Identification and mapping of adult-onset sensitivity to victorin in barley

Victoria blight of oats caused by the fungus Cochliobolus victoriae is of distinct interest due to the link between Victoria blight susceptibility and crown rust resistance. C. victoriae-susceptible oats were introduced into the USA as a source of the Pc2 gene for resistance to the crown rust fungus Puccinia coronata. A dominant gene (Vb) in these oats was found to condition susceptibility to Victoria blight disease and sensitivity to the C. victoriae toxin called victorin. Numerous genetic approaches to separate Vb from Pc2 have failed, suggesting that Pc2 and Vb share identity. Because Victoria blight has only been described in allohexaploid oat, which has a poorly characterized genome of 11,300 Mb, molecular genetic investigations of Vb in oat are not practical. Previously we identified a presumed Vb ortholog in Arabidopsis, called LOV. LOV confers victorin sensitivity and susceptibility to C. victoriae, and encodes a coil-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) protein. Analysis of cereal DNA databases reveals a large array of CC-NBS-LRR genes, but no obvious LOV ortholog. Identifying a cereal ortholog of LOV will require identification and subsequent mapping of victorin sensitivity in a genetically tractable cereal plant. In this work, we surveyed barley for victorin sensitivity and identified adult-onset sensitivity to victorin in eight barley accessions. Evaluation of a doubled haploid (DH) population derived from the cross of sensitive × insensitive parents revealed a single quantitative trait locus (QTL) for victorin sensitivity in a resistance-gene-rich region on the short arm of chromosome 1H. Furthermore, enhanced victorin sensitivity observed in some DH lines suggests a background-dependent enhancement of victorin sensitivity.     

EFFECTS OF HELMINTHOSPORIUM VICTORIAE AND VICTORIN UPON PERMEABILITY

Wheeler, Harry, and Homer S. Black. (Louisiana State U., Baton Rouge.) Effects of Helminthosporium victoriae and victorin upon permeability. Amer. Jour. Bot. 50(7): 686–693. Illus. 1963.—Helminthosporium victoriae, the causal agent of Victoria blight of oats, produces a potent toxin called victorin. All of the symptoms of Victoria blight, both visible and biochemical, can be induced in susceptible plants either by infection with H. victoriae or by treatment with victorin. Among the effects produced both by the fungus and the toxin are marked changes in respiration and cell permeability. These are of special interest because they are characteristic of many plant diseases. This paper is concerned with the nature of changes in permeability and the possible relation of these to changes in respiration observed in tissues infected with H. victoriae or treated with victorin. Suseeptible oat tissues treated with victorin lost electrolyes when suspended and shaken in distilled water more rapidly than control tissues, and the magnitude of the effect varied with the concentration of victorin applied. Similar results were obtained with naturally infected susceptible plants but not with inoculated or victorin-treated resistant plants. The rate of electrolyte loss from victorin-treated tissue had a low temperature coefficient typical of a physical process and was not dependent upon oxygen tension. The concentration of victorin needed to produce an increase in the rate of electrolyte loss was approximately 50-fold less than that required to induce a significant increase in the respiratory rate. Changes in permeability were detected within 5 min after victorin was applied, whereas respiratory changes were not clearlyevident until after 30 min. These results indicate that if a causal relationship between these 2 phenomena exists, changes in permeability are responsible for changes in respiratory activity rather than vice versa.     

STOMATAL BEHAVIOR OF AVENA SATIVA TREATED WITH TWO PHYTOTOXINS,VICTORIN AND FUSICOCCIN

The transpiration and stomatal behavior of blight-susceptible and blight-resistant oats (Avena sativa L.) were followed after treatment with victorin, the host-specific toxin of Helminthosporium victoriae, and fusicoccin, the toxin produced by Fusicoccum amygdali. Victorin, at 0.012 units/ml and greater, significantly decreased the transpiration of the blight-susceptible variety in 3 hr, but had no effect on the transpiration of the blight-resistant variety. The stomatal resistance, measured with a ventilated diffusion porometer, showed that the decrease in transpiration was caused by stomatal closure; the greater the concentration of victorin the earlier the stomata closed. At high concentrations of victorin the stomata reopened in the blight-susceptible variety, and partial stomatal closure was observed in the resistant variety. Inactivated toxin had no effect on the stomata of either oat variety. When fusicoccin and victorin were compared under the same environmental conditions, victorin closed the stomata of the blight-susceptible leaves, whereas fusicoccin opened the stomata in both the blight-susceptible and resistant leaves. It is concluded that the two toxins differ in their effects on the guard cells; their possible modes of action are discussed.     

Molecular Features Affecting the Biological Activity of the Host-Selective Toxins from Cochliobolus victoriae

The structures of the toxins produced by Cochliobolus victoriae, victorin B, C, D, E, and victoricine, have recently been established. These toxins and modified forms of victorin C were tested for their effect on dark CO₂ fixation in susceptible oat (Avena sativa) leaf slices. Half-maximal inhibition of dark CO₂ fixation occurred with the native toxins in the range of 0.004 to 0.546 micromolar. An essential component for the inhibitory activity of victorin is the glyoxylic acid residue, particularly its hydrated aldehyde group. Removal of glyoxylic acid completely abolished the inhibitory activity of victorin, and the reduction of the aldehydo group transformed the toxin into a protectant. Conversion of victorin to its methyl ester resulted in diminution of inhibitory activity to 10% of the original activity of the toxin, whereas derivatization of the ε-amino group of the β-hydroxylysine moiety resulted in a decrease of inhibitory activity to 1% of that of victorin C. However, the derivatized toxin retained its host selectivity. In addition, the opening of the macrocyclic ring of the toxin drastically reduced the inhibitory activity.     

Resistance responses to Phoma lingam of plants regenerated from selected cell and embryogenic cultures of haploid Brassica napus

Summary Resistant plants and plants with reduced susceptibility against the pathogen Phoma lingam could be regenerated from selected callus and embryogenic cultures of haploid rape (Brassica napus) previously treated with mutagens. In the two in vitro selection systems used — absence of fungus growth on the cultures after incubation with parasite spores and resistance to the toxic filtrate — the resistance to the toxin was effective. In addition, some regenerants with increased tolerance were obtained from unselected cultures. Resistance tests on regenerated plants were carried out by inoculation of whole plants in the greenhouse, reproducing as much as possible the infection mechanisms which take place under natural conditions. Preliminary results on resistance of the progeny of single susceptible and tolerant regenerants seem to indicate that the acquired resistances are of a genetic nature.Part of these results was reported in a scientific meeting (Die höhere Pflanzenzelle: Möglichkeiten und Grenzen der Forschung) dedicated to Professor G. Melchers in the Max-Planck-Institut für Zellbiologie (Ladenburg) in January 1981     

In vitro selection for methomyl resistance in CMS-T maize

Summary Many plants resistant to methomyl (Lannate), an insecticide which selectively damages maize with the Texas (T) type of cytoplasmic male sterility (CMS-T), were obtained by in vitro selection and also without selection. The selection procedure used 0.6–0.7mM methomyl and callus from CMS-T versions of several field and sweet corn genotypes (W182BN, Wf9, P39, MDM1, SW1 and hybrids of SW1, IL766A1, IL766A2, and 442 with W182BN-N). Addition of 1 mM methomyl to the regeneration medium greatly reduced recovery of methomyl-sensitive escapes. Resistance was linked with reversion to male fertility and maternally inherited. Most progeny of resistant plants exhibited stable maternally inherited resistance for two generations in field tests. First-generation progeny of seven culture-derived plants segregated for resistance and sensitivity; this suggests that ears of these seven regenerants were cytoplasmically chimeral. Resistance to methomyl was associated with resistance to T toxin from Helminthosporium maydis race T and with changes in mitochondrial physiology. Prolonged culture (14–16 months versus 6–8 months) increased the frequency of resistance among both selected and non-selected regenerants. Little or no resistance was found among regenerants from certain genotypes. Selection with methomyl may be useful for production of improved sweet corn lines and as a source of mitochondrial mutants. This system is also convenient for studies of the effects of nuclear background and of culture and selection systems on the generation of cytoplasmic mutants.     

Identification and characterization of victorin sensitivity in Arabidopsis thaliana

Cochliobolus victoriae is a necrotrophic fungus that produces a host-selective toxin called victorin. Victorin is considered to be host selective because it has been known to affect only certain allohexaploid oat cultivars containing the dominant Vb gene. Oat cultivars containing Vb are also the only genotypes susceptible to C. victoriae. Assays were developed to screen the "nonhost" plant of C. victoriae, Arabidopsis thaliana, for victorin sensitivity. Sensitivity to victorin was identified in six of 433 bulk populations of Arabidopsis. In crosses of Col-4 (victorin-insensitive) x victorin-sensitive Arabidopsis ecotypes, victorin sensitivity segregated as a single dominant locus, as it does in oats. This Arabidopsis locus was designated LOV, for locus orchestrating victorin effects. Allelism tests indicate that LOV loci are allelic or closely linked in all six victorin-sensitive ecotypes identified. LOV was localized to the north arm of Arabidopsis thaliana chromosome I. The victorin-sensitive Arabidopsis line LOV1 but not the victorin-insensitive line Col-4 was susceptible to C. victoriae infection. Consequently, the LOV gene appears to be a genetically dominant, disease susceptibility gene.     

Development of a non-lethal selection system by using the aadA marker gene for efficient recovery of transgenic rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The application of aminoglycoside-3-adenyltransferase (aadA) gene-mediated streptomycin resistance for non-lethal selection of transgenic rice resulted in plant regeneration frequencies under selection pressure as high as those in non-transformed controls without selection. Since streptomycin does not kill non-transgenic cells, and allows plant regeneration from them, a selection procedure was developed that made the visual identification of transgenic calli and regenerants possible. For callus-level selection, a vital pH indicator—Chlorophenol Red—was applied together with streptomycin, making use of the phenomenon that fast-growing cell lines lower the pH in the culture medium. Transgenic plants were selected according to their main distinctive features; their green colour (photomixotrophic assimilation), and more intense growth. At the same time, non-transgenic regenerants were bleached (heterotrophic assimilation), and growth was retarded in the presence of streptomycin and sucrose. The final efficiency of genetic transformation based on streptomycin resistance was found to be double that of transformations where the selective agent was l-phosphinothricin, and nearly three times more compared to transformations resulting in hygromycin-resistant regenerants. To the best of our knowledge, this is the first report on producing nuclear transformed rice plants by using a non-lethal selection strategy based on the chimaeric aadA gene.Communicated by D. Dudits     

Covalent binding sites of victorin in oat leaf tissues detected by anti-victorin polyclonal antibodies

Polyclonal antibodies against victorin, the host-specific toxin produced by Cochliobolus victoriae, were raised in rabbits immunized with a victorin-bovine serum albumin conjugate. The antibodies were purified from serum by protein A column chromatography and characterized by indirect and direct enzymelinked immunosorbent assays (ELISA). The concentration of victorin that inhibited anti-victorin antibody binding by 50% was 10 nanograms per milliliter in an indirect ELISA. The lowest concentration of victorin detectable was 10 picograms per milliliter. In a direct ELISA, 25 nanograms per milliliter of victorin inhibited binding of victorin-horseradish peroxidase conjugate by 50%. In vivo and in vitro covalent binding of victorin to proteins in susceptible and resistant oat (Avena sativa) tissue was examined by western blotting assays using anti-victorin antibody and a second antibody conjugated with ¹²⁵I or alkaline phosphatase. In vivo binding of victorin to proteins of 100 and 45 kilodaltons was observed in both susceptible and resistant cultivars of oats. Victorin also bound in vitro to proteins of 100, 65, and 45 kilodaltons in both susceptible and resistant oats. The data indicate that victorin binds covalently to the same sites in susceptible and resistant genotypes of oats.     

In Vitro selection of groundnut cell lines fromCercosporidium personatum culture filtrates and regeneration of resistant plants through cell culture

Cell suspensions derived from immature leaves of the groundnut (Arachis hypogaea L.) were cultured in the presence and absence ofCercosporidium personatum pathotoxic culture filtrates. Cell viability and reactions of cell lines were determined after exposure to various concentrations (25–100%, v/v) of the filtrates. Cell lines have been selected for resistance to the toxin(s) produced byC. personatum. Selected cell lines were used for plant regeneration on regeneration media containingC. personatum culture filtrates. Plant regeneration frequency was found to be low in long-term cultures, whereas it was high in short-term cultures. The selfed progeny of the plants regenerated from the resistant cell lines showed resistance to the pathogen in the field. Six out of 82 plants exhibited enhanced resistance in the R₂ generation. The culture filtrate stimulated callus proliferation as well as plant regeneration at lower concentrations, a response that could prove to be very useful for obtaining disease resistant plants throughin vitro selection.     

Short- and long-term effects of vinblastine on the rat adrenal medulla

Summary The effects of a single high dose (10mg/kg) of vinblastine (vb) sulfate (Velbe, Lilly) on the ultrastructure, catecholamine (CA) content and activity of CA-synthesizing enzymes of the rat adrenal medulla were studied for up to 120h after intravenous injection of the drug.By 1 h, microtubules were virtually absent from chromaffin cells and preganglionic cholinergic axons, and typical paracrystals had appeared inside the nerve fibers. By 16h microtubules were completely reconstituted and paracrystals had disappeared. From 16h onwards, there was an increasing depletion of storage granules from adrenaline (A) — producing cells, which coincided with biochemical determinations showing a reduction of adrenal A to about 40 % of control levels by 48 h, with noradrenaline (NA) remaining in the range of controls. Both A- and NA-storing cells showed an extensive proliferation of the rough endoplasmic reticulum (ER). Vb caused a marked increase in tyrosine hydroxylase (TH; +113%) and dopamine -hydroxylase (DBH; +82%) activities after 48 h. Splanchnicotomy completely abolished the vb-mediated increase in TH and DBH activities. A smaller increase (+ 47 %) in enzyme activity was observed with phenylethanolamine N-methyltransferase (PNMT). Vb (10^–5M) had no apparent effect on granule content and the amount of rough ER in chromaffin cells, which were cultured for 48 h.The results demonstrate that a single high dose of vb has relatively little short-term effects on the rat adrenal medulla, but causes drastic long-term changes in CA-content and enzyme activities that are mediated by the preganglionic nerves. These changes could be interpreted as an effort to compensate for a loss of CA-stores in peripheral adrenergic nerves (cf. Cheney et al., 1973). The differential long-term effect of vb on adrenal NA and A might be due to the lower induction of PNMT as compared to TH and DBH activities and/or to a preferential release of A versus NA, which may occur at high frequencies of stimulation of the splanchnic nerves.Supported by grants from the Deutsche ForschungsgemeinschaftDedicated to Professor G. Petry in honor of his 65th birthday     

Immunological comparison of the in vitro and in vivo labeled victorin binding protein from susceptible oats

The fungus Cochliobolus victoriae causes victoria blight of oats and produces the host-specific toxin victorin. The reaction of oats to the fungus and its toxin is controlled by a single dominant gene whose product has been hypothesized to function as the site of action (receptor) of the toxin in susceptible oat genotypes. Previously, using a biologically active ¹²⁵I derivative of the toxin, we identified a 100 kilodalton victorin-binding protein (VBP) which binds victorin in a ligand-specific manner and binds in vivo only in susceptible oat genotypes. However, a VBP in both the susceptible and resistant oat genotypes was identified by in vitro binding experiments. One interpretation of the lack of genotype-specific binding in vitro is that the 100 kilodalton protein detected in vitro is not the same 100 kilodalton protein detected in vivo. To clarify the relationship between the 100 kilodalton protein(s) labeled in vivo and in vitro, we developed antisera to the in vitro-labeled VBP from the susceptible genotype and demonstrated that these preparations react with the in vivo-labeled VBP from the susceptible genotype. This finding coupled with previous observations strongly suggest that the VBP observed in vivo is the same protein detected in vitro. Furthermore, the results support our previous observations which suggest that the VBPs labeled in vitro in susceptible and resistant genotypes are closely related or identical.     

In Vitro Selection Against Ascochyta Blight of Chickpea (Cicer arietinum L)

Callus cultures established on MS medium containing 2.0 mg l^-1 2, 4-D were inoculated on the regeneration medium supplemented with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3%, v/v) of culture filtrate (CF) of Ascochyta rabiei infesting chickpea. Out of 486 callus pieces and 270 regenerants obtained from immature embryo derived callus screened, 50 callus lines and 74 regenerants were found resistant. Further, these resistant callus lines and regenerants were subjected to stability test by growing them on a medium containing 3% CF. Seventeen callus lines and 28 regenerants of the selected lines showed normal growth on the selection medium. The regenerated plants were tested in pots under artificial epiphytotic conditions where they showed normal growth behaviour and high degree of resistance.     

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Summary Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt⁶⁰,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via gamma-fusion.     

Characterization of biomass production,cytology and phenotypes of plants regenerated from embryogenic callus cultures of Pennisetum americanum x P. purpureum (hybrid triploid napiergrass)

Summary Five hundred and twenty-four plants of a triploid, sexually sterile hybrid napiergrass (Pennisetum americanum x P. purpureum; 3x=21) were regenerated from embryogenic callus cultures obtained from segments of young inflorescences. Replicated field trials were conducted for two consecutive years to compare the biomass yield, phenotype and cytology of tissue culture regenerants (TC) and vegetatively propagated (V) plants. In the first year total biomass yield of TC plants was significantly greater than V plants but there was no significant difference in the second year. TC plants had more tillers compared to V plants. V plants did not show any morphological variability. The TC population also exhibited a high degree of phenotypic stability (96%). There were 23 phenotypic variants in the TC population of 524, most of them being more dwarf and late-flowering. Detailed morphological analysis of the TC-variant plants suggests that they very likely arose from only a few variant cell lines. Cytological analysis indicated stability of the triploid status in randomly selected regenerants. Two of the morphological variants were hexaploids (6x=42). It is concluded that embryogenic callus cultures can provide useful alternative for the rapid propagation of hybrid napier-grass which is commonly propagated by cuttings.     

Transgenic and herbicide resistant pearl millet (Pennisetum glaucum L.) R.Br. via microprojectile bombardment of scutellar tissue

Four different pearl millet breeding lines were transformed and led to the regeneration of fertile transgenic plants. Scutellar tissue was bombarded with two plasmids containing the bar selectable marker and the -glucuronidase reporter gene (gus or uidA) under control of the constitutive CaMV 35S promoter or the maize Ubiquitin1 promoter (the CaMV 35S is not a maize promoter). For the delivery of the DNA-coated microprojectiles, either the particle gun PDS 1000/He or the particle inflow gun was used. The calli and regenerants were selected for their resistance to the herbicide Basta (glufosinate ammonium) mediated by the bar gene. Putative transformants were screened for enzyme activity by painting selected leaves or spraying whole plants with an aqueous solution of the herbicide Basta and by the histochemical GUS assay using cut leaf segments. PCR and Southern blot analysis of genomic DNA indicated the presence of introduced foreign genes in the genomic DNA of the transformants. Five regenerated plants represent independent transformation events and have been grown to maturity and set seed. The integration of the bar selectable and the gus reporter gene was confirmed by genomic Southern blot analysis in all five plants. All five plants had multiple integrations of both marker genes. To date, the T₁ progeny of three out of four lines generated by the PDS particle gun shows co-segregating marker genes, indicating an integration of the bar and the gus gene at the same locus in the genome.     

Response to NaCl of alfalfa plants regenerated from non-saline callus cultures

Salinity restricts crop productivity in many arid environments. Inadvertent selection for tolerance to osmotic stress may occur under cell or tissue culture conditions and could affect the performance of regenerated plants. The effect of NaCl on forage produced by alfalfa (Medicago sativa L.) plants regenerated from non-saline callus cultures was examined in this study. Plants of Regen-S, which was selected for improved callus growth and regeneration in non-saline cultures, had higher forage weight when grown on SHII medium at NaCl levels up to 100 mM compared to its parental cultivars, Saranac and DuPuits. Five additional original-regenerant plant pairs, each derived from non-saline callus cultures of different alfalfa plants, were evaluated in a solid (soil-like) substrate under saline and non-saline conditions. Weight of forage produced by rooted stem cuttings of regenerated plants was 33% higher at 50 mM NaCl compared to cuttings of explant donor plants. Self progenies from four of five regenerants had higher relative forage weight at 100 mM NaCl (percent of 0 NaCl treatment) than the original plants indicating increased NaCl tolerance.     

Somacional variation and eyespot toxin tolerance in sugarcane

The analysis of sugarcane plants regenerated from culture for their reaction to eyespot (Helminthosporium sacchari) toxin is described. A total of 480 culture-derived plants (somaclones) from cultivar Q101 were characterized. Some of these plants derived from cultures which had been subjected to selection with the eyespot toxin and others were derived without overt selection. Leaves were assayed for their toxin reaction. A very high frequency of toxin-tolerant variants was found. The distribution was even further biased toward resistance in those plants regenerated from cultures exposed to toxin selection.A total of 85 somaclones was analysed for the stability of their increased toxin tolerance to the primary somaclone; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relate to the possibility of using consecutive vegetative segregation.Six tolerant variants were also passed through a second tissue culture cycle and 60 secondary somaclones were assayed. Twenty four (40%) of these plants had a similar tolerance to the primary somacione; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relative to the possibility of using consecutive cycles of culture to stack improved characters into a sugarcane cultivar.     

In Vitro Selection for Improved Plant Resistance to Toxin-Producing Pathogens

Simultaneously with the progress in plant biotechnology since the 1980s, new methods in plant pathology have been developed. This review summarizes papers that cover basic research on the effects of selective agents on in vitro cultures of host plants, as well as applications of agents in regeneration systems that result in lines with increased variability in resistance or susceptibility. The first part of the study deals with theoretical aspects of the interactions between plants and toxin‐producing pathogens, mode of phytotoxic action, and host‐ and non‐host‐selective toxins. The second part lists and describes various agents used for selections in vitro. In the last two decades more than 100 publications focused on these selections for the improvement of resistance to plant pathogens. Over 30 plant species were examined to utilise various selection agents extracted from about 40 plant pathogens. The review covers basic research studies and methods that elucidate the relationships between in vitro and in vivo mechanisms of resistance, but also try to develop practical applications to obtain resistant breeding lines. Such methods often utilise some type of explant cultures of the host plants that are treated with various selective agents (culture filtrates, toxins, elicitors), which then elicit typical reactions that parallel those by the pathogens. Their application successfully resulted in resistant lines in banana, carnation, grapevine, strawberry and wheat. Nowadays, these techniques are an important complement to classical breeding methods.