CONIDIOGENESIS OF DETERIORATED VARIANT OF THE STRAIN Abnc OF Aspergillus nidulans Ágata Cristiane

The Abnc strain of A. nidulans carries the bncA1 gene (binucleated conidia), which induces the formation of binucleate and trinucleate conidia, displaying a chromosome I duplicated area and shifted to the chromosome II (IàII), and bringing forth genetic instability with degenerated sectors. This work has considered in a cytological level the conidiogenesis of the deteriorated variants isolated from the Abnc strain of A. nidulans, observing the event at the level of structural alterations, which composes the conidiophore, and the variations in the number of sterigmata and conidia nuclei. Cytogenetic analyses of conidiogenesis were accomplished in predetermined periods, under Giemsa stain, to observe the nuclei and analysis through the Scanning Electronic Microscopy, and also the structures that composes the conidiophore. The analyzed, deteriorated variants presented alterations in the cell-foot, metulae and phialides structure, conidiophore number and conidias reduction, and the formation of secondary conidiophores. These alterations can be related to genes for the development, bristle, and activities of NIMA e NINX (involved in morphogenesis regulatory cycle) that induce the expression of brislte, establishing the link to the regulation and expression of the genes control throughout the conidiophore development.


INTRODUCTION
The fungus Aspergillus nidulans presents a life cycle three phases, Vegetative Cycle, Asexual Cycle or Conidiogenesis and Sexual Cycle or Ascosporogenesis.The vegetative growth begins with the spore germination that can be a conidia or an ascospore (Lee & Adams, 1994).The ramified air hyphae differs in a conidiophore, a generative structure of the asexual cycle (Adams et al., 1998).This differentiation and development process constitute the Conidiogenesis.The conidiophore morphology shows five development stages (Mims et al., 1988): (i) transformation of a hyphae cell in a cell-foot, the structure that maintains and develops the conidiophore; (ii) cell-foot differentiation in the conidiophore stem, and apex's gradual enlargement, creating a vesicle with several nuclei; (iii) metulae come out as protuberances in the intern surface of the vesicle wall, with nuclear migration from the vesicle apical area to metulae, making it uninucleated and after that occurring a mitosis; (iv) the phialides are formed in metulae apex with nucleus-son migration from metulae to phialides; (v) formation of the Sci.Agric.(Piracicaba, Braz.), v.61, n.4, p.401-406, July/August 2004 phialides apex original primary conidia and arranging conidia chains (Oliver, 1972;Bergen & Morris, 1983).
The structural and temporal development processes are controlled by groups of regulatory genes and the activation sequence involves the genes bristle (brlA), abacus (abaA) and wet-white (wetA), establishing the regulatory pathway and genes expression control during the conidiophore development (Adams et al., 1998).Other two genes, stund (stuA) and medusa (medA), interact with the regulators genes (Miller, 1990;Miller et al., 1992).
The strain A of A. nidulans, with double chromosome segment (chromosome I) and transferred (chromosome II) (Nga & Roper, 1968;1969) presents mitotic instability, which leads to deteriorated sections from total or partial loss of the chromosomic duplication, with subsequent insert of that segment in any of the eight groups of junction of A. nidulans (Azevedo & Roper, 1970).Several factors affect the genetic instability: chemical factors, as the caffeine (Azevedo & Santana, 1975), physical factors, such as temperature (Lieber, 1976), and genetic factor, such as the effect of the bncA1 gene, that originates conidia bi and trinucleted (Pizzirani-Kleiner & Azevedo, 1986a, 1994b;Pascon, 1994) and reduce the mitotic instability in the Abnc strain, with an increase in the frequency of deteriorated sections (Pascon, 1994).The bncA1 gene causes abnormalities during the formation of the conidiophore such as the reduced number and phialides and metulae septa (Pascon et al., 2001).
This article presents the study of the conidiogenisis of the deteriorated variants originated from the Abnc strain of A. nidulans.Structural and temporary alterations were observed in the conidiophore of the deteriorated variants, VB7, VB8, VB9, VB10 and VB11.These structural alterations present structural likeness with mutants bristle.

Medium Culture and Conidiogenesis.
Conidia of the VB 7, VB8, VB9, VB10 and VB11 strain were incubated in complete medium (Pontecorvo et al., 1953), and over dialysis membranes and subsequent incubation at a temperature of 37 o C for 20, 32 and 72 h (Giancoli, 2000).

Stain of nucleus and optical microscopy
After the established periods, the coverslips with adherent hyphae were transferred to etanol and acetic acid, lactic acid solution (6:1:1), and incubated at room temperature for 15 min.Then, coverslips were incubated for 12 min.at 63 o C in 1 mol L -1 HCl solution, rinsed in distilled water and transferred to 100 mmol L -1 phosfhate buffer (pH 7.0) for 1 hour, prior to staining after incubation for 20 min.at room temperature in a Giemsa solution (Tanaka et al., 1979).

Scanning Electron Microscopy (SEM)
After the pre established periods the cover-slips adherent hyphae and conidiophores were transferred to glutaraldehyde 25% solution, paraformaldehyde 10% in caccodilate buffer 0.2% pH 7.2 and CaCl 2 0,1 mol L -1 .After 1 -2 hours the cove-rslips were washed for 10 min with buffer and post fixed to 1 hour in similarly buffered 1% OsO 4 .Cover slips were then washed in distilled water and dehydrated in a graded acetone series.Cover slips were dried to a critical point (Balzers CPD 030), coated with gold (MED 010 -Balzers), and examined under a Scanning Electron Microscope (Leo 435 VP).

RESULTS AND DISCUSSION
Starting from 20 hours of cultivation MSE and Abnc strain, it was possible to observe indifferentiated hyphae, and in some cases differentiated hyphae in cell-foot with the stem of the conidiophore already formed.At 32 hours, ripe phialides with conidia development were detected.These data are similar to those reported by Axelrod et al. (1973) and Champe et al. (1981), demonstrated that the temporary and space development of the conidiophores occurred after 20 hours of cultivation.Starting from 72 hours, it was possible to observe the conidiophores with long conidia chains (Figures 1a and 1b).The Abnc strain bearing of the bncA1 gene (Pizzirani-Kleiner, 1981), besides presenting conidia bi and trinucleated, shows other interesting aspects: the disposition of the conidia chains, where they are just found conidia chains uninucleated and or just binucleated, are rarely found mixed chains, what agrees with the observations obtained by Pascon (1994) and Pascon et al. (2001).The irregular organization of the metulae and phialides along the vesicle of the conidiophore, was noticed in a small number of structures when compared with the strains that don't show the mutation bncA1 (not given presented); the phialides are septate with until three nuclei in its interior (Figure 3a).Clutterbuck (1969;1970;1977) suggested classifications of the genes involved in the differentiation of the conidiophore in: Class I -mutants that affect the acquisition of the competence; and Classe II -mutants that affect the formation of the conidiophore and conidia, inside of the Classe II -are subdivided more four types of genes (bristle, abacus, stund and medusa).The bncA1 gene probably is into the mutants' categories with modification of the conidiophore (Miller et al., 1992;Pascon et al., 2001), which are combined by the genes stuA and medA, that cause specific alterations in the morphology and space organization of the conidiophore, but do not avid the conidia development The deteriorated variants were produced with the approximate frequency of one in ten analyzed colonies (Giancoli, 2000).The deteriorated variants analyzed, VB7, VB8, VB9, VB10 and VB11 presented little conidiation and different degrees pigmentation.
The deteriorated variant VB7 presented time of compatible development with the pattern strain (MSE), with differentiation of the hyphae starting from 20 hours of development.The conidia's growth occurred within 32 hours, being observed small conidia chains uni and binucleated.VB7 presented a smaller amount of conidiophore if compared with the MSE strain.The temporary development of the conidiophore was late, the metulae and phialides demonstrated characteristic alterations, ob-served in the strain with the bncA1 gene (Figure 3a).The metulae and phialides present extended and unlikely structures (Figure 2a) with lack of conidia.Normal conidiophores were observed.We could observe the formation of secondary conidiophore that is a characteristic of the bristle and medusa genes (Figure 2b).
The deteriorated variant VB8 has shown a space and temporary abnormal organization outstanding when compared to the other deteriorated variants.The hyphae differentiation, followed by the vesicles growth was observed starting from 32 hours.There was a characteristic abnormal spatial organization, with extended and unlikely metulae.(Figures 2c, 2d and 3b).Some vesicles present  irregularities in their formats and the amount of conidiophores are small.That deteriorated variant presented an extremely high number of hülle cells, when compared with the MSE strain (data not available).
The cytological analysis of VB9 presented likeness with VB7.The period of temporary development was similar to the established ones for the pattern strain.There was a characteristic abnormal spatial organization, with the extension and diferentiation for metulae (Figures 2a  and 2e).The amount of conidia is reduced.For carrying the mutation in the bncA1 gene it presents septated and smaller amount of metulae and phialides.Normal conidiophores were observed.
The deteriorated variants VB 10 and VB11 presented great likeness.There was a delay in the process of temporary-spatial development of the conidiophore.In these deteriorated variants occurred abnormal spatial organization, with metulae extension (Figures 2f, 2g and  3d and 3e).VB11 presented some vesicles with irregular format (Figure 2f).The beginning of the differentiation of the hyphae occurred about 32 hours and presented a reduced number of conidiophores.
The morphologic alterations observed in the structure of the conidiophore are characteristic for development genes.These genes should cause conidia alterations (wetA); alterations in the conidiophore (stuA and medA), which don't affect the conidia formation; mutants that block the conidia formation (brlA and abaA) and mutants that affect the pigmentation of the conidia (ivoA and ivoB) (Timberlake & Clutterbuck, 1994).The main characteristics of the conidiophore development are due to the central control of the bristle gene.However, the activation of the protein Brl is not well studied, but the occurrence of the interactions is known among the genes stu (stunted) and med (medusa).Ye et al. (1999) and Schier et al. (2001), while studying the relationship between the regulation of the cellular cycle and the morphogenetic development of A. nidulans, they noticed that exists a deep relationship between the two cycles, being the morphogeneses dependent of the increase of the quinase activity of NIMA and NINX cdc2 , so this way can occur the activation and regulation in cascade of genes, that take the normal formation of the conidiophore.The microscopic analyses resulted in the detection of abnormalities formation of the conidiophore, as the presence of secondary conidiophore starting from metulaes abnormal, besides extended metulae and phialide.This phenotype is characteristic so much of mutants light bristle or middleman (leaky) as of medula mutants.These last ones, however, present a pigment decrease of the conidiophore and metulae chains (Miller, 1993), two absent characteristics in the analyzed deteriorated variants.
In analyzing the intermediate conidiophore we always find morphological vesicles and metula alterations.Adams et al. (1998) observed that after the first synthesis of the protein Brl (BrlA), there is a differentiation of the vesicle and the germination of the metulae, so the only nuclei then migrate for the metulae and differentiation is concluded in an autonomous way.These results suggest that the amount of protein Brl that is aimed for each metulae altogether with the cytoplasm during its germination should induce the genes for the final stage of the fiálides differentiation.However, when there is little protein expression Brl in the vesicles, the metulae could receive an insufficient amount for the beginning of the induction, tending to an abnormal behavior, differentiating in a stem.This abnormal stem could originate a new secondary conidiophore.This interpretation would also justify the fact that some vesicles not present differentiated metulae, growing as secondary stem, beside differentiated metulae, with fialides and conidia.The synchronism lack can be the result of different amounts of protein Brl of each metulae.These observations indicate that the synchronism usually found in the differentiation of the structures of the conidiophore depend on the minimum of protein Brl produced in the vesicle, guaranteeing surpluses in the metulae, enough to control small deviations in the distribution of this product during the germination (Adams et al., 1998).
The cytological analyses suggest that the deteriorated variants present the characteristic phenotype of the bristle gene.However, these alterations can be influenced by regulatory genes of the cell cycle avoiding the correct activation of the bristle gene (Ye et al., 1999 andSchier et al., 2001).These were the first steps in the study of this group of deteriorated variants that are going along with the investigation at a molecular level.