Visualizing nuclear migration during conidiophore development in Aspergillus nidulans and Aspergillus oryzae: multinucleation of conidia occurs through direct migration of plural nuclei from phialides and confers greater viability and early germination in Aspergillus oryzae

Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein. Active trafficking of nuclei from a vesicle to a phialide and subsequently into a conidium provided the mechanistic basis for the formation of multinucleate conidia in A. oryzae. In particular, the first direct visual evidence on multinucleate conidium formation by the migration of nuclei from a phialide into the conidium, rather than by mitotic division in a newly formed conidium, was obtained. Interestingly, a statistical analysis on conidial germination revealed that conidia with more nuclei germinated earlier than those with fewer nuclei. Moreover, multinucleation of conidia conferred greater viability and resistance to UV-irradiation and freeze-thaw treatment.     

Observation of EGFP-visualized nuclei and distribution of vacuoles in Aspergillus oryzae arpA null mutant

The arpA gene encoding Arp1 (actin-related protein) was previously cloned and characterized from Aspergillus oryzae. Phenotypes of the arpA null mutant indicate its requirement for normal nuclear distribution and morphology of conidiophores. In this study, we further characterized the function of the arpA gene in distribution of organelles. For further analysis of nuclear migration in living cells, an expression system consisting of a fusion protein of Aspergillus nidulans histone H2B and EGFP (H2B::EGFP) was used. This demonstrated diminished hyphal-tip growth rate and inefficient nuclear transport to apical regions in the arpA null mutant. Expression of H2B::EGFP also revealed an increase in the nuclear number of each conidium in the arpA null mutant, implicating a role for the arpA gene in controlling the nuclear movement into conidia. Furthermore, staining of vacuoles of the arpA null mutant with CMAC (cell tracker blue) suggested that the arpA gene is required for proper vacuolar distribution in addition to its role in normal nuclear distribution.     

Characterization of an Aspergillus nidulans mutant with abnormal distribution of nuclei in hyphae, metulae, phialides and conidia

The V10 deteriorated variant of Aspergillus nidulans has hyphae, metulae, phialides and conidia with abnormal nuclear distributions. The alterations observed were: increase in the number of nuclei in hyphae, metulae and phialides, presence of anucleate, uninucleate and multinucleate conidia, abnormal vegetative growth and defective conidiation. When 0.5 M NaCl was added to the medium, an increase in the number of conidia was observed but their morphology and number of nuclei were not modified. The gene responsible for these alterations was named anuA1. The anuA1 gene is located on linkage group VII and is possibly involved in nuclear migration to hyphae, metulae, phialides and conidia.     

The Aspergillus nidulans bncA1 mutation causes defects in the cell division cycle, nuclear movement and developmental morphogenesis

Wild-type Aspergillus nidulans conidia are uninucleate. The mutation bncA1 (binucleated conidia) was first described as a single mutation located on chromosome IV that caused formation of approximately 25% binucleate and 1% trinucleate conidia. In this study, we show that bncA1 conidia exit G1 arrest earlier than the wild type. Germlings have hyphal elements with abnormal morphology, elevated numbers of randomly distributed nuclei and an irregular septation pattern. Older hyphal elements undergo mitotic catastrophe, suggesting the nuclear division cycle of internal (nonterminal) elements is not arrested. The bncA1 mutation also causes aberrant morphogenesis of the asexual reproductive structure, the conidiophore. Metulae and phialides are elongated and have incorrect numbers of nuclei. Phialides also have internal septation that appears to delineate hyphal-like elements. Heterokaryon analysis using strains with contrasting auxotrophic markers showed that the bncA1 mutation resulted in a higher frequency of diploid and multinucleated prototrophic conidia than control heterokaryons. These results suggest that in bncA1 strains multiple nuclei can move from the conidiophore vesicle to the metulae and/or from the phialide to the conidium. The bncA1 mutant also showed hypersensitivity to the anti-microtubule drugs thiabendazole and nocodazole, which is consistent with the defects in cell cycle regulation and nuclear movement. We propose that bncA has an important role in correctly regulating both the cell division cycle and nuclear movement.     

The Formation, General Characteristics and Production of α-Amylase in Heterocaryons and Diploids of Aspergillus oryzae

S ummary . Heterocaryons and diploids from Aspergillus oryzae were investigated with respect to nuclear number/conidium and to conidial size. Heterocaryons usually had larger conidia and more nuclei/conidium than diploids and the haploid parent mutants. Diploids contained significantly fewer nuclei/conidium than haploids. However, they could not be distinguished from haploids by measurement of conidial size. The strains were examined for the production of α-amylase. All auxotrophic mutants produced less α-amylase than the prototrophic wild type. Heterocaryons gave yields which were intermediate between that of their parent mutants or the same as the best producing parent. Diploids which produced more α-amylase than the best producing parent strain were synthesized. The highest yield from a diploid was of the same order of magnitude as the yield from the wild type.     

Visualization of vacuoles in Aspergillus oryzae by expression of CPY-EGFP

Vacuolar carboxypeptidase Y (CPY) from Aspergillus nidulans was used to construct a CPY-EGFP fusion protein and expressed in A. oryzae to study vacuolar morphology and functions in A. oryzae. While the fluorescence of EGFP was barely detectable in A. oryzae expressing CPY-EGFP grown under normal conditions at pH 5-6, the increase in pH of the growth medium towards alkalinity restored the fluorescence. In accordance with such an observation, the fluorescence of CPY-EGFP fusion protein in cell extract decreased in acidic pH condition, concomitant with lowered content of EGFP detected in A. oryzae grown under acidic pH conditions. The pH sensitivity of EGFP fluorescence and enhanced degradation of proteins in vacuoles under acidic pH conditions are thus proposed to result in the reduction of fluorescence in A. oryzae. Further, visualization of vacuoles revealed the presence of peculiar ring- or tube-like structures as distinct from normal spherical-shaped vacuoles.     

Transformation with green fluorescent protein of Trichoderma harzianum 1051, a strain with biocontrol activity against Crinipellis perniciosa,the agent of witches'-broom disease of cocoa

A plasmid vector for fungal expression of an enhanced, red-shifted variant of the Aequoria victoriae green fluorescent protein was constructed by fusion of the EGFP gene to the highly expressed Aspergillus nidulans gpd promoter and the A. nidulans trpC terminator. This construction was introduced by cotransformation, using benomyl selection, into Trichoderma harzianum strain 1051, a strain being evaluated for the biological control of witches'-broom disease of cocoa caused by Crinipellis perniciosa. Epifluorescence microscopy was used to monitor germination and attachment of stable transformant conidia on the surface of C. perniciosa hyphae.     

Reduction of the number of nuclei per cell ofHelminthosporium euphorbiae by protoplast isolation

Helminthosporium euphorbiae is a pathogen of the weedEuphorbia heterophylla, which causes severe losses in soybean (Glycine max) crops. The fungus causes leaf loss and affects germination, making it a promising biocontrol agent for this weed. In order to start a breeding program for this species, four isolates were examined for number of nuclei in the conidia and hyphae and nuclear behavior at different cultivation stages. The conidia were multinucleated with about 20 nuclei per conidium, and 5 to 7 nuclei were observed in the hyphae compartments. The high number of nuclei makes the genetic manipulation of this species diffucult, so the protoplast formation is an alternative for obtaining cells with a reduced number of nuclei. Thus the experimental conditions for the production and regeneration of protoplasts inH. euphorbiae were determined by assessing three enzymatic complexes and seven osmotic stabilizers. The efficiency of formation and regeneration frequency of the protoplasts varied depending on isolates, stabilizers and enzyme mixture used. The number of nuclei estimated per protoplast was reduced to 1 to 6, depending on the stage of mycelial growth during the protoplast formation process.     

A simple method for enrichment of uninucleate conidia of Aspergillus oryzae

Aspergillus oryzae produces multinucleate conidia, which makes the obtaining of homokaryons labor-intensive. Analysis of conidia by flow cytometry clarified the relationship that conidia of lower nuclear number were smaller in size. Based on this, we have developed a simple way to enrich uninucleate conidia with a membrane filter. Our results also suggest that the method is useful for elimination of heterokaryons.     

Nuclear behavior during the formation of appressoria byAlternaria alternata

Nuclear behavior in the developmental process of appressoria inAlternaria alternata was investigated. In pregerminated conidia, approximately 94% of the conidial cells were uninucleate. The migration of a nucleus into an elongating germ tube from a germinating conidium was confirmed after 2h of incubation at 24±1°C in PDB. Peak frequencies of binucleate and trinucleate germ tubes were detected 1 and 2h after the peak frequency of uninucleate germ tubes, respectively. Four-and five-nucleate germ tubes did not show marked peak frrequencies. A marked peak frequency of the six-nucleate germ tubes occurred about 1 h after the peak frequency of the trinucleate germ tubes, suggesting that the nuclei in the trinucleate germ tubes each divided once within 1 h. The significance of early establishment of multinucleate appressorial cells in the colonization of host plants by pathogenicA. alternata was discussed.     

ELECTRON MICROSCOPY OF PHIALOCONIDIOGENESIS IN METARRHIZIUM ANISOPLIAE

Fine-structure observations with two different fixation procedures showed that phialide necks possessed a thickened electron-transparent wall layer. Phialoconidia developed from a wall layer which originated 1–1.5 μm within phialide necks. After conidium initials blew out of phialide tips and organelles entered, conidia were delimited by transverse septa which did not appear to be plugged by Woronin body-like plugs. Instead, septa appeared to become functionally complete by continued centripetal growth. Conidium-delimiting septa moved distally out of phialide necks as subsequent conidium initials formed. During this distal movement, septa increased in thickness and lamellae appeared on the conidium side; mature conidia had bipolarly lamellate cell walls. Conidial walls had a thin, ridged electron-dense outer wall layer and a thicker electron-transparent inner wall layer which increased in thickness centripetally after septum delimitation. Conidia were usually uninucleate and possessed conspicuous storage vacuoles with lipid and protein contents. Conidia also possessed numerous presumably lipid droplets. Multivesicular bodies were observed near conidium-delimiting septa and conidium walls which were increasing in thickness.     

Maize ribosome-inactivating protein inhibits normal development of Aspergillus nidulans and Aspergillus flavus

The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion.     

Successful induction and recognition of conidiation,conidial germination and chlamydospore formation in pure culture of Morchella

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5–8 μm in diameter, with a vast majority (nearly 99%) 2.5–5 μm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1–2 μm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.     

Conidium differentiation in Aspergillus nidulans wild-type and wet-white (wetA) mutant strains

Conidium (asexual spore) differentiation in wild-type and the wet-white (wetA) mutant of Aspergillus nidulans was compared in intact chains of successively older conidia. Carbohydrate cytochemistry helped define three stages (Stages I, II, and III) of wild-type conidium maturation on the basis of changes in the ultrastructure and composition of the conidium wall. Conidia of the wetA6 mutant strain formed normally but failed to mature during Stages II and III. Specifically, the inner wall layer of wetA6 conidia did not condense during Stage II and two wall layers that stained for carbohydrates did not form during the transition to Stage III. Concomitantly, wetA6 conidia formed large cytoplasmic vacuoles and underwent lysis. The wetA gene appears to have a conidium-specific function for the modification of the conidium wall during Stages II and III. These modifications of the conidium wall are essential for the stability of mature, dormant conidia.     

B-chromosome condensation inPhleum pollen grains

Pollen grains were investigated in 11 specimens ofPhleum Boehmeri Wib. [P. phleoides (L.) Karst.] differing in the number of B-chromosomes (2n=14+0–3B). In some uninucleate pollen grains there were seen large chromocentres, not observed in other tissues. They resembled meiotically and mitotically condensed Bs both in size and shape. They occurred only in plants with 2 or 3 Bs. In the plants with 2 Bs, 2.5% pollen grains had a nucleus with 1 chromocentre; in the plants with 3 Bs, 41.4% of uninucleate pollen grains had 1 chromocentre and 0.2% of grains had 2 chromocentres. Comparison of the frequency of Bs in the metaphase of the first pollen mitosis and that of chromocentres in the nuclei of the uninucleate pollen grains was carried out. The author suggests that in pollen-grain nuclei one B chromosome per genome remains decondensed, and every next one (if it occurs) forms a chromocentre.     

Uniparental Inheritance and Replacement of Mitochondrial DNA in Neurospora Tetrasperma

This study tested mechanisms proposed for maternal uniparental mitochondrial inheritance in Neurospora: (1) exclusion of conidial mitochondria by the specialized female reproductive structure, trichogyne, due to mating locus heterokaryon incompatibility and (2) mitochondrial input bias favoring the larger trichogyne over the smaller conidium. These mechanisms were tested by determining the modes of mitochondrial DNA (mtDNA) inheritance and transmission in the absence of mating locus heterokaryon incompatibility following crosses of uninucleate strains of Neurospora tetrasperma with trichogyne (trichogyne inoculated by conidia) and without trichogyne (hyphal fusion). Maternal uniparental mitochondrial inheritance was observed in 136 single ascospore progeny following both mating with and without trichogyne using mtDNA restriction fragment length polymorphisms to distinguish parental types. This suggests that maternal mitochondrial inheritance following hyphal fusions is due to some mechanism other than those that implicate the trichogyne. Following hyphal fusion, mututally exclusive nuclear migration permitted investigation of reciprocal interactions. Regardless of which strain accepted nuclei following seven replicate hyphal fusion matings, acceptor mtDNA was the only type detected in 34 hyphal plug and tip samples taken from the contact and acceptor zones. No intracellular mtDNA mixtures were detected. Surprisingly, 3 days following hyphal fusion, acceptor mtDNA replaced donor mtDNA throughout the entire colony. To our knowledge, this is the first report of complete mitochondrial replacement during mating in a filamentous fungus.     

Hereditary variation and genetic recombination in Koji-molds (Aspergillus oryzae and Asp. sojae). I. Natural variation

Natural variation in monospore lines of Koji-molds (Asp. oryzae and Asp. sojae), isolated from commercial Koji material or soil and from laboratory stock cultures, has been observed. We can divide the 58 strains of Koji-molds investigated into two groups; one group consists of inconstant strains which are very liable to produce natural variants, and the other consists of strains which remain constant through successive single spore culture. The inconstant strains develop colonies bearing various proportions of conidia and aerial mycelium (X-type). They generally form large conidia (Asp. oryzae var. magnasporus) but sometimes medium sized conidia (Asp. oryzae s. str.), which produce large conidia occasionally. The colonies of the constant strains show abundant conidial formation and smooth surfaces (C-type). The conidia are mostly small (Asp. oryzae var. microsporus) but sometimes medium in size (Asp. oryzae s. str.). The colony types of the variants are as follows: C (Conidial type, whole colony covered with conidia), M (mycelial type), R (restricted in growth rate), St (sterile type, little sporulation on all media tested), Nit (requiring reduced nitrate, very faint growth on Czapek's agar), and LS (semi lethal, growth cease immediately after germination). Pedigree cultures of the 8 inconstant strains have been made, but no definite segregation ratios for each variant type have been recognized through successive generations. The LS and N types commonly occur spontaneously from the M-type.     

Sexual diploids of Aspergillus nidulans do not form by random fusion of nuclei in the heterokaryon

The sexual stage of Aspergillus (Emericella) nidulans consists of cleistothecia containing asci, each with eight ascospores. The fungus completes the sexual cycle in a homokaryotic or a heterokaryotic mycelium, respectively. The common assumption for the last 50 years was that different nuclear types are not distinguishable when sexual development is initiated. When cultured on a medium limited for glucose supplemented with 2% sorbitol, sexual development of A. nidulans is slowed and intact tetrads can be isolated. Through tetrad analysis we found that unlike haploid nuclei fuse preferentially to the prezygotic diploid nucleus. When heterokaryons are formed between nuclei of different genetic backgrounds, then recombinant asci derived from opposite nuclei are formed exclusively. Strains in the same heterokaryon compatibility group with moderate differences in their genetic backgrounds can discriminate between the nuclei of a heterokaryon and preferentially form a hybrid diploid nucleus, resulting in 85% recombinant tetrads. A. nidulans strains that differ at only a single genetic marker fuse the haploid nuclei at random for formation of diploid nuclei during meiosis. These results argue for a genetically determined "relative heterothallism" of nuclear recognition within a heterokaryon and a specific recruitment of different nuclei for karyogamy when available.     

Cellular events in growth and germination of giant conidia of Neurospora crassa

During growth of conidia in 3.22 M ethylene glycol the increase in the number of the nuclei is proportional to the increase in volume only in the phase of maximum growth rate and is lower in the preceding and in the following periods of growth. DNA synthesis similarly initiates later and decreases faster than protein synthesis. The dilution of ethylene glycol is followed by the germination of giant conidia, which is characterized by the absence of a lag phase, a high degree of synchrony and the formation of more than one germ tube per conidium. The number of germ tubes is dependent on the volume reached by conidia at the end of the treatment and does not increase with time. The resuming of DNA synthesis after germination is preceded by a sharp increase in protein synthesis and the division of almost half of the nuclei and shows a synchronized pattern. Results are discussed in the light of models of growth proposed for eukaryotic cells.     

产黄青霉ds RNA病毒通过原生质体融合的转移

将国内青霉素产生菌(Penicillium chrysogenum)的黄孢子系统及绿孢子(包括淡绿,灰绿)系统的十多个菌株,经过病毒提取、电镜观察、奥氏免疫双扩散、凝胶电泳及放射免疫测定,证明黄孢子系统的菌株含有不同滴定度的、直径40nm的球形病毒,而绿孢子系统中检查不出病毒。从营养要求、孢子颜色不同的带病毒和无病毒菌体中分离原生质体,进行不同组合的原生质体的融合杂交,获得营养互补融合的异核体。异核体1中,病毒通过胞质融合转移到原来无病毒的灰绿孢子菌株及细胞核融合后的杂合二倍体中。灰绿孢子的病毒量接近二倍体的1/3。二倍体菌落生长稳定,低温保存二年后经0.01—0.02M对氟苯丙氨酸(PFA)诱发和分离,产生亲本类型的分离子,分离子及二倍体仍然含有病毒。异核体2作亲本性分离,黄孢子仍有病毒,淡绿孢子及细胞核融合后产生的二倍体均无病毒,表明非感染性为显性。此种淡绿孢子的突变体中存在非感病菌系,它不支持病毒的复制。提取各杂交组二倍体内的病毒所特有的dsRNA时,可看出dsRNA的存在和病毒的存在一致。多数杂合二倍体的青霉素产量比亲本高。