Protein accumulation in the germinating Uromyces appendiculatus uredospore

Uromyces appendiculatus is a rust fungus that causes disease on beans. To understand more about the biology of U. appendiculatus, we have used multidimensional protein identification technology to survey proteins in germinating asexual uredospores and have compared this data with proteins discovered in an inactive spore. The relative concentrations of proteins were estimated by counting the numbers of tandem mass spectra assigned to peptides for each detected protein. After germination, there were few changes in amounts of accumulated proteins involved in glycolysis, acetyl Co-A metabolism, citric acid cycle, ATP-coupled proton transport, or gluconeogenesis. Moreover, the total amount of translation elongation factors remained high, supporting a prior model that suggests that germlings acquire protein translation machinery from uredospores. However, germlings contained a higher amount of proteins involved in mitochondrial ADP:ATP translocation, which is indicative of increased energy production. Also, there were more accumulating histone proteins, pointing to the reorganization of the nuclei that occurs after germination prior to appressorium formation. Generally, these changes are indicative of metabolic transition from dormancy to germination and are supported by cytological and developmental models of germling growth.     

Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava

Puccinia psidii sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MS^E was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.     

Changes in ribosomes associated with spore senescence in the bean rust fungus.

Studies were carried out to idenify the cause of the decline in transferase activity and capacity to bind polyuridylic acid which occurs in ribosomes from germinated uredospores of the bean rust fungus, Uromyces phaseoli (Pers.) Wint., aged longer than 6 h on a water surface. We have shown that such ribosomes lose the capacity to respond to added transferase-I and that both subunits were affected by the ageing process. These changes were not accompanied by a significant alteration in the composition of the ribosome. However, deoxycholate had a greater detergent effect on ribosomes from germinated spores than from nongerminated spores as shown both by loss of capacity to polymerize amino acids and loss of protein. Ribonuclease activity did not increase during germination, but the amount found (Imug/g spores) was easily detectable. It was suggested that loss of response to transferase-I was due to an alteration of ribosomal proteins of both subunits.     

Development of Infection Structures by the Direct-Penetrating Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) on Artificial Membranes

The development of infection structures by the directly infecting soybean rust fungus of different artificial membranes was followed by light and scanning electron microscopy. On water agar uredospores developed germ tubes without appressoria. On dialysis membranes more than 80% of the uredospores formed appressoria. With low frequencies (1–7%) also primary hyphae and/or penetration hyphae were present. When cellulose nitrate membrane filters with pore diameters ≤ 0.2 μm were used, uredospores germinated but showed a strongly reduced appressoria formation. Membranes with pores ≥ 0.1 μm allowed a development of infection structures similar to that on dialysis membranes. In experiments with paraffin oil incorporated into collodion membranes more than 90% of the uredospores formed appressoria, about 50% of the appressoria developed hyphae. Ungerminated spores and germ tubes always contained 2 nuclei. In fully developed appressoria 4 nuclei were present. Compared with stomata entering rust fungi appressoria formation by Phakopsora pachyrhizi occurred more frequently and seemed to be less dependent on specific stimuli. Moreover, in most cases only few of the appressoria formed penetration or primary hyphae. The induction of these structures seemed to be dependent on further unknown stimuli.     

Endoplasmic Reticulum Subcompartments in a Plant Parasitic Fungus and in Baker's Yeast: Differential Distribution of Lumenal Proteins

Bachem, U., and Mendgen, K. 1995. ER subcompartments in a plant parasitic fungus and in baker's yeast: Differential distribution of lumenal proteins. Experimental Mycology 19, 137-152. His-Asp-Glu-Leu (HDEL)-bearing proteins were quantified in different endoplasmic reticulum (ER) subcompartments of Saccharomyces cerevisiae and the plant parasite Uromyces viciae-fabae by immuno-electron microscopy (immuno-EM). In both fungi, the immunogold labeling of these proteins within the ER was three times greater than within the nuclear envelope. In U. viciae-fabae, the ER in germinating uredospores differed from the ER in fungal structures produced within the plant, e.g., haustoria. In haustoria, the cisternal ER differentiated large tubular-vesicular complexes (TVC). TVC contained higher levels of HDEL-bearing proteins than ordinary ER cisternae. ELISA readings also indicated an increased concentration of these proteins in isolated haustoria compared to germinating uredospores. In S. cerevisiae, the ER was differentiated into cortical and internal regions. Immuno-EM revealed that labeling of the binding protein (BiP) was lower in the ER of the cell cortex. Heat shock increased BiP signals, but the relative distribution within the ER did not change. Our results suggest that ER subcompartments can be differentiated by immunogold labeling of proteins with a retention signal. In special cases, such as in the parasitic phase of rust fungi, these proteins accumulate to higher levels in ER subcompartments, probably as a response to plant-induced stress.     

The ultrastructure of the uredospore of Puccinia recondita

Dry, fresh uredospores ofPuccinia recondita that have been killed by infra-red radiation showed no striking ultrastructural differences from dry, fresh, viable uredospores. Numerous spherical and elipsoidal mitochondria with distinct and deep cristae, ribosomes, endoplasmic reticulum and convoluted plasmalemma were well shown by both. When moistened and incubated, killed uredospores lost ultrastructural organization, whereas moistened, viable, fresh uredospores imbibed moisture, became more spherical and germination commenced. A more or less centrally located nucleus was seen with the double membrane invaginated at some points. The advance of the germ tube was initially by enzymic degradation and concluded by mechanical disruption of the degraded pore plug. The mitochondria and the endoplasmic reticulum increased in number and the stored lipid bodies were gradually depleted as germination progressed. Features known as the ‘foamy cytoplasm’ and ‘folded membranes’ were seen in the germinating uredospores only. It was suggested that the ‘foamy cytoplasm’ could be functionally similar to the glyoxisome because of the close association of the former to lipid bodies. The ‘folded membranes’ may be accumulated endoplasmic reticulum being transported to the site of wall formation.     

Origin of sterols in uredospores of Uromyces phaseoli

The sterols from healthy bean leaves are β-sitosterol, stigmasterol, campesterol and 28-isofucosterol. An additional sterol observed in bean leaves infected with Uromyces phaseoli was identified as 7,(Z)-24(28)-stigmastadien-3β-ol, which is the major sterol of the uredospores of the fungus. The fungus appears to stimulate sterol synthesis, but most of the increased sterol content of infected leaves can be attributed to the sterol of the uredospores.     

Lectin Binding Studies on the Cell Walls of Soybean Rust (Phakopsora pachyrhizi Syd.)

Walls of uredospores, infection structures, intercellular hyphae and haustoria of the soybean rust fungus (Phakopsora pachyrhizi) were studied by electron microscopy using gold-labeled wheat germ lectin (WGL) and Concanavalin A (ConA) as cytochemical probes. Receptors for WGL (probably chitin) were detected in all fungal walls included in this study. WGL-binding occurred throughout the entire walls (uredospores, appressorial cone, penetration hyphae, haustorial mother cells) or only to the inner wall layers (germ tubes, appressoria, intercellular hyphae).     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

Fixation of carbon dioxide in the dark by the malic enzyme of bean and oat stem rust uredospores

Malic enzyme was found in both bean rust and cat stem rust uredospores. In bean rust uredospores it was shown to catalyze the formation of pyruvic acid from l-malic acid and to synthesize malic acid from pyruvic acid and CO₂. The malic enzyme from bean rust uredospores was specific for NADP and dependent on manganous ions for activity. The specific activity of the bean rust malic enzyme in crude extracts of ungerminated uredospores was approximately 6 times greater than that found in crude extracts obtained from germinated uredospores. The malic enzyme was also found in extracts obtained from healthy and rust-infected bean leaves. The specific activity of the enzyme was approximately 2 to 5 times greater in partially purified extracts obtained from the infected bean tissue at 6 days after inoculation. The specific activity of the malic enzyme in crude extracts obtained from oat stem rust uredospores was 2 times greater than the specific activity of this enzyme in crude extracts obtained from bean rust uredospores. Phosphoenolpyruvate carboxylase activity could not be demonstrated in crude extracts obtained from the ungerminated uredospores of the bean rust fungus.     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Light- and Scanning Electron Microscopic Studies on the Development of the Mycoparasite Verticillium psalliotae Treschow on Uredospores of the Soybean Rust (Phakopsora pachyrhizi Syd.)

Abstract By light and scanning electron microscopy it is shown that a strain of Verticillium psalliotae , originally isolated from pustules of the soybean rust in Thailand, is able to infect uredospores of this rust fungus. In most cases appressoria-like structures were formed at the infection sites. However, only a part of the spores were infected. Most spores appeared heavily damaged without any visible mycelium inside. This indicates that growth of Verticillium psalliotae on uredospores of the soybean rust is probably mainly based on the production of lytic enzymes.     

Degradation of Uredospores of the Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) by Cell-Free Culture Filtrates of the Mycoparasite Verticillium Psalliotae Treschow

Verticillium psalliotae isolates Taiw and Thai C are effective parasites of the soybean rust fungus. Cell-free culture filtrates of these fungi, prepared after growth on autoclaved uredospores, contained β-1,3-glucanase, chitinase and protease activities and caused degradations, when rust spores were treated with them for 24 or 72 h. During these lytic processes carbohydrates, amino compounds and N-acetylhexosamine were released. The carbohydrate fraction was composed of mannitol, arabitol, trehalose, glucose and unidentified substances showing low R_f-values during thin layer chromatography. The amino compounds consisted of 10 amino acids (leucine and/or isoleucine, phenylalanine, glutamic acid, valine, arginine, asparagine, glutamine, serine, aspartic acid, histidine) and 5—7 substances which could not be identified. Verticillium lecanii isolate Konz is a weak parasite of soybean rust. During growth on uredospores the fungus produced culture filtrates without chitinase activity and with low total activities of β-1,3-glucanase and protease. Compared with V. psalliotae, culture filtrates of V. lecanii exerted lower lytic activities on soybean rust uredospores. The results are consistent with the aspect that the rapid growth of V. psalliotae on soybean rust fungus is primarily based on the secretion of lytic enzymes which make nutrients available to the mycoparasite.     

Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation

We used two-dimensional gel electrophoresis (2-DE) to identify the proteins that are induced in the rice blast fungus Magnaporthe grisea during appressorium formation. Proteins were extracted from conidia that had germinated on hydrophilic glass plates or from germinated and appressoria-forming conidia on leaf wax-coated hydrophobic glass plates after 4, 8, and 12 h of incubation. Differentially expressed protein spots during appressorium formation were confirmed from gels after 2-DE analysis where proteins had been labeled with (35)S methionine and stained with silver. Internal amino acid sequencing identified five proteins among several proteins induced during appressorium formation. Two denoted as M. grisea proteasome homolgues (MgP1 and MgP5) were 20S proteasome alpha subunits. The remaining three were scytalone dehydratase (SCD), and serine carboxypeptidase Y (CPY). None of the five have been reported previously in the rice blast fungus apart from SCD. We further investigated the role the alpha subunit of 20S proteasome plays in appressorium formation. We confirmed by Western blot analysis that MgP5 is highly expressed during appressorium formation and found that it is also markedly induced by nitrogen- and carbon-starvation, in particular by the former. These observations suggest that the 20S proteasome may be involved in remobilizing storage proteins, which then help to build the appressorium. Thus, fungal proteome analysis may provide important clues about developmental changes such as the generation of the appressorium.     

Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus

Few studies have been conducted to identify the extracellular proteins and enzymes secreted by filamentous fungi, particularly with respect to dispensable metabolic pathways. Proteomic analysis has proven to be the most powerful method for identification of proteins in complex mixtures and is suitable for the study of the alteration of protein expression under different environmental conditions. The filamentous fungus Aspergillus flavus can degrade the flavonoid rutin as the only source of carbon via an extracellular enzyme system. In this study, a proteomic analysis was used to differentiate and identify the extracellular rutin-induced and non-induced proteins secreted by A. flavus. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. While 15 rutin-induced proteins and 7 non-induced proteins were identified, more than 90 protein spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced.     

Purification of calmodulin from bean rust uredospores

Calmodulin has been isolated from uredospores of the bean rust fungus and purified to apparent homogeneity as judged using sodium dodecyl sulfatepolyacrylamide gels. The protein substituted for bovine calmodulin as an activator of Ca²⁺-dependent cyclic nucleotide phosphodiesterase. Its molecular mass was 15.6 kDa in the presence of Ca²⁺ and 17.0 kDa in its absence. ThepI was 4.4. The amino acid composition was generally similar to those of calmodulin from other fungi.     

A eukaryotic repressor protein, the qa-1S gene product of Neurospora crassa, is homologous to part of the arom multifunctional enzyme

Little is known about the proteins involved in the control of gene expression in eukaryotes. Although some of these proteins have been sequenced, their biochemical functions are not well understood and the identification of homologies to proteins of known activities may give useful clues to the functions of these regulatory proteins. We report here that the qa-1S repressor protein of the fungus Neurospora crassa is strongly homologous to the pentafunctional arom enzyme found in many lower eukaryotes. We believe that this is the first known case of a repressor protein that is unequivocally homologous to metabolic enzymes. These findings allow us to propose likely functions for some regions of the qa-1S protein.     

The development of Puccinia hordei on barley cv. Zephyr

Germination of uredospores of Puccinia hordei was similar on cover-slips and on the first leaves of barley seedlings (cv. Zephyr) at 100 % r.h. over the range 5–25 °C, being greatest at 20 °C. At 15, 20 and 25 °C maximum germination was attained in 6 h. No uredospores germinated on coverslips in humidities below saturation. The numbers of pustules which subsequently developed on plants incubated at 5, 10, 15 or 18 °C and 100 % r.h. for varying periods up to 24 h, were directly related to rise in temperature and length of incubation. The time from inoculation to eruption of pustules (generation time) was 6 days at 25 °C, 8 days at 20 °C, 10 days at 15 °C, 15 days at 10 °C and 60 days at 5 °C. Pustule production on inoculated plants which had been kept at 5 °C was rapidly accelerated when they were transferred to 20 °C. Data obtained at constant temperatures were used to predict generation times of the fungus in the field. The productivity of pustules, determined as weight of uredospores, was examined at 10, 15 and 20 °C. Significantly more spores were produced at 15 than at 10 °C and most were produced at 20 °C. The results are discussed in relation to those obtained by other workers and to the development of brown rust in the field.     

Transient transformation of the rust fungus Puccinia graminis f. sp. tritici

The biotrophic rust fungus Puccinia graminis f. sp. tritici (Pgt) was transformed by particle bombardment. The promoter from the Pgt translation elongation factor 1alpha (EF-1alpha) gene was fused to the bacterial marker genes hygromycin B phosphotransferase (hpt) and beta-glucuronidase (GUS). Transformation constructs were introduced into uredospores of Pgt, an obligate pathogen of wheat, by biolistic bombardment. Uredospores transformed with the construct containing the hpt gene germinated and initiated branching on selective medium, indicating that they had acquired resistance to hygromycin B. However, transformants stopped growing 5 days after bombardment. GUS activity in uredospores and germlings was histochemically detected 4-16 h after bombardment. GUS expression was also obtained using the INF24 promoter from the bean rust fungus Uromyces appendiculatus, demonstrating that heterologous genes can be expressed in P. graminis under the control of regulatory sequences from closely related organisms.     

禾谷镰刀菌基因组中含寄主靶向模体分泌蛋白功能的初步分析

基于生物信息学的方法,以SignalP v3.0、TargetP v1.01、Big-PI Predictor和TMHMM v2.0四个分析软件对禾谷镰刀菌(Fusarium graminearum Schw.)全基因组的11 640个蛋白编码基因的N-端氨基酸序列进行信号肽分析,预测出606个潜在的分泌蛋白编码基因.通过对这些潜在的分泌蛋白进行MEME软件分析,发现其中有157个分泌蛋白的剪切点下游120氨基酸残基范围内具有一个保守的RXLX模体,其中有79个分泌蛋白具有可预测的功能性描述,包括FG00023.1具有与草酸盐氧化酶1有关的功能,FG01588.1具有与1,4-α-葡糖苷酶葡聚糖有关的功能,这些基因可作为禾谷镰刀菌致病相关的候选基因.其中FG04097.1编码的具有与丝氨酸型蛋白酶有关的功能在致病疫霉和疟原虫真核寄生物中具有相似保守的寄主靶标模体的效应蛋白中也观察到,但FG09127.1,FG05287.1编码的蛋白尚未被证明参与致病过程的研究报道.深入研究分泌蛋白将有助于明确植物与病原微生物互作的分子机制.利用禾谷镰刀菌基因组学研究成果,结合计算机技术和生物信息学的方法,分析其分泌蛋白组学,将有助于全面掌握其致病因子的结构与功能.对于这些蛋白功能的比较研究,将有利于对禾谷镰刀菌致病性的分子机制的探索,并为设计新的防治措施提供理论依据.