Group IVA cytosolic phospholipase A 2 (cPLA 2 ␣) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA 2 ␣ (IMLF ؊/؊ ) were reconstituted with wild type and cPLA 2 ␣ mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP 2 ) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA 2 ␣ translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA 2 ␣ to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA 2 ␣ to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF ؊/؊ expressing phosphorylation site (S505A) and PIP 2 binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA 2 ␣, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA 2 ␣ reduced activation by PIP 2 but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA 2 ␣ hydrolytic activity.

Complex suites of proteins that are secreted by plants and phytopathogens into the plant apoplast play crucial roles in surveillance, assault, defense, and counter-defense. High-throughput genome-scale strategies are being developed to better understand the nature of these "secretomes" and the identity of pathogen-derived effector proteins that subvert plant defenses and promote pathogenicity. Although combined bioinformatic and experimental approaches recently have provided comprehensive coverage of secreted proteins from bacterial phytopathogens, far less is known about the secretomes and batteries of effectors of eukaryotic phytopathogens; notably fungi and oomycetes. The yeast secretion trap (YST) represents a potentially valuable technique to simultaneously target pathogen and host secretomes in infected plant material. A YST screen, using a new vector system, was applied to study the interaction between tomato (Solanum lycopersicum) and the oomycete Phytophthora infestans, revealing sets of genes encoding secreted proteins from both pathogen and host. Most of those from the oomycete had no identifiable function and were detectable in planta only during pathogenesis, underlining the value of YST as a tool to identify new candidate effectors and pathogenicity factors. In addition, the majority of the P. infestans proteins had homologs in the genomes of the related oomycetes R. sojae and P. ramorum.