Host protein BSL1 associates with Phytophthora infestans RXLR effector AVR2 and the Solanum demissum Immune receptor R2 to mediate disease resistance.

Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.

Regulation of hematopoietic cell function by inhibitory immunoglobulin G receptors and their inositol lipid phosphatase effectors.

Numerous autoimmune and inflammatory disorders stem from the dysregulation of hematopoietic cell activation. The activity of inositol lipid and protein tyrosine phosphatases, and the receptors that recruit them, is critical for prevention of these disorders. Balanced signaling by inhibitory and activating receptors is now recognized to be an important factor in tuning cell function and inflammatory potential. In this review, we provide an overview of current knowledge of membrane proximal events in signaling by inhibitory/regulatory receptors focusing on structural and functional characteristics of receptors and their effectors Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 and SH2 domain-containing inositol 5-phosphatase-1. We review use of new strategies to identify novel regulatory receptors and effectors. Finally, we discuss complementary actions of paired inhibitory and activating receptors, using Fc gammaRIIA and Fc gammaRIIB regulation human basophil activation as a prototype.

Identification of HLA-CW3, GNAS and IMPA as cytotoxic T-lymphocyte (CTL) target antigens using an allogeneic mixed lymphocyte tumor cell culture (MLTC) system and subsequent cDNA library screening.

Allogeneic mixed lymphocyte tumor cell cultures (MLTCs) were established using lymphocytes from non-small-cell lung cancer (NSCLC) patient UKY-53 and HLA-A2+ NSCLC tumor cells (UKY-29). The tumor cells expressed the lymphocyte costimulatory molecule CD80 (UKY29.7). Cytolytic activity showed the cytotoxic T-lymphocytes (CTL) lysed UKY-29, but not K562 or Daudi. The CTL also lysed: HLA-A2+ and -A24+ tumor cell lines from a number of tumor histologies. The CTL also lysed Epstein Barr virus transformed (EBV) B-cells, UKY-29EBV, autologous to the stimulating cell line, UKY29TC. These data suggested the presence of both tumor-specific and allogeneic reactivities in the bulk CTL population. Subsequent cDNA cloning analysis and sequencing demonstrated that the bulk CTL population was recognizing: (i) allogeneic target HLA-CW3, and two minor histocompatibility antigens; (ii) guanine nucleotide-binding protein, G(S) (GNAS), and (iii) inositol myophosphatase (IMPA). All three antigens, we believe, were restricted by HLA-A2. Whereas the system described was initially intended to identify tumor-associated antigens recognized by CTL, the nature of the allogeneic system provides a unique opportunity for the identification of epitopes that confer both allo and minor antigen recognition.

The isolation and characterization of cDNA encoding human and rat brain inositol polyphosphate 4-phosphatase.

Inositol polyphosphate 4-phosphatase, an enzyme of the inositol phosphate signaling pathway, catalyzes the hydrolysis of the 4-position phosphate of inositol 3,4-bisphosphate, inositol 1,3,4-trisphosphate, and phosphatidylinositol 3,4-bisphosphate. The amino acid sequences of tryptic and CNBr peptides of the enzyme isolated from rat brain were determined. Degenerate oligonucleotide primers based on this sequence were used to amplify a 74-base pair polymerase chain reaction product. This product was used to isolate a 5607-base pair composite cDNA, which had an open reading frame encoding a protein with 939 amino acids with a predicted molecular mass of 105,588 Da. The rat brain polymerase chain reaction product was used as a probe to isolate a human brain cDNA that predicts a protein with 938 amino acids and a molecular mass of 105,710 Da. Remarkably, the human and rat proteins were 97% identical. Recombinant rat protein expressed in Escherichia coli catalyzed the hydrolysis of all three substrates of the 4-phosphatase. Northern blot hybridization indicates that the 4-phosphatase is widely expressed in rat tissues with the highest levels of expression occurring in brain, heart, and skeletal muscle. Polyclonal antiserum directed against the carboxyl terminus of the 4-phosphatase immunoprecipitated > 95% of the 4-phosphatase activity in crude homogenates of rat brain, heart, skeletal muscle, and spleen, suggesting that this enzyme accounts for the 4-phosphate activity present in rat tissues. This antiserum also immunoprecipitated the 4-phosphatase from human platelet sonicates.