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 .
 members of the genus aspergillus are the most common fungi and all reproduce asexually by forming long chains of conidiospores ( or conidia ) . 
 the impact of various aspergillus species on humans ranges from beneficial to harmful . 
 for example , several species including aspergillus oryzae and aspergillus niger are used in industry for enzyme production and food processing . 
 in contrast , aspergillus flavus produce the most potent naturally present carcinogen aflatoxins , which contaminate various plant- and animal - based foods . 
 importantly , the opportunistic human pathogen aspergillus fumigatus has become the most prevalent airborne fungal pathogen in developed countries , causing invasive aspergillosis in immunocompromised patients with a high mortality rate . 
 a. fumigatus produces a massive number of small hydrophobic conidia as the primary means of dispersal , survival , genome - protection , and infecting hosts . 
 large - scale genome - wide expression studies can now be conducted due to completion of a. fumigatus genome sequencing . 
 however , genomics becomes more powerful and informative when combined with genetics . 
 we have been investigating the mechanisms underlying the regulation of asexual development ( conidiation ) and gliotoxin biosynthesis in a. fumigatus , primarily focusing on a characterization of key developmental regulators identified in the model fungus aspergillus nidulans . in this review 
 , i will summarize our current understanding of how conidiation in two aspergilli is regulated . 
Conserved Roles of BrlA-AbaA-WetA in 
BrlA
AbaA and WetA
The BrlA-AbaA-WetA cascade in 
VosA and the 
Upstream regulators of asexual development
Model and Conclusions