Generality of the prerequisite of conidium attachment to a hydrophobic substratum as a signal for germination among Phyllosticta species

It has been shown that conidia of Phyllosticta ampelicida require attachment to a substratum to initiate germination. Furthermore this attachment occurs only on hydrophobic surfaces. This study was initiated to ascertain the breadth of this phenomenon among other species of the genus Phyllosticta. We tested 23 isolates of Phyllosticta representing at least 14 named species. These isolates were collected from North America, Asia and Africa. For 22 of the 23 isolates tested spore attachment occurred at a rate of 60-100% on hydrophobic polystyrene but at 0-5% on hydrophilic polystyrene. The one exception to the preference for a hydrophobic substratum for attachment was an unnamed species of Phyllosticta from Rhus glauca that attached less than 10% on either surface. A similar response was observed when assaying germination and appressorium formation for 17 isolates. Germination and appressorium formation for these isolates proceeded on hydrophobic polystyrene but not on nutrient agar, which is hydrophilic. In five of the tested isolates germination was high on both hydrophobic polystyrene and hydrophilic nutrient media. The isolate from Rhus glauca did not germinate appreciably on either surface. Taken together these results suggest that the requirement for conidium contact/attachment to trigger germination is pervasive to the genus Phyllosticta.     

Effects of Calcium and Calmodulin on Spore Germination and Appressorium Development in Colletotrichum trifolii

Spore germination and appressorium formation are important steps in the process of fungal development and pathogenesis. These prepenetration events, which begin with spore attachment and culminate with appressorium maturation, a common scheme for many pathogenic fungi, are prerequisites for penetration of host external barriers and subsequent colonization. Conditions for in vitro spore germination and appressorium development in Colletotrichum trifolii are described. In addition, effects of Ca(sup2+) and calmodulin on these processes have been examined. Results indicate that, as for other pathogenic fungi, appressorium development is induced on a hard surface. The data suggest that disturbance of calcium homeostasis, by ethylene-bis(oxy-ethylenenitrolo)tetraacetic acid (EGTA) or calcium channel blockers, impairs appressorium development. Moreover, calmodulin inhibitors affect both germination and differentiation, implying that the Ca(sup2+)/calmodulin signal transduction pathway is important in the early development of C. trifolii on the plant host surface.     

Expression of Recombinant Aequorin as an Intracellular Calcium Reporter in the Phytopathogenic Fungus Phyllosticta ampelicida

Conidia of Phyllosticta ampelicida germinate only after they have made contact with a substratum. Previous work has shown that external free calcium must be available to the spore for germination to be initiated. Transgenic strains of P. ampelicida expressing apo-aequorin, a calcium-sensitive luminescent protein, were developed to monitor cytoplasmic free Ca(2+) ([Ca(2+)]c). Transformants were verified by PCR and Southern hybridization. Apo-aequorin production was quantified for each of 21 transformants. The transformant that emitted the most light per unit of protein was found to contain 0.59 mg apo-aequorin/g total protein. To ascertain the feasibility of aequorin-based [Ca(2+)]c quantification, [Ca(2+)]c changes were measured in mycelia during various physiologically perturbing treatments: exposure to high concentrations of external Ca(2+), hypoosmotic shock, and mechanical perturbation. This is the first report of a plant pathogenic fungus for which aequorin-based Ca(2+) measurement protocols have been developed.     

Ca2(+)-channel agonist BAY K8644 mimics 1,25(OH)2-vitamin D3 rapid enhancement of Ca2+ transport in chick perfused duodenum.

To further understand the molecular mechanism by which 1,25(OH)2-vitamin D3 [1,25(OH2D3] rapidly stimulates intestinal calcium transport (termed "transcaltachia"), the effect of the calcium channel agonist BAY K8644 was studied in vascularly perfused duodenal loops from normal, vitamin D-replete chicks. BAY K8644, 2 mu M, was found to stimulate 45Ca2+ transport from the lumen to the vascular effluent to the same extent as physiological levels of 1,25(OH)2D3. The sterol and the Ca2+ channel agonist both increased 45Ca2+ transport 70% above control values within 2 min and 200% after 30 min of vascular perfusion. The effect of the Ca2+ channel agonist was dose dependent. Also, 1,25(OH)2D3-enhanced transcaltachia was abolished by the calcium channel blocker nifedipine. Collectively, these results suggest the involvement of 1,25(OH)2D3 in the activation of basal lateral membrane Ca2+ channels as an early effect in the transcaltachic response.     

cAMP regulation of "pathogenic" and "saprophytic" fungal spore germination

We report on the elucidation of two separate pathways of spore germination in a plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene. Conidia of the fungus can germinate either from one side or from both sides, depending on external conditions. In shake culture that includes an extract made up from fresh peas, the unicellular conidium divides and one of the two cells develops a germ tube. On a solid surface this germ tube differentiates an appressorium. In rich medium without pea extract, germination is highly similar to Aspergillus spore germination: the conidium swells, forms a single germ tube and then divides and forms a second germ tube. Conidia that germinate in a rich medium do not form appressoria even on a solid surface and are non-pathogenic. In rich medium, cAMP stimulates germination in rich liquid cultures and induces appressoria formation on a hard surface. In pea extract cAMP induces swelling and formation of irregular germ tubes and appressoria. Our results suggest that plant surface signals induce pathogenic-specific spore germination in a cAMP-independent manner. cAMP is required for saprophytic germination and for appressorium formation.     

cAMP介导的梨果表皮物化信号对链格孢侵染的调控

采用药理学方法,用cAMP抑制剂阿托品（atropine）处理链格孢Alternaria alternata孢子悬浮液,通过体外试验分析cAMP信号级联通路在链格孢响应梨果皮蜡质疏水性、化学组分和外源乙烯利等刺激后启动孢子萌发、附着胞形成的调控作用,并通过体内试验研究其对链格孢致病性的调控。结果表明,高疏水性表面和梨果蜡涂膜表面及1μmol/L的乙烯利均可显著促进链格孢的孢子萌发和附着胞形成。cAMP信号级联通路抑制剂atropine处理后显著抑制了表皮疏水性、蜡质和外源乙烯介导的链格孢的孢子萌发和附着胞形成,其中抑制剂处理后4h,链格孢在疏水性、果蜡涂膜表面和乙烯等处理中附着胞形成率分别较对照降低了75.3%、63.7%和74.3%,同时抑制剂处理还可抑制损伤接种链格孢早酥梨黑斑病的扩展。外源cAMP可以部分恢复抑制剂的作用,外源cAMP+atropine处理后4h,在高疏水性（108°）和果蜡涂膜表面,链格孢附着胞形成率为抑制剂atropine单独处理的2.4倍和1.6倍,表明cAMP信号级联通路可通过调控侵染结构的形成而影响链格孢对梨果表皮物理化学信号的识别和应答。     

Involvement of ion channels in the induction of proenkephalin A gene expression by nicotine and cAMP in bovine chromaffin cells

The involvement of Na+ and Ca2+ channels in the stimulatory effect of nicotine and cAMP upon proenkephalin A mRNA (mRNA ENK) levels in primary cultures of bovine adrenal chromaffin cells was analyzed. Nicotine (10 microM) caused about a 2-3-fold increase in mRNA ENK which was abolished by the nicotinic receptor antagonist tubocurarine (4 X 10(-7) M), inhibited by the Ca2+ channel antagonist nifedipine (100 nM) abolished by the Ca2+ channel blocker D600 (10 microM), and augmented by the Ca2+ channel agonist BayK 8644 (100 nM). In contrast, blockade of the Na+ channel by tetrodotoxin (1 microM) did not modulate the nicotine-induced increase in mRNA ENK. Incubation of the cells with forskolin (25 microM) and 8-bromo-cAMP (1 mM) also resulted in an increase in mRNA ENK levels that was inhibited by the Ca2+ channel blocker verapamil (50 microM) and nifedipine (100 nM), whereas it was enhanced by BayK 8644 (100 nM). In addition, the effect of forskolin and 8-bromo-cAMP was decreased by the Na+ channel blocker tetrodotoxin (1 microM). These results suggest that the induction of proenkephalin A gene expression by cAMP and nicotine involves the modulation of ion channels. It appears that changes in Ca2+ flux are involved in mediating this induction. The dihydropyridines nifedipine and BayK 8644 and the Ca2+ channel blockers verapamil and D600 all modulate 45Ca uptake. In addition, we show that incubation of the cells with A23187 (10(-7) M), a Ca2+ ionophore, resulted in an increase in mRNA ENK, indicating that changes in intracellular Ca2+ levels may indeed modulate proenkephalin A gene expression. Although it appears that an elevation of mRNA ENK upon nicotinic receptor activation occurs rapidly (an increase could be detected after 2 h incubation), the findings that the rise in mRNA ENK could be abolished by the Ca2+ channel blocker D600 but not affected by tetrodotoxin (1 microM), and that agents such as KCl (20 mM) and veratridine (5 microM) that increase mRNA ENK by activation of voltage-dependent Ca2+ channels do not result in an increase in intracellular cAMP, provide no evidence for a major role of the adenylate cyclase system in the inducing effect of nicotine upon proenkephalin A gene expression.     

Ca2+-permeable channels in the plasma membrane of Arabidopsis pollen are regulated by actin microfilaments

Cytosolic free Ca2+ and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca2+ channels, as well as channel-mediated Ca2+ influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca2+ in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca2+ channel blockers La3+ or Gd3+ and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca2+. Ca2+ fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca2+ levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca2+ increase induced by CD or CB was abolished by addition of Ca2+ channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca2+ currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca2+-permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca2+-permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca2+ channel activity and may consequently regulate cytoplasmic Ca2+.     

Preloading of micromolar intracellular Ca2+ during capacitation of Sicyonia ingentis sperm, and the role of the pHi decrease during the acrosome reaction.

In studying the mechanism controlling the sperm acrosome reaction (AR) in the marine shrimp Sicyonia ingentis, intracellular Ca2+ and pH were measured using the fluorescent indicators Fura-2 and Fluo-3 for Ca2+, and SNARF-1 for pH. Capacitated sperm possessed an apparent resting Ca2+ concentration of 1-2 microM which remained constant upon induction of the AR with egg water. Uncapacitated sperm had extremely low Ca2+ levels and did not respond to egg water. These results suggest that, while in other species the Ca2+ is elevated to micromolar levels during initiation of the AR, S. ingentis sperm are preloaded with Ca2+ during capacitation and the trigger for the AR is downstream of the Ca2+ increase. The notion that Ca2+ influx is not involved at the actual time of the AR in capacitated S. ingentis sperm is supported by the inability of Ca2+ ionophore A23187 to induce the AR and the ineffectiveness of Ca2+ channel antagonists to block egg water-induced AR. Measurements of capacitated sperm pH showed a significant decrease during the first 10-15 min of the AR, which did not correlate temporally to either acrosomal exocytosis (at 5 min post-induction) or filament formation (after 45 min). Inhibition of egg protease activity required for induction of filament formation did not inhibit the pH drop, indicating that intracellular acidification is not the final trigger for filament formation, although it may be required prior to action of the protease.     

Hydrophobicity of contact surface induces appressorium formation in Magnaporthe grisea

Abstract Infection by Magnaporthe grisea , the causal agent of rice blast, requires the formation of a melanized, dome-shaped infection cell, called an appressorium. Little is known about the signals and mechanisms regulating this important developmental process. We have previously observed a correlation between hydrophobicity of the contact surface and appressorium formation. To evaluate this thigmotropic response more precisely, we measured appressorium formation on the surfaces of silicon wafers modified to create various degrees of hydrophobicity. We also examined the effects of artificial ridges created on polystyrene surfaces. Hydrophobic surfaces induced a high level of appressorium formation, whereas hydrophilic surfaces did not. Tips of germ-tubes did not respond to ridges of any particular height, but formed appressoria in a random manner. These results indicate that hydrophobicity of the substratum is a primary determinant and is sufficient to induce appressorium formation in M. grisea .     

Generation and amplification of the cytosolic calcium signal during secretory responses to gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) stimulates characteristic biphasic increases in cytosolic calcium concentration ([Ca2+]i) and in luteinizing hormone (LH) release in cultured gonadotrophs, with an early peak followed by a prolonged plateau in both responses. Analysis of [Ca2+]i by dual-wavelength fluorimetric assay and of LH release at 5-sec intervals in perifused pituitary cells revealed increases in both responses within a few seconds of exposure to GnRH. The maximum elevation of [Ca2+]i occurred within 20 sec, and the peak gonadotropin release in 35 sec; the total duration of the spike phase for both [Ca2+]i and LH release was 2.5 min. Under extracellular Ca2(+)-deficient conditions, the GnRH-induced peak in [Ca2+]i was reduced by about 20% and the plateau phase was abolished. Concomitantly, the magnitude of the acute phase of LH release was reduced by 40% and that of the second phase by about 90%. Recovery of the plateau phase of LH release occurred within 25 sec after addition of 1.25 mM Ca2+ to Ca2(+)-deficient medium. In a dose-dependent manner, the non-selective Ca2+ channel blockers Co2+ and Cd2+ reduced the Ca2+ current measured by whole-cell recording in pituitary gonadotrophs and abolished the extracellular Ca2(+)-dependent component of LH release. The selective calcium channel blocker, nifedipine, decreased the magnitude of the Ca2+ current and reduced the plateau phase of LH release by 50%; conversely, the dihydropyridine agonist methyl, 1,4,dihydro-2,6-dimethyl 3-nitro-4-(2-trifluorome) (Bay K 8644) consistently enhanced the amplitudes of both Ca2+ current and GnRH-induced LH release. These data reveal a close temporal correlation between changes in [Ca2+]i and LH release during GnRH action, with Ca2+ mobilization during the spike phase and Ca2+ influx through dihydropyridine-sensitive and insensitive sets of receptor-operated calcium channels during the spike and plateau phases. In addition, analysis of the magnitudes of the [Ca2+]i and LH responses to a wide range of GnRH concentrations in the presence and absence of extracellular Ca2+ is consistent with amplification of the [Ca2+]i signal in agonist-stimulated gonadotrops.     

苜蓿假盘菌子囊孢子萌发特性及子实体超微结构观察

【背景】苜蓿假盘菌(Pseudopeziza medicaginis)侵染苜蓿叶片引起的褐斑病是苜蓿的重要病害之一。研究表明,该菌以子囊盘越冬,翌年释放子囊孢子完成初侵染,因此观察该菌子实体的超微结构特征,有利于进一步揭示子囊菌的侵染机制。【目的】明确P. medicaginis子囊孢子萌发、Ca²⁺信号途径是否参与调控附着胞形成;观察子囊孢子和子实体的超微结构特征。【方法】采用光学显微镜技术,研究不同碳、氮源和Ca²⁺/CaM信号途径对P. medicaginis子囊孢子萌发、附着胞形成的影响,并采用电镜技术观察子实体和子囊孢子的超微结构。【结果】碳、氮源有利于P. medicaginis子囊孢子的萌发,葡萄糖、麦芽糖、蔗糖、氨基乙酸、酵母粉、尿素、蛋白胨、硝酸铵、硝酸钠等碳、氮源诱导形成附着胞,以尿素诱导效果最明显;Ca²⁺不仅促进子囊孢子的萌发,显著诱导附着胞的形成,而且高于碳、氮源;10mmol/L的Ca²⁺诱导效果最明显,随其浓度的增加,子囊孢子的萌发率和附着胞的形成率下降;低浓度Ca     

Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.     

Genetic requirements for induction of germination of spores of Bacillus subtilis by Ca(2+)-dipicolinate

Dormant Bacillus subtilis spores can be induced to germinate by nutrients, as well as by nonmetabolizable chemicals, such as a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA). Nutrients bind receptors in the spore, and this binding triggers events in the spore core, including DPA excretion and rehydration, and also activates hydrolysis of the surrounding cortex through mechanisms that are largely unknown. As Ca(2+)-DPA does not require receptors to induce spore germination, we asked if this process utilizes other proteins, such as the putative cortex-lytic enzymes SleB and CwlJ, that are involved in nutrient-induced germination. We found that Ca(2+)-DPA triggers germination by first activating CwlJ-dependent cortex hydrolysis; this mechanism is different from nutrient-induced germination where cortex hydrolysis is not required for the early germination events in the spore core. Nevertheless, since nutrients can induce release of the spore's DPA before cortex hydrolysis, we examined if the DPA excreted from the core acts as a signal to activate CwlJ in the cortex. Indeed, endogenous DPA is required for nutrient-induced CwlJ activation and this requirement was partially remedied by exogenous Ca(2+)-DPA. Our findings thus define a mechanism for Ca(2+)-DPA-induced germination and also provide the first definitive evidence for a signaling pathway that activates cortex hydrolysis in response to nutrients.     

Calcium restores prepenetration morphogenesis abolished by polyamines in Colletotrichum gloeosporioides infecting red pepper

Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, to infect its host, red pepper. Polyamines (putrescine, spermidine, and spermine) as well as S-adenosyl methionine inhibitor, methylglyoxal-bis-guanyl hydrazone (MGBG), impaired conidial germination and appressorium formation of C. gloeosporioides. Curtailment of cell differentiation by polyamines and MGBG was more evident in conidial germination than in appressorium development. Exogenous addition of calcium restored conidial germination and appressorium formation and expression of calmodulin-encoding gene (CgCaM) inhibited by polyamines. Taken together, proper regulation of intracellular polyamine concentration is indispensable for conidial germination and appressorium formation, and involved in Ca(2+)/calmodulin-dependent signaling pathways of C. gloeosporioides infecting red pepper.     

Mechanism of Ca2+ and dipicolinic acid requirement for L-alanine induced germination of Bacillus cereus BIS-59 spores

Spores prepared from different sporulating media containing varying amounts of Ca and dipicolinic acid (DPA), exhibited differential responses to germination in L-alanine (0.25 M). Ca-spores with moderately high Ca and DPA contents could be triggered to germination by L-alanine, whereas P-spores with low contents of Ca and DPA could not be germinated by L-alanine unless Ca2+ or DPA was exogenously added. The initiation of L-alanine induced germination by P-spores in the presence of 45CaCl2 was associated with a marked uptake of 45Ca2+. Experiments involving stepwise extraction of 45Ca from prelabelled spores indicated that a part of the spore calcium may be involved in L-alanine induced germination. Both Ca2+ and DPA seemed to have a stimulatory effect on the incorporation of 14C-L-alanine.     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.     

Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme

The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of approximately 9 and a temperature optimum of 60 degrees C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl(2). Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca(2+) almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca(2+) and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca(2+)-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca(2+)-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.     

Conidial germination in the filamentous fungus Fusarium graminearum

The ascomycetous fungus Fusarium graminearum is an important plant pathogen causing Fusarium head blight disease of wheat and barley. To understand early developmental stages of this organism, we followed the germination of macroconidia microscopically to understand the timing of key events. These events, recorded after suspension of spores in liquid germination medium, included spore swelling at 2h, germination tube emergence and elongation from conidia at 8h and hyphal branching at 24h. To understand changes in gene expression during these developmental changes, RNA was isolated from spores and used to interrogate the F. graminearum Affymetrix GeneChip. RNAs corresponding to 5813 genes were detected in fresh spores and 5146, 5249 and 5993, respectively, in spores incubated in germination medium after 2, 8 or 24h (P<0.001). Gene expression data were used to predict the cellular and physiological state of each developmental stage for known processes. Predictions were confirmed microscopically for several previously unreported developmental events such as manifestation of peroxisomes in fresh spores and nuclear division resulting in binuclear cells within macroconidia prior to spore germination. Knowledge of stage-specific gene expression and changes in gene expression levels between developmental stages are an important first step to understanding the molecular mechanisms responsible for spore germination and development.