Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide.

Reactive oxygen intermediates (ROIs) regulate apoptosis during normal development and disease in animals. ROIs are also implicated in hypersensitive resistance responses of plants against pathogens. Arabidopsis lsd1 mutants exhibited impaired control of cell death in the absence of pathogen and could not control the spread of cell death once it was initiated. Superoxide was necessary and sufficient to initiate lesion formation; it accumulated before the onset of cell death and subsequently in live cells adjacent to spreading lsd1 lesions. Thus, runaway cell death seen in lsd1 plants reflected abnormal accumulation of superoxide and lack of responsiveness to signals derived from it.

Induction of defense-related responses in Cf9 tomato cells by the AVR9 elicitor peptide of cladosporium fulvum is developmentally regulated

The AVR9 elicitor from the fungal pathogen Cladosporium fulvum induces defense-related responses, including cell death, specifically in tomato (Lycopersicon esculentum Mill.) plants that carry the Cf-9 resistance gene. To study biochemical mechanisms of resistance in detail, suspension cultures of tomato cells that carry the Cf-9 resistance gene were initiated. Treatment of cells with various elicitors, except AVR9, induced an oxidative burst, ion fluxes, and expression of defense-related genes. Agrobacterium tumefaciens-mediated transformation of Cf9 tomato leaf discs with Avr9-containing constructs resulted efficiently in transgenic callus formation. Although transgenic callus tissue showed normal regeneration capacity, transgenic plants expressing both the Cf-9 and the Avr9 genes were never obtained. Transgenic F1 seedlings that were generated from crosses between tomato plants expressing the Avr9 gene and wild-type Cf9 plants died within a few weeks. However, callus cultures that were initiated on cotyledons from these seedlings could be maintained for at least 3 months and developed similarly to callus cultures that contained only the Cf-9 or the Avr9 gene. It is concluded, therefore, that induction of defense responses in Cf9 tomato cells by the AVR9 elicitor is developmentally regulated and is absent in callus tissue and cell-suspension cultures, which consists of undifferentiated cells. These results are significant for the use of suspension-cultured cells to investigate signal transduction cascades.

Perception and transduction of an elicitor signal in cultured parsley cells.

Treatment of cultured parsley cells or protoplasts with a purified extracellular glycoprotein from Phytophthora megasperma f.sp. glycinea induces the transcription of the same set of defence-related genes as is activated in parsley leaves upon infection. Elicitor activity was shown to reside in a specific portion of the protein moiety which was isolated, sequenced and synthesized. Partial cDNAs encoding the entire mature protein as well as other related proteins have been isolated, indicating the presence of a small gene family. The elicitor-active oligopeptide is located in the C-terminal portion of the deduced amino acid sequence. Binding of the elicitor to target sites on the parsley plasma membrane appears to be the initial event in defence gene activation. The subsequent intracellular transduction of the elicitor signal was shown to involve rapid and transient influxes of Ca2+ and H+, as well as effluxes of K+ and Cl-. Inhibition of elicitor-induced ion fluxes by channel blockers also inhibited phytoalexin synthesis, while stimulation of similar ion fluxes by treatment of cells or protoplasts with the polyene antibiotic, amphotericin B, induced the production of phytoalexins and activated the complete set of defence-related genes in the absence of elicitor.

Reactive oxygen intermediates as mediators of programmed cell death in plants and animals.

Programmed cell death (PCD) is a physiological process occurring during development and in pathological conditions of animals and plants. The cell death program can be subdivided into three functionally different phases: a stimulus-dependent induction phase, an effector phase during which the wide range of death-stimuli are translated to a central coordinator, and a degradation phase during which the alterations commonly considered to define PCD (apoptotic morphology of the nucleus and chromatin fragmentation) become apparent. Recent studies suggest that mitochondrial permeability transition is the central coordinator of PCD and deciding whether or not a cell will die. There is increasing evidence that reactive oxygen intermediates (ROI) serve as direct and indirect mediators of PCD in mammalian and plant cells. Overexpression of genes encoding pro- and antioxidant enzymes in transgenic animals and plants has been informative regarding the function of ROI. Recent data imply a dual role of ROI in the apoptotic process: first, as a facultative signal during the induction phase, and, second, as a common consequence of mitochondrial permeability transition leading to the final destruction of the cell. The present review discusses and compares new insights into the function of ROI during PCD in mammalian cells and in human and plant diseases.

Elicitor-stimulated ion fluxes and O2- from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley.

Fungal elicitor stimulates a multicomponent defense response in cultured parsley cells (Petroselinum crispum). Early elements of this receptor-mediated response are ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Omission of Ca2+ from the culture medium or inhibition of elicitor-stimulated ion fluxes by ion channel blockers prevented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion fluxes. Inhibition of elicitor-stimulated ROS production using diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O2- but not H2O2 stimulated phytoalexin accumulation, without inducing proton fluxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activation of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O2- rather than H2O2 appears to trigger the subsequent reactions.

Signal perception and intracellular signal transduction in plant pathogen defense.

Disease resistance in plant/pathogen interactions requires sensitive and specific recognition mechanisms for pathogen-derived signals in plants. Cultured parsley (Petroselinum crispum) cells respond to treatment with a crude cell wall preparation derived from the phytopathogenic fungus Phytophthora sojae with transcriptional activation of the same set of defense-related genes as are activated in parsley leaves upon infection with fungal spores. A 13 amino acid core sequence (Pep-13) of a 42 kDa fungal cell wall glycoprotein was identified, which stimulates the same responses as the crude cell wall elicitor, namely macroscopic Ca2+ and H(+)-influxes, effluxes of K(+)- and Cl- ions, production of active oxygen species (oxidative burst), defense-related gene activation, and formation of antifungal phytoalexins. Using [125I]Tyr-Pep-13 as ligand in binding assays, a single-class high-affinity binding site in parsley microsomal membranes and protoplasts could be detected. Binding was specific, saturable, and reversible. By chemical crosslinking, a 91 kDa parsley plasma membrane protein was identified to be the receptor of the peptide elicitor. Isolation of this receptor protein involved in pathogen defense in plants is under way.

High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses.

An oligopeptide of 13 amino acids (Pep-13) identified within a 42 kDa glycoprotein elicitor from P. mega-sperma was shown to be necessary and sufficient to stimulate a complex defense response in parsley cells comprising H+/Ca2+ influxes, K+/Cl- effluxes, an oxidative burst, defense-related gene activation, and phytoalexin formation. Binding of radiolabeled Pep-13 to parsley microsomes and protoplasts was specific, reversible, and saturable. Identical structural features of Pep-13 were found to be responsible for specific binding and initiation of all plant responses analyzed. The high affinity binding site recognizing the peptide ligand (KD = 2.4 nM) may therefore represent a novel class of receptors in plants, and the rapidly induced ion fluxes may constitute elements of the signal transduction cascade triggering pathogen defense in plants.