Shiv D. Kale

External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells.

Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.Pathogens of plants and animals produce effector proteins that are transferred into the cytoplasm of host cells to suppress host defenses. One type of plant pathogens, oomycetes, produces effector proteins with N-terminal RXLR and dEER motifs that enable entry into host cells. We show here that effectors of another pathogen type, fungi, contain functional variants of the RXLR motif, and that the oomycete and fungal RXLR motifs enable binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P). We find that PI3P is abundant on the outer surface of plant cell plasma membranes and, furthermore, on some animal cells. All effectors could also enter human cells, suggesting that PI3P-mediated effector entry may be very widespread in plant, animal and human pathogenesis. Entry into both plant and animal cells involves lipid raft-mediated endocytosis. Blocking PI3P binding inhibited effector entry, suggesting new therapeutic avenues.

RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER–containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.

Conserved C-Terminal Motifs Required for Avirulence and Suppression of Cell Death by Phytophthora sojae effector Avr1b

The sequenced genomes of oomycete plant pathogens contain large superfamilies of effector proteins containing the protein translocation motif RXLR-dEER. However, the contributions of these effectors to pathogenicity remain poorly understood. Here, we show that the Phytophthora sojae effector protein Avr1b can contribute positively to virulence and can suppress programmed cell death (PCD) triggered by the mouse BAX protein in yeast, soybean (Glycine max), and Nicotiana benthamiana cells. We identify three conserved motifs (K, W, and Y) in the C terminus of the Avr1b protein and show that mutations in the conserved residues of the W and Y motifs reduce or abolish the ability of Avr1b to suppress PCD and also abolish the avirulence interaction of Avr1b with the Rps1b resistance gene in soybean. W and Y motifs are present in at least half of the identified oomycete RXLR-dEER effector candidates, and we show that three of these candidates also suppress PCD in soybean. Together, these results indicate that the W and Y motifs are critical for the interaction of Avr1b with host plant target proteins and support the hypothesis that these motifs are critical for the functions of the very large number of predicted oomycete effectors that contain them.

A Secreted Effector Protein of Laccaria bicolor Is Required for Symbiosis Development

Soil-borne mutualistic fungi, such as the ectomycorrhizal fungi, have helped shape forest communities worldwide over the last 180 million years through a mutualistic relationship with tree roots in which the fungal partner provides a large array of nutrients to the plant host in return for photosynthetically derived sugars. This exchange is essential for continued growth and productivity of forest trees, especially in nutrient-poor soils. To date, the signals from the two partners that mediate this symbiosis have remained uncharacterized. Here we demonstrate that MYCORRHIZAL iNDUCED SMALL SECRETED PROTEIN 7 (MiSSP7), the most highly symbiosis-upregulated gene from the ectomycorrhizal fungus Laccaria bicolor, encodes an effector protein indispensible for the establishment of mutualism. MiSSP7 is secreted by the fungus upon receipt of diffusible signals from plant roots, imported into the plant cell via phosphatidylinositol 3-phosphate-mediated endocytosis, and targeted to the plant nucleus where it alters the transcriptome of the plant cell. L. bicolor transformants with reduced expression of MiSSP7 do not enter into symbiosis with poplar roots. MiSSP7 resembles effectors of pathogenic fungi, nematodes, and bacteria that are similarly targeted to the plant nucleus to promote colonization of the plant tissues and thus can be considered a mutualism effector.

Transcriptional Programming and Functional Interactions within the Phytophthora sojae RXLR Effector Repertoire

This study presents a broad functional survey of a large sample of candidate RXLR effectors in the oomycete pathogen of soybean (Phytophthora sojae). Suppression of plant defense, transcription patterns, and polymorphisms were assayed. Essential effectors and effector subsets with distinct expression patterns and defense suppression activities were identified. The genome of the soybean pathogen Phytophthora sojae contains nearly 400 genes encoding candidate effector proteins carrying the host cell entry motif RXLR-dEER. Here, we report a broad survey of the transcription, variation, and functions of a large sample of the P. sojae candidate effectors. Forty-five (12%) effector genes showed high levels of polymorphism among P. sojae isolates and significant evidence for positive selection. Of 169 effectors tested, most could suppress programmed cell death triggered by BAX, effectors, and/or the PAMP INF1, while several triggered cell death themselves. Among the most strongly expressed effectors, one immediate-early class was highly expressed even prior to infection and was further induced 2- to 10-fold following infection. A second early class, including several that triggered cell death, was weakly expressed prior to infection but induced 20- to 120-fold during the first 12 h of infection. The most strongly expressed immediate-early effectors could suppress the cell death triggered by several early effectors, and most early effectors could suppress INF1-triggered cell death, suggesting the two classes of effectors may target different functional branches of the defense response. In support of this hypothesis, misexpression of key immediate-early and early effectors severely reduced the virulence of P. sojae transformants.

Copy Number Variation and Transcriptional Polymorphisms of Phytophthora sojae RXLR Effector Genes Avr1a and Avr3a

The importance of segmental duplications and copy number variants as a source of genetic and phenotypic variation is gaining greater appreciation, in a variety of organisms. Now, we have identified the Phytophthora sojae avirulence genes Avr1a and Avr3a and demonstrate how each of these Avr genes display copy number variation in different strains of P. sojae. The Avr1a locus is a tandem array of four near-identical copies of a 5.2 kb DNA segment. Two copies encoding Avr1a are deleted in some P. sojae strains, causing changes in virulence. In other P. sojae strains, differences in transcription of Avr1a result in gain of virulence. For Avr3a, there are four copies or one copy of this gene, depending on the P. sojae strain. In P. sojae strains with multiple copies of Avr3a, this gene occurs within a 10.8 kb segmental duplication that includes four other genes. Transcriptional differences of the Avr3a gene among P. sojae strains cause changes in virulence. To determine the extent of duplication within the superfamily of secreted proteins that includes Avr1a and Avr3a, predicted RXLR effector genes from the P. sojae and the P. ramorum genomes were compared by counting trace file matches from whole genome shotgun sequences. The results indicate that multiple, near-identical copies of RXLR effector genes are prevalent in oomycete genomes. We propose that multiple copies of particular RXLR effectors may contribute to pathogen fitness. However, recognition of these effectors by plant immune systems results in selection for pathogen strains with deleted or transcriptionally silenced gene copies.

Phytophthora sojae Avirulence Effector Avr3b is a Secreted NADH and ADP-ribose Pyrophosphorylase that Modulates Plant Immunity

Plants have evolved pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) to protect themselves from infection by diverse pathogens. Avirulence (Avr) effectors that trigger plant ETI as a result of recognition by plant resistance (R) gene products have been identified in many plant pathogenic oomycetes and fungi. However, the virulence functions of oomycete and fungal Avr effectors remain largely unknown. Here, we combined bioinformatics and genetics to identify Avr3b, a new Avr gene from Phytophthora sojae, an oomycete pathogen that causes soybean root rot. Avr3b encodes a secreted protein with the RXLR host-targeting motif and C-terminal W and Nudix hydrolase motifs. Some isolates of P. sojae evade perception by the soybean R gene Rps3b through sequence mutation in Avr3b and lowered transcript accumulation. Transient expression of Avr3b in Nicotiana benthamiana increased susceptibility to P. capsici and P. parasitica, with significantly reduced accumulation of reactive oxygen species (ROS) around invasion sites. Biochemical assays confirmed that Avr3b is an ADP-ribose/NADH pyrophosphorylase, as predicted from the Nudix motif. Deletion of the Nudix motif of Avr3b abolished enzyme activity. Mutation of key residues in Nudix motif significantly impaired Avr3b virulence function but not the avirulence activity. Some Nudix hydrolases act as negative regulators of plant immunity, and thus Avr3b might be delivered into host cells as a Nudix hydrolase to impair host immunity. Avr3b homologues are present in several sequenced Phytophthora genomes, suggesting that Phytophthora pathogens might share similar strategies to suppress plant immunity.

Entry of oomycete and fungal effectors into plant and animal host cells

Fungal and oomycete pathogens cause many destructive diseases of plants and important diseases of humans and other animals. Fungal and oomycete plant pathogens secrete numerous effector proteins that can enter inside host cells to condition susceptibility. Until recently it has been unknown if these effectors enter via pathogen‐encoded translocons or via pathogen‐independent mechanisms. Here we review recent evidence that many fungal and oomycete effectors enter via receptor‐mediated endocytosis, and can do so in the absence of the pathogen. Surprisingly, a large number of these effectors utilize cell surface phosphatidyinositol‐3‐phosphate (PI‐3‐P) as a receptor, a molecule previously known only inside cells. Binding of effectors to PI‐3‐P appears to be mediated by the cell entry motif RXLR in oomycetes, and by diverse RXLR‐like variants in fungi. PI‐3‐P appears to be present on the surface of animal cells also, suggesting that it may mediate entry of effectors of fungal and oomycete animal pathogens, for example, RXLR effectors found in the oomycete fish pathogen, Saprolegnia parasitica. Reagents that can block PI‐3‐P‐mediated entry have been identified, suggesting new therapeutic strategies.

Sequence variants of the Phytophthora sojae RxLR effector Avr3a/5 are differentially recognized by Rps3a and Rps5 in Soybean

The perception of Phytophthora sojae avirulence (Avr) gene products by corresponding soybean resistance (Rps) gene products causes effector triggered immunity. Past studies have shown that the Avr3a and Avr5 genes of P. sojae are genetically linked, and the Avr3a gene encoding a secreted RXLR effector protein was recently identified. We now provide evidence that Avr3a and Avr5 are allelic. Genetic mapping data from F2 progeny indicates that Avr3a and Avr5 co-segregate, and haplotype analysis of P. sojae strain collections reveal sequence and transcriptional polymorphisms that are consistent with a single genetic locus encoding Avr3a/5. Transformation of P. sojae and transient expression in soybean were performed to test how Avr3a/5 alleles interact with soybean Rps3a and Rps5. Over-expression of Avr3a/5 in a P. sojae strain that is normally virulent on Rps3a and Rps5 results in avirulence to Rps3a and Rps5; whereas silencing of Avr3a/5 causes gain of virulence in a P. sojae strain that is normally avirulent on Rps3a and Rps5 soybean lines. Transient expression and co-bombardment with a reporter gene confirms that Avr3a/5 triggers cell death in Rps5 soybean leaves in an appropriate allele-specific manner. Sequence analysis of the Avr3a/5 gene identifies crucial residues in the effector domain that distinguish recognition by Rps3a and Rps5.

Structural basis for interactions of the Phytophthora sojae RxLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entry.

Oomycetes such as Phytophthora sojae employ effector proteins that enter plant cells to facilitate infection. Entry of some effector proteins is mediated by RxLR motifs in the effectors and phosphoinositides (PIP) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effectors involved in PIP recognition. We have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that Avh5 is helical in nature, with a long N-terminal disordered region. NMR titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate, directly identified the ligand-binding residues. A C-terminal lysine-rich helical region (helix 2) was the principal lipid-binding site, with the N-terminal RxLR (RFLR) motif playing a more minor role. Mutations in the RFLR motif affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Mutations in the RFLR motif or in the basic region both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. Based on these findings, we propose a model where Avh5 interacts with PtdIns(3)P through its C terminus, and by binding of the RFLR motif, which promotes host cell entry.