Plant height affects Fusarium crown rot severity in wheat.

Effects of plant height on Fusarium crown rot (FCR) disease severity were investigated using 12 pairs of near-isogenic lines (NILs) for six different reduced height (Rht) genes in wheat. The dwarf isolines all gave better FCR resistance when compared with their respective tall counterparts, although the Rht genes involved in these NILs are located on several different chromosomes. Treating plants with exogenous gibberellin increased FCR severity as well as seedling lengths in all of the isolines tested. Analysis of the expression of several defense genes with known correlation with resistance to FCR pathogens between the Rht isolines following FCR inoculation indicated that the better resistance of the dwarf isolines was not due to enhanced defense gene induction. These results suggested that the difference in FCR severity between the tall and dwarf isolines is likely due to their height difference per se or to some physiological and structural consequences of reduced height. Thus, caution should be taken when considering to exploit any FCR locus located near a height gene.

Identification of plant defence genes in canola using Arabidopsis cDNA microarrays.

We report the identification of novel defence genes in canola by using a cDNA microarray from Arabidopsis. We examined changes that occur in the abundance of transcripts corresponding to 2375 Arabidopsis expressed sequence tags (selected for defence gene identification) following inoculation of canola plants with the fungal necrotrophic leaf pathogen, Alternaria brassicicola. Microarray data obtained from this cross-hybridisation experiment were compared to expression profiles previously obtained from the equivalent Arabidopsis experiment. Homology searches using a canola expressed sequence tag database with approximately 6000 unique clones led to identification of canola defence genes. Pathogen-responsive transcripts included those associated to known defence genes, reactive oxygen species metabolism, disease resistance and regulatory genes, and cell maintenance/metabolism genes. Using specific primers for quantitative real-time reverse transcriptase PCR, gene expression profiles in canola were obtained that demonstrated coordinated defence responses, including systemic responses in distal tissue and salicylic acid- and methyl jasmonate-mediated signalling against A. brassicicola.

MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins.

MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear ((15)N) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action.

Transfer of a supernumerary chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides.

Two biotypes (A and B) of Colletotrichum gloeosporioides infect the tropical legumes Stylosanthes spp. in Australia. These biotypes are asexual and vegetatively incompatible. However, field isolates of biotype B carrying a supernumerary 2-Mb chromosome, thought to originate from biotype A, have been reported previously. We tested the hypothesis that the 2-Mb chromosome could be transferred from biotype A to biotype B under laboratory conditions. Selectable marker genes conferring resistance to hygromycin and phleomycin were introduced into isolates of biotypes A and B, respectively. A transformant of biotype A, with the hygromycin resistance gene integrated on the 2-Mb chromosome, was cocultivated with phleomycin-resistant transformants of biotype B. Double antibiotic-resistant colonies were obtained from conidia of these mixed cultures at a frequency of approximately 10(-7). Molecular analysis using RFLPs, RAPDs, and electrophoretic karyotypes showed that these colonies contained the 2-Mb chromosome in a biotype B genetic background. In contrast, no double antibiotic colonies developed from conidia obtained from mixed cultures of phleomycin-resistant transformants of biotype B with biotype A transformants carrying the hygromycin resistance gene integrated in chromosomes >2 Mb in size. The results demonstrated that the 2-Mb chromosome was selectively transferred from biotype A to biotype B. The horizontal transfer of specific chromosomes across vegetative incompatibility barriers may explain the origin of supernumerary chromosomes in fungi.

Induction of beta-1,3-glucanase in barley in response to infection by fungal pathogens.

The sequence of a partial cDNA clone corresponding to an mRNA induced in leaves of barley (Hordeum vulgare) by infection with fungal pathogens matched almost perfectly with that of a cDNA clone coding for beta-1,-3-glucanase isolated from the scutellum of barley. Western blot analysis of intercellular proteins from near-isogenic barley lines inoculated with the powdery mildew fungus (Erysiphe graminis f. sp. hordei) showed a strong induction of glucanase in all inoculated lines but was most pronounced in two resistant lines. These data were confirmed by beta-1,3-glucanase assays. The barley cDNA was used as a hybridization probe to detect mRNAs in barley, wheat (Triticum aestivum), rice (oryza sativus), and sorghum (Sorghum bicolor), which are induced by infection with the necrotrophic pathogen Bipolaris sorokiniana. These results demonstrate that activation of beta-1,3-glucanase genes may be a general response of cereals to infection by fungal pathogens.

Differential expression of peroxidase isogenes during the early stages of infection of the tropical forage legume Stylosanthes humilis by Colletotrichum gloeosporioides.

Infection of Stylosanthes humilis by the fungal phytopathogen Colletotrichum gloeosporioides is associated with an increase in peroxidase enzyme activity within 24 h postinoculation. Peroxidase gene expression was investigated as a first step towards understanding the regulation and functional importance of this host response to fungal attack. Four distinct cDNAs Shpx 2, 5, 6, and 12, isolated from a cDNA library of S. humilis contained deduced amino acid (aa) sequence motifs characteristic of peroxidases. Three of these (Shpx 2, 5, and 6) were full-length and their deduced proteins each fell into a different homology group based on comparisons with other plant peroxidases. Each cDNA appeared to hybridize to only one or two genes in S. humilis. mRNAs corresponding to Shpx2, Shpx6, and Shpx12 were expressed relatively abundantly in young leaves, with lesser expression of Shpx2 and Shpx6 and no expression of Shpx12 detected in roots. No expression of these genes was detected in stems or old leaves. The mRNA of Shpx5 was relatively abundant in stems and to a lesser extent in young leaves. However, infection of young leaves with C. gloeosporioides greatly increased expression of the mRNAs of Shpx2 and Shpx6 but not Shpx5 nor Shpx12 compared to mock-inoculated controls. The mRNA of Shpx6 was strongly induced by the pathogen 4 h postinoculation, a time which precedes fungal penetration, while Shpx2 was induced to higher levels than controls at 24 h after inoculation. The mRNAs of both Shpx2 and Shpx6 but not Shpx5 and Shpx12 were also induced by wounding.(ABSTRACT TRUNCATED AT 250 WORDS)

A peroxidase gene promoter induced by phytopathogens and methyl jasmonate in transgenic plants.

The expression of two closely related peroxidase isogenes, Shpx6a and Shpx6b, of the legume Stylosanthes humilis was studied using isogene-specific reverse transcriptase PCR techniques. Results indicated that transcripts of both genes were rapidly induced following inoculation with the fungal pathogen Colletotrichum gloeosporioides, wounding and treatment with the defense regulator methyl jasmonate (MeJA). In contrast treatment of leaves of S. humilis with abscisic acid (ABA) and salicylic acid (SA) did not induce transcripts of either isogene. A genomic clone containing the Shpx6b gene was isolated and 594 bp of 5' sequence upstream of the translation start was fused in frame to the coding region of the uidA reporter gene and introduced into tobacco. Expression from the Shpx6b promoter in transgenic plants was determined by histochemical staining and quantitative assays of beta-glucuronidase (GUS). In transgenic tobacco, GUS expression was detected in cotyledons, vascular cells of young leaves, anthers, pollen, and the stigma and style. Wounding of the tobacco plants produced very localized GUS staining. Much more extensive staining for GUS was observed following inoculation of tobacco leaves with conidia of the fungal pathogen Cercospora nicotianae and the inoculation of wound sites with mycelium of the Oomycete pathogen Phytophthora parasitica var. nicotianae. Treatment of mature leaves with methyl jasmonate induced GUS activity while treatment with ABA, SA, and H2O2 had no effect. A similar strong induction of GUS activity was measured in young transgenic seedlings germinated on MeJA while some, but much weaker, induction of GUS activity was observed in seedlings treated with SA. The sequence of the promoter contained motifs homologous to putative cis elements in other plant genes responsive to MeJA. The Shpx6b gene is the first plant peroxidase gene shown to be induced by both microbial pathogens and MeJA and its promoter will be useful for investigations of signaling processes during fungal infection and for the expression of foreign gene products at infection sites.

CgDN3: an essential pathogenicity gene of colletotrichum gloeosporioides necessary to avert a hypersensitive-like response in the host Stylosanthes guianensis.

A gene of Colletotrichum gloeosporioides that is induced by nitrogen starvation in axenic culture and is expressed at the early stages of infection of the host Stylosanthes guianensis has been identified and its role in pathogenicity tested. The sequence of this gene, named CgDN3, indicated that it encodes a protein of 74 amino acids that contains a predicted 18 amino acid signal sequence for secretion of a basic 54 amino acid mature protein with weak homology to an internal region of plant wall-associated receptor kinases. Mutants of C. gloeosporioides were produced by homologous recombination in which part of the coding sequence and promoter region of the CgDN3 gene was replaced with a hygromycin-resistance gene cassette. Mutations in the CgDN3 gene were confirmed in two independent transformants and Northern (RNA) analysis demonstrated the disrupted CgDN3 gene was not expressed. The mutants had faster mycelial growth rates in vitro but produced spores that germinated to form appressoria normally on the leaf surface. However, the CgDN3 mutants were unable to infect and reproduce on intact host leaves. Microscopic analysis revealed small clusters of necrotic host cells at inoculation sites on leaves, suggesting that these mutants elicited a localized, host hypersensitive-like response. The mutants were able to grow necrotrophically and reproduce on leaves when conidia were inoculated directly onto wound sites. The putative promoter region of the CgDN3 gene was fused to a gene encoding a modified jellyfish green fluorescent protein and introduced into the fungus. Following inoculation, strong expression of green fluorescent protein was observed in primary infection vesicles in infected epidermal cells with weaker expression evident in hyphae growing within infected leaf tissue. These findings indicate that CgDN3 encodes a novel pathogenicity determinant associated with the biotrophic phase of primary infection and required to avert a hypersensitive-like response by a compatible host.

A strain-specific cyclin homolog in the fungal phytopathogen Colletotrichum gloeosporioides.

The fungus, Colletotrichum gloeosporioides, which infects the tropical pasture legume, Stylosanthes guianensis, contains highly variable mini-chromosomes. The transcription of strain-specific genomic DNA clones previously isolated from one variable mini-chromosome was investigated by using these clones to screen a cDNA library prepared from the fungus grown in liquid medium. A cDNA clone was obtained with one of the genomic clones and was sequenced. A single long open reading frame of 259 amino acids (aa) was detected with significant homology to cyclin proteins in other organisms. Northern blot analysis indicated that the cDNA corresponded to a low-abundance mRNA (approximately 0.001% of poly(A)+RNA). Southern blot analysis indicated that genes encoding this mRNA were discontinuously distributed in this fungal species, indicating it encodes a dispensable function. This result suggests that natural populations of fungi may have variable complements of cyclin-encoding genes.

Acquired ventricular septal defect.

The past 9 years' experience with ventricular septal rupture complicating myocardial infarction has been reviewed. Thirty-six patients were treated surgically, with 10 early deaths (28%) and one late death, for an 8 year actuarial survival rate of 63%. The mortality was highest for those defects which followed inferior infarction, 38% compared with 13% following anterior infarction. The infarction-operation interval also greatly influenced mortality; under 2 weeks, 43%; over 2 weeks, 18%. Concomitant coronary artery bypass grafts (13 patients) or left ventricular aneurysmectomy (14 patients) did not carry an increased mortality. Of 17 patients who presented with cardiogenic shock, eight died (47%). The intra-aortic balloon pump (IABP) was used in 16 patients (44%) and helped greatly in the management of the critically ill. With an estimated 17 acquired septal defects occurring each year in persons under 65 years of age in Wessex, awareness of this complication and of the favorable outcome of operation is essential among those who treat the aftereffects of myocardial infarction.

Characterization of an endo-1,3(4)-beta-D-glucanase gene from Cellvibrio mixtus.

An endo-1,3(4)-beta-D-glucanase gene (cwd2) of Cellvibrio mixtus encoding laminarinase activity was cloned on a 3.9-kb PstI fragment. The Cwd2 enzyme, extracted from recombinant Escherichia coli, degraded both beta-1,3 glucans and beta-1,3-1,4 mixed-linkage glucans, was endohydrolytic and so conformed to the enzyme class 3.2.1.6. The pH and temperature optima of the enzyme were approximately 7 and 40 degrees C respectively. The M(r) of specifically labelled Cwd2 was approximately 34,000. This gene was quite distinct from two other C. mixtus beta-1,3 glucanases previously described.

Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance.

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.

A new tube feed: Aminutrin and Calonutrin.

A new tube feed made up from separate sachets of 1-amino acids and saccharides, and mixed with milk, has been given to five patients for a total of 49 days. The preparation, administration and tolerance of the feed has been uncomplicated, and the electrolyte, calorie and nitrogen content could be easily varied according to the patient's needs. A simple metabolic study of the patients was made during the administration of the feed.

Transformation of Stylosanthes spp. using Agrobacterium tumefaciens.

Tumours were incited on leaf sections of Stylosanthes humilis, S. hamata, S. guianensis and S. scalra following infection by Agrobacterium tumefaciens. The suitability of 2 binary vectors (pGA472, BIN6) for gene transfer in S. humilis was tested and kanamycin-resistant tumour tissue was obtained from infected leaf pieces. The presence and expression of the neomycin phosphotransferase (NPT II) gene in the plant cells was demonstrated by hybridization of the coding region of the NPT II gene of the transposon Tn5 to DNA and RNA of kanamycin resistant tumours and by detection of significant NPT II activity in tissue extracts. Tumours also produced teratomatous shoots expressing the NPT II gene, but these could not be rooted.

Cor pulmonale and the Pierre Robin anomaly. Airway management with a nasopharyngeal tube.

An infant with Pierre Robin anomaly was anaesthetised for cardiac catheterisation. There was cor pulmonale with the pulmonary artery pressure at systemic level, a patent foramen ovale and a persistent ductus arteriosus. The effects of alterations in blood gases on the haemodynamics and intracardiac shunts are considered. Subsequent management of the obstructed airway with a nasopharyngeal tube for 4 weeks is described.

Valve replacement in carcinoid syndrome. Anaesthetic management for tricuspid and pulmonary valve surgery.

This report describes the anaesthetic management of a patient with carcinoid syndrome for cardiac catheterisation followed by replacement of the tricuspid and pulmonary valves. Apart from the precautionary use of aprotinin and steroids, routine techniques of anaesthesia and monitoring were used without complications attributable to secretions from tumour tissue.

Strategies for the suppression of peroxidase gene expression in tobacco. I. Designing efficient ribozymes.

Five short hammerhead ribozymes (Rzs) were constructed and tested, using a range of in vitro reaction conditions, for catalytic activity against the mRNA encoding the lignin-forming peroxidase (TPX) of tobacco. Although all 5 Rzs were shown to be able to cleave the RNA substrate, percentage cleavage varied with pre-denaturation of Rz and substrate, incubation temperature, length of incubation and ribozyme (Rz)-to-substrate ratio. One Rz with two catalytic units and 60 nucleotides of complementary sequence in 3 regions was shown to most efficiently cleave the substrate under all in vitro conditions tested. This ribozyme cleaved better than the two single ribozymes from which it was made. The superior cleaving ability of this Rz was shown to be due to the accessibility of the chosen target site and to the increased length of the hybridizing arms spanning this accessible region of the RNA.

The Retec breathing assistor. An application of fluidics to apparatus for inhalation therapy.

The Retec breathing assistor, with its gluidic valve, is an interesting method of providing assisted positive pressure inspiration, a resistive load to expiration and a capability for the administration of drugs by nebuliser. It requires some co-operation but very little training by patients, and is easily cleaned and maintained.

Beta-adrenoceptor blockade and anaesthesia. Beta-adrenoceptor antagonism during anaesthesia for coronary artery surgery.

Twenty-six patients with severe coronary artery disease, receiving long term beta-adrenoceptor blocking drugs were anaesthetised for aorto-coronary bypass operations. Beta-adrenoceptor blocking drugs were withdrawn 2 to 8 days before surgery in ten patients only. In the remaining sixteen patients there were no serious complications due to the presence of a degree of beta-blockade during anaesthesia and surgery. The undesirable cardiovascular responses to laryngoscopy and tracheal intubation were diminished in these patients, and the rise in heart rate/systolic pressure product, and indicator of myocardial oxygen consumption, was less in this group. The need for peripheral vasodilators to treat systemic arterial pressure rises in response to surgery was also reduced. There appeared to be no contraindication to the continuation of beta-adrenoceptor blockade before operation in patients undergoing aorto-coronary bypass procedures when suitable anaesthetic agents were selected and when an appropriate blood volume was maintained.

Magnesium flux during open heart surgery. The effect of St Thomas' Hospital cardioplegia solution.

The Hearse St Thomas' Hospital cardioplegia infusate is one method of preserving the myocardium in the absence of coronary perfusion, during open heart surgery. The infusate contains 16 mmol magnesium/litre and 20 mmol potassium/litre. Peri-operative plasma magnesium levels and urinary excretion of magnesium have been measured, when the infusate was returned to the circulation in 12 patients. The plasma level (+/- SEM) rose to 1.86 mmol/litre (+/- 0.1) 5 minutes after cardiopulmonary bypass commenced, was 1.57 mmol/litre (+/- 0.09) shortly before termination of cardiopulmonary bypass but was normal on the first day after surgery. Urinary excretion of magnesium was 55% of the administered quantity by Day 1 and 77% by the second day. Two patients excreted less than 40% of the administered magnesium within 24 hours probably indicating magnesium depletion. There were no adverse effects from a magnesium load of 16--32 mmol magnesium given during cardiopulmonary bypass.