It has been hypothesized that plants contain respiratory burst oxidase which, upon activation, oxidize NADPH and generate extracellular superoxide, O2.-. These proteins are proposed to play a central role in defence against pathogens. However, plant DNA sequences that encode proteins with similarity to components of respiratory burst oxidase have not previously been reported. This paper describes the complete cDNA and genomic DNA sequence of the rice rbohA (for respiratory burst oxidase homologue) gene. The predicted RbohA product is most similar to the main catalytic subunit, gp91phox, of the respiratory burst oxidase of neutrophils. Reverse transcriptase PCR detects rbohA transcripts in both roots and shoots of healthy rice plants.

Bacterial pathogens have developed sophisticated mechanisms of evading the immune system to survive in infected host cells. Central to the pathogenesis of Mycobacterium tuberculosis is the arrest of phagosome maturation, partly through interference with PtdIns signalling. The protein phosphatase MptpB is an essential secreted virulence factor in M. tuberculosis. A combination of bioinformatics analysis, enzyme kinetics and substrate-specificity characterization revealed that MptpB exhibits both dual-specificity protein phosphatase activity and, importantly, phosphoinositide phosphatase activity. Mutagenesis of conserved residues in the active site signature indicates a cysteine-based mechanism of dephosphorylation and identifies two new catalytic residues, Asp165, essential in catalysis, and Lys164, apparently involved in substrate specificity. Sequence similarities with mammalian lipid phosphatases and a preference for phosphoinositide substrates suggests a potential novel role of MptpB in PtdIns metabolism in the host and reveals new perspectives for the role of this phosphatase in mycobacteria pathogenicity.

These results provide the first evidence that inhibition of phosphatases can be exploited against mycobacterial infections. The cell activity of the inhibitors together with the lack of MptpB human orthologues suggests a strong potential for these compounds to be developed as drug candidates against tuberculosis and promises a new therapeutic strategy to tackle clearance and reduce the persistence of M. tuberculosis infection.

Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E-and L-forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomyces a-factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the a-factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA-E, PHA-L and GNA with heterogenous N-termini, with the majority of the protein containing N-terminal extensions derived from the a-factor prosequence. Polypeptides in which most of the a-factor prosequence was present were also glycosylated. Inclusion of Glu-Ala repeats at the C-terminal end of the a-factor preprosequence led to efficient processing N-terminal to the Glu-Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N-termini still containing N-terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia.

SummaryA novel protein phosphatase in Arabidopsis thaliana was identi®ed by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion`pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.