Elicitor Molecules Research Articles

The Relationship Between Pathogen-induced Systemic Resistance (ISR) and Multigenic (horizontal) Resistance in Plants

Plants have developed mechanisms to successfully co-exist in the presence of pathogenic organisms. Some interactions between plants and pathogens are based on recognition of specific elicitor molecules from avirulent pathogen races (avr gene products), which is described in the gene-for-gene resistance theory. Another type of resistance, multigenic (horizontal) resistance, is a less well-studied phenomenon that depends upon multiple genes in the plant host. All plants possess resistance mechamisms which can be induced upon pre-treatment of plants with a variety of organisms or compounds. This general phenomenon is known as induced systemic resistance (ISR). At least in some plant species, ISR depends on the timely accumulation of multiple gene products, such as hydrolytic enzymes, peroxidases or other gene products related to plant defences. The pre-treatment of plants with an inducing organism or compound appears to incite the plant to mount an effective defense response upon subsequent encounters with pathogens, converting what would have been a compatible interaction to an incompatible one. Our studies in three plant–pathogen systems clearly document that multigenic-resistant plants constitutively express specific isozymes of hydrolytic enzymes that release cell wall elicitors, which in turn may activate other defense mechanisms. ISR induces constitutive accumulation of these and other gene products prior to challenge. ISR is known to function against multiple organisms, and there is no specificity observed in the accumulation patterns of defense-related gene products when ISR is induced. It is therefore hypothesized that the constitutive accumulation of specific isozymes of hydrolytic enzymes, or other defense related gene products, is an integral part of both multigenic resistance and the phenomenon of ISR. Further, plants in which ISR has been activated appear to move from a latent resistance state to one in which a multigenic, non-specific form of resistance is active.

ChemInform Abstract: Synthesis of the Syributins and Formal Total Synthesis of Syringolide 1.

A synthesis of syributins 1 (5) and 2 (6) from D-glyceraldehyde acetonide (12) and furans (14) and (15) is described. Compounds (5) and (6) are cometabolites of the novel non-proteinaceous C-glycosidic elicitor molecules the syringolides 1 (1) and 2 (2). Under a variety of acidic conditions, the intermediate butenolides (9) and (10) provided syributins 1 (5) and 2 (6). Similar results have been reported by Honda and coworkers who converted butenolide (20) into syringolide 1 (1). Since compound (20) was also synthesized by the present new route, a formal synthesis of syringolide 1 (1) has been achieved.

Protein kinases induced by osmotic stresses and elicitor molecules in tobacco cell suspensions: two crossroad MAP kinases and one osmoregulation-specific protein kinase

Two protein kinases displaying mitogen-activated protein kinase (MAPK) properties are activated both by an hypoosmotic stress and by oligogalacturonides in tobacco cell suspensions [Cazalé et al. (1999) Plant J. 19, 297–307]. Using specific antibodies, they were identified as the salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK). The SIPK was also activated by an hyperosmotic stress, indicating that the same kinase may play a role both in hypo- and hyperosmotic signalling pathways, in addition to its involvement in the transduction of elicitor signals. Using immunoprecipitation followed by two-dimensional in-gel kinase assay, three molecular forms of the SIPK were observed, suggesting that additional modifications of the activated kinase may occur. In contrast to WIPK and SIPK, which are located at the crossroad of several transduction pathways initiated by elicitor or osmotic stimuli, a 44 kDa kinase, that would not belong to the MAPK family, appeared more specific to osmotic stress.

In situ detection of a fungal glycoprotein-elicitor in stem rust-infected susceptible and resistant wheat using immunogold electron microscopy

Immunoelectron microscopy (IEM) was used to analyze the compatible and incompatible host-pathogen interaction between the obligate, biotroph stem rust (Puccinia graminis f.sp. tritici; Pgt) and primary leaves of wheat (Triticum aestivum L.). The investigation was focused on the subcellular localization of a fungal elicitor glycoprotein of stem rust (Pgt-elicitor). Uredospores as well as fungal infection structures of stem rust on wheat leaves were probed with a specific monoclonal antibody, in order to determine the in situ distribution pattern of the antigen. Binding to the anti-elicitor antibody was observed over the cell wall and the germ pore of germinating uredospores. Immunogold staining was found over the infection structures of stem rust within the wheat leaf tissue of both the compatible and incompatible plant-pathogen interaction. Distinct cell wall layers of the intercellular mycelium, of the haustorial mother cells, as well as of the haustoria were clearly labeled. Gold particles were also detected over the intercellular space and the extrahaustorial matrix in between the extrahaustorial membrane and the haustorial cell wall which indicated a release of elicitor molecules from the fungal cell wall. No labeling was observed over the host cell cytoplasm of the compatible and incompatible interaction, respectively. The immunocytochemical detection of elicitor epitopes over the hyphal cell walls of in vitro grown axenic cultures of P. graminis f.sp. tritici confirmed the occurrence of elicitor molecules in young hyphal material. Elicitor molecules were released by the hyphae of axenic cultures of stem rust in vitro.

Use of Molecular Cytology to Study the Structure and Biology of Phytopathogenic and Mycorrhizal Fungi

Molecular cytology, that is, thein situlocalization of selected molecules by labeling with lectins, enzymes, and antibodies, has made a major contribution to our understanding of the structure and biology of fungi and is increasingly becoming an integral part of molecular, genetic, and biochemical studies. The review presented in this article concentrates on recent advances in the application of molecular cytology in investigations of the structure and biology of phytopathogenic and mycorrhizal fungi and of the molecular basis of their infection of host plants. The review examines details of the structure and molecular composition of fungal cell walls revealed by lectin, enzyme, and antibody labeling. Molecular composition is shown to vary according to taxonomic relationships and as a reflection of differences in cell type, location within the cell, and within thickness of the wall. Sites of synthesis and secretion of wall components are also detected through the labeling of selected molecules.In situlabeling of cytoskeletal elements, microtubules and actin microfilaments, has provided much information on the role of these elements in tip growth, organelle distribution, and spore development. Molecular cytology, particularly through the generation of monoclonal antibodies, has also revealed new and exciting information on specialized infection structures formed by fungi in order to infect host plants. The sites of storage and secretion of adhesives and degradative enzymes have been documented, as have surface specializations that may be associated with avoidance of detection by the host. In addition,in situlabeling with enzymes and antibodies has aided studies of the host defense response, including mechanisms of detection of fungal elicitor molecules, changes in wall composition, and the secretion of antifungal compounds. With the increasing production of monoclonal antibodies to fungal molecules, molecular cytology promises to continue to make an important contribution to our understanding of fungal cell structure and function in the future.

Increase of defense gene transcripts by cytoplasmic acidification in tobacco cell suspensions

Cytoplasmic acidification has been previously characterized as a common intracellular response of tobacco cells to elicitors (Y. Mathieu et al. 1996, Planta 199: 416–424). The possibility that cytosolic protons may activate plant defense reactions through the increased mRNA transcript levels of specific genes was investigated in suspension-cultured tobacco (Nicotiana tabacum L. cv. Xanthi) cells. Transcript levels of phenylalanine ammonia-lyase and 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the first enzymes of phenylpropanoid and tobacco phytoalexin pathways, respectively, were quantified in a variety of conditions leading to intracellular acidification. Three artificial ways of inducing intracellular acidification, acid loading, inorganic phosphate uptake and inhibition of the plasma-membrane ATPase were able to increase transcript levels of the two genes in certain conditions. The combination of two parameters of the intracellular pH decrease, intensity and duration, was shown to be crucial for the induction of the two kinds of transcript. In particular, experiments using addition of two signals showed that a cytosolic acidification that was too long and/or too intense no longer allowed the increase in mRNA transcripts to occur. Together, the results indicate that cytosolic protons take part in the accumulation of the two defense gene transcripts and may be considered as one of the second messengers involved in the transduction of elicitor molecules in tobacco cells.

PREPARAÇÕES DE Saccharomyces cerevisiae ELICITORAS DE FITOALEXINAS EM MESOCÓTILOS DE SORGO

A levedura Saccharomyces cerevisiae estimula o acúmulo de fitoalexinas e tem potencial para ser utilizada como agente de controle alternativo no tratamento de doenças fúngicas em sorgo. São descritos aqui os procedimentos iniciais para a purificação de elicitores de fitoalexinas em sorgo, os quais são extraídos das células da levedura S. cerevisiae por autoclavagem, indicando serem termoestáveis. Após precipitacão com etanol, em concentrações finais de 50 e 80%, as moléculas elicitoras permanecem em solução. O acúmulo de fitoalexinas nos mesocótilos é mais elevado quanto maiores os teores de proteínas das amostras elicitoras.

The oxidative burst protects plants against pathogen attack: Mechanism and role as an emergency signal for plant bio-defence — a review

Various aspects, mechanisms and functions of the oxidative burst with generation of O 2 − superoxide anions in plant cells, which is stimulated by active defence-inducing agents such as fungal infection or elicitor treatment, were reviewed mainly on the basis of experimental evidence obtained in a system of Solanaceae plants and Phytophthora spp. The oxidative burst may be due to an O 2 −-generating NADPH oxidase in the plasma membrane, which is activated with combinations of cytosolic proteins, Ca 2+, calmodulin and protein kinase, following stimulation by elicitor molecules. The oxidative burst may play the role of an internal emergency signal for induction of the metabolic cascade for active defence.

Specificity in the immobilisation of cell wall proteins in response to different elicitor molecules in suspension-cultured cells of French bean (Phaseolus vulgaris L.)

A characteristic of the defence response is the immobilisation of wall proteins possibly through the formation of covalent cross-links and the subsequent barrier formation against pathogens. A requirement for this is the generation of active oxygen species, particularly hydrogen peroxide. In the present work, we examine in depth the requirement for H2O2 and the specificity of the immobilisation with respect to particular wall proteins. Salt-extractable wall proteins were analysed for hydroxyproline content and the subset of proteins with this post-translational modification was found to be small. About 50 proteins were found to be easily salt-extractable and in response to elicitor treatment about 5 were found to be specifically immobilised. Immobilisation was very rapid and completed within 15 min after elicitation, and dependent upon the type of elicitor and the intensity of the production of active oxygen species. N-terminal sequencing and amino acid analysis revealed that, apart from one polypeptide, all immobilised proteins were (hydroxy)proline-containing glycoproteins with O-linked oligosaccharide side chains. In contrast, N-linked glycoproteins were not immobilised. N-terminal protein sequencing revealed the immobilised HRGPs to be novel, but both extensin and PRP-like. Implications of these findings for both pathogenic and symbiotic processes are also discussed.

1H and 15N resonance assignment and secondary structure of capsicein, an alpha-elicitin, determined by three-dimensional heteronuclear NMR.

The backbone 1H and 15N resonance assignments and solution secondary structure determination of capsicein, a protein of 98 residues with a molecular mass of 10161 Da, are presented. Capsicein belongs to the elicitin family, elicitor molecules having toxic and signaling properties that are secreted by Phytophthora fungi, responsible for the incompatible hypersensitive reaction of diverse plant species leading to resistance against fungal or bacterial plant pathogens. The protein was uniformly labeled with 15N to overcome spectral overlap of the proton resonances. A combination of 3D HOHAHA-HMQC and 3D NOESY-HMOC experiments allowed the identification of spin systems with through-bond correlations, which were in turn correlated by through-space connections. The sequential assignment was obtained for main- and side-chain resonances and led to the identification of all secondary structures. A 3D HMQC-NOESY-HMQC experiment was performed which characterized the NH(i)-NH(i+1) connections specific to alpha-helical structures. This proved particularly useful for the assignment of degenerate amide protons of successive residues in alpha-helical structures. The data show five alpha-helical regions comprising residues 5-18, 26-33, 44-58, 59-67, and 86-98 and a two-stranded antiparallel beta-sheet involving residues 70-75 and 80-85, packed around a hydrophobic core grouping all of the aromatic residues. The C-terminal secondary structure motifs of capsicein evoke phospholipase structural features, which suggests that elicitins might interact with the lipidic molecules of the plasma membrane.

Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells.

GTP-binding proteins have been shown to serve as second messengers in the transduction of hormone signals across animal cell plasma membranes. We present here three lines of evidence to demonstrate that GTP-binding proteins are also involved in the elicitation of the defense response of cultured soybean cells. First, the antigen-binding fragment (Fab) of an antibody that specifically recognizes GTP-binding proteins in plants and animals was delivered into soybean cells using a non-destructive biotin-mediated delivery technique developed previously. Internalization of this Fab enhanced up to 10-fold the rapid oxidative burst induced by elicitor molecules, whereas internalization of its heat-denatured counterpart or unrelated proteins had no effect. Because the antibody recognizes a protein of molecular mass approximately 45 kDa in soybean cell membranes that is protected from ADP-ribosylation by GTP gamma S (guanosine 5'-O-(thiotriphosphate), we propose the 45-kDa GTP-binding protein is responsible for these effects. Second, mastoparan, a specific activator of GTP-binding proteins, was shown to induce the defense-related oxidative burst in the absence of elicitor stimulation, thus mimicking an activated receptor as it is thought to do in mammalian systems. Finally, but admittedly less convincing, the A subunit of cholera toxin, an activator of certain stimulatory GTP-binding proteins (Gs), was found to weakly enhance the conventional elicitor-induced oxidative burst. Taken together, these data argue for the involvement of GTP-binding proteins in elicitor signal transduction in soybean cells.

Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures.

To deter pathogenic microorganisms and herbivores, plants have developed an inducible chemical defense system. It is known that the induced synthesis of low molecular weight compounds can be provoked by exposing cultured cells to fungal cell wall fragments. In this study we show that endogenous jasmonic acid and its methyl ester accumulate rapidly and transiently after treatment of plant cell suspension cultures of Rauvolfia canescens and Eschscholtzia californica with a yeast elicitor. Thirty-six plant species tested in cell suspension culture could be elicited with respect to the accumulation of secondary metabolites by exogenously supplied methyl jasmonate. Addition of methyl jasmonate initiates de novo transcription of genes, such as phenylalanine ammonia lyase, that are known to be involved in the chemical defense mechanisms of plants. These data demonstrate the integral role of jasmonic acid and its derivatives in the intracellular signal cascade that begins with interaction of an elicitor molecule with the plant cell surface and results, ultimately, in the accumulation of secondary compounds.

Endocytosis in plants

Endocytosis in animal cells has been heavily documented. Both fluid‐phase and receptor‐mediated modes of uptake have been frequently studied, and the endocytic pathway is well defined. This contrasts markedly with the situation in plants where our knowledge of this process is still rudimentary. Partly responsible for this situation has been the view, widely held among plant physiologists, that because of turgor, endocytosis cannot occur in plant cells. As discussed below, the case against endocytosis is no longer water‐tight.Endocytosis is a fact in protoplasts. It can be demonstrated with electron‐dense tracers as well as with membrane impermeant dye Lucifer Yellow CH. The latter has also been used with success on both suspension‐cultured and tissue cells of higher plants, suggesting that fluid‐phase endocytosis is also a feature of cells with walls. Through the application of fluorescently labelled elicitor molecules, which specifically bind to the cell surface of suspension‐cultured cells, it has also been possible to provide convincing evidence for the operation of receptor‐mediated endocytosis in plants. A number of studies on protoplasts and cells clearly indicate that endocytosis in plants can be mediated by coated pits in the plasma membrane. At least one of the organelles that lie on the endocytic pathway in plants has a structurally similar counterpart in animal cells: the multivesicular body. The first recipient of internalized molecules is the partially coated reticulum, although its relationship to the Golgi apparatus and Golgi function remain to be clarified. The final target for endocytosis in plants appears to be the vacuole.

Tobacco mosaic virus coat protein: An elicitor of the hypersensitive reaction but not required for the development of mosaic symptoms in Nicotiana sylvestris

Specific nucleotide changes in the coat protein gene of tobacco mosaic virus (TMV) have been identified as responsible for the induction of the hypersensitive reaction (HR) in Nicotiana sylvestris. Each of these nucleotide changes resulted in amino acid substitutions in the coat protein. To determine if the altered viral RNA or the altered protein acted directly to elicit the HR, the coat protein translational starts were removed from full-length cDNA clones of the HR-inducing mutant TMV 25 and the systemically infecting TMV U1 strain. Infectious transcripts of these altered genomes failed to induce HR in inoculated leaves of N. sylvestris. These free-RNA mutants moved poorly out of inoculated leaves and produced a systemic mosaic symptom 9 to 12 weeks after inoculation. Infectious viral RNA, from both mutants, was recovered from inoculated and systemic mosaic leaves. Western blot analysis of both inoculated and noninoculated leaves revealed the presence of TMV-encoded 126-kDa protein and the absence of coat protein for both mutants. This study demonstrates that the coat protein of TMV 25 is an elicitor molecule responsible for the induction of HR in N. sylvestris and that the TMV coat protein is not required for the development of systemic mosaic symptoms.

Elicitor induction of the synthesis of a novel lectin-like arabinosylated hydroxyproline-rich glycoprotein in suspension cultures of Phaseolus vulgaris L.

A novel lectin-like glycoprotein which accumulates in response to fungal elicitor action has been characterised in endomembranes from suspension cultures of French bean (Phaseolus vulgaris L.). The lectin, which has specificity towards N-acetylglucosamine oligomers, consists of a polypeptide of apparent molecular weight (Mr) 31 000 which is rich in glycine and contains 6.7% hydroxyproline O-linked to arabinose-containing oligosaccharides to give a glycoprotein of Mr 42500. A dual-labelling technique has been used to identify changes in the synthesis of the glycoprotein in cells exposed to fungal elicitor molecules. Thus, incorporation of [(14)C]proline into membranes in vivo and of [1(-3)H]arabinose from uridine 5'-diphosphate [1(-3)H]arabinose in vitro and analysis by isoelectric focussing-polyacrylamide gel electrophoresis gave absolute correspondence of the labelled isoform of the glycoprotein. Having established the absence of contaminating polypeptides, subsequent analysis of microsomal fractions bysodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the peak of sythesis of the Mr-42500 glycoprotein occurred 4 h after the addition of fungal elicitor. The changes in the level of incorporation into the glycoprotein monomers were concomitant with increases in the activity of prolyl hydroxylase (EC 1.14.11.2)Incorporation of [(14)C]proline and its subsequent post-translational modification to hydroxyproline in microsomal polypeptides was followed by rapid transfer into the wall with an average t 1/2 of about 7 min. The Mr-42500 glycoprotein was rapidly transferred out of the endomembrane fraction with a t 1/2 of 2 min and could be detected in wall fractions where it became progressively less extractable. The glycoprotein, which clearly differs from bean extensin, accounts for up to 40% of the hydroxyproline newly exported in response to elicitor action. The lectin, which resembles those found in the Solanaceae and which is coinduced with enzymes of phytoalexin synthesis, may play some role in disease resistance.

Rapid changes in gene expression in response to microbial elicitation

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 K m 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 PHYTOALEXIN RESPONSE: ELICITATION, SIGNALLING AND CONTROL OF HOST GENE EXPRESSION

Summary1. Induced resistance of plants to incompatible races of their microbial pathogens is often characterized by rapid cell death (the hypersensitive response) and the accumulation of low‐Mr antimicrobial compounds termed phytoalexins. There is much indirect evidence to support a major role for phytoalexin induction as an event determining host resistance, and genetical analysis of race‐specific pathogen‐host interactions predicts that induced resistance occurs via interaction between a pathogen avirulence gene product and a host resistance gene product. Elicitors (inducing agents) of phytoalexin accumulation are possible candidates for the active products directly or indirectly resulting from the expression of microbial avirulence genes. The present review considers the process of phytoalexin induction, at the molecular level, from the fungal elicitor to the early changes in host gene expression associated with its action.2. A number of microbial polysaccharides, glycoproteins, pectic enzymes, peptides and fatty acids have potent elicitor activity.3. The results of studies on the purification and characterization of microbial elicitors depend on the nature of the bioassay used. Methods for elicitor extraction may lead to artifacts or loss of race specificity.4. Elicitors isolated from plant pathogens may be race‐specific or race‐non‐specific. In cases where only race‐non‐specific elicitors can be shown, race‐specific induction of phytoalexins may result from the action of enhancer or suppressor molecules. Fungal glucans have been proposed as candidates to act in both these roles.5. Molecular genetic approaches to the identification of phytopathogenic bacterial avirulence genes may help to prove or disprove the role of elicitors of the phytoalexin response as agents responsible for the induction of host resistance. Similar analyses, involving genetic transformation, should soon be possible for phytopathogenic fungi.6. Studies on the nature of host receptors for microbial elicitors are still in their infancy. Such receptors are probably localized in the plant plasma membrane, and elicitation results in often striking changes in host membrane properties.7. Cyclic 3′,5′‐adenosine monophosphate and polyamines do not appear to act in plants as intracellular transducers of the phytoalexin response. Interest is now being shown in a possible role for calcium in intracellular signalling.8. Plant cells contain endogenous elicitor molecules whose synthesis or release may play a role in the intercellular transmission of the phytoalexin response. The main candidates for endogenous elicitors are pectic fragments of the host cell wall, although no direct evidence for their involvement in plant‐pathogen interactions is available. Pectic fragments may act as synergists in parallel with, rather than as couplers in series with, microbial elicitors. The role of ethylene as a response coupler for induced resistance phenomena is difficult to assess.9. Some elicitors may themselves move to the host cell nucleus, although whether this is their effective site of action remains unclear.10. Induction of the phytoalexin response is associated with specific changes in host gene expression related to the selective induction of new mRNA species and enzyme activities.11. Progress is now being made in the characterization of enzymes specific for phytoalexin biosynthesis. Enzyme induction and/or infection appears to involve rapid modulation of gene transcription, although some post‐translational events may also be involved in determining induction patterns.12. Work is now commencing on the sequencing of the genes encoding elicitor‐inducible enzymes. Multigene families have been identified for phenylalanine ammonia‐lyase and chalcone synthase.13. Phytoalexin accumulation may be accompanied by the rapid induction of ethylene biosynthesis and the expression of genes encoding activities involved in the synthesis of hydroxyproline‐rich glycoproteins.14. Pectic endogenous elicitors of phytoalexin accumulation are also active as inducers of systemic proteinase inhibitor synthesis in some solanaceous species.15. Results of studies on the mechanisms of signal transmission and differential gene expression in relation to active defence reactions should be of relevance to many other aspects of the plant's response to environmental stimuli at the physiological, biochemical and molecular genetical levels.

Experimental Studies of Haustorium Initiation and Early Development in Agalinis purpurea (L.) Raf. (Scrophulariaceae)

In parasitic angiosperms the haustorium, an organ specialized for attachment and penetration of host tissue, functions in the transport of water and nutrients from the host to the parasite. In Agalinis purpurea (L.) Raf. (Scrophulariaceae) these organs are initiated laterally along its roots, opposite a primary xylem pole. Analyses of haustoria distribution and cellular root profiles show that the portion of the root which is most sensitive to haustorial elicitor molecules is the area distal to the zone of elongation and near the root meristem. Sectioned material supports this finding and, further, indicates that the cells which are the first to respond to haustorial elicitors are located in the inner cortex. Haustoria develop rapidly in response to a host root or to isolated chemical elicitors (xenognosins) normally contained in host root exudate. By 6 hr, vacuolation and radial cellular enlargement are observed in the cortex, and a lateral swelling along the root is visible. By 12 hr, cells of the epidermis divide anticlinally to establish a group of densely cytoplasmic cells at the apex of the haustorial swelling. Accompanying these divisions is the differentiation of specialized hair cells which elongate from epidermal cells flanking the presumptive haustorial apex. Next, the internal, radially enlarged cortical cells divide periclinally. Periclinal divisions are subsequently initiated in the pericycle as early as 18 hr post-induction. Cellular division and enlargement continue so that by 24–36 hr a mature pre-contact haustorium is formed. There is a reduction in root elongation concomitant with haustorial initiation. Depending upon the number of haustoria produced, elongation typically returns to the preinduction level within 2 or 3 days.

EXPERIMENTAL STUDIES OF HAUSTORIUM INITIATION AND EARLY DEVELOPMENT IN AGALINIS PURPUREA (L.) RAF. (SCROPHULARIACEAE)

In parasitic angiosperms the haustorium, an organ specialized for attachment and penetration of host tissue, functions in the transport of water and nutrients from the host to the parasite. In Agalinis purpurea (L.) Raf. (Scrophulariaceae) these organs are initiated laterally along its roots, opposite a primary xylem pole. Analyses of haustoria distribution and cellular root profiles show that the portion of the root which is most sensitive to haustorial elicitor molecules is the area distal to the zone of elongation and near the root meristem. Sectioned material supports this finding and, further, indicates that the cells which are the first to respond to haustorial elicitors are located in the inner cortex. Haustoria develop rapidly in response to a host root or to isolated chemical elicitors (xenognosins) normally contained in host root exudate. By 6 hr, vacuolation and radial cellular enlargement are observed in the cortex, and a lateral swelling along the root is visible. By 12 hr, cells of the epidermis divide anticlinally to establish a group of densely cytoplasmic cells at the apex of the haustorial swelling. Accompanying these divisions is the differentiation of specialized hair cells which elongate from epidermal cells flanking the presumptive haustorial apex. Next, the internal, radially enlarged cortical cells divide periclinally. Periclinal divisions are subsequently initiated in the pericycle as early as 18 hr post‐induction. Cellular division and enlargement continue so that by 24–36 hr a mature pre‐contact haustorium is formed. There is a reduction in root elongation concomitant with haustorial initiation. Depending upon the number of haustoria produced, elongation typically returns to the preinduction level within 2 or 3 days.

Response of tomato to leaf injection with conidia of virulent and avirulent races of Cladosporium fulvum

Injection of tomato leaflets of various cultivars with conidia of various races of Cladosporium fulvum produced the expected disease reactions. In all interactions, histological study of leaflets sampled up to 6 or 8 days post-inoculation revealed localized development of wal appositions (composed of aniline blue fluorescent material, most likely callose) in those host cells in direct contact with a conidium or a young germ tube. In compatible interactions (Potentate, no known Cf gene, with race 1, 7 or 12; Vinequeen, Cf genes 2 and 4, with race 12) and a delayed incompatible interaction (Stirling Castle, gene Cf 1, with race 7), germ tubes eliciting wall apposition formation often continued to develop, usually by producing a new branch which failed to elicit a visible host response. In incompatible interactions (Vinequeen with race 1 or 7), germ tubes either did not develop further on encountering the wall apposition response or they evoked a response in the next cells contacted and ceased growth. In vitro, the outer surface of germ tubes was readily stained with fluorescein labelled concanavalin A, supporting the possibility that previously described non-specific glycopeptide elicitor molecules are localized on the surface. This leaf-injection method may be useful in studies concerning the molecular interactions of C. fulvum and tomato.