Figure 1.
Schematic of Intracellular Infection of Plasmodium and Phytophthora Parasites
(A) A human erythrocyte (pink) infected by P. falciparum (blue). Invasion by the extracellular merozoite stage leads to formation of a host-derived PVM within which the parasite resides. Proteins (brown squares) secreted by the parasite must cross the PVM to reach and mediate virulence and structural changes in the erythrocyte.
(B) Plant cells (green) infected by P. infestans (blue). P. infestans parasite colonizes the host intracellular spaces and forms haustoria (yellow). The host-derived haustorial membrane must be crossed by pathogenic effectors (brown squares) released into cells to mediate virulence and plant hypersensitive responses.
Figure 2.
Conserved, Plant Pathogenic Oomycete Motif Functions as a Host-Targeting Signal in P. falciparum–Infected Erythrocytes
(A) Sequence alignment of six effectors from P. falciparum HT-secretome (upper panel) and five Avr proteins from the oomycete pathogens Phytophthora and Hyaloperonospora (lower panel). Each sequence contains an 11 amino acid region (black bar) centered on RxL residues (blue). Negatively charged residues (E or D) downstream of the RxL are colored red. Seven residues upstream of the RxL are underlined purple and boxed in the case of PfHRPII.
(B) Live cells expressing secretory GFP chimeras of AVR3a (residues 21 to 69) with no change (i–iii), or where RRLLRK was replaced by AASTAI (iv–vi), where (ii) and (v) indicate fluorescence images, (i) and (iv) corresponding brightfield images, and (iii) and (vi) the respective merges. Constructs contain SS (black) followed by indicated sequences of AVR3a (orange) and GFP (green). For quantitative analyses, two hundred fluorescent images were analyzed as described in Materials and Methods. Fraction of GFP exported to the erythrocyte cytosol is indicated in (vii). As expected, all parasitized cells express and export the transgene (viii). Standard deviations are shown in pink. In all cases: p, parasite; e, erythrocyte; nucleus is Hoechst-stained (blue); scale bar represents 2 μm.
Figure 3.
HT Motif Has High Predictive Value for Phytophthora Effectors
(A) Sequence logos derived from 59 predicted P. infestans secretory proteins and the P. falciparum HT-secretome (boxed inset). Amino acids are represented by one-letter abbreviations and color-coded as follows: blue, basic; red, acidic; black, hydrophobic; and green, polar. Height of amino acids indicates their frequency at that position.
(B) Live cells expressing secretory GFP chimeras of PH001D5 (residues 19 to 88) with no change (i–iii) or where DRQLRGF was replaced with ISAATAI (iv–vi), where (ii) and (v) indicate fluorescence images, (i and iv) corresponding brightfield images, and (iii and vi) show respective merges. The asterisk (*) in panel (ii) indicates intraerythrocytic loop structure that excluded GFP. Constructs contain SS (black), followed by indicated sequences of PH001D5 (orange) and GFP (green). For quantitative analyses, 220 fluorescent images were analyzed as described in Materials and Methods. Fraction of GFP exported to the erythrocyte cytosol is indicated in (vii) in a culture where all parasites are transformed (as expected) and export the transgene (viii). Standard deviations are shown in pink. Export of green fluorescence to erythrocyte is quantitatively blocked on replacement of the P. infestans motif (as indicated in the bar chart in vii [standard deviation show in pink]). p, parasite; e, erythrocyte; nucleus is Hoechst-stained (blue), scale bar is 2 μm.
Figure 4.
Comparative Analyses of Secretory and Cytosolic RxLR–Containing Sequences in Phytophthora: In Silico and Functional Evidence for the Requirement of Downstream E, D Residue Sequences in HT Consensus
(Ai) Secretory: logos generated from sequences containing RxLR in the first 100 residues after the SS cleavage site for predicted secretory proteins (~10% of the SS set).
(Aii) Cytosolic: logos from sequences containing RxLR in the first 100 residues of predicted cytosolic proteins (~5% of the cytosolic set).
(B) Live cells expressing secretory GFP chimeras of Avr3a (residues 21–69) with no change (i–iii), replacement of D/E residues downstream of RxLR with hydrophobic residues (iv–vi), replacement of downstream sequence KNEENEETSEERAPNFNLANLN by NQYSHTALVKIQGLTDKKDYLGK found downstream of RxLR from a non-secretory Phytophthora protein AAY43422.1 (vii–ix).
(C) Live cells expressing secretory GFP chimeras of PH001D5 (residues 19–88) with no change (i–iii), replacement of D/E residues downstream of RxLR with hydrophobic residues (iv–vi), replacement of downstream sequence GFYATENTDPVNNQDTAHEDGEERV by VGPAGGAAAVGTGSGNAASNTAPHAG found downstream of RxLR from a non-secretory Phytophthora protein 83742 (vii–ix).
(B and C) Panels (ii, v, and viii) indicate fluorescence micrographs; (i, iv, and vii), brightfield images; (iii, vi, and ix), respective merges. Constructs contain SS (black) followed by indicated AVR3a or PH001D5 wild-type and modified sequences (orange) and GFP (green). Fraction of GFP exported to the erythrocyte cytosol is indicated in Bx and Cx. Standard deviations are shown in pink. Export of green fluorescence to erythrocyte is quantitatively blocked on replacement of the regions downstream of the Avr3a and PH001D5 motifs, as indicated in the bar charts in (xi). Standard deviations are shown in pink. p, parasite; e, erythrocyte; nucleus is Hoechst-stained (blue); scale bar is 2 μm. In both (ii) panels, the asterisk (*) indicates intraerythrocytic loop structure that excluded GFP. Note, in the case of PH001D5 (Cviii), a small amount of GFP is detected in tubovesicular elements in the erythrocyte. However, export to the erythrocyte cytoplasm or periphery is never detected.
Table 1.
List of Proteins in the Predicted Phytophthora sp. RxLR HT-Secretomes
Figure 5.
Requirement of Sequences Downstream of HT Motif in Protein Export to Erythrocyte Cytosol
(i) Images of live cells exporting a secretory GFP chimera containing the five amino acid HT core (blue) followed by 16 amino acids downstream sequence from PfHRPII (red).
(ii) Removal of the terminal nine amino acids (VHHAHHADV) blocked export to the erythrocyte.
(iii) Replacement of VHHAHHADV with VGMMSMMDV restored export of GFP to the erythrocyte. For quantitative analyses, two hundred fluorescent images were analyzed as described in Materials and Methods.
(iv) Fraction of GFP exported to the erythrocyte cytosol is indicated, and all parasitized cells export the transgene (as expected for stable transfections; unpublished data).
Standard deviations are as shown. Constructs contain SS (black), upstream region (purple), followed by sequence containing core host-targeting motif (blue), the downstream spacer region (red), and GFP (green).
In all cases: p, parasite; e, erythrocyte; nucleus is Hoechst-stained (blue); scale bar is 2 μm.
Figure 6.
Analysis of Upstream Region of RxL Motifs in the P. falciparum HT-Secretome
Logos derived from eight positions upstream of the P. falciparum HT core.
(A) Logo was derived from sequences in the predicted HT-secretome.
(B) Logo was derived from all proteins containing an SS and RxL alone, but excluded from the HT-secretome. Sequences were aligned without gaps using the RxL motif as an anchor. Amino acids are represented by one-letter abbreviations and color-coded as follows: blue, basic; red, acidic; black, hydrophobic; and green, polar. Height of amino acids indicates their relative frequency at that position.
(C) Live cells expressing secretory GFP chimeras of wild-type (i), and mutations in the region upstream of HT core of PfHRPIIGFP IGDN (ii), IVDI (iii). For quantitative analyses, 230 fluorescent images were analyzed as described in Materials and Methods. Fraction of GFP exported to the erythrocyte cytosol is indicated in (iv), and all parasitized cells export the transgene (unpublished data). Standard deviations are as shown. Constructs contain SS (black), upstream region (purple), with indicated point mutations (pink), followed by sequence containing HT core motif (in blue), downstream region (red), and GFP (green). p, parasite; e, erythrocyte; nucleus is Hoechst-stained (blue), scale bar is 2 μm.
Figure 7.
Positional Equivalence of HT Motifs in Phytophthora and P. falciparum
Histograms plotting the lengths of the upstream regions of proteins in the HT secretomes of P. ramorum (i, 147 sequences), P. sojae (ii, 176 sequences), and P. falciparum (iii, 112 sequences). Sequence distribution data from P. infestans is not shown because the complete sequencing of this genome is still under way.
Table 2.
Sequences of Primers Used for Cloning