Measuring PLD Activity In Vivo

Phospholipase D (PLD) hydrolyzes structural phospholipids like phosphatidylcholine (PC) and phosphatidylethanolamine (PE) into phosphatidic acid (PA) and free choline/ethanolamine. In plants, this activity can be stimulated by a wide variety of biotic and abiotic stresses (Li et al., Biochim Biophys Acta 1791:927-935, 2009; Testerink and Munnik, J Exp Bot 62(7):2349-2361, 2011). This chapter describes a protocol for the measurement of PLD activity in vivo. The protocol takes advantage of a unique property of PLD, i.e., its ability to substitute a primary alcohol, such as 1-butanol, for water in the hydrolytic reaction. This transphosphatidylation reaction results in the formation of phosphatidylbutanol (PBut), which is a specific and unique reporter for PLD activity. The assay is highly sensitive for detecting PLD activity in vivo, following stimulation of intact plant cells, seedlings, and tissues, being a valuable method for studying the regulation of plant PLD activity in vivo

Differential expression of G protein alpha and ß subunit genes during development of Phytophthora infestans

A G protein subunit gene (pigpa1) and a G protein subunit gene (pigpb1) were isolated from the oomycete Phytophthora infestans, the causal agent of potato late blight. Heterotrimeric G proteins are evolutionary conserved GTP-binding proteins that are composed of ,, and subunits and participate in diverse signal transduction pathways. The deduced amino acid sequence of both pigpa1 and pigpb1, showed the typical conserved motifs present in G or G proteins from other eukaryotes. Southern blot analysis revealed no additional copies of G or G subunit genes in P. infestans, suggesting that pigpa1 and pigpb1 are single copy genes. By cross-hybridization homologues of gpa1 and gpb1 were detected in other Phythophthora species. Expression analyses revealed that both genes are differentially expressed during asexual development, with the highest mRNA levels in sporangia. In mycelium, no pigpa1 mRNA was detected. Western blot analysis using a polyclonal GPA1 antibody confirmed the differential expression of pigpa1. These expression patterns suggest a role for G-protein-mediated signaling during formation and germination of asexual spores of P. infestans, developmental stages representing the initial steps of the infection process