Gene identification in the obligate fungal pathogen Blumeria graminis by expressed sequence tag analysis

Powdery mildew of barley is caused by the obligate fungal pathogen Blumeria graminis f. sp. hordei. Haploid conidia of B. graminis, landing on the barley leaf, germinate to form first a primary germ tube and then an appressorial germ tube. The appressorial germ tube differentiates into a mature appressorium from which direct penetration of host epidermis occurs. Here we present data on 4908 expressed sequence tags obtained from B. graminis conidia. The combined sequences represent 2676 clones describing 1669 individual genes. Comparison with sequences from other pathogenic and nonpathogenic fungi defines hypotheses on the genes required for pathogenicity and growth on the host. The putative roles of some of the identified genes are discussed.     

cAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea

Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive fungal pathogens of rice throughout the world. Infection of rice by M. grisea requires the formation of an appressorium, a darkly pigmented, dome-shaped structure. The germ tube tip differentiates into an appressorium following germination of conidia on a leaf surface. When conidia germinate on growth medium or other noninductive surfaces, the emerging germ tube does not differentiate and continues to grow vegetatively. Little is known about the endogenous or exogenous signals controlling the developmental process of infection structure formation. We show here that a hydrophobic surface was sufficient for the induction of the appressorium. Furthermore, we demonstrate that the addition of cAMP, its analogs (8-bromo cAMP and N6-monobutyryl cAMP), or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) to germinating conidia or to vegetative hyphae induced appressorium formation on noninductive surfaces. The identification of cAMP as a mediator of infection structure formation provides a clue to the regulation of this developmental process. Elucidation of the mechanism involved is not only of biological interest but may also provide the basis for new disease control strategies.     

A mitogen-activated protein kinase kinase required for induction of cytokinesis and appressorium formation by host signals in the conidia of Colletotrichum gloeosporioides

Differentiation of fungal conidia of phytopathogens into the infection structure, appressorium, requires contact with a hard surface and host signals. The molecular signaling involved in the induction of this differentiation is poorly understood. We report the cloning of a mitogen-activated protein kinase kinase (MEK), CgMEK, from Colletotrichum gloeosporioides and its role in the induction of these developmental processes involved in pathogenesis. Disruption of CgMEK1 resulted in the loss of its ability to form appressoria in response to the host's signals and a loss of virulence. Results of confocal microscopic examination of germinating conidia of the gene-disrupted mutants were similar to those for wild-type conidia treated with an MEK inhibitor, suggesting that CgMEK1 is involved in two developmental processes in the differentiation into appressorium: (1) polarized cell division, with the preferential increase in F-actin in one of the daughter nuclei after nuclear division and the formation of septum; and (2) differentiation of the germ tube into an appressorium. CgMEK1 is required for the differentiation.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

Electron microscopic observation of cell wall structure during appressorium formation in Colletotrichum lagenarium

Summary The formation of cell walls during the appressorium formation inColletotrichum lagenarium was observed by electron microscope on the materials prepared by replicas and sectioning. The outer layer of conidia cell walls ruptured at the time of germination and the inner layer bulged out to form a germ tube. The germ tubes and primordia of appressoria had smooth surface and were consisted of one-layered cell wall. However, as the appressorium matured, the electron dense materials appeared on the outer surface of the cell wall which grew into granules. These granules are believed to form the outer layer of appressoria. The under side of the appressorium in contact with the glass surface showed a round pore.Contribution No. 191.     

Appressorium morphogenesis and cell cycle progression are linked in the grass powdery mildew fungus Blumeria graminis

Conidial germination and differentiation – the so-called prepenetration processes – of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) are essential prerequisites for facilitating penetration of the host cuticle. Although the cell cycle is known to be pivotal to cellular differentiation in several phytopathogenic fungi there is as yet no information available concerning the relationship between cell cycle and infection structure development in the obligate biotroph B. graminis. The timing of specific developmental events with respect to nuclear division and morphogenesis was followed on artificial and host leaf surfaces by 4′,6-diamidino-2-phenylindole (DAPI) staining in combination with a pharmacological approach applying specific cell cycle inhibitors. It was found that the uninucleate conidia germinated and then underwent a single round of mitosis 5–6 h after inoculation. During primary germ tube formation the nucleus frequently migrated close to the site of primary germ tube emergence. This nuclear repositioning was distinctly promoted by very-long-chain aldehydes that are common host cuticular wax constituents known to induce conidial differentiation. The subsequent morphogenesis of the appressorial germ tube preceded mitosis that was spatially uncoupled from subsequent cytokinesis. Blocking of S-phase with hydroxyurea did not inhibit formation of the appressorial germ tube but prevented cytokinesis and appressorium maturation. Benomyl treatment that arrests the cell cycle in mitosis inhibited nuclear separation, cytokinesis, and formation of mature appressoria. Thus, we conclude that a completed mitosis is not a prerequisite for the formation and swelling of the appressorial germ tube, which normally provides the destination for one of the daughter nuclei, while appressorium maturation depends on mitosis.     

The mitogen-activated protein kinase gene MAF1 is essential for the early differentiation phase of appressorium formation in Colletotrichum lagenarium

Colletotrichum lagenarium, the causal agent of cucumber anthracnose, invades host plants by forming a specialized infection structure called an appressorium. In this fungus, the mitogen-activated protein kinase (MAPK) gene CMK1 is involved in several steps of the infection process, including appressorium formation. In this study, the goal was to investigate roles of other MAPKs in C. lagenarium. The MAPK gene MAF1, related to Saccharomyces cerevisiae MPK1 and Magnaporthe grisea MPS1, was isolated and functionally characterized. The maf1 gene replacement mutants grew normally, but there was a significant reduction in conidiation and fungal pathogenicity. The M. grisea mps1 mutant forms appressoria, but conidia of the C. lagenarium maf1 mutants produced elongated germ tubes without appressoria on both host plant and glass, on which the wild type forms appressoria, suggesting that MAF1 has an essential role in appressorium formation on inductive surfaces. On a nutrient agar, wild-type conidia produced elongated germ tubes without appressoria. The morphological phenotype of the wild type on the nutrient agar was similar to that of the maf1 mutants on inductive surfaces, suggesting repression of the MAF1-mediated appressorium differentiation on the nutrient agar. The cmk1 mutants failed to form normal appressoria but produced swollen, appressorium-like structures on inductive surfaces, which is morphologically different from the maf1 mutants. These findings suggest that MAF1 is required for the early differentiation phase of appressorium formation, whereas CMK1 is involved in the maturation of appressoria.     

Evidence that the cAMP pathway controls emergence of both primary and appressorial germ tubes of barley powdery mildew

Development of conidia of barley powdery mildew involves the formation of a primary germ tube (PGT), an appressorial germ tube (AGT), and an appressorium. Previously, it was found that cyclic AMP (cAMP) was involved in these developmental processes. Comparison of development on the host surface with two types of cellulose membrane revealed that frequency of PGT emergence was surface independent. On one type of cellulose, where the frequencies of both AGT and appressorial differentiation were similar to that on the host surface, cAMP levels and protein kinase A (PKA) activities had a biphasic pattern with peaks at 15 min and 4 h after inoculation (prior to PGT and AGT emergence, respectively). The effect of manipulating cAMP levels was tested on another type of cellulose membrane, which stimulated a lower degree of AGT and appressorial formation than the host surface. Cholera toxin and forskolin, activators of adenylyl cyclase, significantly increased PGT emergence, but cAMP did not. Cholera toxin, forskolin, and cAMP increased the frequency of AGT and appressorial formation, but in a time-dependent manner.     

Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea

A mitogen-activated protein (MAP) kinase gene, PMK1, is known to regulate appressorium formation and infectious hyphal growth in the rice blast fungus Magnaporthe grisea. In this study, we constructed a green fluorescent protein gene-PMK1 fusion (GFP-PMK1) to examine the expression and localization of PMK1 in M. grisea during infection-related morphogenesis. The GFP-PMK1 fusion encoded a functional protein that complemented the defect of the pmk1 deletion mutant in appressorium formation and plant infection. Although a weak GFP signal was detectable in vegetative hyphae, conidia, and germ tubes, the expression of GFP-Pmk1 was increased in appressoria and developing conidia. Nuclear localization of GFP-Pmk1 proteins was observed in a certain percentage of appressoria. A kinase-inactive allele and a nonphosphorylatable allele of PMK1 were constructed by site-directed mutagenesis. Expression of these mutant PMK1 alleles did not complement the pmk1 deletion mutant. These data confirm that kinase activity and activation of PMK1 by the upstream MAP kinase kinase are required for appressorium formation and plant infection in M. grisea. When overexpressed with the RP27 promoter in the wild-type strain, both the kinase-inactive and nonphosphorylatable PMK1 fusion proteins caused abnormal germ tube branching. Overexpression of these PMK1 mutant alleles may interfere with the function of native PMK1 during appressorium formation.     

Scanning electron and light microscopy of appressorium development in Piptocephalis unispora (Mucorales)

Appressorium development in the mycoparasite Piptocephalis unispora was studied by means of scanning electron microscopy using the techniques of critical point drying, sputter coating and light microscopy. The germ tube which contacts both the young host hypha or a germinating spore swells at the tip to form an appressorium closely adpressed to the surface of the host. Lateral proliferation of hyphae may occur from the mature appressorium. Factors affecting the sites of appressorium development are suggested and their significance discussed.     

努利虫疠霉感染桃蚜过程的扫描电镜观察

利用扫描电镜观察了努利虫疠霉Pandora nouryi (Remaudière & Hennebert) Humber初生分生孢子接种桃蚜Myzus persicae (Sulzer)后孢子萌发、入侵以及菌体突破虫体的整个侵染过程。结果表明：附着于虫体表面的初生分生孢子在3～5 h后即有60%以上的萌发率,萌发的孢子形成芽管或产生球形或棍棒状的附着胞;12 h后大部分孢子均已萌发,并成功入侵寄主虫体;接种60 h后,呈掌状分枝的假根首先从桃蚜胸部的腹面突破虫体长出体外,明显区别于新蚜虫疠霉Pandora neoaphidis (Remaudière & Hennebert) Humber假根突破虫体的位置;假囊状体不多见,且仅分布于蚜虫身体两侧,这可在一定程度上解释努利虫疠霉产孢对湿度条件要求较高的生物学现象。     

An important role for secreted esterase in disease establishment of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici

The activity of esterase secreted by conidia of wheat powdery mildew fungus, Blumeria graminis f.?sp. tritici, was assayed using indoxyl acetate hydrolysis, which generates indigo blue crystals. Mature, ungerminated, and germinating conidia secrete esterase(s) on artificial media and on plant leaf surfaces. The activity of these esterases was inhibited by diisopropyl fluorophosphate, which is selective for serine esterases. When conidia were inoculated on wheat leaves pretreated with diisopropyl fluorophosphate, both appressorial germ tube differentiation and symptom development were significantly impaired, indicating an important role of secreted serine esterases in wheat powdery mildew disease establishment.     

Evidence that the appressorial development in barley powdery mildew is controlled by MAP kinase activity in conjunction with the cAMP pathway

Development of the barley powdery mildew fungus involves the sequential formation of a primary germ tube, an appressorial germ tube, and an appressorium. Previously, we have shown that the cAMP pathway controls the emergence of the two germ tubes. Following identification of two MAP kinase genes in an EST database from developing conidia we studied the role of the MAP kinase pathway and its interaction with the cAMP pathway. Fungal MAP kinase activity increased rapidly during mildew development, reaching a maximum between 2 and 8h after inoculation. Sphingosine or PAF-16, activators of the MAP kinase pathway, increased activity and appressorial development whilst an inhibitor, PD 98059, decreased both. Studies on the interaction between the cAMP and MAPK pathways revealed that several effectors of the MAPK pathway had no effect on cAMP levels. However upstream effectors of the cAMP pathway, such as cholera toxin and pertussis toxin (activators of G(alpha) proteins) increased MAPK activities whereas downstream effectors such as forskolin (adenylyl cyclase activator) or H89 (PKA inhibitor) had no effect. Combined application of forskolin and sphingosine produced a rise in appressorial germ tube and appressorial formation higher than when either pathway was stimulated individually. These results suggest that the two pathways cooperate in appressorial development.     

昆虫病原真菌粉虱座壳孢对烟粉虱侵染行为的初步研究

粉虱座壳孢(Aschersonia aleyrodis)是粉虱的重要病原真菌,可用于世界性害虫烟粉虱(Bemisia tabaci)的生物防治。本文通过生物测定的方法,发现1-3龄幼虫易受真菌侵染,其中以2龄幼虫的侵染率为最高,达到98%;侵染率随处理时间延长、接种浓度增加而增大;被侵染粉虱幼虫死亡通常发生在处理后的下一个龄期。本文还应用扫描电镜和光学显微镜对粉虱座壳孢侵染烟粉虱的过程进行了研究,分生孢子在昆虫体表萌发、形成芽管后可产生附着胞或直接侵入表皮,昆虫节间膜等薄弱处是其侵染的主要部位,随后菌丝在昆虫体内形成,着胞或直接侵入表皮,昆虫节间膜等薄弱处是其侵染的主要部位如湿度足够高,菌丝穿出体表产孢再侵染新害虫。     

Involvement of cell surface sugars in recognition, attachment, and appressorium formation by a mycoparasite

Fluorescein isothiocyanate labeled lectin binding techniques have revealed differences in the distribution pattern of glycosyl residues at the cell wall level between fungi that are hosts and those that are nonhosts of the mycoparasite Piptocephalis virginiana, and at the protoplast level between compatible and incompatible hosts. The cell wall of the compatible hosts (Choanephora cucurbitarum and Mortierella pusilla) and an incompatible host (Phascolomyces articulosus), as well as that of the mycoparasite itself, contains glucose and N-acetylglucosamine. However, the cell wall of a nonhost (Mortierella candelabrum) tested positive with lectins specific for various sugars, including not only glucose and N-acetylglucosamine, but also fucose, N-acetylgalactosamine, and galactose. These latter sugars could also be exposed at the surfaces of hosts and of the mycoparasite, but only after mild treatment with proteinase or when grown in a liquid culture. Pretreatment of the mycoparasite with glucose and N-acetylglucosamine inhibited its attachment to the host cell surface, but had no obvious effect on appressorium formation. On the other hand, appressorium formation was inhibited by heat treatment of host cell wall fragments which still permitted attachment, thus indicating that the factors responsible for attachment and for appressorium formation are different. The protoplast surfaces of compatible hosts contained all the sugars listed above and these protoplasts could attach to the germ tube of the mycoparasite. Only lectins specific for N-acetylglucosamine and for glucose were bound at the protoplast surface of the incompatible host; these protoplasts did not attach to the mycoparasite germ tube. Key words: mycoparasite, appressorium formation, lectins, host cell surface, attachment, protoplast surface.     

Complementation of the Magnaporthe grisea deltacpkA mutation by the Blumeria graminis PKA-c gene: functional genetic analysis of an obligate plant pathogen.

Obligate plant-pathogenic fungi have proved extremely difficult to characterize with molecular genetics because they cannot be cultured away from host plants and only can be manipulated experimentally in limited circumstances. Previously, in order to characterize signal transduction processes during infection-related development of the powdery mildew fungus Blumeria graminis (syn. Erysiphe graminis) f. sp. hordei, we described a gene similar to the catalytic subunit of cyclic AMP-dependent protein kinase A (here renamed Bka1). Functional characterization of this gene has been achieved by expression in a deltacpkA mutant of the nonobligate pathogen Magnaporthe grisea. This nonpathogenic M. grisea deltacpkA mutant displays delayed and incomplete appressorium development, suggesting a role for PKA-c in the signal transduction processes that control the maturation of infection cells. Transformation of the deltacpkA mutant with the mildew Bka1 open reading frame, controlled by the M. grisea MPG1 promoter, restored pathogenicity and appressorium maturation kinetics. The results provide, to our knowledge, the first functional genetic analysis of pathogenicity in an obligate pathogen and highlight the remarkable conservation of signaling components regulating infection-related development in pathogenic fungi.     

Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium.

Using a green fluorescent protein (GFP)-tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two alpha-tubulin genes of C. lagenarium were isolated, and GFP-alpha-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation which is distinct from their random orientation inside appressoria.     

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co‐regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin‐dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi‐nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild‐type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium‐delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium‐like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium‐like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.     

Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis

We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the DeltaMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the DeltaMgatg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in M. grisea.