Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. II. Effect of the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis

We have studied the effect of a change in the endogenous hormone equilibria on the competence of tomato (Lycopersicon esculentum) cells to defend themselves against the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Calluses from cvs Davis and Red River, respectively resistant and susceptible to Fusarium and transgenic for an auxin- or cytokinin-synthesizing gene from Agrobacterium tumefaciens, were used. The integration of Agrobacterium hormone-related genes into susceptible cv Red River can bring the activation of defense processes to a stable competence as assessed by the inhibition of mycelial growth in dual culture and gem-tube elongation of Fusarium conidia, the determination of callose contents, peroxidase induction and ion leakage in the presence of fusaric acid. This is particularly true when the transformation results in a change of phytohormone equilibria towards an higher cytokin in concentration. On the contrary, in resistant cv Davis the inhibition of both fungal growth in dual culture and conidia germination is higher when the hormone balance is modified in favour of the auxins. No significant effect was observed for ion leakage and peroxidase induction, probably because of a constitutive overproduction of cytokinins in Davis cells.     

The in vitro physiological phenotype of tomato resistance to Fusarium oxysporum f. sp. lycopersici

Summary With the aim of dissecting host-parasite interaction processes in the system Lycopersicon aesculentum-Fusarium oxysporum f. sp. lycopersici we have isolated plant cell mutants having single-step alterations in their defense response. A previous analysis of the physiological phenotypes of mutant cell clones suggested that recognition is the crucial event for active defence, and that polysaccharide content, fungal growth inhibition, peroxidase induction in in vitro dual culture and ion leakage induced by cultural filtrates of the pathogen can be markers of resistance. In this paper we present the results of a similar analysis carried out on cell cultures from one susceptible (Red River), one tolerant (UC 105) and three resistant (Davis UC 82, Heinz, UC 90) tomato cultivars. Our data confirm that the differences in the parameters considered are correlated with resistance versus susceptibility in vivo. Therefore, these parameters can be used for early screening in selection programmes. These data, together with those obtained on isolated cell mutants, suggest that the selection in vitro for altered fungal recognition and/or polysaccharide or callose content may lead to in vivo — resistant genotypes. The data are thoroughly discussed with particular attention paid to the importance of polysaccharides in active defense initiation.     

Fusicoccin activates pathogen-responsive gene expression independently of common resistance signalling pathways,but increases disease symptoms in<Emphasis Type="Italic"> Pseudomonas syringae</Emphasis>-infected tomato plants

Fusicoccin (FC), an activator of the plant plasma membrane H⁺-ATPase, induces several components of plant pathogen resistance responses, including defence hormone biosynthesis and pathogenesis-related (PR) gene expression. The mechanism by which these responses occur, and the effect they have on plant–pathogen interactions is unknown. Here, we show that PR gene expression in response to FC in tomato (Lycopersicon esculentum Mill.) plants does not strictly require the common defence hormones, salicylic acid, jasmonic acid and ethylene. We also show that FC-induced PR gene expression requires neither Ca²⁺ nor reactive oxygen species, typical early pathogen-resistance response signals. The possibility that PR gene expression is related to FC-induced dehydration stress is also discounted. Finally, we show that the defence responses elicited by FC in tomato are not sufficient to confer resistance to the bacterial pathogen Pseudomonas syringae. Rather, FC increases the rate and severity of disease symptom formation in an ethylene-dependent manner.Abbreviations DPI Diphenylene iodonium - EGTA Ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FC Fusicoccin - HR Hypersensitive response - INA 2,6-Dichloroisonicotinic acid - JA Jasmonic acid - PM Plasma membrane - ROS Reactive oxygen species - PR Pathogenesis-related - SA Salicylic acid     

Induced net Ca2+ uptake and callose biosynthesis in suspension-cultured plant cells

In suspension-cultured cells of Glycine max and Catharanthus roseus, marked callose synthesis can be induced by digitonin and chitosan. Leakage of a limited pool of electrolytes precedes callose formation, K⁺ representing the major cation lost. Poly-L-ornithine, as well as the ionophores A 23187 and ionomycin, also induces some callose synthesis but to a lesser extent. Digitonin increases the net uptake of Ca²⁺ from the external buffer with a time course parallel to callose synthesis but lagging behind the leakage of K⁺. Nifedipine partly blocks callose synthesis as well as the digitonin-induced increase in net Ca²⁺ uptake. Taken together, the data support the hypothesis that addition of the various substances might indirectly lead to membrane perturbation causing the common event of an increase in net Ca²⁺ uptake which results in callose deposition by a direct activition of the Ca²⁺-dependent and plasma-membane-located 1,3--glucan synthase.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases.

Summary Treatment of tomato seeds with ethyl methanesulphonate (EMS) followed by allyl alcohol selection of M₂ seeds has led to the identification of one plant (B15-1) heterozygous for an alcohol dehydrogenase (Adh) null mutation. Genetic analysis and expression studies indicated that the mutation corresponded to the structural gene of the Adh-1 locus on chromosome 4. Homozygous Adh-1 null mutants lacked ADH-1 activity in both pollen and seeds. Using an antiserum directed against ADH from Arabidopsis thaliana, which crossreacts with ADH-1 and ADH-2 proteins from tomato, no ADH-1 protein was detected in seeds of the null mutant. Northern blot analysis showed that Adh-1 mRNA was synthesized at wild-type levels in immature seeds of the null mutant, but dropped to 25% in mature seeds. Expression of the Adh-2 gene on chromosome 6 was unaffected. The potential use of the Adh-1 null mutant in selecting rare transposon insertion mutations in a cross with mutable Adh-1 ⁺ tomato lines is discussed.     

Enhanced rates of P700<Superscript>+</Superscript> dark-reduction in leaves of<Emphasis Type="Italic"> Cucumis sativus</Emphasis> L. photoinhibited at chilling temperature

The changes in electron transport within photosystem I (PSI) were studied in detached leaves of Cucumis sativus L. during the course of irradiation with moderate white light (300 mol photons m^–2 s^–1) at 4°C. When intact leaves were exposed to the combination of moderate light and low temperature, the amplitude of far-red light-induced P700 absorbance changes at 820 nm (A₈₂₀), a relative measure of PSI, progressively decreased as the light treatment time increased. Almost no oxidation of P700 was noticeable after 5 h. Methyl viologen accelerated the oxidation of P700 to a steady-state level and also increased the magnitudes of A₈₂₀ changes in photoinhibited leaves, reflecting the rapid removal of electrons from native carriers. Photoinhibition under moderate light and chilling temperature also accelerated the rate of P700⁺ reduction after far-red light excitation as the half-times of the two exponential components of P700⁺ decay curves decreased relative to the control ones. A detailed analysis of the kinetics of P700⁺ reduction using diuron alone or the combination of diuron and methyl viologen strongly favours an increased rate of electron donation from stromal reductants to PSI through the plastoquinone pool following photoinhibitory treatment. Importantly, the marked acceleration of P700⁺ re-reduction is the consequence of the irradiation of leaf segments at low temperature and not caused by chilling stress alone.Abbreviations A ₀ and A ₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - FR Far-red light - F _X , F _A , and F _B Iron–sulfur centers - MT Multiple-turnover flash - MV Methyl viologen - Ndh NAD(P)H-dehydrogenase - PQ Plastoquinone - PS Photosystem - P700 Reaction-center chlorophyll of PSI - ST Single-turnover flash     

Cytology of induced systemic resistance of tomato to Phytophthora infestans

The infection of tomato leaves by Phytophthora infestans was followed using cytological methods. Fungal ingress and plant reactions in untreated and induced resistant plants were studied. Systemic disease resistance was induced by a local pre-infection with the same fungus. Induction retarded fungal progress at the leaf surface, epidermis and in the mesophyll. The reduced numbers of germinated cysts indicate the presence of fungitoxic substances on the leaf surface of induced plants. Frequency of fungal penetration through the outer epidermal cell wall was reduced, but only in plants exhibiting a high level of induced resistance. Autofluor-escent material, indicating the presence of lignin-like substances, accumulated rapidly beneath some of the appressoria, but this plant response was similar in induced and non-induced plants. Staining with aniline blue indicated that callose deposition was not involved in induced resistance. Thus, none of the cytologically investigated plant reactions correlated with the reduced penetration frequency observed. In the mesophyll, however, the cytological picture corresponding to a hypersensitive reaction occurred more often in induced plants. It is concluded that reduction of disease severity by induction is the result of the combined action of several successive defence reactions.Dedicated to the memory of Professor H. Grisebach     

Accumulation and turnover of 2-tridecanone in Tetranychus urticae and its consequences for resistance of wild and cultivated tomatoes

In this study we assessed the dynamic changes of 2-tridecanone in a herbivorous mite (Tetranychus urticae) on tomato (Lycopersicon esculentum, cv. Moneymaker), a plant with methyl ketones in the tetracellular tips of the glandular trichomes (Type VI). We showed that spider mites accumulate 2-tridecanone when foraging on cultivated tomato. Thus, the rate of mite–trichome contact multiplied by the amount of toxin per trichome tip exceeded the relative rate of toxin turnover multiplied by the amount of toxin per mite. The relative rate of toxin turnover was estimated to be 1.1 per day on cucumber, a plant without this toxin. The amount per trichome tip varied from 0.33 ng for middle-leaf trichomes to 1.26 ng for main-stem trichomes. Hence, to achieve a static level of 2-tridecanone equal to 8–17 ng per mite – representing the level we found in mites on middle leaves – the rate of mite–trichome contact should be 26–57 per day. Because methyl ketone apparently accumulates in the spider mites on tomato, the rate of mite–trichome contact is probably higher than that. We expect the accumulation of ketones to occur especially on the stems of cultivated tomato, since this is the area most densely occupied with glandular hairs and because here the hairs have higher levels of the methyl ketones.Using dose–response relationships assessed earlier (Chatzivasileiadis and Sabelis, 1997, 1998), we estimated that the number of mite–trichome contacts causing 50% mortality per day is equal to 88 on a tomato stem, whereas it equals 70 for another strain of spider mites collected from cucumber. On wild tomato, L. hirsutum f. glabratum (PI 134417), just one to two contacts would suffice to cause 50% mortality per day. We suggest that methyl ketones from glandular hairs on tomato are an important mortality factor for spider mites on wild tomato and probably also on cultivated tomato.     

Callose deposition during gravitropism ofZea mays andPisum sativum and its inhibition by 2-deoxy-D-glucose

In etiolated corn (Zea mays L.) and etiolated pea (Pisum sativum L.) seedlings, a gravitropic stimulation induces the deposition of callose. In the corn coleoptiles this occurs within 5 min of gravity stimulation, and prior to the beginning of curvature. Both gravitropic curvature and callose deposition reach their maxima by 12 h. Within the first 2 h more callose is deposited on the upper (concave) side, but after 2–3 h, this deposition pattern is reversed. An inhibitor of protein glycosylation, 2-deoxy-d-glucose (DDG), inhibits callose production and considerably retards gravitropic bending in both species of plants. Mannose can relieve the inhibition of gravitropic bending by DDG. The pea mutant Ageotropum, which does not respond to gravity when etiolated, also fails to produce callose in response to a gravitic stimulus. These correlations indicate that callose deposition may be a biochemical component of gravitropism in plant shoots.Abbreviation DDG 2-deoxy-d-glucose     

Genetics of selection-induced mutations: I. uvrA,uvrB, uvrC,and uvrD are selection-induced specific mutator loci

Selection-induced mutations, sometimes called directed, adaptive, or Cairnsian mutations, are spontaneous mutations that occur as specific responses to environmental challenges, usually during periods of prolonged stress, and that occur more often when they are selectively advantageous than when they are selectively neutral. In this study I show that lesions in uvrA, uvrB, uvrC, or uvrD increase the mutation rate from trpA46 to trpA ⁺ by 10²– to 10⁴–fold during tryptophan starvation, but those same lesions do not affect random mutation rates in growing cells when tryptophan is present. The increased selection-induced mutation rates remain specific to the gene that is under selection in that no increase in the mutation rate from trpA46 to trpA ⁺ is detected during proline starvation.Evidence is presented showing that proline starvation produces a state of cellular stress which results in a burst of mutations from trpA46 to trpA ⁺ when proline-starved cells are plated onto medium lacking tryptophan but containing proline.These results are consistent with the hypermutable state model for selection-induced mutagenesis.     

A novel callose synthase from pollen tubes of Nicotiana

Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)--linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3--d-glucan 3--d-glucosyl transferase, EC 2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca²⁺ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (K _m 1.5–2.5 mM); it was activated by -glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca²⁺, and in the presence of 2 mM Ca²⁺ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (K _m 0.45 mM); it also required a -glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)--d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.Abbreviations Ara l-arabinose - CHAPS 3-[(3-cholamidopropyl)dimethylammonia]-1-propane sulphonic acid - DAP diphenylamine-aniline-phosphoric acid stain - Gal d-galactose - Glc d-glucose - Man d-mannose - Mes 2-(N-morpholino)ethane sulphonic acid - Rha d-rhamnose - Rib d-ribose - TFA trifluoroacetic acid - UDPGlc uridine-diphosphate glucose - Xyl d-xylose This research was supported by funds from a Special Research Centre of the Australian Research Council. H.S. was funded by a Melbourne University Postgraduate Scholarship and an Overseas Postgraduate Research Studentship; S.M.R. was supported by a Queen Elizabeth II Research Fellowship. We thank Bruce McGinness and Susan Mau for greenhouse assistance, and Deborah Delmer and Adrienne Clarke for advice and encouragement throughout this project.     

Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: transgenic plants with normal phenotypes harbor binary vector T-DNA,but no Ri-plasmid T-DNA

Summary Cultivated tomato was genetically transformed using two procedures. In the first procedure, punctured cotyledons were infected with disarmed Agrobacterium tumefaciens strain LBA4404 or with A. rhizogenes strain A4, each containing the binary vector pARC8. The chimeric neomycin phosphotransferase (NPT II) gene on pARC8 conferred on transformed plant cells the ability to grow on medium containing kanamycin. Transformation reproducible yielded kanamycin-resistant transformants in different tomato genotypes. NPT II activity was detected in transformed calli and in transgenic plants. All of these plants were phenotypically normal, fertile and set seeds. Using the second procedure, inverted cotyledons, we recovered transformed tomato plants from A. rhizogenes-induced hairy roots. In this case, all of the transgenic plants exhibited phenotypes similar to hairy root-derived plants reported for other species. Southern blot analysis on these plants revealed that the plant DNA hybridized with both probes representing pARC8-T-DNA, and the T-DNAs of the A4 Ri-plasmid. However, southern analysis on those phenotypically normal transgenic plants from the first procedure revealed that only the pARC8-T-DNA was present in the plant genome, thus indicating that the pARC8-T-DNA integrated into the plant genome independently of the pRi A4-T-DNA. Genetic analysis of these phenotypically normal transgenic plants for the kanamycin-resistance trait showed Mendelian ratios, 31 and 11, for selfed (R1) and in crossed progeny, respectively.     

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K⁺ up to 3.6 M. This creates a very large K⁺ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K⁺ but the residual K⁺ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K⁺ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K _m of between 1 to 2 mM. The driving force for K⁺ transport has been explored. Internal K⁺ concentration is not in equilibrium with m suggesting that K⁺ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K⁺ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K⁺ accumulation has not yet been clearly demonstrated. K⁺ transport in H. volcanii can be compared with K⁺ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium     

Growth of catalase A and catalase T deficient mutant strains of Saccharomyces cerevisiae on ethanol and oleic acid

The parental strain (A⁺T⁺) of Saccharomyces cerevisiae and mutants, deficient in catalase T (A⁺T^–), catalase A (A^–T⁺) or both catalases (A^–T^–), grew on ethanol and oleic acid with comparable doubling times. Specific activities of catalase were low in glucose- and ethanol-grown cells. In the two oleic acid-grown A⁺-strains (A⁺T⁺ and A⁺T^–) high catalase activities were found; catalase activity invariably remained low in the A^–T⁺ strain and was never detected in the A^–T^– strain. The levels of -oxidation enzymes in oleic acid-grown cells of the parental and all mutant strains were not significantly different. However, cytochrome C peroxidase activity had increased 8-fold in oleic acid grown A^– strains (A^–T⁺ and A^–T^–) compared to parental strain cells. The degree of peroxisomal proliferation was comparable among the different strains. Catalase A was shown to be located in peroxisomes. Catalase T is most probably cytosolic in nature and/or present in the periplasmic space.     

Race cultivar-specific differences in callose deposition in soybean roots following infection with Phytophthora megasperma f.sp. glycinea

Primary roots of soybean (Glycine max (L.), Merrill, cv. Harosoy 63) seedlings were inoculated with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea and total callose was determined at various times after inoculation. From 4 h onward, total callose was significantly higher in roots showing the resistant rather than the susceptible response. Local callose deposition in relation to location of fungal hyphae was determined in microtome sections by its specific fluorescence with sirofluor and was quantified on paper prints with an image-analysis system. Callose deposition, which occurs adjacent to hyphae, was found soon after inoculation (2, 3 and 4 h post inoculation) only in roots displaying the resistant response, and was also higher at 5 and 6 h after inoculation in these resistant roots than in susceptible roots. Early callose deposition in the incompatible root-fungus reaction could be a factor in resistance of soybean against P. megasperma.Abbreviation pi post inoculation     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.