The genetics of phytotoxin production by plant pathogenic fungi

Little is known about the genetic control of phytotoxin production by plant pathogenic fungi. The production of host-selective toxins known to play a role in disease development has been genetically analyzed in three species ofCochliobolus. InC. heterostrophus, a single genetic locus with two alleles has been identified controlling the production of HMT-toxin. This locus appears to be at or near the breakpoint of a chromosome rearrangement. Single genetic loci have also been identified controlling the production of HC-toxin byC. carbonum and HV-toxin byC. victoriae. The locus inC. carbonum may be a cluster of tightly linked genes.     

Host recognition by toxigenic plant pathogens

Certain fungal pathogens release host-selective (or host-specific) toxins (HST) as a host recognition factor during spore germination at the infection site on plants. Prior to penetration of the pathogen into its host, the released toxin specifically binds to a putative receptor on the host cells and initiates signaling mechanisms leading to pleiotropic effects on cells. Of these, the crucial one negates the general and inducible defense reactions of the cells. This is accomplished by a signal from the HSt, which is transduced through a path way at or near the step of plasma membrane modulation, which is directly or indirectly triggered by the HST. This mechanism operates even though the toxin may affect mitochondria or chloroplasts as the primary target organelle. The fungal spore is able to penetrate the so-called narcotized cell and completes the initial colonization of the host. The host recognition process may take place without necessitating host cell death, even in the case of perthophytic parasites. At the molecular level, HST-mediated recognition of the host by a pathogen requires strict stereochemical precision like a lock and key.     

Characterization of victorin C,the major host-selective toxin fromCochliobolus victoriae: Structure of degradation products

Summary Several host-selective toxins have been isolated in pure form culture filtrates ofCochliobolus victoriae. Acid hydrolysis of the major toxin, victorin C (apparent mol.wt 796), produced five fragments to which structures1–5 have been assigned. Spectroscopic techniques revealed that the toxin contains an additional subunit corresponding to6; thus all the components of victroin C are accounted for.     

Development of DNA probes for cytotoxin and enterotoxin genes in enteric bacteria

Summary DNA probes to identify the genes encoding toxins in enteric bacteria have been developed. Use of these probes reduces the number of animals required for toxicity testing, as suspect bacteria can be directly tested for the presence of toxin. We have augmented the gene probes available by developing probes against theEscherichia coli enterotoxin LTII and shiga toxin fromShigella dysenteriae 1.The LTII gene fromE. coli 357900 was identified and characterised and a suitable internal probe was obtained. The LTII gene was found not to be common among enterobacteriae from various geographical locations. Isolates predominately of animal origin from Nigeria and Thailand hybridized with the probe.The shiga toxin gene was isolated fromS. dysenteriae 1 by a combination of in vivo and in vitro methods. An internal probe was identified and used against different serogroups ofShigella andE. coli isolated. The probe was found to hybridize withS. dysenteriae 1 isolates and also someS. flexneri andS. sonnei strains. Representatives were tested for toxin production and found to produce toxin at low levels.     

Structure of victorin C,the major host-selective toxin fromCochliobolus victoriae

Summary The predominant host-selective toxin fromCochliobolus victoriae, victorin C, is a peptide with an apparent mol. wt of 796, representing a cyclic array of the subunits1–6. The structure of the toxin has now been established as in16 through analysis of the degradation products generated by enzymic and non-enzymic partial hydrolysis. The presence of a hydrated aldehydo group requires for victorin C the composition C₃₁H₄₅O₁₃N₆Cl₃ with an amended mol. wt of 814, for which independent experimental support has been secured.     

Studies on the fungal phytotoxin victorin: Structures of three novel amino acids from the acid hydrolyzate

Summary The host-selective phytotoxin victorin, produced by the fungusCochliobolus victoriae, was found to be at least partially peptidic in nature, and did not contain victoxinine. The exact mass of the M-H ion was measured by FABMS as 795.1877. Derivatives of three major acid hydrolysis products were isolated. The structures of the corresponding amino acids were assigned as 2S,3R-3-hydroxyleucine, 5,5-dichloroleucine, and 3-hydroxylysine. A into victorin by the fungus in vivo.     

Genetic and molecular approaches to synthesis and action of the yeast killer toxin

Summary The K1 killer toxin ofSaccharomyces cerevisiae is a secreted, virally-coded protein lethal to sensitive yeasts. Killer yeasts are immune to the toxin they produce. This killer system has been extensively examined from genetic and molecular perspectives. Here we review the biology of killer yeasts, and examine the synthesis and action of the protein toxin and the immunity component. We summarise the structure of the toxin precursor gene and its protein products, outline the proteolytic processing of the toxin subunits from the precursor, and their passage through the yeast secretory pathway. We then discuss the mode of action of the toxin, its lectin-like interaction with a cell wall glucan, and its probable role in forming channels in the yeast plasma membrane. In addition we describe models of how a toxin precursor species functions as the immunity component, probably by interfering with channel formation. We conclude with a review of the functional domains of the toxin structural gene as determined by site-directed mutagenesis. This work has identified regions associated with glucan binding, toxin activity, and immunity.     

Protein-O-mannosyltransferases in virulence and development

Protein-O-mannosyltransferases (Pmt proteins) catalyse the addition of mannose to serine or threonine residues of secretory proteins. This modification was described first for yeast and later for other fungi, mammals, insects and recently also for bacteria. O-mannosylation depends on specific isoforms of the three Pmt1, 2 and 4 subfamilies. In fungi, O-mannosylation determines the structure and integrity of cell walls, as well as cellular differentiation and virulence. O-mannosylation of specific secretory proteins of the human fungal pathogen Candida albicans and of the bacterial pathogen Mycobacterium tuberculosis contributes significantly to virulence. In mammals and insects, Pmt proteins are essential for cellular differentiation and development, while lack of Pmt activity causes Walker-Warburg syndrome (muscular dystrophy) in humans. The susceptibility of human cells to certain viruses may also depend on O-mannosyl chains. This review focuses on the various roles of Pmt proteins in cellular differentiation, development and virulence. Received 6 September 2007; received after revision 3 October 2007; accepted 5 October 2007     

Genetics of toxin production and resistance in phytopathogenic bacteria

Genes for phytotoxin production have been identified and cloned from several phytopathogenic pseudomonads. These genes comprise physically linked clusters that have been located both on the chromosome and on endogenous plasmids. Contained within these genetic regions are resistance genes specific to those toxins that have a bactericidal component to their activity. DNA sequences required for toxin production are often conserved among bacteria with divergent host specificities, suggesting the ability of toxin genes to be transferred between bacteria. Toxins are usually modulators of plant pathogenicity, their production causing a significant increase in disease severity. In one case, however, toxin production appears to be a major contributor to the basic pathogenicity of a plant pathogenic bacterium.     

Gel-electrophoretic description of European populations ofTerellia virens (Loew) (Diptera,Tephritidae); implications for its use as an agent for the biological control ofCentaurea spp. (Asteraceae) in North America

Allozyme frequencies of 15 enzyme loci, 14 of which were polymorphic, were used to characterize sevenTerellia virens populations originating from three allopatrically distributedCentaurea species. The two populations whose origins were geographically furthest apart, from Israel (onC. iberica) and from Switzerland (onC. vallesiaca), showed relatively high values of genetic distance from the 5 populations sampled in Austria and Hungary (onC. maculosa) (Nei's D>0.07). The latter five displayed a high degree of genetic similarity. No diagnostic (fixed) allelic differences were observed between these three groups ofT. virens populations, but they could be well characterized by significant differences in allelic frequencies at 9 enzyme loci. Independently of this study, the populations from Switzerland (C. vallesiaca) and eastern Austria (C. maculosa) were selected as potential source populations for future introductions into North America for the biological control of introducedC. maculosa andC. diffusa. Based on the observed genetic differences and results from field experiments on the host specificity of these two potential source populations, it is argued that host specificity screening tests should be conducted separately for local (host plant) populations, as such populations might accept a different set of hosts. Biotype mismatch and the risk of spill-overs to native species could thus possibly be reduced.     

Host recognition by toxigenic plant pathogens

Certain fungal pathogens release host-selective (or host-specific) toxins (HST) as a host recognition factor during spore germination at the infection site on plants. Prior to penetration of the pathogen into its host, the released toxin specifically binds to a putative receptor on the host cells and initiates signaling mechanisms leading to pleiotropic effects on cells. Of these, the crucial one negates the general and inducible defense reactions of the cells. This is accomplished by a signal from the HST, which is transduced through a path way at or near the step of plasma membrane modulation, which is directly or indirectly triggered by the HST. This mechanism operates even though the toxin may affect mitochondria or chloroplasts as the primary target organelle. The fungal spore is able to penetrate the so-called 'narcotized cell' and completes the initial colonization of the host. The host recognition process may take place without necessitating host cell death, even in the case of perthophytic parasites. At the molecular level, HST-mediated recognition of the host by a pathogen requires strict stereochemical precision like a lock and key.     

Competitiveness and communication for effective inoculation byRhizobium,Bradyrhizobium and vesicular-arbuscular mycorrhiza fungi

After a short summary on the ecology and rhizosphere biology of symbiotic bacteria and vesicular-arbuscular (VA) mycorrhiza fungi and their application as microbial inocula, results on competitiveness and communication are summarized. Stress factors such as high temperature, low soil pH, aluminium concentrations and phytoalexins produced by the host plants were studied withRhizobium leguminosarum bv.phaseoli andRhizobium tropici onPhaseolus beans. Quantitative data for competitiveness were obtained by usinggus ⁺ (glucoronidase) labelled strains, which produce blue-coloured nodules. ForPhaseolus-nodulating rhizobia, a group specific DNA probe was also developed, which did not hybridize with more than 20 other common soil and rhizosphere bacteria. Results from several laboratories contributing to knowledge of signal exchange and communication in theRhizobium/Bradyrhizobium legume system are summarized in a new scheme, including also defense reactions at the early stages of legume nodule initiation. Stimulating effects of flavonoids on germination and growth of VA mycorrhiza fungi were also found. A constitutive antifungal compound in pea roots, -isoxazolinonyl-alanine, was characterized.     

β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome

Staphylococci have two mechanisms for resistance to β-lactam antibiotics. One is the production of β-lactamases, enzymes that hydrolytically destroy β-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by β-lactam antibiotics. Strains of S. aureus exhibiting either β-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism. Received 2 April 2005; received after revision 15 July 2005; accepted 25 July 2005     

Fungal competition and mycotoxin production on corn

Summary Aflatoxin and secalonic acid D production in corn in laboratory and field by mixed cultures ofPencillium oxalicum andAspergillus flavus orA. parasiticus was lower than production by the pure cultures. However, mixed culture of these molds withFusarium spp. did not affect mycotoxin production.     

Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells?

Cry proteins, produced by Bacillus thuringiensis (Bt), are widely used for the control of insect pests in agriculture as spray products or expressed in transgenic crops, such as maize and cotton. Little was known regarding the mechanism of action of these toxins when the first commercial Bt product was introduced fifty years ago. However, research on the mechanism of action over the last two decades has enhanced our knowledge of toxin interaction with membrane receptors and their effects in insect midgut cells. All this information allowed for the rational design of improved toxins with higher toxicity or toxins that overcome insect resistance, which could compromise Bt use and effectiveness in the field. In this review we discuss and evaluate the different models of the mode of action of Cry toxins, including a discussion about the role of various receptors in toxin action. Received 13 June 2008; received after revision 05 November 2008; accepted 11 November 2008     

Variation in regulation of aflatoxin biosynthesis among isolates ofAspergillus flavus

Two new phenotypes ofAspergillus flavus which exhibit novel patterns of aflatoxin production have been identified and characterized. In one of the new variants ofA. flavus, aflatoxin is made in the absence of carbohydrate and concomitantly with growth, without a lag period. A second variant did not produce aflatoxin in the presence or absence of carbohydrate. Chemical mutagenesis of this nonaflatoxigenic strain resulted in mutant strains which produced aflatoxin on carbohydrate-containing media. The aflatoxin production pattern observed in these mutants resembled the typical production scheme, with a lag period through log phase growth.     

Occurrence of tetrodotoxin in the starfishAstropecten latespinosus

Summary A toxin causing paralysis was detected in the starfishAstropecten latespinosus. The toxin was purified by a method consisting of charcoal treatment and chromatography on CM-Sephadex C-25 and Bio-Rex 70. The toxin was identified as tetrodotoxin by its behavior in thin-layer chromatography and electrophoresis and its¹H-NMR spectrum.Acknowledgment. This work was in part supported by a research fund from the Ministry of Agriculture, Forestry and Fisheries of Japan.     

Anthraquinone pigments from a conidiating mutant ofTrichoderma viride

Summary Two pigments responsible for the yellow-orange color of a brown conidiating mutant of the deuteromyceteTrichoderma viride were isolated and spectroscopically identified as 1,3,6,8-tetrahydroxyanthraquinone (I) and 1-acetyl-2,4,5,7-tetrahydroxy-9,10-anthracenedione (II). Both compounds are known substances but were not yet reported as metabolites of this fungal species. Their relationship to other anthraquinones produced byT. viride is discussed.Dedicated to Dr Z. Vanek, on the occasion of his 60th birthday.Upper case letters denote multiplicity arising from the directly bonded protons, lower case letters that from the geminal and vicinal couplings.     

Biochemical approaches to the study of plant-fungal interactions in arbuscular mycorrhiza

This communication compares some biochemical methods for quantifying colonization by arbuscular mycorrhizal (AM) fungi. The degree of mycorrhizal colonization can conveniently be measured by determining fungal specific sterols. AM-colonized plants show a specific synthesis of 24-methylene cholesterol and an enhanced level of campesterol (=24-methyl cholesterol). A gene probe for nitrate reductase, the key enzyme for nitrogen assimilation, has been developed, which allows the monitoring of the distribution of this enzyme in fungi. Among the phytohormones tested, only abscisic acid (ABA) is found at a considerably higher level in AM-colonized plants than in controls. The concentration of ABA is about twenty times higher in spores and hyphae of the AM fungusGlomus than in maize roots. Other phytohormones (auxins, cytokinins) do not show such alterations after mycorrhizal colonization. The roots of gramineous plants become yellow as a result of mycorrhizal colonization. The yellow pigment(s) formed is (are) deposited in larger quantities in the vacuole(s) of the root parenchyma and endodermis cells during the development of the gramineous plants. A substance isolated from such roots has now been identified as a C-14 carotenoid with two carboxylic groups, and named mycorradicin.     

Total synthesis of orellinine,a minor toxic component of the fungusCortinarius orellanus fries

Summary The 3,3,4,4-tetrahydroxy-2,2-bipyridyl-N-oxide has been synthesized by dealkylation of the corresponding tetramethyl derivative. The chemical properties of this compound are identical to those reported for the minor fungal toxin ofCortinarius orellanus, orellinine.This work is part of the Tesi di Laurea of Miss Cinzia Pierucci.