Identification and characterization of Cercospora beticola necrosis-inducing effector CbNip1

Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin-initiated formation of reactive oxygen species is light-dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light-dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1-triggered necrosis, as light exposure delayed CbNip1-triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola–sugar beet disease process.     

Cercospora beticola toxins. IX. Relationship between structure of beticolins, inhibition of plasma membrane H+-ATPase and partition in lipid membranes

Beticolins are yellow toxins produced by the fungus Cercospora beticola . The effect of one of them, beticolin-1. has been investigated on corn root plasma membrane H⁺-ATPase (EC 3.6.1.35) at different purification levels (plasma membrane fraction, partially, or highly purified enzyme). The results obtained demonstrated that (1) the purified proton pump was inhibited directly by low amounts of the toxin (I₅₀= 1.62 ± 0.18 μM), (2) the biological effects of beticolin-1 were similar to those of CBT ( Cercospora beticola toxin). Furthermore, it was established that the efficiency of the different beticolins was clearly related to their ability to interact with the lipid bilayers, determined by fluorometric studies: the toxins that exhibited the lower I₅₀ (50% inhibitory concentrations) values were the molecules that had the lowest partition coefficient to liposomes.     

Beticolins, nonpeptidic, polycyclic molecules produced by the phytopathogenic fungus Cercospora beticola, as a new family of ion channel-forming toxins

Beticolins are toxins produced by Cercospora beticola, a phytopathogenic fungus responsible for the leaf spot disease of sugar beet. They form a family of 20 nonpeptidic compounds (named B0 to B19) that share the same polycyclic skeleton but differ by isomeric configuration (ortho- or para-) and by a variable residue R (bridging two carbons in one of the six cycles). It has been previously shown that B0 assembles itself into a multimeric structure and forms ion channels into planar lipid bilayers (C. Goudet, A.-A. Very, M.-L. Milat, M. Ildefonse, J.-B. Thibaud, H. Sentenac, and J.-P. Blein, Plant J. 14:359-364, 1998). In the present work, we investigate pore formation by three ortho-beticolins, B0, B2, and B4, and their related (i.e., same R) para-isomers, B13, B1, and B3, respectively, using planar lipid bilayers. All beticolins were able to form ion channels with multiple conductance states, although the type of cyclization (ortho- or para-) and residue (R) result in variations of channel conductance and ionic permeability, respectively. Channel formation by beticolins is likely to be involved in the biological activity of these toxins.     

De novo genome assembly of Cercospora beticola for microsatellite marker development and validation

Cercospora leaf spot caused by Cercospora beticola is a significant threat to the production of sugar and table beet worldwide. A de novo genome assembly of C. beticola was used to develop eight polymorphic and reproducible microsatellite markers for population genetic analyses. These markers were used, along with five previously described microsatellite loci to genotype two C. beticola populations from table beet fields in New York, USA. High allelic and genotypic diversity and low population differentiation were found between fields. Linkage disequilibrium of loci after clone-correction of datasets was attributed to the presence of two distinct clonal lineages within the populations. Linkage equilibrium of loci in one of the clusters supported the presence of sexual reproduction. The draft de novo genome assembly will help elucidate the reproductive system of C. beticola through investigating evidence of recombination in the C. beticola genome.     

Glutathione and N-acetylcysteinylglycine: protonation and Zn2+ complexation

The speciation study of the Zn(2+)/glutathione (GSH, H(3)G) and Zn(2+)/N-acetylcysteinylglycine (NAcCG, H(2)L) was performed in aqueous solution by means of potentiometry and ESI mass spectrometry. The ligand N-acetylcysteinylglycine was synthesized by protection/activation strategies. (1)H NMR data for the Zn(2+)/NAcCG system at different pH were also collected, to gain insight in the coordination modes for the ligand. The information collected for the NAcCG model ligand were used to propose the structure in solution for the Zn(2+)/GSH complexes. Dinuclear complexes of GSH with Zn(2+), which have never been proposed previously in the literature, were identified in solution and a model of their structure was proposed. Moreover, the Zn(2+) promoted deprotonation of the cysteinyl peptidic NH with formation of five membered (S,N(Cys)(-)) chelating rings was evidenced. The speciation study of the ternary Zn(2+)/GSH/NAcCG system was also performed, showing that the Zn(2+) does not bind preferentially to GSH in presence of NAcCG. The (1)H NMR protonation studies of both GSH and NAcCG were also performed, and a novel proton dissociation microconstant calculation procedure has been proposed and applied to GSH equilibria.     

Analysis of β-tubulin Gene Fragments from Benzimidazole-sensitive and -tolerant Cercospora beticola

Cercospora leaf spot of sugar beet, caused by the fungus Cercospora beticola, is a major foliar pathogen on sugar beet. Fungicide sprays have been used extensively to manage Cercospora leaf spot, including the benzimidazole fungicides. Resistance to benzimidazoles has been observed in isolates of C. beticola. The precise genetics of this resistance is not known in this fungus. We tested benzimidazole‐tolerant and ‐sensitive isolates and found a single mutation in the β‐tubulin gene of benzimidazole‐tolerant isolates that corresponds to a mutation known to confer benzimidazole tolerance in other ascomycetes. This mutation is predicted to cause a change from glutamic acid to alanine in the protein product. Isolates containing this mutation further show an increased sensitivity to an N‐phenylcarbamate, as would be predicted based on the mutant phenotype found in other filamentous fungi. Only a single mutation was found in isolates from different regions of the United States, isolated in different growing seasons.     

Host range of Cercospora apii and C. beticola and description of C. apiicola, a novel species from celery

The genus Cercospora is one of the largest and most heterogeneous genera of hyphomycetes. Cercospora species are distributed worldwide and cause Cercospora leaf spot on most of the major plant families. Numerous species described from diverse hosts and locations are morphologically indistinguishable from C. apii and subsequently are referred to as C. apii sensu lato. The importance and ecological role that different hosts play in taxon delimitation and recognition within this complex remains unclear. It has been shown that Cercospora leaf spot on celery and sugar beet are caused respectively by C. apii and C. beticola, both of which are part of the C. apii complex. During this study we characterized a new Cercospora species, C. apiicola, which was isolated from celery in Venezuela, Korea and Greece. The phylogenetic relationship between C. apiicola and other closely related Cercospora species was studied with five different gene areas. These analyses revealed that the C. apiicola isolates cluster together in a well defined clade. Both C. apii and C. beticola sensu stricto form well defined clades and are shown to have wider host ranges and to represent distinct species.     

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1)

Influxes of 13NH4+ across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Two kinetically distinct uptake systems for NH4+ were identified. In N-deprived plants, a Michaelis-Menten-type high-affinity transport system (HATS) operated in a 2.5 to 350 [mu]M range of external NH4+ concentration ([NH4 +]o). The Vmax of this HATS was 1.9 to 2.4 [mu]mol g-1 h-1, and the Km was 20 to40 [mu]M. At [NH4+]o from 500 [mu]M to 50 mM, a linear low-affinity system (LATS) was apparent. Both HATS and LATS were constitutive. A time-dependence study of NH4+ influx in previously N-deprived seedlings revealed a small transient increase of NH4+ influx after 24 h of exposure to 100 [mu]M [NH4+]o. This was followed by a decline of influx to a steady-state value after 4 d. In seedlings exposed to 100 [mu]M external NO3- concentration for 3 d, the Vmax for NH4+ uptake by HATS was increased approximately 30% compared to that found in N-deprived seedlings, whereas LATS was down-regulated. The present study defines the much higher uptake capacity for NH4+ than for N03- in seedlings of this species.     

Development of polymorphic microsatellite and single nucleotide polymorphism markers for Cercospora beticola (Mycosphaerellaceae)

The plant pathogenic fungus, Cercospora beticola, causes the most important foliage disease of sugar beet. A previous study has shown that isolates of opposite mating types are present in equal proportions in natural populations; therefore, the aim of this study was to develop highly reproducible polymorphic markers for analysing populations of C. beticola. Five microsatellite and four single nucleotide polymorphism (SNP) markers were developed that allow rapid screening of genetic diversity in C. beticola. Six populations were screened with these markers and all were found to be in gametic equilibrium, indicating random mating in C. beticola.     

Differential response of a sensitive and tolerant sugarbeet line to Cercospora beticola infection and UV-B radiation

Increased ultraviolet-B (UV-B, 280–320 nm) radiation, due to depletion of stratospheric ozone, is an increasing threat to living organisms. Furthermore, increased ground level temperatures as a consequence of global warming may favour development of pathogens, such as Cercospora beticola , that thrive at high temperatures. This study evaluates the effect of combined UV stress and Cercospora leaf-spot disease on young sugarbeet plants ( Beta vulgaris L . ). An inoculum consisting of twelve European isolates of C. beticola Sacc. was used in the experiments. One Cercospora -sensitive and one Cercospora -tolerant sugarbeet line were analysed from growth regimes where plants were grown either under visible radiation alone or with supplemental UV-B. Photosynthetic pigments and partial reactions of photosynthesis, including potential yield and quantum yield under illumination, non-photochemical quenching (q_NPQ) and photochemical quenching (q_P), were measured to assess plant response. The combination of Cercospora and supplemental UV-B radiation in the sensitive line resulted in a decreased photosynthetic efficiency, shown by q_NPQ and quantum yield under illumination as compared with that for either stress applied alone. The F_v/F_m was unchanged for plants subjected to UV-B radiation without infection, although the q_NPQ decreased. The Cercospora -tolerant line showed no significant differences under the different treatments. Thus, the line tolerant to Cercospora infection also proved to be tolerant to UV-B radiation alone and in combination with the infection.     

Symptomatological and Morphological Study of the Resistance of Wild Beet Species of the Patellares Section to Cercospora beticola Sacc.

The reactions of Beta procumbens C. Sm. and Beta webbiana Moq. were compared to those of Beta vulgaris L. with regard to an infection by Cercospora beticola Sacc. The fleck reaction obsrved in B. webbiana may be interpreted as hypersensitivity based on symptomatological, light microscopical, fluorescent microscopical and electron microscopical data. The B. procumbens clone was found to show resistance characteristics similar to those of B. webbiana and B. vulgaris, as it reacted both by flecks (B. webbiana) and leaf spots (B. vulgaris) to a C. beticola infection.     

Tautomeric and microscopic protonation equilibria of some alpha-amino acids

Although there is a wealth of structural and theoretical data relating to palindromic sequences in genomes, the mechanisms of extrusion of cruciform structures during various biological processes in the presence of intercalating agents are still poorly understood. The current study addresses the effects of temperature and intercalator on cruciform extrusion from plasmids and also considers the effects of divalent metal ions on cruciform extrusion. It presents evidence that the cytotoxic effects of certain DNA binding drugs in vivo occur over concentration ranges corresponding to those that modulate cruciform extrusion in vitro. The results confirm earlier studies showing an inverse relationship between the effects of negative superhelicity and temperature on cruciform extrusion. By extrapolation, divalent metal ions facilitate cruciform extrusion by increasing superhelicity. The results allow the concentrations that preclude cruciform extrusion in DNA to be determined, and these are potentially informative about the relationships among temperature, DNA helical winding, cruciform formation, and intercalation. Overall, we provide new and interesting insights into the potential role of cruciform structures in biology and, by implication, cancer therapy.     

Radiation studies of aryl glycosides : Part VI. Radicals derived from Phenyl β-d-glucopyranosides

Radicals formed by γ-irradiation of o-, m-, and p-substituted-phenyl β-d-glucopyranosides have been studied in the polycrystalline state, in glassy methanol, and in frozen, aqueous solution. Substituted cyclohexadienyl radicals and radicals derived from the d-glucopyranosyl group are evident after irradiation of the compounds in the solid state and in frozen, aqueous solutions. Cyclohexadienyl radicals are the more stable during thermal annealing and are present in 2–3 times greater yield than the sugar radicals. p-Hydroxy- and methoxy-phenyl β-d-glucopyranosides yield phenoxy radicals, which can be transformed into substituted cyclohexadienyl radicals by thermal annealing. Hydrogen abstraction and inter- and intra-molecular hydrogen-transfer are the most likely processes leading to the radicals which have been identified.     

Microbes and microbial toxins: paradigms for microbial-mucosal interactions. VI. Entamoeba histolytica: parasite-host interactions

The protozoan intestinal parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. E. histolytica causes two major clinical syndromes, amebic colitis and amebic liver abscess. Recent advances in the development of in vitro and in vivo models of disease, new genetic approaches, the identification of key E. histolytica virulence factors, and the recognition of crucial elements of the host response to infection have led to significant insights into the pathogenesis of amebic infection. E. histolytica virulence factors include 1) a surface galactose binding lectin that mediates E. histolytica binding to host cells and may contribute to amebic resistance to complement, 2) amebapores, small peptides capable of lysing cells, which may play a role in killing intestinal epithelial cells, hepatocytes, and host defense cells, and 3) a family of secreted cysteine proteinases that play a key role in E. histolytica tissue invasion, evasion of host defenses, and parasite induction of gut inflammation. Amebae can both lyse host cells and induce their suicide through programmed cell death. The host response is also an important factor in the outcome of infection, and neutrophils may play a key role in contributing to the tissue damage seen in amebiasis and in controlling amebic infection.