Plant and animal PR1 family members inhibit programmed cell death and suppress bacterial pathogens in plant tissues.

A role for programmed cell death (PCD) has been established as the basis for plant-microbe interactions. A functional plant-based cDNA library screen identified possible anti-PCD genes, including one member of the PR1 family, designated P14a, from tomato. Members of the PR1 family have been subject to extensive research in view of their possible role in resistance against pathogens. The PR1 family is represented in every plant species studied to date and homologues have been found in animals, fungi and insects. However, the biological function of the PR1 protein from plants has remained elusive in spite of extensive research regarding a role in the response of plants to disease. Constitutive expression of P14a in transgenic tomato roots protected the roots against PCD triggered by Fumonisin B1, as did the human orthologue GLIPR1, indicating a kingdom crossing function for PR1. Tobacco plants transformed with a P14a-GFP fusion construct and inoculated with Pseudomonas syringae pv. tabaci revealed that the mRNA was abundant throughout the leaves, but the fusion protein was restricted to the lesion margins, where cell death and bacterial spread were arrested. Vitus vinifera grapes expressing the PR1 homologue P14a as a transgene were protected against the cell death symptoms of Pierce's disease. A pull-down assay identified putative PR1-interacting proteins, including members of the Rac1 immune complex, known to function in innate immunity in rice and animal systems. The findings herein are consistent with a role of PR1 in the suppression of cell death-dependent disease symptoms and a possible mode of action.

Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or, alternatively, it has a different mechanism of substrate binding than other polygalacturonases characterized to date.

Leptosphaeria maculans elicits apoptosis coincident with leaf lesion formation and hyphal advance in Brassica napus.

Programmed cell death, with many of the morphological markers of apoptosis, is increasingly recognized as an important process in plant disease. We have investigated the involvement and potential role of apoptosis during the formation of leaf lesions by the fungus Leptosphaeria maculans on susceptible Brassica napus cv. Westar. There were no signs of host cell damage until 7 to 8 days postinoculation (dpi), when trypan-blue-stained leaf mesophyll cells were first detected. Hyphae were visible in the intercellular spaces of the inoculated area from 5 dpi and were associated with trypan-blue-stained cells at 8 to 9 dpi. Hallmarks of apoptosis, observed coincident with or immediately prior to the formation of leaf lesions at 8 to 10 dpi, included membrane shrinkage of the mesophyll cell cytoplasm, loss of cell to cell contact in mesophyll cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of nuclei in apparently "healthy" tissue immediately adjacent to dead areas. Hyphae were highly branched and prolific in the "healthy" tissue immediately adjacent to dead areas 9 to 10 dpi, and formed pycnidia inside dead areas 11 to 12 dpi. Coinfiltration of the tetrapeptide caspase inhibitor Ac-DEVD-CHO with spores of the pathogen significantly suppressed development of leaf lesions but did not affect fungus viability. We hypothesize that L. maculans elicits apoptosis as a dependent component of pathogenesis in susceptible B. napus, and that the fungus uses apoptotic cells as a source of nutrition for reproduction and further growth.

Programmed cell death suppression in transformed plant tissue by tomato cDNAs identified from an Agrobacterium rhizogenes-based functional screen.

The genetic regulation of programmed cell death (PCD) is well characterized in animal systems, but largely unresolved in plants. This research was designed to identify plant genes that can suppress PCD triggered in plants by Fumonisin B1 (FB1). Agrobacterium rhizogenes was used to transform individual members of a cDNA library into tomato roots, which were then screened for resistance to FB1. Cellular changes elicited during FB1-induced PCD include chromatin condensation, fragmentation into pycnotic DNA bodies, TUNEL positive reactions, ROS accumulation, and eventual loss of membrane integrity. Several cDNA library members collectively overexpressed in a transformed root population revealed PCD suppressive action and were recovered by PCR. One of the FB1 suppressive genes was homologous to metallothionein, and shared sequence homology to the animal ortholog reported to suppress PCD through interference with formation or activity of reactive oxygen species (ROS). The metallothionein recovered in this screen suppressed ROS accumulation in FB1-treated roots and prevented symptoms of PCD. Anti-PCD genes recovered by this screen represent potential sources of resistance to PCD-dependent plant diseases, while the screen should be useful to identify genes capable of suppressing PCD triggered by other effectors, including those expressed by root pathogens during infection.

Fas signaling induces raft coalescence that is blocked by cholesterol depletion in human RPE cells undergoing apoptosis.

PURPOSE: To investigate whether the signaling events occurring in Fas-mediated apoptosis alter raft membrane formation in human RPE cells. METHODS: Formation of lipid rafts in cultured human retinal pigment epithelial cells (ARPE-19) was studied by confocal microscopy, with fluorescein-labeled cholera toxin subunit B binding protein (BODIPY)-labeled ganglioside GM1 lipid after Fas-L induction of apoptosis. Apoptosis was assessed by fluorescein-labeled annexin V detection of phosphatidylserine externalization and quadrant analysis with flow cytometry. Membrane rafts were localized into membrane vesicles by passing BODIPY-labeled GM1 RPE cells through a 2-microm-pore polycarbonate membrane using an extruder device. The labeled fractions, containing vesicles enriched in GM1, were detected by flow cytometry and then analyzed for the presence of Fas protein. RESULTS: Differential punctate staining of membrane rafts was demonstrated in normal and FasL-induced apoptotic human ARPE-19 cells in culture by confocal microscopy, using cholera toxin B and GM1 labeling of extruded vesicles. The lipid raft-associated vesicles were derived by plasma membrane dissociation, via a newly developed whole-cell extrusion technique that produced 2-microm vesicles with both GM1 lipid and Fas protein abundance enriched in a subpopulation of the membrane-derived vesicles. The amount of Fas protein in the vesicles containing raft domains markedly increased in FasL-treated cells. Treatment of human ARPE 19 cells with methyl beta-cyclodextrin after FasL induction of apoptosis resulted in cellular cholesterol depletion and markedly reduced the incidence of Fas-receptor localization in GM1 rafts. CONCLUSIONS: Human ARPE-19 cells in culture contain membrane rafts with apoptotic signaling effectors uniformly distributed in the native state. The cells stimulated to undergo apoptosis appear to use membrane rafts in the death-signaling process by mobilization of rafts to localized regions of the membrane that are now enriched with apoptotic signaling effectors. Fas signaling induces apoptotic raft formation that results in polar condensation, or capping, of the rafts in the late stages of apoptosis. A novel extrusion technique is described that allows localization and enrichment of rafts into membrane vesicles, which can be assayed by flow cytometry. Cholesterol depletion, after Fas ligand activation of apoptosis, reduced raft formation in cells induced to undergo apoptosis. Therapeutic implications for the treatment of retinal disorders are discussed.

Expression of the antiapoptotic baculovirus p35 gene in tomato blocks programmed cell death and provides broad-spectrum resistance to disease.

The sphinganine analog mycotoxin, AAL-toxin, induces a death process in plant and animal cells that shows apoptotic morphology. In nature, the AAL-toxin is the primary determinant of the Alternaria stem canker disease of tomato, thus linking apoptosis to this disease caused by Alternaria alternata f. sp. lycopersici. The product of the baculovirus p35 gene is a specific inhibitor of a class of cysteine proteases termed caspases, and naturally functions in infected insects. Transgenic tomato plants bearing the p35 gene were protected against AAL-toxin-induced death and pathogen infection. Resistance to the toxin and pathogen co-segregated with the expression of the p35 gene through the T3 generation, as did resistance to A. alternata, Colletotrichum coccodes, and Pseudomonas syringae pv. tomato. The p35 gene, stably transformed into tomato roots by Agrobacterium rhizogenes, protected roots against a 30-fold greater concentration of AAL-toxin than control roots tolerated. Transgenic expression of a p35 binding site mutant (DQMD to DRIL), inactive against animal caspases-3, did not protect against AAL-toxin. These results indicate that plants possess a protease with substrate-site specificity that is functionally equivalent to certain animal caspases. A biological conclusion is that diverse plant pathogens co-opt apoptosis during infection, and that transgenic modification of pathways regulating programmed cell death in plants is a potential strategy for engineering broad-spectrum disease resistance in plants.