Temporal and Spatial Patterns of Gene Expression around Sites of Attempted Fungal Infection in Parsley Leaves.

We analyzed the expression patterns of several pathogen defense-related genes in primary leaf buds of parsley by in situ RNA hybridization. Labeled antisense RNA probes were generated from seven selected cDNAs detecting transcripts from genes that are rapidly and strongly activated in cultured parsley cells upon treatment with fungal elicitor. These genes encode two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, a furanocoumarin-specific bergaptol O-methyltransferase, one pathogenesis-related protein, and three less well characterized proteins, designated as ELI 3, ELI 5, and ELI 7. In uninfected tissue, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNA levels were high in epidermal cells, oil-duct epithelial cells, and cells of the developing xylem; bergaptol O-methyltransferase mRNA was confined to oil-duct epithelial cells; and the pathogenesis-related protein and ELI 3, ELI 5, and ELI 7 mRNAs were undetectable. All seven mRNAs accumulated transiently and locally around infection sites caused by the soybean-pathogenic fungus Phytophthora megasperma f. sp. glycinea, to which parsley is nonhost-resistant. The observed late appearance of bergaptol O-methyltransferase mRNA, as compared with all other mRNAs, is in accord with a similar relative timing of transient gene activation in elicitor-treated cell cultures. Sharp borders were observed between the infection center, where hypersensitive cell death had occurred in response to fungal penetration, the surrounding area of local gene activation, and the remainder of the tissue not showing any apparent response. Some of the genes were also activated, although less sharply localized, upon wounding of parsley leaves.

Biphasic Temporal and Spatial Induction Patterns of Defense-Related mRNAs and Proteins in Fungus-Infected Parsley Leaves.

Previous experiments using in situ RNA hybridization have shown that the mRNAs encoding phenylalanine ammonia-lyase, 4-coumarate:coenzyme A ligase, and pathogenesis-related protein 1 accumulated transiently around fungal infection sites in parsley (Petroselinum crispum) leaf buds. These studies have now been extended by (a) analyzing different stages of the infection process and (b) monitoring the timing of appearance and the spatial distribution of the proteins as well as the corresponding mRNAs. An early and short period of mRNA induction throughout a large portion of the infected leaf was followed by a longer period, during which the mRNA levels remained high in a more localized area around the site of fungal penetration with sharp borders toward the surrounding tissue. This biphasic pattern of mRNA accumulation was followed after some delay by the same pattern of protein accumulation.

Correlation of Rapid Cell Death with Metabolic Changes in Fungus-Infected, Cultured Parsley Cells.

To study in detail the hypersensitive reaction, one of the major defense responses of plants against microbial infection, we used a model system of reduced complexity with cultured parsley (Petroselinum crispum) cells infected with the phytopathogenic fungus Phytophthora infestans. Experimental conditions were established to maintain maximal viability of the cultured cells during co-cultivation with fungal germlings, and a large proportion of the infected parsley cells responded to fungal infection with rapid cell death, thereby exhibiting major features of the hypersensitive reaction in whole-plant-pathogen interactions. Rapid cell death clearly correlated with termination of further growth and development of the fungal pathogen. Thus, the system fulfilled important prerequisites for investigating cell-death-related metabolic changes in individual infected cells. Using cytochemical methods, we monitored the increase of mitochondrial activity in single infected cells and the intracellular accumulation of reactive oxygen species prior to the occurrence of rapid cell death. We obtained strong correlative evidence for the involvement of these intracellularly accumulating reactive oxygen species in membrane damage and in the resulting abrupt collapse of the cell.

Structural analysis and activation by fungal infection of a gene encoding a pathogenesis-related protein in potato.

The structure, genomic organization, and temporal pattern of activation of a gene encoding a pathogenesis-related protein (PR1) in potato (Solanum tuberosum) have been analyzed. The gene is rapidly activated in leaves from the potato cultivar Datura, containing the resistance gene R1, in both compatible and incompatible interactions with appropriate races of the late-blight fungus Phytophthora infestans. Activation is also observed in leaves treated with fungal elicitor. The gene occurs in multiple, very similar copies and encodes a polypeptide (Mr = 25,054; pI = 5.5) that is classified as a PR protein by several criteria. Small fragments with great sequence similarity to portions of the two exons were found closely linked to the expressed gene, which altogether represents a simple case of genome organization in potato. The coding sequence of the prp1 gene and the deduced amino acid sequence are strikingly similar to the corresponding sequences of a 26-kDa heat shock protein from soybean.

Expression in Escherichia coli of catalytically active phenylalanine ammonia-lyase from parsley.

In parsley (Petroselinum crispum), phenylalanine ammonia-lyase (PAL) is encoded by 4 structurally similar genes. The nucleotide sequence of a near full-length cDNA and the deduced amino acid sequence of PAL-4 are presented and compared with the corresponding sequences of PAL-1, a previously described representative of the gene family. Transformation of Escherichia coli cells with PAL-1 or PAL-4 cDNA yielded catalytically active PAL, suggesting that the catalytic center of the enzyme is formed spontaneously rather than by a plant-specific mechanism.

Mutational analysis of 4-coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes.

4-Coumarate:coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for a large variety of important plant secondary products, such as lignin, flavonoids, or phytoalexins. To identify amino acids important for 4CL activity, eight mutations were introduced into Arabidopsis thaliana At4CL2. Determination of specific activities and K(m) values for ATP and caffeate of the heterologously expressed and purified proteins identified four distinct classes of mutants: enzymes with little or no catalytic activity; enzymes with greatly reduced activity but wild-type K(m) values; enzymes with drastically altered K(m) values; and enzymes with almost wild-type properties. The latter class includes replacement of a cysteine residue which is strictly conserved in 4CLs and had previously been assumed to be directly involved in catalysis. These results substantiate the close relationship between 4CL and other adenylate-forming enzymes such as luciferases, peptide synthetases, and fatty acyl-CoA synthetases.

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.

Differential wound activation of members of the phenylalanine ammonia-lyase and 4-coumarate:CoA ligase gene families in various organs of parsley plants.

We analyzed the developmental regulation and the activation by wounding of several stress-related genes in various parsley organs. The genes encode phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), two enzymes of general phenylpropanoid metabolism; a flavonoid specific enzyme, chalcone synthase (CHS); a furanocoumarin specific enzyme, bergaptol O-methyltransferase (BMT); and a pathogenesis-related protein (PR 1). All genes or gene families exhibited high levels of expression in roots and during certain stages of leaf development. PAL, 4CL and CHS were preferentially expressed in young leaves, BMT and PR 1 in old leaves. An appreciable increase in CHS mRNA levels was observed in wounded leaves. By contrast, root wounding led to a decrease in the existing CHS mRNA levels. A biphasic response (a decrease followed by an increase) to wounding was seen for BMT and PR 1 mRNAs in roots and for BMT mRNA in attached leaves. Using gene-specific oligonucleotide probes to measure the expression rates of three of the four PAL genes and of the two 4CL genes separately we observed a differential behavior of the individual family members under many of the conditions tested. While PAL-3 was preferentially activated in wounded leaves and 4CL-1 in wounded roots, PAL-2 and 4CL-2 were primarily responsible for the high constitutive expression levels in roots and flowering stems respectively. Despite the differential expression of their individual members, the PAL and 4CL gene families displayed very similar changes in the overall patterns of expression, reflecting their closely related functions in phenylpropanoid metabolism.

Further studies on the relationship between the rates of nitrate uptake, growth and conductivity changes in the medium of plant cell suspension cultures.

The changes in packed cell volume and in nitrate content and conductivity of the medium during the growth cycle of cell suspension cultures from Petroselinum hortense Hoffm., Glycine max Merr., and Haplopappus gracilis A. Grey in a chemically defined medium were compared. In all three cases sigmoidal curves obtained for large decreases in the conductivity were paralleled by similar curves for the rates of nitrate depletion from the medium until this nutrient was completely exhausted. Further decreases in the conductivity subsequent to nitrogen starvation proceeded at relatively slow rates and ceased when the cultures entered into the stationary phase of the growth cycle. Thus the previously reported method of deriving growth curves indirectly from conductivity measurements (Hahlbrock and Kuhlen, Planta 108: 271-278, 1972; Hahlbrock et al., Planta 118: 75-84, 1974) might be generally applicable for this particular medium. The method seems to be based on a continuous uptake by the cells of ionic constituents throughout all stages of actual growth, even beyond the stage of nitrate exhaustion.Cell suspension cultures from Cicer arietinum L. and Acer pseudoplatanus L. in two different, more complex media were used for similar experiments, in which the changes in packed cell volume and in the conductivity of the medium were recorded. As with the results obtained with the fully synthetic medium, the mirror-images of the curves obtained for the decline in conductivity initially paralleled the growth curves. However, the two curves became incongruous after a certain growth stage was reached. These results are discussed with respect to the composition of the media used and to the apparent limitations of the method of determining specific growth stages by monitoring conductivity changes in the medium.