conidiation in aspergillus involves many common developmental themes including spatial and temporal control of gene expression , specialized differentiation of cells , and intra and intercellular communications . conidiation is regulated by an asexual developmental signaling pathway that directs expression control elements and other genes required for conidiophore assembly . as developmental mechanisms in a. nidulans have been intensively studied and results have provided important clues for understanding conidiation in other aspergilli [ 2 , 4 , 7 ] , what is known in a. nidulans ( ani ) will be described first followed by what has been found in a. fumigatus ( afu ) . in ani , a key and essential step for conidiophore development is the activation of brla encoding a c2h2 zinc finger transcription factor ( tf ) , which induces expression of other genes required for conidiation . loss - of - function brla mutants form structures that resemble conidiophore stalks ( thus named " bristle " ) , except that they grow indeterminately and fail to produce vesicles , metuale , phialides , and conidia , indicating that brla controls the initiation of conidiophore development ( fig . , brla overexpression in vegetative cells causes termination of polar growth coupled with the commencement of abnormal sporulation leading to the formation of viable spores from hyphae . no environmental signals , including nutrient limitations or various ( osmotic and oxidative ) stresses , can bypass the brla requirement for conidiation , indicating that activation of brla expression early in conidiophore development represents a key and essential control point for commencing conidiation . the anibrla ( but not afubrla ) gene is a compound gene consisting of two overlapping transcriptional units designated brlaa and brlab . important information on the complex regulation of the two overlapping brla transcription units is well described elsewhere . afubrla , the afu brla homologue , shows 68% identity and 77% similarity to the ani brla . mah and yu   tested whether brla - dependent conidiation is conserved in these two aspergilli using genetics and found that afubrla deletion completely eliminated asexual development in afu , resulting in elongated aerial hyphae and increased hyphal mass in the colonies . these studies clearly demonstrated that afubrla is essential for conidiophore development in afu , and that the role of the core downstream tf brla in conidiation is conserved in these aspergilli ( fig . brla is a putative tf ( activator ) with two c2h2 zinc finger motifs at the c - terminus . disruption of either one of the fingers by mutating the key cysteine to serine results in complete loss of brla activity . chang and timberlake   further demonstrated that brla expression in the budding yeast saccharomyces cerevisiae results in brla dependent activation of aspergillus genes . furthermore , they proposed the consensus brla binding sites ( brla response elements [ bres ] ; 5'-(c / a)(g / a)aggg(g / a)-3 ' ) . although direct binding of brla to bres remains to be verified in vitro , a number of developmental genes including abaa , weta , roda , and ya have multiple bres in their promoter regions . importantly , tao and yu   reported that multiple bres are present in the promoter regions of afu brla , abaa , weta , roda and the velvet regulators , implying a potentially conserved regulatory circuit in the two aspergilli . the abaa gene , activated by brla during the middle stages of conidiation when metulae and phialides are being formed , functions in the differentiation of phialides . abacus ( abaa ) mutants have nearly normal conidiophores bearing abacus - like structures with swellings at intervals instead of chains of conidia . morphological studies have revealed that the metulae of abaa mutants produce supernumerary tiers of cells with properties of metulae , not phialides , indicating that abaa is essential for the differentiation and functionality of phialides as conidiogenous cells . abaa expression is dependent on brla activity , and bres are found in the abaa promoter region , suggesting that brla directly activates abaa . the abaa gene encodes a developmental regulator , which begins to be expressed when phialides form at 15~18 hr after developmental induction ( fig . 3 ) , and loss of abaa function results in abnormal expression of a number of developmentally regulated genes . abaa overexpression in vegetative hyphae causes growth cessation and enhances cellular vacuolization but not spore formation in ani . abaa overexpression activates the expression of weta and brla , and it is thought that abaa induces the expression of brla at certain times of development . however , the genetic interaction between abaa and brla seems more complex , as brla mrna levels increase in abaa null mutants [ 15 , 20 ] , which might involve vosa ( fig . 3 ) . the abaa protein contains an atts / tea dna - binding motif [ 21 , 22 ] and a leucine zipper for potential dimerization . expression studies in both ani and s. cerevisiae have demonstrated that abaa binds to the cis - regulatory elements upstream of the ya gene . results of a gel mobility shift assay indicate that abaa binds to the consensus sequence 5'-cattcy-3 ' ( abaa response element [ are ] where y is pyrimidine ) . in fact , multiple ares are found in the promoter regions of developmentally regulated genes , including brla , weta , ya , roda , and abaa . importantly , park et al .   demonstrated that abaa binds to the chitin synthase gene promoter , chsc , suggesting that abaa also regulates chitin biosynthesis during conidiophore development by controlling the expression of certain chitin synthetases . wet - white ( weta ) mutants produce colorless conidia that completely autolyze within a few days , leading to the formation of liquid droplets ( wet ) on the tops of conidial heads . sewall et al .   have shown that the weta gene is required for the synthesis of crucial cell wall components late in the development of the inner c4 layer ( fig . the ani weta gene encodes two transcription units , and weta transcription is predicted to be complex and subjected to developmental control . abaa , but not brla , activates weta expression , as abaa overexpression activates weta in the absence of wild type brla . furthermore , weta is auto - activated , as it is not expressed in weta temperature - sensitive mutants ( fig . the ani weta gene is predicted to encode a 60 kda protein rich in serine ( 14% ) , threonine ( 7% ) , and proline ( 10% ) . while no known dna binding domains are present in weta , it has been proposed to function as a regulator of spore - specific gene expression . this is based on observations that weta alone is sufficient to activate many sporulation - specific genes , and that the weta mutants fails to accumulate many sporulation - specific mrnas . furthermore , weta overexpression in vegetative cells inhibits hyphal growth , resulting in excessive hyphal branching and the activation of spore - specific genes . however , weta overexpression does not result in brla or abaa activation or lead to precocious conidiation . taken together , weta is proposed to activate a set of genes required for spore formation and maturation , which may function to complete the final two conidial wall layers and/or direct their assembly ( fig . , the brlaabaaweta cascade has been proposed to define a central regulatory pathway that controls temporal and spatial expression of conidiation - specific genes during conidiophore development and spore maturation ( fig . vosa is a multifunctional regulator that acts in concert with the central regulatory genes , couples trehalose biogenesis and conidia maturation , and exerts negative feedback regulation of brla , thereby completing conidiation in ani ( fig . 3 ) . afuabaa ( eal88194 , 60% identity , 74% similarity to aniabaa ) and afuweta ( eal89470 , 58% identity , 68% similarity ) were previously identified through a genome search . the afuabaa open reading frame ( orf ) is composed of 2,518 bp with three exons and two introns and is predicted to encode a 797 aa - length protein containing an atts / tea dna - binding domain . the afuweta orf comprises 1,701 bp with no introns and is predicted to encode a 566 aa - length protein with a conserved c - terminal domain . tao and yu   examined the levels of the afuabaa and afuweta transcripts throughout the lifecycle and found that afuabaa and afuweta are highly expressed during asexual development . afuabaa mrna starts to accumulate at 6 hr post - developmental induction , reaches its highest level at 12 hr , and disappears at 48 hr . afuweta mrna begins to accumulate at 12 hr post - developmental induction , reaches its highest level at 48 hr post induction , and is present in conidia . deletion of afuabaa results in the formation of aberrant conidiophores exhibiting reiterated cylinder - like terminal cells without conidia , indicating that , as in ani , afuabaa is essential for differentiation and functionalization of afu phialides as conidiogenous cells . importantly , tao and yu   found that the most distal cells of the afuabaa conidiophores are capable of apical hyphal growth , whereas the fractions of cylinder - like elongated phialides do not undergo vegetative growth . they also examined hyphal dry weights and found that the absence of afuabaa causes delayed hyphal mass loss compared to that in the wild type . when cell viability was examined by determining the percent alamar blue reduction , which represents living cell mitochondrial activity , the afuabaa mutant clearly exhibited prolonged cell viability compared to the wild type . tao and yu   then examined the effects of afuabaa overexpression and found that it caused accelerated hyphal fragmentation , disintegration , a dramatic reduction of mycelial mass , and precocious cell death . the afuweta gene is subsequently activated by afuabaa in the middle to late phases of conidiation . furthermore , tem analyses of the afuweta mutant revealed that weta conidiophores exhibit an interconnected conidia phenotype , i.e. , incomplete conidial separation and maturation . as afuweta plays an essential role in completion of the conidial wall , tao and yu   hypothesized that the absence of afuweta may affect spore viability and/or integrity , and found that the afuweta mutant exhibits dramatically reduced viability starting from day 10 , whereas wild type conidia maintain high viability until day 20 . moreover , tem studies revealed that , even at day 2 , about 20% of the afuweta conidia appear to lack cytoplasm and exhibit sheared conidial walls . tao and yu   then examined the effects of afuweta on spore tolerance to various stressors and found that the afuweta conidia were much more sensitive to heat , and oxidative and osmotic stresses . as trehalose is essential for long - term spore viability and stress resistance [ 15 , 27 ] , tao and yu   determined that the afuweta conidia do not contain any trehalose , whereas wild type conidia harbor about 3.4 pg of trehalose per conidium . these results indicate that afuweta plays an essential role in trehalose biogenesis in conidia , which probably affects their viability and stress tolerance . this was the first report that weta is essential for trehalose biogenesis in fungal conidia . additionally , given that the afuweta gene is activated during the late stage of conidiation , and that its mrna accumulates preferentially in mature conidia , tao and yu   further determined whether the absence of afuweta had an effect on spore germination and early vegetative growth . they found that after a 8-hr incubation in liquid submerged culture , the afuweta mutant conidia formed only one unidirectional germ tube without branching , which resulted in loosened mycelial aggregates , whereas conidia of wild type and complemented strains produced bi- or multi - directional germ tubes that formed branches . moreover , after a 14-hr incubation , the afuweta mutant clearly showed a five - fold reduction in hyphal branching . these results suggest that afuweta is associated with proper germ tube formation and vegetative growth . this is consistent with the previous finding that aniweta overexpression in hyphae causes excessive branching in ani . tao and yu   further dissected the genetic interactions among the central regulatory genes in afu . the absence of afubrla eliminates the expression of all conidiation - specific genes tested , indicating that afubrla functions upstream of afuabaa , afuweta , afuvosa , and afuroda . as afuabaa is necessary for activating afuweta , afuweta mrna accumulation is not detected in the afuabaa mutant . moreover , levels of afuvosa mrna decrease considerably in both afuabaa and afuweta strains , indicating the role of these genes in proper afuvosa expression . however , as afuvosa is activated before afuweta in the wild type , it is proposed that afuabaa primarily activates afuvosa with the assistance of afuweta . importantly , afubrla is highly upregulated in the afuabaa and afuweta mutants , particularly in conidia ( aberrant conidiophores in afuabaa ) and early ( 6 hr ) and late ( 24 hr ) phases of vegetative growth , suggesting that afuabaa and afuweta are necessary for proper negative regulation of afubrla upon completion of conidiation and during certain phases of vegetative growth . afuroda is a small , moderately secreted hydrophobic polypeptide that forms the outermost rodlet layer of conidia . conidia of afuroda mutants lack the external rodlet layer and are hydrophilic . as no afuroda mrna is detected in the afubrla mutant and afuroda levels are low in the afuabaa mutant , tao and yu   speculated that afuroda is primarily activated by afubrla with the assistance of afuabaa . a recent genetic study identified the novel regulator vosa , which functions in maturation of conidia and completion of ani development . importantly , vosa deletion results in the lack of trehalose in both types of spores , resulting in a rapid disappearance ( evaporation ) of the cytoplasm , disintegration of cellular organelles , and loss of long - term spore viability . moreover , the vosa mutant spores exhibit dramatically reduced tolerance to heat and oxidative stress . as vosa mainly localizes in the nucleus of mature conidia and contains a potential transcriptional activation domain at the c terminus , it may be a tf that primarily controls the late process of sporulation , including trehalose biogenesis . the vosa protein expressed in metulae and phialides and later localized to the nucleus of the conidia plays two chief roles : 1 ) activation of genes involved in spore maturation ; 2 ) negative feedback regulation of brla and developmental specific genes ( fig . 3 ) . vosa studies clearly divide conidiophore formation and conidia maturation into two distinct genetic phases . vosa has been identified with three other ani proteins sharing high levels of similarity ; vea , velb ( ef540815 ) , and velc ( ef540816 ) . these , together with vosa , define the velvet regulators , and they are highly conserved in many filamentous and dimorphic fungi , sharing at least one highly conserved domain [ 15 , 30 ] . importantly , recent studies have shown that vea , velb , and vosa bridge light - responding development and secondary metabolism by forming trimeric complexes with the nuclear master regulator of secondary metabolism , laea , in ani [ 30 , 31 ] . the afuvosa gene shows an mrna accumulation pattern almost identical to that of anivosa . to begin to understand the roles of the afu velvet proteins , we generated a fu mutants lacking afuvea , afuvelb , afuvosa , and afuvelc , and found that , while the deletion of afuvosa caused an approximate 50% reduction in the spore trehalose content and viability , it did not result in uncontrolled activation of conidiation in afu ( park and yu , unpublished ) . our recent unpublished data suggest that the feedback regulation of conidiation in afu is primarily exerted by velb and vea , whereas trehalose biogenesis is conferred by both afuvosa and afuvelb ( additive roles ) ( fig . identification and characterization of six upstream genes ( flug , flba , flbb , flbc , flbd , and flbe ) required for proper expression of brla in ani have illuminated genetic regulatory cascades for activating conidiation . among these , flbb , flbc , flbd , and flbe are defined by mutants exhibiting the fluffy delayed conidiation phenotypes . flbb , flbc , and flbd are putative tfs containing a basic leucine zipper ( b - zip ) , two c2h2 zinc fingers , and a cmyb - dna binding domain , respectively . thus , it has been thought that flbb , flbb , and flbd likely function as dna binding proteins that may control the transcriptional activation of other developmental regulators , such as brla , in response to sporulation signals . detailed functional studies of these potential tfs have demonstrated that flbb / c / d activate brla expression [ 33 - 38 ] . moreover , b - zip tf flbb is necessary for the activation of flbd expression , while flbb and flbd cooperatively activate brla . they found that flbc mrna is present throughout the lifecycle at relatively high levels during vegetative growth and during early asexual and late sexual developmental phases . deletion of flbc causes a delay / reduction in sporulation , brla and vosa expression , and conidial germination . while flbc overexpression does not cause conidiophore development , it inhibits hyphal growth and activates expression of brla , abaa , and vosa , but not weta . further reported that flbc contains two c2h2 zinc - fingers at the c - terminus and a putative activation domain at the n - terminus . they also found that flbc localizes to the nuclei of both hyphae and developmental cells . localization and expression of flbc is not affected by the absence of flbb or flbe and vice versa . importantly , as flbc overexpression inhibits growth and activates abaa and vosa in the absence of brla and abaa , respectively , kwon et al .   proposed that flbc plays a direct activating role in the expression of these genes . consistent with this idea , an in vitro dna binding assay revealed that flbc binds to the promoter regions of brla , abaa , and vosa , but not to that of weta . the aniflbe gene is predicted to encode a 201 aa - length polypeptide with two conserved yet uncharacterized domains , and it has been demonstrated that flbe and flbb are functionally interdependent , that they physically interact in vivo , and co - localize to the hyphal tip in ani . our recent studies   revealed that both deletion and overexpression of flbe in ani results in developmental defects , enhanced autolysis , precocious cell death , and delayed expression of brla / vosa , suggesting that balanced flbe activity is crucial for proper growth and development . the n - terminal portion of flbe exhibits a transactivation ability in budding yeast , whereas the c - terminal half negatively affects this activity . site - directed mutagenesis of certain conserved n - terminal amino acids abolishes transactivation ability , overexpression - induced autolysis , and complements the null mutation . these results suggest that the conserved n - terminal domain might be crucial for the functionality of flbe . finally , flbd overexpression , but not that of flbb or flbc , restores conidiation in ani dflbe , generally supporting the current genetic model of developmental regulation . in a series of recent studies , we characterized the functions of flbe and flbb in afu [ 39 , 40 ] . the predicted afu flbe protein is composed of 222 aa in length . while flbe is transiently expressed during the early growth phase in ani , deletion of afuflbe causes reduced conidiation and delayed expression of brla and vosa in both species . moreover , afuflbe is necessary for salt - induced development of afu in a liquid submerged culture . as the ani flbe null mutation can be fully complemented by afuflbe , the developmental function of flbe appears to be conserved in aspergilli . conducted a series of molecular analyses and found that afuflbb produces two transcripts predicted to encode two b - zip polypeptides , afuflbbb ( 420 aa ) and afuflbba ( 390 aa ) . afuflbb deletion results in delayed and reduced conidiation , precocious cell death , the absence of conidiophore development in liquid submerged culture , altered expression of afubrla and afuabaa , and the lack ( or a reduction ) in gliotoxin production . importantly , they found that while introducing the wild type afuflbb allele fully complemented these defects , disruption of the atg start codon to either one of the afuflbb polypeptides led to partial complementation , indicating that both polypeptides are needed for wild type level sporulation and gliotoxin biogenesis . consistent with these observations , introducing ani flbb encoding one polypeptide ( 426 aa ) into the afuflbb null mutant partially restores asexual and chemical development . .   also found that the presence of 0.6 m kcl in a liquid submerged culture suppresses the defects caused by the lack of one , but not both , of the afuflbb polypeptides , suggesting a genetic prerequisite for afuflbb in afu development . northern blot analyses revealed that both afuflbb and afuflbe are necessary for afuflbd expression , suggesting that flbd functions downstream of flbb and flbe in both aspergilli . further suggested that afubrla might be necessary for gliotoxin biosynthesis , which was based on the observations that the afubrla mutant lacks gliotoxin production , and that multiple bres are present in the promoter regions of many gliotoxin biosynthetic and regulatory genes . regarding the upstream region of flbb / c / d / e , mah and as discovered in ani , levels of the afuflug transcript are relatively constant throughout the lifecycle . the afuflug deletion mutant conidiated normally , similar to wild type on solid medium , indicating that activation of afu conidiation in the presence of air does not require afuflug . however , the afuflug deletion mutant did not produce conidiophores in liquid submerged culture , whereas afu wild type strains sporulated abundantly at approximately 24 hr . moreover , the afuflug deletion mutant showed reduced conidiation levels and delayed afubrla expression upon induction of synchronized asexual development . these results led mah and yu   to conclude that while the presence of air bypasses the need for afuflug in conidiophore development , afuflug plays a particular role in afu conidiation and afubrla expression . these findings led to the hypothesis that afu has multiple pathways to activate afubrla expression ( fig . in ani , a key and essential step for conidiophore development is the activation of brla encoding a c2h2 zinc finger transcription factor ( tf ) , which induces expression of other genes required for conidiation . loss - of - function brla mutants form structures that resemble conidiophore stalks ( thus named " bristle " ) , except that they grow indeterminately and fail to produce vesicles , metuale , phialides , and conidia , indicating that brla controls the initiation of conidiophore development ( fig . , brla overexpression in vegetative cells causes termination of polar growth coupled with the commencement of abnormal sporulation leading to the formation of viable spores from hyphae . no environmental signals , including nutrient limitations or various ( osmotic and oxidative ) stresses , can bypass the brla requirement for conidiation , indicating that activation of brla expression early in conidiophore development represents a key and essential control point for commencing conidiation . the anibrla ( but not afubrla ) gene is a compound gene consisting of two overlapping transcriptional units designated brlaa and brlab . important information on the complex regulation of the two overlapping brla transcription units is well described elsewhere . afubrla , the afu brla homologue , shows 68% identity and 77% similarity to the ani brla . mah and yu   tested whether brla - dependent conidiation is conserved in these two aspergilli using genetics and found that afubrla deletion completely eliminated asexual development in afu , resulting in elongated aerial hyphae and increased hyphal mass in the colonies . these studies clearly demonstrated that afubrla is essential for conidiophore development in afu , and that the role of the core downstream tf brla in conidiation is conserved in these aspergilli ( fig . brla is a putative tf ( activator ) with two c2h2 zinc finger motifs at the c - terminus . disruption of either one of the fingers by mutating the key cysteine to serine results in complete loss of brla activity . chang and timberlake   further demonstrated that brla expression in the budding yeast saccharomyces cerevisiae results in brla dependent activation of aspergillus genes . furthermore , they proposed the consensus brla binding sites ( brla response elements [ bres ] ; 5'-(c / a)(g / a)aggg(g / a)-3 ' ) . although direct binding of brla to bres remains to be verified in vitro , a number of developmental genes including abaa , weta , roda , and ya have multiple bres in their promoter regions . importantly , tao and yu   reported that multiple bres are present in the promoter regions of afu brla , abaa , weta , roda and the velvet regulators , implying a potentially conserved regulatory circuit in the two aspergilli . the abaa gene , activated by brla during the middle stages of conidiation when metulae and phialides are being formed , functions in the differentiation of phialides . abacus ( abaa ) mutants have nearly normal conidiophores bearing abacus - like structures with swellings at intervals instead of chains of conidia . morphological studies have revealed that the metulae of abaa mutants produce supernumerary tiers of cells with properties of metulae , not phialides , indicating that abaa is essential for the differentiation and functionality of phialides as conidiogenous cells . abaa expression is dependent on brla activity , and bres are found in the abaa promoter region , suggesting that brla directly activates abaa . the abaa gene encodes a developmental regulator , which begins to be expressed when phialides form at 15~18 hr after developmental induction ( fig . 3 ) , and loss of abaa function results in abnormal expression of a number of developmentally regulated genes . abaa overexpression in vegetative hyphae causes growth cessation and enhances cellular vacuolization but not spore formation in ani . abaa overexpression activates the expression of weta and brla , and it is thought that abaa induces the expression of brla at certain times of development . however , the genetic interaction between abaa and brla seems more complex , as brla mrna levels increase in abaa null mutants [ 15 , 20 ] , which might involve vosa ( fig . 3 ) . the abaa protein contains an atts / tea dna - binding motif [ 21 , 22 ] and a leucine zipper for potential dimerization . expression studies in both ani and s. cerevisiae have demonstrated that abaa binds to the cis - regulatory elements upstream of the ya gene . results of a gel mobility shift assay indicate that abaa binds to the consensus sequence 5'-cattcy-3 ' ( abaa response element [ are ] where y is pyrimidine ) . in fact , multiple ares are found in the promoter regions of developmentally regulated genes , including brla , weta , ya , roda , and abaa . importantly , park et al .   demonstrated that abaa binds to the chitin synthase gene promoter , chsc , suggesting that abaa also regulates chitin biosynthesis during conidiophore development by controlling the expression of certain chitin synthetases . wet - white ( weta ) mutants produce colorless conidia that completely autolyze within a few days , leading to the formation of liquid droplets ( wet ) on the tops of conidial heads . sewall et al .   have shown that the weta gene is required for the synthesis of crucial cell wall components late in the development of the inner c4 layer ( fig . the ani weta gene encodes two transcription units , and weta transcription is predicted to be complex and subjected to developmental control . abaa , but not brla , activates weta expression , as abaa overexpression activates weta in the absence of wild type brla . furthermore , weta is auto - activated , as it is not expressed in weta temperature - sensitive mutants ( fig . the ani weta gene is predicted to encode a 60 kda protein rich in serine ( 14% ) , threonine ( 7% ) , and proline ( 10% ) . while no known dna binding domains are present in weta , it has been proposed to function as a regulator of spore - specific gene expression . this is based on observations that weta alone is sufficient to activate many sporulation - specific genes , and that the weta mutants fails to accumulate many sporulation - specific mrnas . furthermore , weta overexpression in vegetative cells inhibits hyphal growth , resulting in excessive hyphal branching and the activation of spore - specific genes . however , weta overexpression does not result in brla or abaa activation or lead to precocious conidiation . taken together , weta is proposed to activate a set of genes required for spore formation and maturation , which may function to complete the final two conidial wall layers and/or direct their assembly ( fig . together , the brlaabaaweta cascade has been proposed to define a central regulatory pathway that controls temporal and spatial expression of conidiation - specific genes during conidiophore development and spore maturation ( fig . vosa is a multifunctional regulator that acts in concert with the central regulatory genes , couples trehalose biogenesis and conidia maturation , and exerts negative feedback regulation of brla , thereby completing conidiation in ani ( fig . afuabaa ( eal88194 , 60% identity , 74% similarity to aniabaa ) and afuweta ( eal89470 , 58% identity , 68% similarity ) were previously identified through a genome search . the afuabaa open reading frame ( orf ) is composed of 2,518 bp with three exons and two introns and is predicted to encode a 797 aa - length protein containing an atts / tea dna - binding domain . the afuweta orf comprises 1,701 bp with no introns and is predicted to encode a 566 aa - length protein with a conserved c - terminal domain . tao and yu   examined the levels of the afuabaa and afuweta transcripts throughout the lifecycle and found that afuabaa and afuweta are highly expressed during asexual development . afuabaa mrna starts to accumulate at 6 hr post - developmental induction , reaches its highest level at 12 hr , and disappears at 48 hr . afuweta mrna begins to accumulate at 12 hr post - developmental induction , reaches its highest level at 48 hr post induction , and is present in conidia . deletion of afuabaa results in the formation of aberrant conidiophores exhibiting reiterated cylinder - like terminal cells without conidia , indicating that , as in ani , afuabaa is essential for differentiation and functionalization of afu phialides as conidiogenous cells . importantly , tao and yu   found that the most distal cells of the afuabaa conidiophores are capable of apical hyphal growth , whereas the fractions of cylinder - like elongated phialides do not undergo vegetative growth . they also examined hyphal dry weights and found that the absence of afuabaa causes delayed hyphal mass loss compared to that in the wild type . when cell viability was examined by determining the percent alamar blue reduction , which represents living cell mitochondrial activity , the afuabaa mutant clearly exhibited prolonged cell viability compared to the wild type . tao and yu   then examined the effects of afuabaa overexpression and found that it caused accelerated hyphal fragmentation , disintegration , a dramatic reduction of mycelial mass , and precocious cell death . these results indicate that afuabaa functions in afu autolysis and cell death . the afuweta gene is subsequently activated by afuabaa in the middle to late phases of conidiation . furthermore , tem analyses of the afuweta mutant revealed that weta conidiophores exhibit an interconnected conidia phenotype , i.e. , incomplete conidial separation and maturation . as afuweta plays an essential role in completion of the conidial wall , tao and yu   hypothesized that the absence of afuweta may affect spore viability and/or integrity , and found that the afuweta mutant exhibits dramatically reduced viability starting from day 10 , whereas wild type conidia maintain high viability until day 20 . moreover , tem studies revealed that , even at day 2 , about 20% of the afuweta conidia appear to lack cytoplasm and exhibit sheared conidial walls . tao and yu   then examined the effects of afuweta on spore tolerance to various stressors and found that the afuweta conidia were much more sensitive to heat , and oxidative and osmotic stresses . as trehalose is essential for long - term spore viability and stress resistance [ 15 , 27 ] , tao and yu   determined that the afuweta conidia do not contain any trehalose , whereas wild type conidia harbor about 3.4 pg of trehalose per conidium . these results indicate that afuweta plays an essential role in trehalose biogenesis in conidia , which probably affects their viability and stress tolerance . this was the first report that weta is essential for trehalose biogenesis in fungal conidia . additionally , given that the afuweta gene is activated during the late stage of conidiation , and that its mrna accumulates preferentially in mature conidia , tao and yu   further determined whether the absence of afuweta had an effect on spore germination and early vegetative growth . they found that after a 8-hr incubation in liquid submerged culture , the afuweta mutant conidia formed only one unidirectional germ tube without branching , which resulted in loosened mycelial aggregates , whereas conidia of wild type and complemented strains produced bi- or multi - directional germ tubes that formed branches . moreover , after a 14-hr incubation , the afuweta mutant clearly showed a five - fold reduction in hyphal branching . these results suggest that afuweta is associated with proper germ tube formation and vegetative growth . this is consistent with the previous finding that aniweta overexpression in hyphae causes excessive branching in ani . tao and yu   further dissected the genetic interactions among the central regulatory genes in afu . the absence of afubrla eliminates the expression of all conidiation - specific genes tested , indicating that afubrla functions upstream of afuabaa , afuweta , afuvosa , and afuroda . as afuabaa is necessary for activating afuweta , afuweta mrna accumulation is not detected in the afuabaa mutant . moreover , levels of afuvosa mrna decrease considerably in both afuabaa and afuweta strains , indicating the role of these genes in proper afuvosa expression . however , as afuvosa is activated before afuweta in the wild type , it is proposed that afuabaa primarily activates afuvosa with the assistance of afuweta . importantly , afubrla is highly upregulated in the afuabaa and afuweta mutants , particularly in conidia ( aberrant conidiophores in afuabaa ) and early ( 6 hr ) and late ( 24 hr ) phases of vegetative growth , suggesting that afuabaa and afuweta are necessary for proper negative regulation of afubrla upon completion of conidiation and during certain phases of vegetative growth . afuroda is a small , moderately secreted hydrophobic polypeptide that forms the outermost rodlet layer of conidia . conidia of afuroda mutants lack the external rodlet layer and are hydrophilic . as no afuroda mrna is detected in the afubrla mutant and afuroda levels are low in the afuabaa mutant , tao and yu   speculated that afuroda is primarily activated by afubrla with the assistance of afuabaa . a recent genetic study identified the novel regulator vosa , which functions in maturation of conidia and completion of ani development . importantly , vosa deletion results in the lack of trehalose in both types of spores , resulting in a rapid disappearance ( evaporation ) of the cytoplasm , disintegration of cellular organelles , and loss of long - term spore viability . moreover , the vosa mutant spores exhibit dramatically reduced tolerance to heat and oxidative stress . as vosa mainly localizes in the nucleus of mature conidia and contains a potential transcriptional activation domain at the c terminus , it may be a tf that primarily controls the late process of sporulation , including trehalose biogenesis . the vosa protein expressed in metulae and phialides and later localized to the nucleus of the conidia plays two chief roles : 1 ) activation of genes involved in spore maturation ; 2 ) negative feedback regulation of brla and developmental specific genes ( fig . 3 ) . vosa studies clearly divide conidiophore formation and conidia maturation into two distinct genetic phases . vosa has been identified with three other ani proteins sharing high levels of similarity ; vea , velb ( ef540815 ) , and velc ( ef540816 ) . these , together with vosa , define the velvet regulators , and they are highly conserved in many filamentous and dimorphic fungi , sharing at least one highly conserved domain [ 15 , 30 ] . importantly , recent studies have shown that vea , velb , and vosa bridge light - responding development and secondary metabolism by forming trimeric complexes with the nuclear master regulator of secondary metabolism , laea , in ani [ 30 , 31 ] . the afuvosa gene shows an mrna accumulation pattern almost identical to that of anivosa . to begin to understand the roles of the afu velvet proteins , we generated a fu mutants lacking afuvea , afuvelb , afuvosa , and afuvelc , and found that , while the deletion of afuvosa caused an approximate 50% reduction in the spore trehalose content and viability , it did not result in uncontrolled activation of conidiation in afu ( park and yu , unpublished ) . our recent unpublished data suggest that the feedback regulation of conidiation in afu is primarily exerted by velb and vea , whereas trehalose biogenesis is conferred by both afuvosa and afuvelb ( additive roles ) ( fig . identification and characterization of six upstream genes ( flug , flba , flbb , flbc , flbd , and flbe ) required for proper expression of brla in ani have illuminated genetic regulatory cascades for activating conidiation . among these , flbb , flbc , flbd , and flbe are defined by mutants exhibiting the fluffy delayed conidiation phenotypes . flbb , flbc , and flbd are putative tfs containing a basic leucine zipper ( b - zip ) , two c2h2 zinc fingers , and a cmyb - dna binding domain , respectively . thus , it has been thought that flbb , flbb , and flbd likely function as dna binding proteins that may control the transcriptional activation of other developmental regulators , such as brla , in response to sporulation signals . detailed functional studies of these potential tfs have demonstrated that flbb / c / d activate brla expression [ 33 - 38 ] . moreover , b - zip tf flbb is necessary for the activation of flbd expression , while flbb and flbd cooperatively activate brla . they found that flbc mrna is present throughout the lifecycle at relatively high levels during vegetative growth and during early asexual and late sexual developmental phases . deletion of flbc causes a delay / reduction in sporulation , brla and vosa expression , and conidial germination . while flbc overexpression does not cause conidiophore development , it inhibits hyphal growth and activates expression of brla , abaa , and vosa , but not weta . further reported that flbc contains two c2h2 zinc - fingers at the c - terminus and a putative activation domain at the n - terminus . they also found that flbc localizes to the nuclei of both hyphae and developmental cells . localization and expression of flbc is not affected by the absence of flbb or flbe and vice versa . importantly , as flbc overexpression inhibits growth and activates abaa and vosa in the absence of brla and abaa , respectively , kwon et al .   proposed that flbc plays a direct activating role in the expression of these genes . consistent with this idea , an in vitro dna binding assay revealed that flbc binds to the promoter regions of brla , abaa , and vosa , but not to that of weta . the aniflbe gene is predicted to encode a 201 aa - length polypeptide with two conserved yet uncharacterized domains , and it has been demonstrated that flbe and flbb are functionally interdependent , that they physically interact in vivo , and co - localize to the hyphal tip in ani . our recent studies   revealed that both deletion and overexpression of flbe in ani results in developmental defects , enhanced autolysis , precocious cell death , and delayed expression of brla / vosa , suggesting that balanced flbe activity is crucial for proper growth and development . the n - terminal portion of flbe exhibits a transactivation ability in budding yeast , whereas the c - terminal half negatively affects this activity . site - directed mutagenesis of certain conserved n - terminal amino acids abolishes transactivation ability , overexpression - induced autolysis , and complements the null mutation . these results suggest that the conserved n - terminal domain might be crucial for the functionality of flbe . finally , flbd overexpression , but not that of flbb or flbc , restores conidiation in ani dflbe , generally supporting the current genetic model of developmental regulation . in a series of recent studies , we characterized the functions of flbe and flbb in afu [ 39 , 40 ] . the predicted afu flbe protein is composed of 222 aa in length . while flbe is transiently expressed during the early growth phase in ani , deletion of afuflbe causes reduced conidiation and delayed expression of brla and vosa in both species . moreover , afuflbe is necessary for salt - induced development of afu in a liquid submerged culture . as the ani flbe null mutation can be fully complemented by afuflbe , the developmental function of flbe appears to be conserved in aspergilli . xiao et al .   conducted a series of molecular analyses and found that afuflbb produces two transcripts predicted to encode two b - zip polypeptides , afuflbbb ( 420 aa ) and afuflbba ( 390 aa ) . afuflbb deletion results in delayed and reduced conidiation , precocious cell death , the absence of conidiophore development in liquid submerged culture , altered expression of afubrla and afuabaa , and the lack ( or a reduction ) in gliotoxin production . importantly , they found that while introducing the wild type afuflbb allele fully complemented these defects , disruption of the atg start codon to either one of the afuflbb polypeptides led to partial complementation , indicating that both polypeptides are needed for wild type level sporulation and gliotoxin biogenesis . consistent with these observations , introducing ani flbb encoding one polypeptide ( 426 aa ) into the afuflbb null mutant partially restores asexual and chemical development . .   also found that the presence of 0.6 m kcl in a liquid submerged culture suppresses the defects caused by the lack of one , but not both , of the afuflbb polypeptides , suggesting a genetic prerequisite for afuflbb in afu development . northern blot analyses revealed that both afuflbb and afuflbe are necessary for afuflbd expression , suggesting that flbd functions downstream of flbb and flbe in both aspergilli . xiao et al .   further suggested that afubrla might be necessary for gliotoxin biosynthesis , which was based on the observations that the afubrla mutant lacks gliotoxin production , and that multiple bres are present in the promoter regions of many gliotoxin biosynthetic and regulatory genes . regarding the upstream region of flbb / c / d / e , mah and as discovered in ani , levels of the afuflug transcript are relatively constant throughout the lifecycle . the afuflug deletion mutant conidiated normally , similar to wild type on solid medium , indicating that activation of afu conidiation in the presence of air does not require afuflug . however , the afuflug deletion mutant did not produce conidiophores in liquid submerged culture , whereas afu wild type strains sporulated abundantly at approximately 24 hr . moreover , the afuflug deletion mutant showed reduced conidiation levels and delayed afubrla expression upon induction of synchronized asexual development . these results led mah and yu   to conclude that while the presence of air bypasses the need for afuflug in conidiophore development , afuflug plays a particular role in afu conidiation and afubrla expression . these findings led to the hypothesis that afu has multiple pathways to activate afubrla expression ( fig . our further studies suggest that afuflbd is essential for proper asexual development in afu ( xiao and yu , unpublished data ) . to examine potential genetic interactions among upstream developmental regulators in afu , xiao et al . conducted a series of expression studies and proposed that afu has an upstream regulatory cascade slightly different from the one proposed for ani ( fig . afuflbe expression is independent , and both are required for proper afuflbd expression , xiao et al . proposed that afuflbb and afuflbe function upstream of afuflbd and cooperatively activate afuflbd , which subsequently activates afubrla . while the transcriptional activation of afuflbb and afuflbe is independent , it may be that the afuflbb and afuflbe proteins interact and form a functional complex as in ani . furthermore , as afuflbc expression is independent of afuflbb and afuflbe , afuflbc is predicted to function in a separate pathway . because the afubrla mutant is defective in gliotoxin production and multiple bres are present in the promoter regions of many gliotoxin biosynthetic and regulatory genes , xiao et al . further functional and molecular studies of afuflbc and the identification of additional developmental regulators , including afusfga and afuwetb , are in progress in my laboratory . in this review , i have briefly summarized our current understanding of the upstream and downstream regulation of aspergillus conidiation focusing on key tfs . in summary , these two aspergilli have common essential downstream activators of conidiophore development ; brla , abaa , and weta . moreover , velvet regulator functions are generally conserved in these aspergilli . however , as clear differences in the upstream regulation of conidiation between the two species exist , much remains to be learned . further genetic and genomics studies aimed at identifying and characterizing the unique regulators and control networks in afu will illuminate the complex mechanisms of development in this opportunistic pathogenic fungus .