Conidiation Genes

Mutants of A. nidulans were obtained that are normal in hyphal growth and sexual reproduction but are defective in conidiophore development (Clutterbuck, 1969). Since conidia are not formed, the conidiation mutants are detected by the lack of the wild green color and by their inability to be replica plated—a simple technique that allows colonies producing loose conidia in a petri dish to be sampled by pressing a velvet cloth secured over the top of a cylinder and onto the surface of new medium in a petri dish. The procedure transfers each conidiating colony in a way that the pattern of colonies is maintained on the replica plate. The colonies that are missing because of non-conidiation are identified in the master

Figure 7.2 Conidiation in a strain of Neurospora crassa. (A) Fusion of conidial germ tubes and formation of mycelium. (B) A conidiophore in surface-grown mycelium bearing macroconidia formed by budding. (From Maheshwari (1991), with permission from Society of General Microbiology.) (C) A conidiophore in surface-grown mycelium producing arthroconidia by septation. (D) Microcyclic conidiation in the same strain grown in submerged shake culture.

Figure 7.2 Conidiation in a strain of Neurospora crassa. (A) Fusion of conidial germ tubes and formation of mycelium. (B) A conidiophore in surface-grown mycelium bearing macroconidia formed by budding. (From Maheshwari (1991), with permission from Society of General Microbiology.) (C) A conidiophore in surface-grown mycelium producing arthroconidia by septation. (D) Microcyclic conidiation in the same strain grown in submerged shake culture.

plate. In one study, the number of conidiation-specific genes was estimated by comparing the frequency of conidiation mutants with the frequency of auxotrophic mutants:

number of conidiation genes =

frequency of conidiation mutants frequency of auxotrophic mutants

X number of auxotrophic genes

It was estimated that approximately 100 genes specifically affect conidiation (Martinelli and Clutterbuck, 1971). A larger number of geners (about 1000) was estimated based on the diverse messenger RNA that accumulated specifically during conidiation (Timberlake, 1980). Based on the mutant phenotypes and the comparison of gene expression in wild and conidiation mutants, three genes were identified as essential regulators of conidiophore development: the bristle (brl) gene controls the swelling of conidiophore stalks into vesicles; the abacus (abaA) gene controls the budding of conidia by the conidiophore stalk; and the wet-white (wetA) gene controls the hydrophobicity that is essential for the aerial dissemination of conidia. The wetA mutants produce normal conidiophores but the conidia lack hydrophobicity and autolyze, forming droplets at the ends of conidial chains. These three genes are themselves not specific to conidiation—rather, they integrate the expression of other genes that determine the structure of the conidiophore. The genes are therefore considered to be regulatory genes that play a key role in conidiophore development and act in the sequence brlA ^ abaA ^ wetA. Using a genomic library, the wild brlA, abaA and wetA genes were isolated by complementation of the mutant strains. The nucleotide sequence of brlA showed that it has a short open reading frame (ORF) that regulates transcription. The deduced amino acid sequence of brlA protein product can form a secondary structure with a loop of the polypeptide chain, to which a Zn atom can bind.

Figure 7.3 Diagram of microcycle conidiation in Aspergillus niger. The mycelial phase was bypassed when conidia were subjected to a regime of heat shock. (From Anderson and Smith (1971).)

Figure 7.3 Diagram of microcycle conidiation in Aspergillus niger. The mycelial phase was bypassed when conidia were subjected to a regime of heat shock. (From Anderson and Smith (1971).)

Since this type of structure is common in DNA-binding proteins, the brlA gene is thought to encode a transcription regulator that activates genes required for conidiophore formation (Adams et al., 1990).

To determine whether the conidiation-specific genes occur in a close physical order that reflects their order of action or are distributed randomly in the genome, thirty cDNA clones containing approximately 1.5 kb long DNA inserts were hybridized to poly(A)+ RNA (Orr and Timberlake, 1982). On the basis of the random distribution of conidiation genes, the majority of clones were expected to have only one spore-specific transcript. However, the experimental finding was that many clones hybridized to several spore-specific transcripts of different molecular weights, indicating that the spore-specific genes in A. nidulans may be clustered and constitute a functional unit of developmental gene regulation.

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