Two novel genes induced by hard-surface contact of Colletotrichum gloeosporioides conidia

Germinating conidia of many phytopathogenic fungi must differentiate into an infection structure called the appressorium in order to penetrate into their hosts. This differentiation is known to require contact with a hard surface. However, the molecular basis for this requirement is not known. Induction of this differentiation in the avocado pathogen, Colletotrichum gloeosporioides, by chemical signals such as the host's surface wax or the fruit-ripening hormone, ethylene, requires contact of the conidia with a hard surface for about 2 h. To study molecular events triggered by hard-surface contact, we isolated several genes expressed during the early stage of hard-surface treatment by a differential-display method. The genes that encode Colletotrichum hard-surface induced proteins are designated chip genes. In this study, we report the characterization of CHIP2 and CHIP3 genes that would encode proteins with molecular masses of 65 and 64 kDa, respectively, that have no homology to any known proteins. The CHIP2 product would contain a putative nuclear localization signal, a leucine zipper motif, and a heptad repeat region which might dimerize into coiled-coil structure. The CHIP3 product would be a nine-transmembrane-domain-containing protein. RNA blots showed that CHIP2 and CHIP3 are induced by a 2-h hard-surface contact. However, disruption of these genes did not affect the appressorium-forming ability and did not cause a significant decrease in virulence on avocado or tomato fruits suggesting that C. gloeosporioides might have genes functionally redundant to CHIP2 and CHIP3 or that these genes induced by hard-surface contact control processes not directly involved in pathogenesis.     

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.     

胶孢炭疽菌附着胞形成过程中核相的动态变化

通过DAPI荧光染料染色观察胶孢炭疽菌Colletotrichumgloeosporioides附着胞发育过程中的核相动态变化,结果显示,第2次有丝分裂发生的部位在分生孢子产生芽管的一端中;分裂后,最接近芽管的一个子核移入芽管顶端,或通过芽管移入附着胞中。0.10μg/mL的三环唑可完全抑制附着胞中黑色素形成,但不影响核的分裂。三环唑处理12h后,发生2次有丝分裂数量约为73%,而发生3次有丝分裂的数量约为23.9%;绝大多数附着胞中是单核,双核数量小于5%。     

Isolation of defense-related genes differentially expressed in the resistance interaction between pepper fruits and the anthracnose fungus Colletotrichum gloeosporioides

Unripe mature green fruits of pepper (Capsicum annuum) are susceptible to Colletotrichum gloeosporioides, whereas ripe red fruits are not. We established this pepper-C. gloeosporioides interaction as a model system to study the fungal resistance that develops during ripening of nonclimacteric fruit. Histochemical examination of transverse sections suggested that fungal invasion 24 h after inoculation (HAI) and colonization 48 HAI are critical events that differentiate between resistant and susceptible interactions. Based on this observation, we used messenger RNA differential display to isolate defense-related genes differentially expressed at 24 and 48 HAI. RNA gel blot analysis showed that six out of eighty cloned cDNAs were differentially expressed after infection of ripe fruit. The proteins encoded by these six clones, ddP1, ddP3, ddP4, ddP6, ddP13, and ddP47, showed significant homology to aldehyde dehydrogenase, P23 protein, NP24 protein, cytochrome P450 protein, esterase, and MADS-box protein, respectively, and may be involved in the resistance of ripe fruit to C. gloeosporioides infection.     

Isolation and characterization of a clay-dispersing polysaccharide produced by the phytopathogenic fungus, Colletotrichum gloeosporioides

The fungus, Colletotrichum gloeosporioides, which is pathogenic to peppers produced an extracellular polysaccharide in liquid culture which possessed clay-dispersing activity. The polysaccharide could bind cationic dyes, Ruthenium Red and Alcian Blue, indicating it to be polyanionic. The polysaccharide dispersed kaolin in water and the dispersion was maintained for more than 7 days at 25 °C. Kaolin dispersion by the polysaccharide was stable from pH 3 to 10 but the addition of divalent metals at 1 mM inhibited half of the dispersion activity comparing to the control. The polysaccharide could disperse bentonite, calcium carbonate and other fine particles but did not possess emulsifying activity.     

Dispersal of the conidia of Colletotrichum gloeosporioides by rain and the development of anthracnose on onion

The conidia of Colletotrichum gloeosporioides were found to be dispersed during rainfall by wash-off and splash mechanisms. The initiation and development of onion anthracnose was found to depend on the frequency of rainfall and the movement of conidial inoculum during rainfall. Experiments conducted under controlled conditions in the laboratory employing splash and wash-off assemblies showed that impacting incident water drops (splash) and flowing water (wash-off) liberated the conidia from the anthracnose lesions of the onion leaf/peduncle. Peak liberation of conidia occurred with 3 to 5 water drops and most of the conidia were removed from the source within 90 seconds. A possibility of the dispersal of conidia of C. gloeosporioides from soil to lower leaf by splash mechanisms and then from the leaves to the neck of the onion bulb and to the bulb by wash-off mechanisms is indicated.     

Purification and characterization of an endochitinase produced by Colletotrichum gloeosporioides

The phytopathogenic fungus Colletotrichum gloeosporioides was analyzed for chitinase activity, the best production occurring at the fourth day. A 43 kDa endochitinase with specific activity of 413 U microg(-1) protein was purified corresponding to a 75% yield. The optima of temperature and pH for the enzyme were 50 degrees C and pH 7.0, respectively. The enzyme showed a high stability at 50 degrees C and pH 7.0. Values of pH from 5.0 up to 7.0 gave, at least, 50% of maximum activity, suggesting a biotechnological application. Further studies are in progress to determine the possible use of this endochitinase in biological control.     

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.     

A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.     

Purification and partial characterization of phytotoxic glycopeptides secreted by Colletotrichum gloeosporioides

Colletotrichum gloeosporioides secretes a phytotoxic compound that causes anthracnose in Hevea brasiliensis: as a first step we attempted to follow mycelium growth, pH and toxin production by this fungus. The compound was isolated and partially purified by ultrafiltration, acetone precipitation and affinity and gel-permeation chromatography. Material purified was largely carbohydrate with a small protein fraction. Galactose, mannose and rhamnose were the neutral sugars encountered, and serine and threonine the major amino acids found. We propose a molecular mass for this substance of 50333 Da.     

蓝光诱导的胶孢炭疽菌(Colletotrichum gloeosporioides)类胡萝卜素积累

胶孢炭疽菌(Colletotrichumgloeosporioides)为一种丝状真菌,蓝光照射可诱导类胡萝卜素的积累。光镜下观察表明,蓝光可诱导胶孢炭疽菌菌丝积累色素颗粒,而黑暗和红光处理却无此现象。类胡萝卜素的积累受蓝光光照强度的影响。28℃且蓝光为6.5μmol.m-2.s-1时,类胡萝卜素积累量可随光照时间延长呈增长趋势,在第5天达到最高峰为71.8μg/g FW,随后含量下降。此外,胶孢炭疽菌在黑暗中预培养的时间也影响蓝光的诱导反应。     

Glucosylceramides in Colletotrichum gloeosporioides are involved in the differentiation of conidia into mycelial cells

Glucosylceramides (GlcCer) were extracted from the plant pathogen Colletotrichum gloeosporioides and purified by several chromatographic steps. By using electrospray ionization mass spectrometry and nuclear magnetic resonance, GlcCer from C. gloeosporioides were identified as N-2'-hydroxyoctadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and N-2'-hydroxyoctadecenoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against these structures were produced and used as tools for the evaluation of the role of GlcCer in the morphological transition of C. gloeosporioides. In the presence of antibodies to GlcCer, the differentiation of conidia into mycelia was blocked. Since GlcCer is present in several plant pathogens, the inhibitory activity of external ligands recognizing these structures may be applicable in other models of fungal infections.     

Participation of Oryza sativa leaf wax in appressorium formation by Pyricularia oryzae

Appressorium formation of Pyricularia oryzae P2 on cover-glass coated with each of the components of rice leaf wax was examined. Wax esters, aldehydes and alcohols, having polar groups and low contact angles, promoted appressorium formation, but alkanes, non-polar molecules having high contact angles, had no effects. Germination of conidia, however, was not aftected with those constituents.     

Isolation and characterization of genes that are expressed during Ciona intestinalis metamorphosis

In ascidians, the events of metamorphosis transform the non-feeding, mobile tadpole larva into a filter-feeding, fixed juvenile, and the process involves rearrangements of cells, two organs and physiological changes. Differential screening was used to isolate two genes that are not expressed in swimming larvae but are expressed immediately after the initiation of metamorphosis in Ciona intestinalis. One of the genes, Ci-meta1, encodes a polypeptide with a putative secretion signal sequence, 6 epidermal growth factor (EGF)-like repeats and 13 calcium-binding EGF-like repeats. The gene begins to be expressed immediately after the beginning of metamorphosis in the adhesive organ and is likely to be associated with the signal response for metamorphosis. Another gene named Ci-meta2 encodes a protein with a putative secretion signal and three thrombospondin type-1 repeats. Ci-meta2 gene expression begins at the larval stage and is upregulated in the metamorphosing juveniles. Ci-meta2 expression is found in three regions; the adhesive organ which is also associated with settlement, the neck region between the trunk and the tail of the larva which is associated with tail resorption, and dorsal regions of the trunk which correspond to the location of the siphon primordium. This gene may be involved in the dynamic arrangement of cells during ascidian metamorphosis.