We have analysed three nearly full-length cDNAs complementary to mRNAs encoding two PR1 (pathogenesis-related, class 1) proteins in parsley (Petroselinum crispum). Furthermore, one selected genomic clone containing the PcPR1-1 gene was investigated in detail. The structural organization and possible regulatory elements in the 5' flanking region of this gene are presented. In situ RNA hybridization in fungus-infected parsley leaf tissue demonstrated rapid and massive PR1 mRNA accumulation around infection sites.

Treatment of cultured parsley (Petroselinum crispum) cells with fungal elicitor rapidly activates ftransription of many genes encoding specific steps in pathogen defenserelated pathways. We report evidence that three cDNAs corresponding to such genes represent two key enzymes of the activated methyl cycle. Two cDNAs are derived from distinct members of the S-adenosyl-L-methionine synthetase gene family, based on extensive similarity of the deduced polypeptides with authentic enzymes from Arabidopsis thaliana, rat, yeast, and Escherichia coi. The third cDNA exhibits large sim it with a functionally related gene, encoding S-adenosyl-Lhomocysteine hydrolase, from rat and a slime mold. Marked differences in the mRNA levels occurred in different organs of parsley plants. Elicitor treatment strongly induced both mRNAs in cultured cells as well as intact leaves and led to marked increases in S-adenosyl-L-homocysteine hydrolase enzyme activity. These results suggest a close metabolic link between pathogen defense and an increased turnover of activated methyl groups.In all organisms investigated, S-adenosyl-L-methionine (AdoMet) serves as the major methyl-group donor for numerous highly specific methyl-transferase reactions. These reactions involve a large variety of acceptor molecules, such as phenylpropanoid derivatives, cyclic fatty acids, proteins, polysaccharides, and nucleic acids (1). In addition, AdoMet has regulatory functions-e.g., in the allosteric stimulation of threonine synthase (2). In plants, AdoMet (6,15,17,18), and only two cDNA clones encoding a rat and a Dictyostelium discoideum SHH have been reported (19,20).Here we describe the identification of parsley cDNAs for SMS and SHHt and demonstrate that fungal elicitor induces the corresponding mRNAs in cultured cells as well as leaves of this plant. MATERIALS AND METHODSCell Culture/Plants/Elicitor Treatment. Cell suspension cultures of parsley (Petroselinum crispum L.) were grown in the dark for 6 days and were then treated with elicitor at 50 ,ug/ml derived from the fungus Phytophthora megasperma f. sp. glycinea (21, 22).Nondetached leaves of greenhouse-grown parsley plants were treated with elicitor solution (50 jug/ml) using a 1-ml syringe for pressure infiltration via the stomata. RNA Isolation and Blot Hybridization. Total RNA from parsley plants and cell cultures was prepared according to Lois et al. (23). Ten micrograms of total RNA was denatured and separated on formaldehyde-agarose gels. Conditions for denaturing, electrophoresis, and capillary blotting were as described (24). The RNA was transferred to Hybond-N nylon membrane (Amersham) and cross-linked by UV light. Prehybridization (30 min) and hybridization (16 hr) were carried out at 650C in 1 M NaCl/1% SDS/10% dextran sulfate containing salmon sperm DNA at 100 ,ug/ml. Blots were washed twice for 30 min with 2x standard saline citrate/0.5% SDS and once for 30 min with 0.5 x standard saline citrate/ 0.5% SDS at 650C. The cDNA probes were 32P-labeled by random priming (25). Total RN...

We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabidopsis thaliana. The predicted product shares no homology to known sequences. ELI3 mRNA accumulates in A. thaliana leaves in response to challenge with phytopathogenic Pseudomonas syringae strains. The timing and magnitude of this response are dictated by the genetics of the plant‐pathogen interaction being analyzed. During incompatible interactions, where resistance in the plant genotype Col‐0 is dictated by the dominant RPM1 locus, ELI3 mRNA accumulates to high levels 5–10 h post‐inoculation. This kinetic behavior is also generated by the presence of a cloned bacterial avirulence gene, in otherwise virulent bacteria, which triggers resistance mediated via RPM1 action. The phenotypic outcome is a hypersensitive resistance reaction visible 8–15 h post‐infiltration. Thus, the induction kinetics of ELI3 mRNA accumulation are consistent with a functional role for the ELI3 gene product in establishing the resistant phenotype. In contrast, during compatible interactions with the susceptible plant genotype Nd‐0, which is homozygous recessive at the rpm1 locus, ELI3 mRNA accumulates significantly only after 15 h. We show genetically that ELI3 activation is strictly dependent on the presence of dominant alleles at RPM1 using an assay generalizable to any pathogen induced plant defense phenomena.

Ubiquitin is an omnipresent protein found in all eukaryotes so far analysed. It is involved in several important processes, including protein turnover, chromosome structure and stress response. Parsley (Petroselinum crispum) contains at least two active polyubiquitin (ubi4) genes encoding hexameric precursor proteins. The deduced amino acid sequences of the ubiquitin monomers are identical to one another and to ubiquitin sequences from several other plant species. Analysis of the promoter region of one ubi4 gene revealed putative regulatory elements. In parsley plants, the ubi4 mRNAs were the predominant ubiquitin mRNAs and were present at comparable levels in all plant organs tested. In cultured parsley cells, high levels of ubiquitin gene expression remained unaffected by heat shock, elicitor or light treatment.

Two recently isolated cDNAs representing genes that are transcriptionally activated in fungus-infected parsley leaves or elicitor-treated, cultured parsley cells are shown to encode a hydroxyproline-rich glycoprotein (HRGP) and an anionic peroxidase. The deduced HRGP protein is rich in tyrosine residues, a feature also found in other pathogen- and wound-induced plant HRGPs. Expression of the peroxidase gene(s) is induced rapidly upon elicitation and precedes that of the HRGP gene. In situ hybridization experiments demonstrate the presence of HRGP and peroxidase mRNAs in parsley tissue around fungal infection sites. Peroxidase mRNA accumulation is particularly sharply restricted to plant cells directly adjacent to fungal hyphae. These results provide further evidence for an important role of specific cell wall modifications in plant defense.