Engagement of the T cell antigen receptor (TCR)1 results in the sequential activation of the Src (p56 lck /p59 fyn ) and Syk (Syk/ZAP-70) families of protein-tyrosine kinases (PTKs) (1-3). Both families of PTKs are required for normal T cell development and function (4 -9). In resting T cells, the TCR chain is constitutively phosphorylated and associated with 11). Tyrosine phosphorylation of the receptor-associated ZAP-70 by p56 lck is a requisite modification resulting in the up-regulation of ZAP-70 catalytic activity (12, 13). Therefore, the recruitment of the CD4 and CD8 co-receptors into the TCR complex positions p56 lck to trans-phosphorylate ZAP-70. Phosphorylation and activation of ZAP-70, in turn, is required for both an increase in phosphoinositide metabolism and activation of the ras pathway.2 The integration of these downstream signals gives rise to transcriptional activation of cytokine genes and a resultant elevation in cytokine synthesis and secretion (15). While the up-regulation of ZAP-70 catalytic activity is required for TCR function, little is known about the cellular proteins which serve as substrates for this PTK.SLP-76 is a recently identified molecule which undergoes tyrosine phosphorylation upon TCR cross-linking (16 -19). This protein associates with both the SH3 domain of Grb2 and an SH2 domain of PLC␥. SLP-76 is structurally characterized by a C-terminal SH2 domain, a region enriched in proline residues which probably serves as the site for Grb2 binding, and an N-terminal motif which contains three tandemly repeated DYE(S/P)P sequences. We demonstrate here that SLP-76 is phosphorylated by ZAP-70 and that phosphorylation of these repeated tyrosine motifs is required for optimal generation of IL-2 in response to TCR ligation. In addition, overexpression of SLP-76 augments TCR-mediated transcriptional activation of the IL-2 gene, while mutation of the SH2 domain attenuates this response. Together, these studies identify SLP-76 as a physiologic substrate for ZAP-70 and suggest a mechanism by which TCR-induced activation of ZAP-70 regulates both the calcium and ras pathways. EXPERIMENTAL PROCEDURESCells and Antibodies-Jurkat and Sf9 cells (Pharmingen) were maintained as described previously (12). The mouse monoclonal antibody (mAb) H3 was generated against the SLP-76 SH2 domain. SLP-76 polyclonal antisera (22652) was generated against a peptide spanning amino acids 301-318 of human SLP-76. C305 is an anti-Jurkat Ti␣/␤-mAb (20); 4G10 (UBI) and PY20 (Santa Cruz) are anti-phosphotyrosine mAbs; 2F3.2 is an anti-ZAP-70 mAb (UBI), and 9E10 is an anti-myc mAb. Anti-GST mAb was purchased from Santa Cruz.Construction of Plasmids-A full-length SLP-76 cDNA was generated by reverse transcription PCR from murine T-cell mRNA. This cDNA was appended with a myc-epitope at the 3Ј end of the coding cDNA. Mutations were generated by PCR-directed mutagenesis and confirmed using standard dideoxy sequencing methods. These constructs were then subcloned into the pApuro vector (21) and the baculoviral vector pVL13...

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.

Immune response during pathogen infection requires extensive transcription reprogramming. A fundamental mechanism of transcriptional regulation is histone acetylation. However, how pathogens interfere with this process to promote disease remains largely unknown. Here we demonstrate that the cytoplasmic effector PsAvh23 produced by the soybean pathogen Phytophthora sojae acts as a modulator of histone acetyltransferase (HAT) in plants. PsAvh23 binds to the ADA2 subunit of the HAT complex SAGA and disrupts its assembly by interfering with the association of ADA2 with the catalytic subunit GCN5. As such, PsAvh23 suppresses H3K9 acetylation mediated by the ADA2/GCN5 module and increases plant susceptibility. Expression of PsAvh23 or silencing of GmADA2/GmGCN5 resulted in misregulation of defense-related genes, most likely due to decreased H3K9 acetylation levels at the corresponding loci. This study highlights an effective counter-defense mechanism by which a pathogen effector suppresses the activation of defense genes by interfering with the function of the HAT complex during infection.

Necrosis- and ethylene-inducing-like proteins (NLP) are widely distributed in eukaryotic and prokaryotic plant pathogens and are considered to be important virulence factors. We identified, in total, 70 potential Phytophthora sojae NLP genes but 37 were designated as pseudogenes. Sequence alignment of the remaining 33 NLP delineated six groups. Three of these groups include proteins with an intact heptapeptide (Gly-His-Arg-His-Asp-Trp-Glu) motif, which is important for necrosis-inducing activity, whereas the motif is not conserved in the other groups. In total, 19 representative NLP genes were assessed for necrosis-inducing activity by heterologous expression in Nicotiana benthamiana. Surprisingly, only eight genes triggered cell death. The expression of the NLP genes in P. sojae was examined, distinguishing 20 expressed and 13 nonexpressed NLP genes. Real-time reverse-transcriptase polymerase chain reaction results indicate that most NLP are highly expressed during cyst germination and infection stages. Amino acid substitution ratios (Ka/Ks) of 33 NLP sequences from four different P. sojae strains resulted in identification of positive selection sites in a distinct NLP group. Overall, our study indicates that expansion and pseudogenization of the P. sojae NLP family results from an ongoing birth-and-death process, and that varying patterns of expression, necrosis-inducing activity, and positive selection suggest that NLP have diversified in function.

Phytophthora pathogens secrete an array of specific effector proteins to manipulate host innate immunity to promote pathogen colonization. However, little is known about the host targets of effectors and the specific mechanisms by which effectors increase susceptibility. Here we report that the soybean pathogen Phytophthora sojae uses an essential effector PsAvh262 to stabilize endoplasmic reticulum (ER)-luminal binding immunoglobulin proteins (BiPs), which act as negative regulators of plant resistance to Phytophthora. By stabilizing BiPs, PsAvh262 suppresses ER stress-triggered cell death and facilitates Phytophthora infection. The direct targeting of ER stress regulators may represent a common mechanism of host manipulation by microbes.