposition of callose at the cell wall. Several T3SEs have been shown to suppress accumulation of PAMP-triggered callose deposition (Hauck et al., 2003; DebRoy et al., 2004). It has also been shown that flg22-induced callose deposition is dependent on AtRbohD, which is at least partially required for flg22-triggered ROS production (Zhang et al., 2007). The processing-dependent suppression of flg22-triggered ROS production and callose deposition by AvrRPS4 suggest that the cleaved AvrRPS4 product might act at or upstream of ROS generation. However, it was shown that agroinfiltration of AvrRPS4 does not suppress PTI in N. benthamiana (Hann and Rathjen, 2007). As the virulence function of AvrRPS4 is processing-dependent, the expression of full-length AvrRPS4 at 48 h post-infiltration by Hann and Rathjen (2007) was probably too late to suppress PTI phenotypes, which are normally activated at the very early stage of bacterial infection. In contrast, expression and delivery of AvrRPS4 from a naturally pathogenic strain of P. syringae enhances pathogen virulence in N. benthamiana in our experiments. This corroborates our previous work showing that the translocation and processing of AvrRPS4 in plant cells occurs within at least 10 h of infection when AvrRPS4 is expressed in Pst DC3000 (Sohn et al., 2007).

It is interesting to note that AvrRPS4 enhances the virulence of bacterial pathogens on both N. benthamiana and Arabidopsis, as AvrRPS4 is found predominantly in P. syringae strains that are pathogenic on legume plants (Hinsch and Staskawicz, 1996). This suggests that AvrRPS4 might promote plant susceptibility by targeting very well conserved host protein(s) involved in PTI. However, it is unclear how AvrRPS4 suppresses PTI and promotes pathogen growth in plant cells. Further experiments, such as yeast two-hybrid screening, to identify a virulence target protein(s) of AvrRPS4 will better enable us to understand the molecular mechanism underlying AvrRPS4 function. In addition, identification of AvrRPS4 target protein(s) will be necessary to understand how RPS4 recognizes AvrRPS4 and initiates subsequent defence responses.

Experimental procedures

Bacterial strains and plasmid mobilizations

The bacterial strains used in this study were Escherichia coli DH5α, Pseudomonas syringae pv. tabaci 6606, and Pseudomonas syringae pv. tomato DC3000 wild-type, ΔhrcC and ΔhrcU (Deng et al., 1998). E. coli DH5α or P. syringae strains were grown in low-salt Luria–Bertani broth at 37 or 28°C, respectively. P. syringae cultures were centrifuged at 2500 g for 5 min at room temperature to recover bacteria, washed briefly and resuspended in sterile water for infection assays. The plasmid constructs were mobilized to P. syringae strains by standard electroporation methods.

Plasmid constructions

Plasmids pV316-1A (avrRPS4) and pMH10 (avrRPS4-Flag) were kindly provided by Brian Staskawicz (Department of Plant and Microbial Biology, University of California at Berkeley, CA). To construct AvrRPS4 wild-type and N-terminal deletions (M53, M80, M120, M132, M133, M134, M135, M136, M137, M138, M139, M140, M141 and M153), standard polymerase chain reactions were performed using Pfu polymerase (Promega, http://www.promega.com/) with pV316-1A as template DNA and AvrRPS4-specific primers containing ClaI-ATG (forward primer) and BamHI (reverse primer) sequences. PCR products were treated with ClaI and BamHI and cloned into ClaI- and BamHI-treated pBIN19g to create 35S:avrRPS4:HA constructs. For site-directed mutagenesis of avrRPS4, standard overlapping PCR was performed to generate various AvrRPS4 mutants (K135R, R136K, KR135–136AA, RV136–137AA, VY137–138AA, YQ138–139AA, KRVY135–138AAAA, Q139A and I140A), and cloned in PstI- and BamHI-treated pBIN19g for agroinfiltration. For expression of AvrRPS4 mutants in P. syringae, pV316-1A plasmid was treated with HindIII and PstI (from the native AvrRPS4 promoter and the PstI site of avrRPS4 ORF) and cloned in pUC19 to create pUC19AN. avrRPS4 mutants cloned in pBIN19g were amplified, treated with PstI and EcoRI, and cloned in pUC19AN. Subsequently, pUC19:avrRPS4 mutant constructs were treated with HindIII and EcoRI and ligated into HindIII- and EcoRI-treated pVSP61. The full-length XopO ORF was amplified and cloned into the ClaI and BamHI sites of pBIN19g for transient expression experiments. For the RPS2p:avrRPS4:HA construct, the 1.4 kb native promoter of RPS2 was amplified from Col-0 genomic DNA and cloned into the EcoRI and ClaI sites of avrRPS4:pBIN19g. All primer sequences are available upon request.

Secretion assay

Pst DC3000 strains grown overnight on NYGA (5 g/L bactopeptone, 3 g/L yeast extract and 20 m/L glycerol) at 28°C were resuspended in 1× MinA (10.5 g/L K2HPO4, 4.5 g/LKH2PO4 and 1g/L (NH4)2SO4) (pH 7.0). Cells were diluted to 1 × 108 cells ml−1 in 10 ml of fresh 1× MinA (pH 7.0) containing appropriate antibiotics. Cultures were grown at 28°C until late log-phase growth. Cells were collected by centrifugation at 2500 g for 5 min, washed with 1 mm MgCl2, and resuspended in 1 mm MgCl2. Bacteria were then diluted to 2 × 108 cells ml−1 in 10 ml of 1× MinA (pH 5.7), lacking antibiotics but containing 50 μg ml−1 BSA. Cultures were shaken for 5–6 h at 22°C until they reached a cell density of 6–7 × 108 cells ml−1. Cellular lysate fractions were obtained by resuspending the cells from 1 ml culture in 100 μl of 1× Laemmli buffer. Culture fluid fractions were obtained by removing cells from the remaining culture by centrifugation at 2500 g for 5 min, and then directly filtering supernatants through a 0.22 μm filter (Ministart; Sartorius, http://www.sartorius.com). Filtrates (1 ml) were precipitated with trichloroacetic acid on ice for 30 min. Proteins were collected by centrifugation at 13 000 g for 10 min, washed with acetone, and resuspended in 100 μl of 1× Laemmli buffer containing 0.1 m NaOH. The cellular lysate fraction (5 μl) and the culture fluid fraction (15 μl) were analysed by SDS–PAGE.

Generation of transgenic plants

The transgenic Arabidopsis plants were generated using the floral-dip method (Clough and Bent, 1998). Briefly, flowering Arabidopsis accession Col-0 (rps4-2) plants were dipped into Agrobacterium tumefaciens GV3101 carrying pTA7002:AvrRPS4 (wild-type or R112L), and the seeds were harvested to select transformants. The same procedure was repeated to generate Arabidopsis accession RLD carrying RPS2p:avrRPS4:HA.

Plant pathology experiments

Leaves of 4–5-week-old Arabidopsis or plants were hand-infiltrated with 5 × 105 cfu ml−1 using a 1 ml syringe, or dip-inoculated with 5 × 108 cfu ml−1 of bacterial suspensions. For N. benthamiana plants, 5 × 104 cfu ml−1 or 107 cfu ml−1 of bacteria were used for hand infiltration or dip inoculation, respectively. Bacterial growth was measured at 0 and 4 dpi by extracting bacteria from leaf discs and plating a series of dilutions on the NYGA medium supplemented with appropriate antibiotics. Plants were kept in a growth cabinet at 22°C with a 10/14 h day/night cycle. For dip-inoculated N. benthamiana plants, 90% humidity was maintained for 3 days immediately after inoculation.

Agrobacterium-mediated transient expression assays

Agrobacteria cells from 5 ml overnight cultures grown at 28°C in low-salt Luria–Bertani medium were harvested by centrifugation at 3500 g and resuspended in a buffer containing 10 mm MES (pH 5.6), 10 mm MgCl2 and 150 μm acetosyringone to a final OD600 of 0.5. The cultures were incubated at room temperature for 3 h and then hand-infiltrated onto leaves of turnip, N. benthamiana or Arabidopsis using a 1 ml needle-less syringe.

Protein analysis

Frozen plant tissue was ground and mixed with an equal volume of cold protein isolation buffer [20 mm Tris/HCl pH 7.5, 1 mm EDTA pH 8.0, 5 mm DTT, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 10% glycerol, 1× protease inhibitor cocktail (Sigma, http://www.sigmaaldrich.com/)]. The mixture was spun down and the supernatant was transferred to a new tube and SDS loading buffer (300 mm Tris/HCl pH 6.8, 8.7% SDS, 5%β-mercaptoethanol, 30% glycerol, 0.12 mg ml−1 bromophenol blue) was added. Proteins were separated by SDS–PAGE, electroblotted onto PVDF membrane (Bio-Rad, http://www.bio-rad.com/), and probed with horseradish peroxidase-conjugated anti–HA antibody (Sigma). Bands were visualized using ECL+ (Amersham, http://www5.amershambiosciences.com/).

Immunopurification of AvrRPS4–HA for N-terminal protein sequencing

The AvrRPS4–HA protein was transiently expressed in N. benthamiana by agroinfiltration and in Col-0 (Dex-AvrRPS4–HA) by spraying with 40 μm dexamethasone. Leaf samples were collected after 48 h for N. benthamiana and after 24 h for Col-0 (Dex-AvrRPS4–HA). Isolation of AvrRPS4–HA protein was performed as described above. Immunoprecipitation of AvrRPS4–HA protein was performed as described by Moffett et al. (2002), except that the transferred AvrRPS4–HA protein on PVDF membrane (Bio-Rad) was stained with Coomassie brilliant blue and the stained bands were sliced and subjected to N-terminal protein sequencing at the Department of Biochemistry, University of Cambridge (http://www.bioc.cam.ac.uk/pnac/proteinsequencing.html) (Moffett et al., 2002).

Measurement of ROS production

A total of 96 leaf discs from 5-week-old rps4-2 [Dex-AvrRPS4(WT)–HA] or [Dex-AvrRPS4(R112L)–HA] plants were taken and floated on water or 20 μm dexamethasone solution for 24 h prior to flg22 treatment. ROS production was measured by adding luminol (final concentration 20 μm; Sigma-Aldrich), 1 μg of horseradish peroxidase (Sigma-Aldrich) and flg22 (final concentration 100 nm) to a 96-well plate containing leaf discs (48 samples for each pre-treatment). Luminescence was measured every 2 min after treatment with flg22 using a Photek camera system (http://www.photek.co.uk).

Callose staining and microscopic analysis

Leaves of 5-week-old rps4-2 [Dex-avrRPS4(WT)–HA] or [Dex-avrRPS4(R112L)–HA] plants were sprayed with dexamethasone (20 μm, in 0.02% Silwet-77) and subsequently hand-infiltrated with flg22 (1 μm) after 24 h. Samples were taken at 8 h after flg22 treatment. A total of eight leaf discs were taken from eight leaves for callose staining. Harvested leaf samples were cleared three times (overnight incubation at room temperature) with 100% MeOH, washed three times (2 h for each wash) with sterile water, and stained with aniline blue (0.05% in phosphate buffer, pH 8.0) for 24 h. Leaf disc samples stained with aniline blue were examined using a Zeiss Axiophot Photomicroscope with an A3 fluorescence cube (http://www.zeiss.com/), and the images were analysed using the image-pro plus software program (Media Cybernetics, http://www.mediacy.com).

Acknowledgements

We are grateful to Matthew Metz and Brian Staskawicz (Department of Plant and Microbial Biology, University of California at Berkeley) for providing α-AvrRPS4 antibody and plasmid constructs. We thank Alan Collmer (Cornell University, Ithaca, NY) for the bacterial strains, and Mary Beth Mudgett (Department of Biological Science, Stanford University) for XopO clone. We also thank Matthew Smoker and Jodie Pike for generating Arabidopsis transgenic plants, Alexandre Robert-Seilaniantz, Matthew Smoker and Adnane Nemri for critical reading of the manuscript, and Sara Perkins, Tim Glister and Damian Alger for taking care of the plants. This work was funded by the Gatsby Charitable Foundation.

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