Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. I. Selection from a susceptible cultivar for high and low polysaccharide content

Plant cell walls play a major role in the outcome of host-parasite interactions. Wall fragments released from the plant, and/or the fungal pathogen, can act respectively as endogenous and exogenous elicitors of the defence response, and other wall components, such as callose, lignin, or hydroxyproline-rich glycoproteins, can inhibit pathogen penetration and/or spreading. We have previously demonstrated that calli from tomato cultivars resistant in vivo to Fusarium oxysporum f.sp. lycopersici show a high amount of polysaccharides in vitro. The aim of the present work was to assess the possible role of polysaccharide content and/or synthetic capacity in determining the competence of plant cells for active defence. For this purpose, tomato cell clones with increased and decreased polysaccharide (FL⁺, FL^-) and callose (A⁺, A^-) content have been selected by means of specific stains as visual markers and tested for the effect of these changes on the extent of response to Fusarium. The analysis of several parameters known to be indicative of active defence (cell browning after elicitor treatment, peroxidase and -glucanase induction and inhibition of fungal growth in dual culture) clearly shows that FL⁺ and A⁺ clones have acquired an increased competence for the activation of defence response. The results are thoroughly discussed in terms of an evaluation of the relative importance of constitutive and/or inducible polysaccharide synthetic capacity for plant response to pathogens, and their possible regulation by plant physiological backgrounds.     

Selection of higher regenerative callus and change in isozyme pattern in rice (Oryza sativa L.)

Summary Calli were initiated from seedling roots in rice (Oryza sativa L. var. Tadukan) and subcultured at 45-day intervals on MS medium supplemented with 2 mg/l 2,4-D. Sectors of callus which differentiated shoot meristems (green spots) under the same 2,4-D concentration were selected from the calli subcultured 90 days after initiation. The selection was continued for about 2 years. Responses to 2,4-D between original and selected lines differed considerably, although differentiation was not generally seen in rice callus in the presence of 2 mg/l 2,4-D. After 180 days, calli of the selected line differentiated into numerous shoot-bud primordia and grew out new callus tissues under 2 mg/l 2,4-D concentration; the frequency of the differentiation exceeded 90%. On the other hand, no calli of non-selected line differentiated into shootbuds under 2 mg/l 2,4-D, and the frequency of the shootbud was only about 50% under lower 2,4-D concentration (0.1 mg/l). The pattern and activity of peroxidase isozyme varied markedly between calli of the selected and non-selected lines. First, two strong peroxidase bands which show fast mobility and one intermediate peroxidase band with slow mobility were detected only in the calli of selected line. Secondly, changes in band pattern of proteins separated by SDS-PAGE were observed. In the calli of selected line, there was a loss of the polypeptide bands with molecular weight of 24 and 42 K in the selected calli, but they were clearly present in the unselected line. The appearance of new peroxidase isozyme bands and loss of polypeptide bands, change in response to auxin and increased ability for shoot bud differentiation are closely correlated to each other.     

In vitro selection for 2,4-D tolerance in red clover

Summary In vitro, selection is a viable method of selecting herbicide-tolerant crops. This research was to evaluate in vitro selection techniques for enhancing 2,4-D [(2,4-dichlorophenoxy) acetic acid] tolerance in red clover (Trifolium pratense L.). In vivo and in vitro responses to 2,4-D of eight diverse red clover populations were correlated (r=0.77), justifying in vitro selection for 2,4-D tolerance. Suspension cultures of a red clover genotype capable of regeneration were plated onto agar-based nutrient media supplemented with 0.18 mM 2,4-D for selection experiments. After two cycles of selection, 16 2,4-D tolerant callus lines were identified based on visual growth assessment. These lines were evaluated for 2,4-D tolerance (based on 2,4-D content), using a 2,4-D bioassay procedure which consisted of placing selected callus tissue pieces on top of oat (Avena sativa L.) coleoptile or internode sections. The relative amount of 2,4-D in the callus tissue was estimated by the amount of oat section elongation after 24 h. Two of the more tolerant callus lines had 61% and 83% less 2,4-D in their tissues than the susceptible control tissue. These studies indicated that in vitro selection can enhance the levels of 2,4-D tolerance in red clover callus tissue.Florida Agricultural Experiment Station Journal Series No. 8943     

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Effect of auxins and ectomycorrhizal elicitors on wall-bound proteins and enzymes of spruce [Picea abies (L.) Karst.] cells

Summary Elicitors of the ectomycorrhizal fungus Hebeloma crustuliniforme and auxins (IAA, NAA and 2,4-D) were tested for their effects on apoplastic proteins and enzymes of suspension cultured cells of Picea abies (L.) Karst. The ectomycorrhizal elicitor increased the amount of some ionically wall-bound proteins (36, 28, 24, 21 kDa) and decreased the amount of others (61, 22 kDa). The elicitor triggered an H₂O₂ burst and enhanced the peroxidase (EC 1.11.1.7) activity of the Picea cells by increasing one of the two wall-bound peroxidase isoforms. Auxins significantly suppressed the elicitor induction of peroxidase but did not influence the elicitor-triggered H₂O₂ burst. The elicitors and auxin did not change the amount and the pattern of wall-bound invertase isoforms (EC 3.2.1.26) of spruce cells. However, auxin reduced the uptake of glucose by spruce cells and increased the acidification of the cell culture medium. Since Hebeloma lacks apoplastic invertase as well as a sucrose uptake system, utilization of plant-derived sucrose depends on the apoplastic plant invertase activity. Although the host invertase is constitutive, the fungus might be able to increase this invertase activity within a mycorrhiza by lowering the pH of the interface towards the pH optimum of the enzyme via the action of auxin. This fungus-released hormone could increase the H⁺ extrusion of plant cells by activation of the plant membrane H⁺-ATPases. Additionally, an auxin-dependent suppression of glucose uptake by cortical root cells could improve the glucose supply for the fungus. Furthermore, the fungal auxin might suppress the elicitor induced formation of defense enzymes, such as peroxidase.     

Two purified oligosaccharide elicitors,N-acetylchitohepatose and tetraglucosyl glucitol, derived fromMagnaporthe grisea cell walls, synergistically activate biosynthesis of phytoalexin in suspension-cultured rice cells

Two purified oligosaccharide elicitors generatable from fungal cell walls, N-acetylchitoheptaose and a tetraglucosyl glucitol from rice blast fungus (Magnaporthe grisea), synergistically activated phytoalexin biosynthesis in cultured rice cells. Inhibition experiments for the binding of radiolabeled N-acetylchitooligosaccharide elicitor to the plasma membrane from rice cells indicate that the two elicitors are recognized by different receptors. These results also indicate the presence of a positive interaction between the signal transduction cascade downstream of each elicitor/receptor, which enhances resistance against pathogens.     

Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells by Phytophthora capsici

Cell suspension cultures of three varieties of Capsicum annuum L., each with a different degree of sensitivity to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate with a hypersensitive reaction. They showed the synthesis or accumulation of PR-proteins with peroxidase (EC 1.11.1.7) activity and the accumulation of lignin-like polymer (as measured by derivatization with thioglycolic acid). The cultivation medium was optimised for both plant and fungus growth in order to avoid any stress during their combination. The resistant pepper variety, Smith-5, showed a more intense response to the elicitor preparations than the sensitive varieties, Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate. After elicitation, the cell walls thickened through the accumulation of lignin, as can be observed by staining microscope preparations with methylene blue. Elicitation also reduced the level of total peroxidase activity in the susceptible varieties, while such activity increased in resistant varieties, and was accompanied by de novo expression of acidic peroxidase isoenzymes in the extracellular and cell wall fractions. Of note was the PR protein of pI 5.7 showing peroxidase activity, which was induced by both elicitor types in the elicited cell suspensions of the resistant variety alone, making it a marker of resistance. The increases in the activity of these peroxidases in the resistant variety are in concordance with the accumulation of lignin observed 24 h after inoculation by both elicitors from the fungus. The possible role of these isoenzymes in lignin biosynthesis, used to reinforce the cell walls against fungal penetration of the cells, is discussed. These results are in accordance with those previously observed in plant stem sections.     

Relation between auxin autotrophy and tryptophan accumulation in cultured plant cells

Auxin autotrophy was studied in cultured carrot (Daucus carota L.), tobacco (Nicotiana tabacum L.), and potato (Solanum tuberosum L.) cell lines. Of 10 carrot lines resistant to 5-methyltryptophan (5MT), which accumulate free tryptophan (trp) because of an altered control enzyme, 5 were auxinautotrophic while the normal parent line was not. Carrot lines selected from the same parent line as resistant to other amino-acid analogs were not auxinautotrophic, like the parent. The only 5MT-resistant potato line studied was also auxin-autotrophic while the normal line was only partially auxin-autotrophic. The tobacco lines which accumulated free trp were not auxin-autotrophic, and no auxin-autotrophic tobacco lines were selected by screening for growth in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D). Several auxin-autotrophic carrot and potato lines were selected from the normal lines and none of these lines were resistant to 5MT. Length of time in culture and difficulty in selecting auxin-autotrophic lines were correlated on the 3 normal carrot lines studied. The addition of trp or indole to the culture medium would partially alleviate the auxin requirement of the normal lines studied. 2,4-D (0.4 mg/l) stimulated the growth of all auxin-autotrophic carrot lines.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PEP DL-p-fluorophenylalanine - IAA indole-3-acetic acid - 5MT DL-5-methyltryptophan - trp L-tryptophan     

Is the Phytophthora citrophthora culture filtrate a reliable tool for the in vitro selection of resistant Citrus variants?

Summary Nucellar calli from four Citrus cultivars with known resistance to the Phytophthora citrophthora pathogen were chosen as experimental material to test the pathogen's response to culture filtrate (CF). Sensitivity of the four calli to CF of the fungus was in reverse order to what is known on the susceptibility of the cultivars in vivo. Sensitivity of protoplasts derived from the same four calli to 2,4-dichlorophenoxyacetic acid (2,4-D) was in the same order as that of calli to CF. Protoplasts derived from calli selected for tolerance to CF showed a higher plating efficiency with increasing concentration of CF in the medium. TLC and GLC determinations showed the presence of indole acetic acid in the culture filtrate. Results indicate that CF of P. citrophthora cannot be used as a selection tool in vitro.Contribution No. 1655-E, 1986 series, from the Agricultural Research Organization, Bet-Dagan, Israel     

The phytotoxicity ofFusarium metabolites: An update since 1989

The present article summarises the published phytotoxic effects of severalFusarium metabolites (mycotoxins, phytotoxins, antibiotics and pigments) since 1989. The phytotoxicity of many of the commonly isolated metabolites cannot be disputed, but their role in pathogenesis ofFusarium-induced plant diseases is uncertain. Plant species/varieties differ in their susceptibililty resistance to these toxinsin vitro, as well as toFusarium pathogens under field conditions. Such variations in plant response may reflect resistance mechanisms that operate at several levels, including an initial ability to prevent fungal invasion; prevention of fungal spread and toxin tolerance or degradation. Little is known about the mode of action of most of these metabolites on either animal or plant cells. Several novelFusarium metabolites have been isolated in the past few years. Many are toxic to animals and cell lines, but assessment of their phytotoxicity has largely been neglected. Since many plant pathogenic Fusaria produce a plethora of metabolites, the additive or synergistic actions of toxins in combination must be considered in plant pathology.     

Recovery of plants from cryopreserved embryogenic cell suspensions of Pinus caribaea

Summary Embryogenic cell suspension cultures of Pinus caribaea var. hondurensis have been cryopreserved in liquid nitrogen for up to four months, using sucrose and dimethylsulfoxide as cryoprotectants. Post-thaw growth was obtained after a short lag phase. Removal of the remaining liquid around the cells using a filter disc favoured subsequent regrowth of the cells. These reestablished cultures maintained an embryogenic potential similar to non-frozen cultures. The embryos produced were able to regenerate into plants, which are now growing in a greenhouse.Abbreviations BA 6-benzyladenine - DMSO dimethylsulfoxide - FDA fluorescein diacetate - MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - PAR photosynthetically active radiation     

Analysis of mycorrhizal associations formed by Cistus incanus transformed root clones with Terfezia boudieri isolates

One clone (M-2), out of several Agrobacterium rhizogenes transformed root clones of Cistus incanus, formed ecto- or endomycorrhiza in vitro with two isolates of Terfezia boudieri collected in Israel. All other clone-fungal isolate combinations formed ectomycorrhiza. The endomycorrhiza-forming isolate secreted smaller amounts of auxin than an ectomycorrhiza-forming isolate. Addition of 2,4-dichlorophenoxyacetic acid (2,4-D) led to ectomycorrhiza formation by the M-2 clone on low P medium. Endomycorrhizas were formed by both M-2 and a control clone with the same T. boudieri isolates on high P medium with 2,4-D. The M-2 clone of C. incanus exhibited greater sensitivity to exogenous auxins (IAA and 2,4-D) than other clones, and clonal sensitivity to auxin was increased tenfold under low P conditions. Results are discussed in relation to phosphate and auxin influence on T. boudieri–C. incanus interaction.     

Peroxidase activity in calluses and cell suspension cultures of radishRaphanus sativus var. Cherry Bell

Calluses ofRaphanus sativus var. Cherry Bell were induced in a medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine. The biomass and peroxidase activities were determined, both in agar cultures and cell suspension cultures. Different growth-regulator concentrations induced different responses measured as peroxidase activity in callus. The suspended-cell cultures showed the importance of selecting the cell line, in order to obtain an optimal response in extracellular peroxidase activity. The commercial production of this enzyme by utilizing plant cell tissue cultures is discussed.     

Phaseollin production in cell suspensions and whole plants of Phaseolus vulgaris

Production of phaseollin was measured in cell suspension cultures and whole plants of Phaseolus vulgaris. In suspension cultures phaseollin appeared when there was no further increase in cell mass. Cells transferred to a medium without auxins yielded three times higher phaseollin concentrations than cells grown in their presence. Addition of autoclaved fungal mycelia or polysaccharides as elicitors resulted in an increased phaseollin concentration in the cell suspension.In whole plants phaseollin could be detected only after the plants were challenged by a fungus which caused lesions (browning) of the upper root neck region, Rhizoctonia solani. Treatment of non-infected plants with autoclaved fungal mycelia or other elicitors did not induce phaseollin production. However, when they were added before or together with the pathogenic fungus, the elicitors further increased phaseollin concentration in the root neck regions of the plants. This indicated that the pathogenic fungus was important for the penetration of the elicitors to inner plant tissues where phaseollin (and probably other phytoalexins) is produced.     

In vitro assay for 2,4-D resistance in transgenic cotton

Summary 2,4-Dichlorophenoxyacetic acid (2,4-D) resistant plants of transgenic cotton (Gossypium hirsutum L.) were produced using Agrobacterium tumefaciens containing a plasmid carrying the neomycin phosphotransferase II (npt II) and 2,4-D monooxygenase (tfd A) genes. An in vitro assay was performed to determine the sensitivity of seed germination, and the growth of seedlings of transgenic and non-transgenic cotton to various concentrations of kanamycin and 2,4-D. The results indicated that kanamycin caused the cotyledons of non-transgenic plants to turn white, but transgenic plants grew normally. Seed germination and seedling growth of non-transgenic plants were strongly inhibited by 2,4-D, but only slightly for transgenic plants. Transgenic plants and non-transgenic plants can be clearly distinguished by the use of 2 mg l⁻¹ 2,4-D in seed germination medium. There was a high correlation between the response of seed germination and the growth of seedlings to kanamycin or 2,4-D, based on the germination ration, albino ratio, dry weight or fresh weight. On this basis, we development a rapid method for identifying transgenic plants that has been verified in the field. These findings will allow identification of cotton transformants at an early stage of plant development, saving time and improving cultivars containing the 2,4-D resistance trait.     

Variation in structure and plant regeneration of Agrobacterium rhizogenes transformed and control roots of the potato cv. Bintje

Agrobacterium rhizogenes transformed and control roots of the tetraploid potato cv. Bintje were compared. Transformed roots were obtained after infection by A. rhizogenes 15834 or 1855. Both in leaf and stem segments, more roots were formed at the basal side of the segments, indicative for a polarity in root formation. As compared to control roots the transformed roots are characterized by smaller and more densely stained cells, a zone of cell division, and smaller statoliths. These characteristics are correlated with vigorous growth, high branching incidence and diminished geotropism. The plant regeneration procedure according to Ooms et al. [1] was modified. The transformed roots required less 2,4-D than control roots for the induction of shoot-competent calli. The callus and shoot induction phases were reduced from 8 and 6 weeks to 3 and 3 weeks, respectively. Upon induction, 25%, 58% and 61% of the root clones originating from tuber, stem and leaf, respectively, produced shoots, whereas all of the control roots produced shoots. Shoot outgrowth occurred on liquid MS medium in the absence of hormones.Abbreviations Ri-root Agrobacterium rhizogenes transformed root - BAP benzylaminopurine - IAA indoleacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid - 2,4-D 2,4 dichlorophenoxyacetic acid     

Effect of chitinase antisense RNA expression on disease susceptibility of Arabidopsis plants

Chitinases accumulate in higher plants upon pathogen attack are capable of hydrolyzing chitin-containing fungal cell walls and are thus implicated as part of the plant defense response to fungal pathogens. To evaluate the relative role of the predominate chitinase (class I, basic enzyme) of Arabidopsis thaliana in disease resistance, transgenic Arabidopsis plants were generated that expressed antisense RNA to the class I chitinase. Young plants or young leaves of some plants expressing antisense RNA had <10% of the chitinase levels of control plants. In the oldest leaves of these antisense plants, chitinase levels rose to 37–90% of the chitinase levels relative to vector control plants, most likely because of accumulation and storage of the enzyme in vacuoles. The rate of infection by the fungal pathogen Botrytis cinerea was measured in detached leaves containing 7–15% of the chitinase levels of control plants prior to inoculation. Antisense RNA was not effective in suppressing induced chitinase expression upon infection as chitinase levels increased in antisense leaves to 47% of levels in control leaves within 24 hours after inoculation. Leaves from antisense plants became diseased at a slightly faster rate than leaves from control plants, but differences were not significant due to high variability. Although the tendency to increased susceptibility in antisense plants suggests that chitinases may slow the growth of invading fungal pathogens, the overall contribution of chitinase to the inducible defense reponses in Arabidopsis remains unclear.