Cerato-ulmin Research Articles

Plant Defense Elicitation by the Hydrophobin Cerato-Ulmin and Correlation with Its Structural Features.

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic-hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein's loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a β-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability.

Characterization of Submicron Bubbles Formed by the Hydrophobin Cerato-ulmin.

Cerato-ulmin is a fungal hydrophobin protein with a high surface activity due to its amphipathic nature. When aqueous dispersions are gently agitated by hand, cerato-ulmin (CU) assembles into cylindrical bubbles visible in an optical microscope. After approximately 1 h the larger micron-sized bubbles rise out of the solution, leaving only submicron particulates, which persist indefinitely. Dynamic light scattering experiments show that these persistent particles shrink when positive air pressure is applied to the suspension and expand when vacuum is applied. Small-angle X-ray scattering at ambient pressure suggests an extended core-shell structure, consistent with small air-filled bubbles stabilized by a protein film. A comparison between the SAXS of the persistent submicron bubbles and AFM of the buoyant larger bubbles found immediately after agitation show that both have similar film thickness of 13-15 nm or five protein molecules. The extended shapes confirm the solid-like properties of these CU membranes, even in submicron particulate structures, consistent with microtensiometry results on interfacial CU membranes.

Electric Field Deformation of Protein-Coated Droplets in Thin Channels.

High-strength droplet interfaces are attractive for many applications, specifically in cases where droplets are channeled through fluidic devices and manipulated by electromagnetic fields. Using models and experiments, we study the deformation of droplets and capsules with protein interfaces in an electric field in thin and wide electrode gaps. Proteins are chosen from candidates expected to display qualitatively different interfacial interactions and strengths: a globular protein (bovine serum albumin), a reversible cross-linking peptide (AFD4), and a hydrophobin (cerato ulmin). Dilute protein additives can lead to over 1 order of magnitude stronger oil-water interfaces than those stabilized by small surfactants. We develop small deformation models to evaluate a protein membrane's interfacial elasticity, notably accounting for the electric field perturbation encountered in a gap and a careful treatment of a generalized elastic interface with both surface tension and interfacial elasticity. Results indicate that globular proteins, which typically have comparable surface tension and interfacial elasticity, can be modeled well by this generalized elastic interface. We further find that when in a gap, droplets and capsules migrate toward one electrode, deform asymmetrically, exhibit polar spreading on the electrode, and predictably stretch more than in the infinite gap scenario at constant field strength.

Formation of a Rigid Hydrophobin Film and Disruption by an Anionic Surfactant at an Air/Water Interface.

Hydrophobins are amphiphilic proteins produced by fungi. Cerato-ulmin (CU) is a hydrophobin that has been associated with Dutch elm disease. Like other hydrophobins, CU stabilizes air bubbles and oil droplets through the formation of a persistent protein film at the interface. The behavior of hydrophobins at surfaces has raised interest in their potential applications, including use in surface coatings, food foams, and emulsions and as dispersants. The practical use of hydrophobins requires an improved understanding of the interfacial behavior of these proteins, alone and in the presence of added surfactants. In this study, the adsorption behavior of CU at air/water interfaces is characterized by measuring the surface tension and interfacial rheology as a function of adsorption time. CU is found to adsorb irreversibly at air/water interfaces. The magnitude of the dilatational modulus increases with adsorption time and surface pressure until CU eventually forms a rigid film. The persistence of this film is tested through the sequential addition of strong surfactant sodium dodecyl sulfate (SDS) to the bulk liquid adjacent to the interface. SDS is found to coadsorb to interfaces precoated with a CU film. At high concentrations, the addition of SDS significantly decreases the dilatational modulus, indicating disruption and displacement of CU by SDS. Sequential adsorption results in mixed layers with properties not observed in interfaces generated from complexes formed in the bulk. These results lend insight to the complex interfacial interactions between hydrophobins and surfactants.

Protein-Assisted Assembly of π-Conjugated Polymers

In an aqueous suspension process, protein dispersions facilitated improved alignment and organization of poly(3-hexylthiophene) (P3HT) chains into highly ordered crystalline structures. A solution of P3HT in 1,2,4-trichlorobenzene (TCB) was added to an aqueous dispersion of the hydrophobin, Cerato ulmin (CU). Upon gentle agitation, the semiconductor solution was readily confined within CU membrane-stabilized microstructures, often with extended shapes. UV–vis and polarized micro-Raman spectroscopy suggested complex, enhanced molecular alignment due to a transition from isotropic to liquid crystalline fluid to polycrystalline states. Grazing-incidence X-ray diffraction corroborates this interpretation. On aging, the initial CU:P3HT/TCB structures develop dendritic architectures that slowly release polymer-containing capsules. The counterintuitive evolution from large structures to smaller ones suggests the initial structures were nonequilibrium, and it opens the door to latex-like processing of semiconduct...

Establishment of invasive and non-invasive reporter systems to investigate American elm–Ophiostoma novo-ulmi interactions

Dutch elm disease (DED), caused by ascomycete fungi in the Ophiostoma genus, is the most devastating disease of American elm (Ulmus americana) trees. Cerato ulmin (CU), a hydrophobin secreted by the fungus, has been implicated in the development of DED, but its role in fungal pathogenicity and virulence remains uncertain and controversial. Here, we describe reporter systems based on the CU promoter and three reporter proteins (GFP, GUS and LUC), developed as research tools for quantitative and qualitative studies of DED in vitro, in vivo and in planta. A strain of the aggressive species Ophiostoma novo-ulmi was transformed with the reporter constructs using Agrobacterium-mediated transformation and the fungal transformants, namely M75-GFP, M75-GUS and M75-LUC, were examined for mitotic stability after repeated subcultures. The intensity of GFP fluorescence was strong in M75-GFP spores and hyphae, allowing microscopic investigations of spore structure, fungal morphogenesis and fungal development. The interaction of M75-GFP and U. americana callus cells was explored with scanning laser confocal microscopy facilitating qualitative studies on fungal strategies for the invasion and penetration of elm cells. M75-GUS was generated to provide an invasive, yet quantitative approach to study fungal–plant interactions in vitro and in planta. The generation of M75-LUC transformants was aimed at providing a non-destructive quantitative approach to study the role of CU in vivo. The sensitivity, low background signal and linearity of LUC assays all predict a very reliable approach to investigate and re-test previously claimed roles of this CU in fungal pathogenicity. These reporter systems provide new tools to investigate plant–pathogen interactions in this complex pathosystem and may aid in better understanding the development of DED.

Progressive pearl necklace collapse mechanism for cerato-ulmin aggregation film

Cerato-ulmin (CU) is a fungal toxin class II hydrophobin, involved in Dutch elm disease. The formation of hydrophobin films at the air-water interface is a key mechanism which plays a role of paramount importance at different stages of the fungal development. We present a study on the precursor stages of growth towards the self-assembly aggregation film of CU. Atomic force microscopy images of CU dropped on mica substrates indicate that the system self-organizes in almost one-dimensional pearl-necklace-like chains, which subsequently collapse and possibly merge to form extended and rather compact planar films. We propose and verify a simple model to describe the self-aggregation mechanism in terms of progressive thickening of the pearl chains due to the successive merging and collapse of the elementary constitutive units.

Isolation of the orthologue of the cerato-ulmin gene in Ophiostoma quercus and characterization of the purified protein

Ophiostoma quercus is an ophiostomatoid fungus strictly related to the Ophiostoma's ( O. ulmi, O. novo-ulmi, and O. himal-ulmi) that cause Dutch elm disease (DED). O. quercus has a number of morphological characteristics in common with the DED pathogens, and is a well-known and economically important sapstaining fungus occurring worldwide on hardwoods and commercially produced pines, and causes typical cankers on oak stems. In elm trees O. quercus can survive for months without causing any disease symptoms. DED fungi produce cerato-ulmin (CU), a class II hydrophobin, which is generally considered as the main toxin potentially involved in various phases of the DED pathogenesis. In the present work we isolated and sequenced the orthologue of the cu gene in the O. quercus isolates H988, H1042, and H2053. Moreover the CU protein from O. quercus isolate H988 was also purified and characterized. Sequence analysis showed that there is a pronounced difference between the whole cu gene region of O. quercus and the homologous fragments of the DED-causing species O. ulmi, O. novo-ulmi, and O. himal-ulmi. It also appeared that differences in the structural conformation of the promoter were unlikely to play a role in the modulation of the transcript level and that, for O. quercus, differences in CU production did not result from the potential different regulation levels. Clear differences were shown in the transcriptional unit of the cu genes and in the amino acid sequences among all the CUs. The purified O. quercus CU was separated using matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) spectrometry into seven forms of increasing molecular weight from 7190 to 7724 Da. The hydrophobicity profiles indicated that two regions of the O. quercus CU protein were more hydrophobic than the corresponding regions of the CUs of the DED fungi. The O. quercus CUs had theoretical isoelectric point values similar to those of the DED fungi. Finally, the contradiction between the consistent differences between these four Ophiostoma species in the cu gene region and in the CU proteins and their strict phylogenetic relationship is discussed.

Real time RT-PCR quantification and Northern analysis of cerato-ulmin ( CU) gene transcription in different strains of the phytopathogens Ophiostoma ulmi and O. novo-ulmi

Cerato-ulmin is a surface protein that belongs to the class of fungal proteins known as hydrophobins. This class II hydrophobin is produced throughout the life cycle and in all developmental stages of Ophiostoma novo-ulmi and O. ulmi; the aggressive and non-aggressive pathogens responsible for Dutch elm disease. Since yeast/mycelial transitions are often important to pathogenesis in dimorphic fungi such as Ophiostoma, we have examined the levels and abundance of cu mRNA in the yeast and mycelial stages of this fungus. The fungus contains one copy of the cu gene per haploid genome, located on chromosome IV. Our studies have been done using phosphoimager-based Northern analysis and real-time quantitative RT-PCR (qRT-PCR) to measure levels of cu mRNA. These measurements were made in both yeast-like and mycelial stages of the pathogen. Two wild-type, aggressive, strains of O. novo-ulmi (VA30 and H327) and one wild type non-aggressive strain of O. ulmi (H5) were analysed. As controls, we have utilized two types of mutants that we had previously generated, the null cu mutants THEK5-8 and THEK5-8-1, and a cu over-expression mutant, H5-tf16. Data generated by both Northern hybridization and real-time qRT-PCR analyses demonstrate that there is no cu mRNA transcription in the null mutants. The Northern analysis clearly showed that the over-expressing mutant H5-tf16 produces much more cu mRNA than the non-aggressive or aggressive strains. The quantitative data generated using qRT-PCR demonstrated that mycelium generally had 20-60% more cu mRNA than the yeast form. The non-aggressive strain of O. ulmi (H5) produces one-tenth as much cu mRNA as the aggressive strains (VA30 and H327). When transformed with additional copies of the cu gene, this same non-aggressive strain (H5-tf16) expressed about 20 times more cu mRNA than the wild type H5 strain. These data were consistently generated in multiple real-time qRT-PCR experiments with different RNA preparations, clearly demonstrating that the quantitative abundance values obtained were reproducible. Our study represents the first report on the use of real-time qRT-PCR to compare and quantify gene transcription in different growth phases of a fungal pathogen.

Two natural cerato‐ulmin (CU)‐deficient mutants of Ophiostoma novo‐ulmi: one has an introgressed O. ulmi cu gene, the other has an O. novo‐ulmi cu gene with a mutation in an intron splice consensus sequence

Summary The nucleotide sequences of the cerato-ulmin (cu) genes of two naturally occurring pathogenic CU-deficient mutants, PG470 and MAFf8, of the Dutch elm disease fungus, Ophiostoma novo-ulmi, were determined. The PG470 cu gene sequence was identical to that of CU-secreting isolates of O. novo-ulmi, except for a G to A mutation in the GT splice consensus sequence at the start of intron 1, suggesting that the CU deficiency was due to a splicing defect in the premRNA. In contrast, the MAFf8 cu gene showed a 99.1% sequence identity with cu genes of O. ulmi isolates, but only 92.8% sequence identity with cu genes of CU-secreting isolates of O. novo-ulmi, and in a dendrogram clustered with cu gene sequences of O. ulmi isolates with 100% bootstrap support. Restriction fragment length polymorphisms of the ribosomal RNA region, random amplified polymorphic DNA markers, and many biological properties of MAFf8, including pathogenicity, were typical of O. novo-ulmi. It is therefore likely that the cu gene of MAFf8 has been introgressed from O. ulmi, probably as a result of rare hybrid formation between O. ulmi and O. novo-ulmi, followed by backcrossing of the hybrid with O. novo-ulmi. The presence of an O. ulmi-like cu gene in MAFf8 is consistent with its CU deficiency, since the O. ulmicu gene is known to be poorly expressed and O. ulmi isolates secrete little or no CU in culture.

Functional expression of the gene cu, encoding the phytotoxic hydrophobin cerato-ulmin, enables Ophiostoma quercus, a nonpathogen on elm, to cause symptoms of Dutch elm disease.

We studied the involvement of the phytotoxic hydrophobin cerato-ulmin (CU) in pathogenesis and virulence of Dutch elm disease (DED) by expressing its encoding gene (cu) in Ophiostoma quercus, a nonpathogenic species on elm closely related to the DED pathogens O. ulmi and O. novo-ulmi. The production of the toxin was quantitatively determined in culture filtrates and in mycelial extracts of the transformants. Production of CU in vitro was associated with the ability to cause typical DED symptoms, consisting of foliar yellow and wilting and vascular tissue discoloration on a moderately resistant elm genotype. The presence of CU was monitored by enzyme-linked immunosorbent assay in symptomatic leaves of plants inoculated with O. quercus transformants expressing CU and found to be associated with wilt symptoms. In general, the virulence of the cu-expressing transformants, as measured in terms of vascular discoloration and percentage of defoliation, was lower than that of the mildly pathogenic isolate E2 of O. ulmi. However, one transformant (C39) displayed a virulence level intermediate between that of E2 and 182, a highly virulent isolate of O. novo-ulmi. Our results indicate that CU production influences virulence in nonaggressive strains of Ophiostoma fungi.

Cerato-ulmin, a Hydrophobin Secreted by the Causal Agents of Dutch Elm Disease, Is a Parasitic Fitness Factor

Dutch elm disease is caused by the aggressiveOphiostoma novo-ulmiand the nonaggressiveO. ulmi.Both secrete the protein cerato-ulmin (CU). To determine what role CU plays in the pathology of Dutch elm disease, we constructed a CU overexpression mutant of the nonaggressiveO. ulmiH5. Stable integration of a single copy of thecugene from the aggressiveO. novo-ulmiinto the genome of the nonaggressive isolate resulted in increased secretion of CU protein. Trials with American elm,Ulmus americana,suggested no alteration of virulence of this overexpressing transformant. Using aggressive and nonaggressive wild types, thecuoverexpressing mutant, and our cu−mutant (Bowdenet al.,1996), we have demonstrated that CU production is correlated with an altered phenotype and more hydrophobic and adherent yeast-like cells. Our results also demonstrate that CU has a role in protecting infectious propagules from desiccation. These biological roles for CU would affect transmission of Dutch elm disease, and we therefore propose that this hydrophobin acts as a parasitic fitness factor.

Comparative determination of cerato-ulmin on cell surface and in mycelial extracts of pathogenic and non-pathogenic Ophiostoma species

Cerato-ulmin (CU) presence was monitored on cell surface and quantitatively determined in mycelial extracts of the elm pathogens Ophiostoma novo-ulmi (races EAN and NAN) and O. ulmi and of the non-pathogenic O. piceae . CU was detected on the surfaces of Ophiostoma novo-ulmi (races EAN and NAN) and, for the first time, of the weak Dutch elm disease pathogen O. ulmi and the non-pathogen O. piceae . Quantitative determination of CU content in the mycelial extracts of the three species showed that high CU cellular content is associated with high CU content in culture filtrates. The content of CU in biomasses and in culture filtrates was influenced by temperature, growth phase and fungal species or race. CU synthesis occurred during the stationary phase and in the late logarithmic phase when fungi were grown at 23° and 32°C, respectively. High temperatures of growth (32°) did not have a negative effect on the cellular CU content but severely hampered CU secretion in high CU-producers O. novo-ulmi isolates.

Dutch elm disease progression and quantitative determination of cerato-ulmin in leaves, stems and branches of elms inoculated withOphiostoma novo-ulmiandO. ulmi

The progression of disease and infection was monitored following inoculation of elm plants with the Dutch elm disease (DED) agentsOphiostoma novo-ulmiorOphiostoma ulmi.The disease was more severe in plants inoculated withO. novo-ulmi,with a rapid appearance of foliar symptoms just 10 days after inoculation. The degree of progression of the two pathogens in elms was similar except for the plant sections below the inoculation point, whereO. novo-ulmigrew more thanO. ulmi.Neither pathogen could be reisolated from symptomatic leaves. The concentrations of cerato-ulmin (CU) in stems, branches and leaves was quantitatively determined by enzyme-linked immunosorbent assay (ELISA). Foliar symptoms were associated with high CU contents. The contents of CU were not significantly different in branches bearing symptomless or wilted leaves. These findings support the hypothesis that CU is produced by the pathogens in xylem vessels of stems and branches, and is then translocated into leaves where it accumulates, causing DED symptoms.

Comparison of the cerato-ulmin ( cu) gene sequences of the Himalayan Dutch elm disease fungus Ophiostoma himal-ulmi with those of O. ulmi and O. novo-ulmi suggests that the cu gene of O. novo-ulmi is unlikely to have been acquired recently from O. himal-ulmi

Cerato-ulmin (CU) is a low molecular weight hydrophobin protein secreted at high levels in vitro by the highly aggressive Dutch elm disease pathogen, O. novo-ulmi, but at much lower levels by the less aggressive O. ulmi. The recent discovery of an endemic Dutch elm disease fungus in the Himalayas, Ophiostoma himal-ulmi, which is a separate species from O. ulmi and O. novo-ulmi, yet like O. novo-ulmi produces high levels of CU in vitro, suggested that O. novo-ulmi may have acquired its cu gene from O. himal-ulmi. To investigate this possibility we have compared the nucleotide sequences of the cu gene isolates of O. himal-ulmi with those of O. ulmi and of the EAN and NAN races of O. novo-ulmi. Alignments of nucleotide sequences and their derived amino acid sequences are presented, allowing estimations of evolutionary relationships to be made. These comparisons indicate that the cu gene of O. himal-ulmi is less closely related to the cu gene of either O. ulmi or O. novo-ulmi than the cu gene of O. novo-ulmi is to that of O. ulmi; hence the cu gene of O. novo-ulmi is unlikely to have been acquired recently from O. himal-ulmi.

Isolation and characterization of non cerato-ulmin producing laboratory induced mutants of Ophiostoma novo-ulmi

Five laboratory mutants of Ophiostoma novo-ulmi EAN isolate H328 were obtained from uv-irradiated and unirradiated blastoconidia. Although the mutants were initially selected on the basis of various characteristics, including failure to produce cerato-ulmin (CU) in the culture filtrate, growth-temperature responses and colony morphology, they all were eventually also found unable to produce CU when grown in liquid shake culture at 23 °C and 33°, or they produced CU only in negligible amounts. This association suggests possible linkages between CU production and other fungal metabolic pathways. Genetic analysis of crosses between the mutants and a wild type isolate of EAN O. novo-ulmi suggested that each mutant involved a change at a single-locus, at least as far as segregations for colony morphology were concerned. Pathogenicity trials using the five mutants were carried out on four-year old Ulmus carpinifola and U. procera . Two out of the five mutants showed a significant reduction in pathogenicity in comparison to the O. novo-ulmi wild type isolate H328.

Lack of association between cerato-ulmin production and virulence in Ophiostoma novo-ulmi.

Cerato-ulmin (CU), a hydrophobin produced by Ophiostoma novo-ulmi, has been implicated in the pathogenicity of this fungus on elm. We have generated a CU- mutant by transformation-mediated gene disruption of a highly virulent (aggressive) strain of O. novo-ulmi. The inability of the mutant to synthesize CU was confirmed by transcript analysis as well as turbidity and immunological measurements. Bioassay of the CU- strain in highly susceptible elm trees indicated no difference in the virulence parameters, percent vascular discoloration, and percent foliar wilting, when compared with the wild type. Our results indicate that the inability to produce CU had no measurable effect on the ability of O. novo-ulmi to produce symptoms of Dutch elm disease on inoculated elms.

Isolation and characterization of the cerato-ulmin toxin gene of the Dutch elm disease pathogen, Ophiostoma ulmi.

The hydrophobic protein cerato-ulmin (CU), produced by Ophiostoma ulmi, has been implicated in the pathogenicity of this fungus on elm. Primers were designed based on the nucleotide sequence deduced from the published CU amino-acid sequence, and a DNA fragment of the cu gene was amplified using the polymerase chain reaction. The amplified cu fragment was used as a hybridization probe to identify and isolate the cu gene from a genomic DNA library of an aggressive isolate of O. ulmi ( = O. novo-ulmi). The cu coding region is interrupted by two introns and encodes a 100 amino-acid prepro-CU polypeptide that is processed to a 75 amino-acid mature protein upon secretion. CU shows significant sequence similarity to hydrophobins secreted by certain other fungi.

Effect of temperature on growth and cerato-ulmin production of Ophiostoma novo-ulmi and O. ulmi

Ophiostoma novo-ulmi and O. ulmi , previously named ‘aggressive’ and ‘non-aggressive’ subgroups of the old species O. ulmi , are distinguished by a wide range of morphological, molecular, genetical and physiological characters, observed both in vivo and in vitro . They show different virulence on elms of moderate resistance, that seems to be correlated with the ability to synthesize phytotoxic compounds, of which the most important is cerato-ulmin (CU). To date, O. novo-ulmi isolates have been considered the greatest producers of CU in contrast to O. ulmi isolates, that synthesize very little or no toxin. We demonstrate that the synthesis of CU is temperature-dependent and that the two species seem to have different temperature optima for its production. However, while the difference in CU production is linked to temperature, it is not a consequence of differences in fungus growth in liquid shake culture caused by these temperature differences.

Production of cerato-ulmin in white elm following artificial inoculation with Ceratocystis ulmi

In vivo production of cerato-ulmin (CU) in white elm was investigated following artificial inoculation with Ceratocystis ulmi. Conditions for inoculation and incubation were controlled and water extracts obtained from the infected host tissue assayed for CU by immuno-double diffusion. Detached host (white elm) branch sections were individually inoculated with C. ulmi by flushing the sections with a spore suspension; 4-day or 10-day incubation periods followed. The interim liquid was flushed out with water then concentrated and assayed for CU. Confirmation that CU was produced in the detached host following the 10-day incubation period only indicates that detectable levels of CU were produced during the prolonged incubation period. Intact host (white elm) trees were also artificially inoculated with C. ulmi by pressure injection of a spore suspension via the root system. When 50% of the crown became wilted, the brown-streaked outermost xylem wood, which comprised the major site for host-pathogen interaction, was removed and the concentrated water extract obtained from the processed wood powder was assayed for CU. It was confirmed that CU was produced in the intact host when external symptoms of wilting and internal symptoms of brown-streaking were expressed. These trials clearly support previous evidence for in vivo production of CU in white elm naturally infected with C. ulmi, and further strengthen the hypothesis that CU is involved in the development of Dutch elm disease.