Plants exhibit natural resistance to disease which has been exploited by breeders to reduce crop losses and hence increase yield. Disease resistance involves not only static protection, but also inducible defence mechanisms including: (i) accumulation of host-synthesized phytoalexins; (ii) deposition of lignin-like material; (iii) accumulation of hydroxyproline-rich glycoproteins and (iv) increases in the activity of certain hydrolytic enzymes such as chitinase and glucanase [1]. Although the genetics, physiology and cytology of plant:pathogen interactions have been extensively studied, until recently relatively little was known at the biochemical level about how plants respond to infection to activate these defence responses.

Phenylalanine ammonia-lyase and chalcone synthase catalyze the first reaction of phenylpropanoid biosynthesis and the first reaction of a branch pathway specific for flavonoid-isoflavonoid biosynthesis, respectively. These enzymes are key control elements in the synthesis of kievitone, phaseollin, and related isoflavonoid-derived phytoalexins. RNA blot hybridization with 32P-labeled cDNA sequences was used to demonstrate marked accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs in excision-wounded hypocotyls of Phaseolus vulgaris L. (dwarf French bean) and during race-cultivar-specific interactions between hypocotyls of P. vulgaris and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant), early concomitant accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs, localized mainly but not entirely in tissue adjacent to the site of infection, was observed prior to the onset of phytoalexin accumulation and expression of localized, hypersensitive resistance. In contrast, in a compatible interaction (host susceptible) there was no early accumulation of these transcripts; instead, there was a delayed widespread response associated with phytoalexin accumulation during attempted lesion limitation. Two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in vitro by translation of isolated polysomal RNA demonstrated stimulation of the synthesis of characteristic sets of phenylalanine ammonia-lyase and chalcone synthase isopolypeptides in directly infected tissue and distant, hitherto uninfected tissue in both compatible and incompatible interactions. Our data show that specific accumulation of plant defense gene transcripts is a key early component in the sequence of events leading to expression of defense responses in wounded tissue and in infected tissue during race-cultivar-specific interactions and that an elicitation signal is transmitted intercellularly in response to infection.

Treatment of cell suspension cultures of French bean (Phaseolus vulgaris) with polysaccharide elicitor molecules from cell walls of the anthracnose fungus, Colletotrichum lindemuthianum, results in the rapid accumulation of isoflavonoid phytoalexins, deposition of wall-bound phenolic compounds and synthesis of hydroxyproline-rich glycoproteins. These changes are dependent upon a highly selective induction of gene products, including the enzymes L-phenylalanine ammonia-lyase, cytochrome P450-dependent cinnamic acid 4-hydroxylase, chalcone synthase, chalcone isomerase, prolyl hydroxylase and protein: arabinosyl transferase. Use of in vivo labelling, in vitro translation and RNA blot hybridization techniques has shown that these elicitormediated changes arise from rapid but transient induction of enzyme synthesis, resulting from the accumulation of specific mRNAs. Similar phenomena are observed in bean hypocotyls at the onset of phytoalexin synthesis in response to infection by incompatible and compatible races of C. lindemuthianum. In bean, both L-phenylalanine ammonia-lyase and chalcone synthase are encoded by multigene families and, at the protein level, both exhibit subunit and intact enzyme polymorphism. A number of less than full-length phenylalanine ammonialyase copy DNAs containing identical open reading frames have been produced from mRNA from elicitor-induced bean cells. Analysis of phenylalanine ammonia-lyase genomic clones predicts the presence of enzyme forms of differing amino acid sequence. In cultured bean cells, elicitor differentially induces the two apparent phenylalanine ammonia-lyase iso-forms with the lowest Km values. In addition to transcriptional control of the appearance of specific gene products, post-translational processes may result in increased subunit polymorphism for phenylalanine ammonia-lyase, and in the activation of chalcone isomerase. Changes in endogenous phenylpropanoid intermediate pools may signal the rapid removal of phenylalanine ammonia-lyase activity, in addition to exerting less specific inhibitory effects on the formation and/or activity of the mRNAs encoding phenylalanine ammonia-lyase and other phytoalexin biosynthetic enzymes.

The pattern of messenger RNA synthesis in suspension-cultured bean cells ( Phaseolus vulgaris L.) was analyzed by blot hybridization and in vitro translation of newly synthesized messenger RNA. The RNA was separated from preexisting RNA by organomercurial affinity chromatography after in vivo labeling with 4-thiouridine. The elicitor induced the synthesis of messenger RNA's encoding phenylalanine ammonia-lyase, chalcone synthase, and chalcone isomerase, three enzymes of phenylpropanoid metabolism involved in the synthesis of isoflavonoidderived phytoalexins. This is part of a rapid and extensive change in the pattern of messenger RNA synthesis directing production of a set of proteins associated with expression of disease resistance.

Changes in the rates of synthesis of three enzymes of phenyl‐propanoid biosynthesis in Phaseolus vulgaris L. (dwarf French bean) have been investigated by immunoprecipitation of [35S]methionine‐labeled enzyme subunits with mono‐specific antisera. Elicitor causes marked, rapid but transient co‐ordinated increases in the rate of synthesis of phenyl‐alanine ammonia‐lyase, chalcone synthase and chalcone isomerase concomitant with the phase of rapid increase in enzyme activity at the onset of accumulation of phenyl‐propanoid‐derived phytoalexin antibiotics in suspension cultures of P. vulgaris. Co‐ordinate induction of enzyme synthesis is also observed in hypocotyl tissue during race:cultivar‐specific interactions with Colletotrichum lindemuthianum, causal agent of anthracnose. In an incompatible interaction (host resistant) there are early increases apparently localized to the initial site of infection prior to the onset of phytoalexin accumulation and expression of hypersensitive resistance. In contrast, in a compatible interaction (host susceptible) there is no induction of synthesis in the early stages of infection, but a delayed widespread response at the onset of lesion formation associated with attempted lesion limitation. It is concluded that expression of the phytoalexin defense response in biologically stressed cells of P. vulgaris characteristically involves co‐ordinate induction of synthesis of phytoalexin biosynthetic enzymes.

DNAs complementary to poly(A)(+) RNA present in elicitor-treated cells of Phaseolus vulgaris L. were inserted into pBR325 and used to transform Escherichia coli strain JA221. A clone was identified that contained sequences complementary to mRNA encoding chalcone synthase, a regulatory enzyme of phenylpropanoid biosynthesis, which catalyzes the first reaction of a branch pathway specific to flavonoid and isoflavonoid biosynthesis. Rapid, marked but transient increases in chalcone synthase mRNA in response to elicitor treatment were observed by RNA blot hybridization with (32)P-labeled chalcone synthase cDNA sequences. Induction of chalcone synthase mRNA governs the rate of enzyme synthesis throughout the phase of rapid increase in enzyme activity at the onset of accumulation of isoflavonoid-derived phytoalexins. The data are consistent with the hypothesis that elicitor causes a rapid transient stimulation of transcription of chalcone synthase gene(s) as an early event in the expression of the phytoalexin defense response.