Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing the hypersensitive response.

The hypersensitive response (HR), elicited when resistant hosts are infected by incompatible races of biotrophic fungi, has been researched extensively. New studies on host responses to necrotrophic fungi are beginning to show that when the HR occurs in hosts colonized by necrotrophs, fungal growth is accelerated rather than retarded. We review current knowledge about how necrotrophs survive in host plants in which the HR is expressed. We discuss how necrotrophs cope with the environmental factors formed as a result of the HR. Necrotrophs contain an array of enzymes, which can help in exploiting the hostile environment in order to colonize the host and to remove or inactivate active oxygen species (AOS). Among this array of enzymes are superoxide dismutase (SOD), peroxidases, catalase, and perhaps laccases and polyphenol oxidases. Of these, only SOD and catalase have been studied in any detail. The precise significance of SOD and catalase in host invasion and fungal resistance is still not adequately known. The importance of different peroxidases is also still far from clear. We speculate that AOS species may trigger the response of necrotrophs to the host environment.

Effect of cucurbitacins on mRNA coding for laccase in Botrytis cinerea.

The effect of cucurbitacin and of Ecballium extract on the formation of mRNA coding for laccase was examined in cultures of Botrytis cinerea grown with inducers of laccase formation, in the presence or absence of the inhibitory compounds. RNA was isolated from the cultures and probed with specific DNA probes for laccase. As an internal control, the RNA was probed for Botrytis beta-tubulin mRNA. From an analysis of the results it is clear that cucurbitacin I and Ecballium extract specifically repress the amount of mRNA coding for laccase. This could account for the previously observed repression of laccase formation by cucurbitacins.

Superoxide dismutase: a differentiation protein expressed in Uromyces germlings during early appressorium development.

Germlings of the bean rust fungus Uromyces appendiculatus detect penetration sites on the surface of the host leaf by thigmosensing topographical features. Within 2-4 min after the apex of a urediospore germ tube encounters the cuticular lip of a stomate, the germling ceases polarized growth and begins to swell over the aperture. The mechanism by which the cells detect topographical signals is not understood; however, previous experiments indicated that the initiation process does not involve de novo gene expression. In order to detect posttranslational modifications, the protein profiles of induced and noninduced germlings were compared at the earliest stages of appressorium formation, and a 21-kDa differentiation protein was identified by a shift in isoelectric point. The N-terminal amino acid sequence exhibited homology with superoxide dismutase (SOD), and antibodies to a synthetic peptide fragment of the respective sequence recognized cooper/zinc isozymes of SOD in electroblots of native gels. Electroelution of the active enzyme bands and separation by SDS-PAGE indicated that the 21-kDa protein is a component of a tetrameric 85-kDa SOD.

INF56 represents a family of differentiation-specific genes from Uromyces appendiculatus.

In germlings of U. appendiculatus, a gene designated INF56 is preferentially expressed during development of the infection structures. A comparison of sequences between INF56 genomic DNA and a cDNA revealed several differences, randomly distributed throughout the coding region, which resulted in RFLPs at ASpI, HphI, NruI and ScaI sites. This observation, along with DNA-blot analysis, which revealed multiple copies of INF56 in uredospore genomic DNA, indicated that INF56 represents a multigene family. All copies of INF56 examined contain the same 67-bp intron reported previously (Xuei et al. 1992).

Cloning and regulatory analysis of starvation-stress gene, ssgA, encoding a hydrophobin-like protein from the entomopathogenic fungus, Metarhizium anisopliae.

The nucleotide (nt) sequence of a starvation-stress gene (ssgA) of the entomopathogenic fungus, Metarhizium anisopliae, and its deduced amino acid (aa) sequence were determined. The primary structure of the SSGA (96 aa; deduced M(r) = 9925; pI = 4.1) protein shares extensive similarities with fungal wall proteins of the 'hydrophobin' class, and the eight Cys residues and putative signal sequences are conserved. Secondary structure predictions suggest an additional resemblance to low-M(r) toxins and agglutinins. Northern (RNA) blot analysis and nuclear run-on assays demonstrated transcriptional control of expression of ssgA during nutrient deprivation and during formation of infection structures. Hybridizations of M. anisopliae genomic DNA indicate that there is only one form of ssgA in the genome.

Effects of growth hormone-releasing factor and feed intake on energy metabolism in growing beef steers: net nutrient metabolism by portal-drained viscera and liver.

Effects of growth hormone-releasing factor (GRF) and intake on net nutrient metabolism by portal-drained viscera (PDV) and liver were measured in six growing Hereford x Angus steers fed a 75% concentrate diet at two intakes in a split-plot design with 4-wk saline or GRF injection periods within 8-wk intake periods. Daily rations were fed as 12 equal meals delivered every 2 h. Steers were injected s.c. for 21 d with either saline or 10 micrograms/kg of (1-29)NH2 human GRF at 12-h intervals. Six hourly measurements of net nutrient flux (venous-arterial concentration different [VA] x blood flow) across PDV and liver were obtained 8 to 10 d after injections began. Energy and N balances were measured using respiration calorimetry during the last week of injections. Greater intake increased blood flow (P less than .01) and net visceral release or removal of most nutrients (P less than .10). Exceptions included a decrease (P less than .10) in net PDV glucose release with greater intake in saline-treated steers and a decrease (P less than .01) in net liver removal of lactate with greater intake. Treatment of steers with GRF decreased net liver removal of alpha-amino N (AAN; P less than .05) and ammonia N (NH3N; P less than .10) and release of urea N (UN; P less than .05), increased liver release of glutamate (P less than .05), and decreased net PDV release of NH3 N (P less than .10). Decreased liver extraction ratio for AAN in GRF-treated steers (P less than .01) implies a direct effect of GRF treatment on liver metabolism separate from changes in liver AAN supply. Proportions of body N retention not accounted for by net total splanchnic AAN release increased with GRF treatment. This suggests a change in peripheral utilization of dietary AAN supply or an increase in total splanchnic N retention.

Molecular cloning and regulatory analysis of the cuticle-degrading-protease structural gene from the entomopathogenic fungus Metarhizium anisopliae.

The proteinaceous insect cuticle is an effective barrier against most microbes, but entomopathogenic fungi can breach it using extracellular proteases. We report here the isolation and characterization of a cDNA clone of the cuticle-degrading protease (Pr1) of Metarhizium anisopliae. The cDNA sequence revealed that Pr1 is synthesized as a large precursor (40.3 kDa) containing a signal peptide, a propeptide and the mature protein predicted to have a molecular mass of 28.6 kDa. The primary structure of Pr1 has extensive similarity with enzymes of the subtilisin subclass of serine endopeptidases and the serine, histidine and aspartate components of the active site in subtilisins are preserved. Proteinase K demonstrated the closest sequence similarity to Pr1 (61%) but Pr1 was twofold more effective than proteinase K at degrading isolated cuticles of Manduca sexta and 33-fold more effective at degrading structural proteins bound to the cuticle by covalent bonds. We postulate that the additional positively charged residues on the surface of the Pr1 molecule, as determined using proteinase K, may facilitate electrostatic binding to cuticle proteins which is a prerequisite for activity. Northern-blot analysis of RNA and nuclear run-on assays demonstrated transcriptional control of the expression of Pr1 during nutrient deprivation and during the formation of infection structures. Southern-blot analysis demonstrated that genes with significant homologies to Metarhizium Pr1 were present in the entomopathogens Aspergillus flavus and Verticillium lecanii but not Zoophthora (= Erynia) radicans.

Characterization of INF56, a gene expressed during infection structure development of Uromyces appendiculatus.

The bean rust fungus, Uromyces appendiculatus, undergoes thigmotropic differentiation to produce infection structures. Six differentiation-specific genes have been isolated and one, INF24, has been characterized [Bhairi et al., Gene 81 (1989) 237-243]. Here, we report the structure of a second gene, INF56, which was subcloned on a 2.6-kb fragment and sequenced. The location of the 1.0-kb INF56 transcript was determined by S1 nuclease protection and primer extension. A TATA box was found 38 bp upstream and a CAAT box 130 bp upstream from the major transcription start point (tsp). The gene contains two open reading frames: ORF2 is nested within ORF1; they share a 67-bp intron. ORF1 encodes a 14.1-kDa polypeptide which has an amino acid sequence rich in Gly, Pro and Ser. It has sequence similarity to a functional domain (V2) of mammalian cytokeratin type II. ORF2 encodes a 10.1-kDa polypeptide which is rich in Pro. It shares similarity with the cell-surface recognition region of chicken fibronectin. Hybrid selection and in vitro translation of the INF56 mRNA yielded two polypeptides of 15.5 and 23 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. INF56 is constitutively expressed at a low level, but the abundance of its steady-state transcript is upshifted 4.5 h after spore hydration during the period that infection structures are formed.

Effect of dietary carbohydrate on liver and kidney enzyme activities and plasma amino acids in the LA/N-cp rat.

Twenty obese and 20 lean LA/N-cp male rats and 20 male Sprague-Dawley rats were fed a diet containing either 54 percent sucrose or starch for six weeks. After a 14-16 hour fast, rats were killed. Liver and kidney enzyme activities were determined in the LA/N-cp rats while plasma urea and selected amino acids were determined in all rats. Liver glucose-6-phosphatase (G6PASE), fructose-1,6-bisphosphatase (FBPASE), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME), glucokinase (GK), pyruvate kinase (PK), phosphofructokinase (PFK), glutamic-oxaloacetic-transaminase (GOT), glutamic-pyruvic transaminase (GPT), arginase (ARGASE), arginine-synthase (ARG-SYN) and ornithine transcarbamylase (OTC) levels were significantly affected by phenotype (obese greater than lean). All the above changes in enzyme levels were exaggerated by sucrose-feeding with the exception of PK, PFK, GOT, GPT, ARGASE and ARG-SYN. Kidney cortex G6PASE, PEPCK and ARGASE activities were higher in the obese rats as compared to the lean littermates. Sucrose feeding resulted in higher cortex G6PASE, FBPASE and PEPCK as compared to starch-fed rats. A phenotype effect was noted with plasma glutamate, urea, leucine, isoleucine and valine (obese greater than lean) and a diet effect was seen with aspartate, phenylalanine, leucine and valine (sucrose greater than starch) concentration. Sprague-Dawley rats had higher plasma urea and lower alanine than lean LA/N-cp males. Metabolic obesity in the LA/N-cp rat appears to involve an elevated capacity for pathways of glycolysis, gluconeogensis, lipogenesis and amino acid catabolism in the liver.

Isolation and identification of actin from the phytopathogenic filamentous fungus uromyces appendiculatus.

Crude protein extracts of Uromyces appendiculatus contain a polypeptide that resembles actin in several ways. This protein eluates from DEAE-cellulose with concentrations of KCl known to release actin of other species from the cation. The polypeptide is recognized by polyclonal antibodies directed to sodium dodecyl sulfate-denatured actin of chicken gizzard as well as by a monoclonal antibody also made to gizzard actin from chicken, but not by antibodies made against rabbit skeletal muscle actin. Western blot analysis after electrophoresis of the protein on polyacrylamide revealed that the protein has an electrophoretic mobility very similar to that of rabbit skeletal muscle actin. We were unable either to isolate actin by affinity chromatography using immobilized DNase-I, or to identify bean rust actin using DNase-I inhibition assays. Nevertheless, large quantities of the protein sedimented by high speed centrifugation. The sedimented protein resisted attempts to solubilize it under conditions normally used to depolymerize actin filaments. Both of the latter findings indicate unusual features of bean rust actin. Immunocytochemical studies of actin localization in germlings of the fungus using two chicken gizzard actin antibodies revealed actin-containing sites which were similar to those previously observed with fluorescently tagged phallotoxin derivatives.

Novel GTP-binding proteins in plasma membranes of the fungus Metarhizium anisopliae.

We report the existence of several families of GTP-binding proteins in plasma membranes of Metarhizium anisopliae. Two proteins (18.4 and 24 kDa) resemble mammalian Gn-proteins in their being toxin insensitive, binding [alpha-32P]GTP on nitrocellulose blots of sodium dodecyl sulfate (SDS)-polyacrylamide gels, and also in their immunological properties. Four other proteins (31-38.2 kDa) were similar except that they did not bind [alpha-32P]GTP after treatment with sodium dodecyl sulfate. An 18.2 kDa cholera toxin substrate and three toxin insensitive bands (18.6, 18.8, and 24 kDa) are novel proteins antigenically related both to mammalian G-proteins and ras gene products. An additional 23 kDa pertussis toxin substrate (the major G-protein in a crude mycelial extract) reacted strongly with antisera to G-proteins but not with anti-ras serum. Other substrates ADP ribosylated by cholera toxin or botulinum D toxin were immunologically unreactive. Analysis of the structural and functional characteristics of these multiple GTP-binding proteins will promote a better understanding of signal transduction in fungi.

Characterization of an infection structure-specific gene from the rust fungus Uromyces appendiculatus.

Uredospores of the plant pathogen, Uromyces appendiculatus, infect leaves of the bean plant, Phaseolus vulgaris, through stomata. Physical stimuli provided by the stomate induce differentiation of the germ tube to form a series of infection structures involved in host colonization. Contact between the uredospores and the oil-collodion membranes induces formation of infection structures in the absence of the host. This report describes the characterization of a Uromyces gene, INF24, that is induced by the physical stimulus of an oil-collodion membrane. INF24 contains a 450-bp open reading frame which encodes a 16.4-kDa polypeptide. The N terminus of the INF24-encoded protein, and the C terminus of human single-stranded DNA-binding protein are both glycine-rich and share homology.

Signaling for growth orientation and cell differentiation by surface topography in uromyces.

The dimensions of the topographical signals for growth orientation and infection structure formation, a cell differentiation event that includes nuclear division, were determined for the stomatal penetrating rust fungus Uromyces appendiculatus. The differentiation signal was found to be a simple ridge on the substrate surface that had a markedly optimum height of 0.5 micrometer. Such ridges were microfabricated on silicon wafers by using electron-beam lithography. A similar ridge, in the form of a stomatal lip, was found associated with the stomatal guard cells of the bean (Phaseolus vulgaris) leaf. Ridge elevations greater than 1.0 micrometer or less than 0.25 micrometer did not serve as effective signals. Germ tubes of the fungus were highly oriented by ridge spacings of 0.5 to 6.7 micrometers. The data indicate that the fungus is able to distinguish uniquely minute differences in leaf surface topography in order to infect the host plant.

The development of infection structures by the rusts and other fungi.

Germlings of the rust fungi develop infection structures in response to specific cues from the host's surfaces. Features such as the stomatal guard cell, which directs placement of the appressorium over the stomate, serve to optimize pathogen entry. A wide range of plant parasitic fungi develop infection structures.

Inositol and sugars in adaptation of tomato to salt.

Tomato (Lycopersicon esculentum Mill. cv New Yorker) plants subjected to 100 millimolar NaCl plus Hoagland nutrients exhibited a pattern of wilting, recovery of turgor, and finally recovery of growth at a reduced level, which required 3 days. During the nongrowing, adaptation phase there were immediate increases in free hexoses and sucrose which declined to near control levels as growth resumed. There was a steady increase in myo-inositol content which reached its maximal level at the time of growth resumption. The myo-inositol level then remained elevated for the remainder of the experiment. Myo-inositol constituted two-thirds of the soluble carbohydrate in leaves and three-fourths of the soluble carbohydrate in roots of salt-adapted plants. Plants which were alternated daily between salt and control solutions accumulated less myo-inositol and exhibited less growth than the continuously salt-treated plants. In L. pennellii and in salt-tolerant and salt-sensitive breeding lines selected from L. esculentum x L. pennellii BC(1) and F(8), myo-inositol content was highest in the most tolerant genotypes, intermediate in the normal cultivar, and lowest in the sensitive genotype after treatment with salt.

Visualization of actin in situ by rhodamine-conjugated phalloin in the fungus Uromyces phaseoli.

Rhodamine-conjugated phalloin, a derivative of phalloidin which binds to F-actin, was associated with three types of structures in uredospore germlings of the bean rust fungus, Uromyces phaseoli. The structures were filaments, peripheral plaques, and intranuclear inclusions. The filaments, located throughout the germ tube but especially in the more basipetal regions, were observed as either barely perceivable, fine elements or as easily detectable, coarser structures. The plaques, which we suggest to be equivalent to filasomes, occurred near the periphery of the cell's cytoplasm. They were most numerous in the hyphal tip regions. Nuclear inclusions occurred within the nucleoplasm subjacent to the spindle pole body. Treatments with KI and phalloidin substantiated that the fluorescently labelled sites were F-actin. Treatment of the germlings with cytochalasin E caused the intranuclear inclusions to become extended often with branched, fine filaments. Similar treatments led to a disappearance of cytoplasmic filaments, but had no perceivable effect on the peripheral plaques.