AvrRxv is a member of a family of pathogen effectors present in pathogens of both plant and mammalian species. Xanthomonas campestris pv. vesicatoria strains carrying AvrRxv induce a hypersensitive response (HR) in the tomato cultivar Hawaii 7998. Using a yeast two-hybrid screen, we identified a 14-3-3 protein from tomato that interacts with AvrRxv called AvrRxv Interactor 1 (ARI1). The interaction was confirmed in vitro with affinity chromatography. Using mutagenesis, we identified a 14-3-3-binding domain in AvrRxv and demonstrated that a mutant in that domain showed concomitant loss of interaction with ARI1 and HR-inducing activity in tomato. These results demonstrate that the AvrRxv bacterial effector recruits 14-3-3 proteins for its function within host cells. AvrRxv homologues YopP and YopJ from Yersinia do not have AvrRxv-specific HR-inducing activity when delivered into tomato host cells by Agrobacterium. Although YopP itself cannot induce HR, its C-terminal domain containing the catalytic residues can replace that of AvrRxv in an AvrRxv-YopP chimera for HR-inducing activity. Phylogenetic analysis indicates that the sequences encoding the C-termini of family members are evolving independently from those encoding the N-termini. Our results support a model in which there are three functional domains in proteins of the family, translocation, interaction, and catalytic.

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1. We have used two experimental approaches to examine regulation of intracellular calcium ion levels in fish retinal ganglion cells. In the first set of experiments, we ratio-imaged fura-2 emission intensity to estimate the concentration of free intracellular calcium ions ([Ca2+]i) in isolated goldfish retinal ganglion cells depolarized by increases in extracellular levels of potassium ions ([K+]o), in the presence and absence of extracellular sodium ions (Na+). Stepwise increases in [K+]o from 5 mM to as high as 60 mM produced stepwise increases in [Ca2+]i. These increases were sustained in the absence of external Na+, but transient and smaller in the presence of external Na+. The decline of [Ca2+]i in high-K, Na(+)-containing saline could be reversed by application of the ionophore monensin, or by replacement of external Na+ with either N-methyl-D-glucamine or lithium. In Na(+)-containing saline, [Ca2+]i fell to control levels after [K+]o was restored to control levels. 2. In the second set of experiments, we assessed Na(+)-Ca2+ exchanger-like immunoreactivity in goldfish retinal ganglion cells with the use of a polyclonal antiserum directed against Na(+)-Ca2+,K+ exchanger purified from bovine rod outer segments. This antiserum specifically stained the somata, neurites, and growth cones of isolated ganglion cells, the outer segments of rod photoreceptors, and (on Western blots prepared from mechanically isolated rods) protein displaying an apparent molecular mass of 210 kDa. 3. These results provide measurements of changes in [Ca2+]i of retinal ganglion cells depolarized in Na(+)-containing saline, and the distribution and apparent molecular weight of Na(+)-Ca2+ exchanger-like immunoreactivity in teleost retina.(ABSTRACT TRUNCATED AT 250 WORDS)

Although single-channel and whole-cell patch-clamp recordings have demonstrated the presence of Na+ currents in retinal ganglion cell somata, it has not previously been reported that an anti-Na+-channel antiserum stains both retinal ganglion cell somata and proteins with molecular weights corresponding to complexes of alpha and beta subunits. We probed adult goldfish retinas for Na+ channel-like immunoreactivity with a polyclonal antibody directed against the EOIII segment of vertebrate voltage-gated Na+ channels. In vertical sections and whole mounts, this antibody consistently stained the somata, axons, and proximal dendrites of retinal ganglion cells. Some somata in the proximal third of the inner nuclear layer were also stained. In Western blots, this antibody specifically stained multiple protein bands from retina and optic nerve, all with apparent molecular weights between 200 and 315 kDa. The largest of these molecular weights agrees with that reported previously for complexes of alpha and beta subunits in mammalian neurons, including retinal ganglion cells. The intermediate and lowest molecular weights are consistent with the presence of multiple Na+ channel alpha subunits, either in individual proximal retinal neurons or in different morphological subtypes.