RXLR and dEER Motifs Are Required for Avr1b Function in P. sojae Transformants.
(A) Sequences of mutations in the RXLR1, RXLR2, and dEER motifs. Blue represents wild-type amino acids targeted for replacement by Ala residues (red).
(B) PstI restriction analysis of PCR products amplified from Avr1b-1 transformants using primers specific for the HAM34 promoter and terminator regions. PstI restriction profiles of Avr1b(RXLR1^AAAA), Avr1b(RXLR2^AAAA), Avr1b(RXLR1^AAAA, 2^AAAA), Avr1b(dEER^A6), and wild-type Avr1b are distinguished from each other because the mutations introduce a PstI site. Avr1b(dEER^A6)-9 was confirmed by sequencing the PCR product.
(C) Detection of Avr1b mRNA in P. sojae stable transformants by RT-PCR. Top panel shows amplification with primers internal to the Avr1b C terminus. Bottom panel shows amplification with P. sojae actin primers. P. sojae stable transformants were the same as for (B) except that an amplification reaction is also shown from RNA from a P. sojae transformant containing a GUS gene. No amplification was observed when reverse transcriptase was omitted from the reactions.
(D) Distributions of HMM scores of RXLR flanking regions for all RXLR-containing secreted proteins from P. sojae and P. ramorum (nonpermuted), for all secreted proteins retaining an RXLR string after sequence permutation (permuted), and for all high-quality RXLR-effector candidates identified by Jiang et al. (2008) (curated). The locations on the distribution of the HMM scores of the RXLR strings of known avirulence proteins and HpAvh341 are shown by the arrows.
(E) Phenotype of L77-1863 (Rps1b) seedlings inoculated on the hypocotyls with transformants carrying the indicated wild-type or mutant Avr1b-1 genes and photographed 4 d later.

Secretion and Reentry of Avr1b-GFP Fusion Proteins Expressed in Onion Cells.
DNA encoding various fusions of Avr1b with A. coerulescens GFP was bombarded into onion epidermal cells using the Gene Gun without the double-barrel attachment. Cells were photographed under both UV and white light illumination 12 to 24 h after bombardment. a, sites where secreted GFP has begun to spread into the apoplast between pairs of neighboring cells; n, nuclei visualized under white light; p, plasma membrane in plasmolyzed cells; DAPI, cells stained with 4′,6-diamidino-2-phenylindole and photographed under UV illumination.
(A) Fusion of GFP to the secretory leader of Avr1b alone.
(B) GFP with no secretory leader (native GFP). DAPI-stained cells are shown instead of white light–illuminated cells.
(C) GFP fused to the Avr1b RXLR2^AAAA mutant, including its secretory leader. In the top right panel, a white trace of the plasma membrane from the panel below has been added. The right-side panels show photographs of plasmolyzed cells.
(D) GFP fused to Avr1b dEER^A6 mutant, including its secretory leader.
(E) GFP fused to wild-type Avr1b, including its secretory leader. The right-side panels show photographs of plasmolyzed cells.
(F) GFP fused to mature Avr1b lacking its secretory leader. DAPI-stained cells are shown instead of white light–illuminated cells.

P. sojae Stable Transformants Show That Two Other Avh Proteins Can Replace the RXLR-dEER Region of Avr1b.
(A) Sequences of the N termini of wild-type and mutant Avr1b proteins and of fusions with two other Avh proteins. Purple, secretory leader; blue, RXLR motif; red, dEER motif. The C-terminal sequence of Avr1b is underlined.
(B) PCR analysis of DNA from P. sojae stable transformants. WT: pl, pHamAvr1b plasmid DNA; T17 and T20, two transformants with wild-type Avr1b-1 transgenes. HpAvh341-Avr1bCt: pl, pHamAvh341 plasmid DNA (encoding Hp Avh341-Avr1bCt); 13 and 17, two transformants containing pHamAvh341. PsAvr4/6-Avr1bCt: pl, pHamAvh171 plasmid DNA (encoding Ps Avr4/6-Avr1bCt); 3 and 19, two transformants containing pHamAvh171. mAvr1bCt: pl, pHamAvr1bCt plasmid DNA (encoding mAvr1bCt protein); 4 and 5, two transformants containing pHamAvr1bCt. The sizes of the PCR products for Avr1b-1, pHamAvh341, pHamAvh171, and pHamAvr1bCt are 577, 721, 748, and 385 bp, respectively.
(C) Detection of Avr1b mRNA in P. sojae stable transformants by RT-PCR. Top panel shows amplification with primers internal to the Avr1b C terminus. Bottom panel shows amplification with P. sojae actin primers. P. sojae stable transformants were the same as for (B) except that an amplification reaction is also shown from RNA from a P. sojae transformant containing GUS. Pl, pHamAvr1b plasmid DNA as template. No amplification was observed when reverse transcriptase was omitted from the reactions.
(D) Phenotype of L77-1863 (Rps1b) seedlings inoculated on the hypocotyls with the indicated transformants carrying wild-type or mutant Avr1b-1 genes and photographed 4 d later. HpAvh341-Avr1b-17, PsAvr4/6-Avr1b-3, and mAvr1bCt-5 gave similar results to HpAvh341-Avr1b-13, PsAvr4/6-Avr1b-19, and mAvr1bCt-4 (Table 1).

Summary of Avr1b-1 Mutations and Their Phenotypes in P. sojae Stable Transformants and Soybean Transient Expression Assays.
A, avirulent; V, virulent; NT, not tested; Y, significantly fewer blue (GUS-positive) tissue patches from GUS expression resulting from Avr1b-induced cell death; N, not significantly fewer blue tissue patches; P, partial reduction in blue tissue patches; SP, signal peptide.

RXLR and dEER Functions Confirmed by Particle Bombardment Assay.
Soybean leaves were bombarded using a double-barreled device that delivered Avr1b-1 DNA-bearing particles to one side of the leaf and control (empty vector) DNA to the other; both sides received GUS DNA.
(A) Ratio of blue spots in the presence of Avr1b-1 compared with the control. sAvr1b indicates a gene encoding secretory Avr1b, and mAvr1b indicates one encoding mature Avr1b (lacking the secretory leader). WT indicates wild-type RXLR motif, RXLR2^AAAA indicates the four Ala replacement of the RXLR2 motif, and dEER^A6 indicates the six Ala replacement of the dEER motif. Averages and se are from 16 pairs of shots. P values comparing results from cultivars with Rps1b (L77-1863) or without (rps; Williams) were calculated using the Wilcoxon rank sum test.
(B) Direct comparison of bombardment with mature Avr1b.
(C) Direct comparison of bombardment with secretory Avr1b.
In both (B) and (C), DNA encoding wild-type (left) and RXLR2^AAAA (right) versions of mAvrb or sAvr1b, respectively, were bombarded onto the same leaf of L77-1863 (Rps1b). The dashed lines indicate the positions of a divider that prevents particles from the two shots from overlapping. In both photographs, the brightness and contrast were adjusted uniformly to improve the visibility of the blue spots.

RXLR-dEER-GFP Fusion Proteins Isolated from E. coli Can Enter Soybean Cells in the Absence of the Pathogen.
GFP fusion proteins were expressed in E. coli, partially purified, and incubated with soybean root tips for 12 h. The root tips were then washed for 4 h and photographed under UV and white light illumination.
(A) Protein gel electrophoresis analysis of GFP fusion proteins partially purified from E. coli cells: lane 1, Arg₉-GFP; lane 2, GFP fused to the N-terminal 44 amino acids of mature wild-type Avr1b protein (RXLR1⁺,RXLR2⁺-dEER-GFP); lane 3, same as lane 2 with both RXLR1 and RXLR2 mutations (RXLR1^AAAA,RXLR2^AAAA-dEER-GFP); lane 4, same as lane 2 except with dEER mutation (RXLR1⁺,RXLR2⁺-dEER^AAAAAA-GFP). The left lane contained molecular mass markers; the sizes of the markers are shown on the left (in kD). All expressed GFP proteins fluoresce normally under UV illumination.
(B) to (F) UV (left panels) and back-lit white light (right panels) illumination of roots after incubation with the indicated GFP protein fusion. The UV photographs represent longitudinal optical sections taken using the confocal microscope as illustrated by the dashed line in the inset of (C). The GFP concentration, illumination, and exposure of the UV photographs was identical in all 10 panels shown.
(B) Buffer alone with no fusion protein.
(C) RXLR1⁺,RXLR2⁺-dEER-GFP.
(D) Arg₉-GFP.
(E) RXLR1^AAAA,RXLR2^AAAA-dEER-GFP.
(F) RXLR1⁺,RXLR2⁺-dEER^AAAAAA-GFP.
(G) and (H) Higher-magnification photographs after the root tips were gently squashed following washing, showing nuclear accumulation of GFP.
(G) RXLR1⁺,RXLR2⁺-dEER-GFP.
(H) Arg₉-GFP.

Functional Replacement of Avr1b Host Targeting Signal with Protein Transduction Motifs and Plasmodium Host Targeting Signals.
(A) Sequences of modified Avr1b proteins. PfGBP, PfHRP, and Pf1615c refer to the Plasmodium Pf GBP-130, Pf HRPII, and Pf PFE1615c proteins (Bhattacharjee et al., 2006). All nonnative Avr1b sequences are underlined, Avr1b RXLR2 and Plasmodium RXLX^E/_Q motifs are in bold, and acidic residues in the dEER region are in italics. The Avr1b secretory leader was used in all constructs. Details of all constructs are given in Supplemental Table 2 online.
(B) Ratio of blue spots in the presence of Avr1b-1 compared with the control, assayed as described in Figure 2. Constructs are as in (A). Averages and se are from eight pairs of shots.
(C) Secretion and reentry of GFP protein fused to Avr1b protein containing Arg₉ in place of RXLR2, expressed in onion cells.
(D) Secretion and reentry of GFP protein fused to Avr1b protein containing the TAT protein transduction signal in place of RXLR2, expressed in onion cells.
In (C) and (D), DNA encoding the fusion proteins was bombarded into onion epidermal cells using the Gene Gun without the double-barrel attachment. Cells were photographed 24 h after bombardment.

Common Host Targeting Mechanism in Oomycetes and Plasmodium.
(A) Features and functional exchange of host targeting signals in P. sojae Avr1b, Plasmodium falciparum HRPII, and P. infestans Avr3a. Common RXL(R) and dEER-like motifs are shown in blue and pink, respectively, and were defined experimentally (this article; Marti et al., 2004; Hiller et al., 2004; Whisson et al., 2007). Flanking regions inferred also to be required are shown in gray. In Ps Avr1b, the flanking regions are defined as the 19 residues upstream and 16 residues downstream of RXLR2 shown to be sufficient for translocation in the GFP fusion experiments. In Pf HRPII, the eight residues upstream and 13 residues downstream were defined experimentally (Bhattacharjee et al., 2006). In Pi Avr3a, the flanking regions are defined by the region tested in Plasmodium (Bhattacharjee et al., 2006). The region of Pi Avr3a tested in Plasmodium and the region of Pf HRPII tested in P. sojae (this article) are shown by the brackets.
(B) Anatomical contexts of oomycete and Plasmodium effector entry are similar. The haustorium is a specialized invagination of the plant cell formed by oomycete (and fungal) pathogens. The plant cell wall is pierced during formation of the haustorium, while the oomycete cell wall is retained but differentiates into the haustorial wall. Both the haustorial membrane and the parasitiphorous vacuolar membrane are derived from the host plasma cell membrane during pathogen invasion.