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Annu. Rev. Phytopathol. 2005. 43:43758doi: 10.1146/annurev.phyto.43.040204.140214
Copyright c 2005 by Annual Reviews. All rights reserved
REGULATION OF SECONDARY METABOLISMIN FILAMENTOUS FUNGI
Jae-Hyuk Yu1 and Nancy Keller1,21Department of Food Microbiology and Toxicology, 2Department of Plant Pathology,
University of Wisconsin, Madison, Wisconsin 53706;
email: [email protected], [email protected]
Key Words fungi, mycotoxins, transcriptional control, G proteins, RGS proteins
Abstract Fungal secondary metabolites are of intense interest to humankind dueto their pharmaceutical (antibiotics) and/or toxic (mycotoxins) properties. In the pastdecade, tremendous progress has been made in understanding the genes that are associ-ated with production of various fungal secondary metabolites. Moreover, the regulatorymechanisms controlling biosynthesis of diverse groups of secondary metabolites havebeen unveiled. In this review, we present thecurrent understanding of thegenetic regula-tion of secondary metabolism from clustering of biosynthetic genes to global regulators
balancing growth, sporulation, and secondary metabolite production in selected fungiwith emphasis on regulation of metabolites of agricultural concern. Particularly, theroles of G protein signaling components and developmental regulators in the mycotoxinsterigmatocystin biosynthesis in the model fungus Aspergillus nidulans are discussedin depth.
INTRODUCTION
Secondary metabolite production in fungi is a complex process coupled withmorphological development (reviewed in 27). Secondary metabolites often have
obscure or unknown functions in the producing organism but have tremendous
importance to humankind in that they display a broad range of useful antibiotic
and pharmaceutical activities as well as less desirable immunosuppressant and
toxic activities.
In most cases, the function of secondary metabolites for the producing fungus is
unknown but is inferred from a few studies using mutants or enzyme inhibitors. The
most obvious fungal natural products are the pigmentstypically brown and black
pigments referred to as melaninsgiving color to spores, appressoria, sclerotia,sexual bodies, and other developmental structures. Studies of pigment function
in these structures have shown that they act as plant (58, 71) and animal (114)
virulence factors or that they are required for general survival, presumably as UV
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438 YU KELLER
protectants (72, 77), antigrowth deterrents (98), or ROS scavengers (37). Fungal
phytotoxins are proven pathogenicity or virulence factors that cause significant
disease on agricultural crops (5, 54, 127). Another notorious group of agriculturally
important secondary metabolites are the mycotoxins, which are excreted by fungias they grow in various commodities (8). The primary aim of this review is to
present our current understanding of how secondary metabolites are regulated in
fungi, with emphasis on regulation of metabolites of agricultural concern.
BIOSYNTHETIC GENE CLUSTERS
The inherent properties of secondary metabolites, both desirable and destructive,
spurred efforts toward identifying genes involved in their synthesis. Accumulatingdata from studies of known secondary metabolite biosynthetic genes dispelled an
original premise that fungal metabolic genes would be scattered throughout the
genome. Rather, the hallmark of secondary metabolite genesin contrast to genes
involved in primary metabolismis that they are clustered in fungal genomes
(reviewed in 68, 135). As described below, the contiguous clustering of metabolic
genes specific to one product has considerable bearing on the regulation of these
genes.
Aflatoxin and SterigmatocystinWith the possible exception of the penicillin metabolic cluster, the most thor-
oughly examined fungal secondary metabolite gene clusters are those involved
in mycotoxin biosynthesis, particularly the aflatoxin (AF) and sterigmatocystin
(ST) biosynthetic clusters found in several Aspergillus spp. (Figure 1). Both car-
cinogenic metabolites are products of the same lengthy pathway where ST is
the penultimate precursor of AF (16, 129). The AF cluster in A. parasiticus and
A. flavus contains genes that constitute a cluster spanning more than 70 kb. Among
these genes, 21 have been verified or predicted to encode biosynthetic enzymes, in-
cluding fatty acid synthases, a polyketide synthase, monooxygenases, reductases,dehydrogenases, methyltransferases, an esterase, a desaturase, and an oxidase (85,
129). One gene in the cluster, aflR, encodes a binuclear zinc cluster (Zn(II)2Cys6)
Figure 1 Order and direction of transcription of genes in the sterigmatocystin (ST)
and aflatoxin (AF) gene clusters. Orthologous genes in the two clusters are indicated
by the same bar pattern. Solid black bars represent ST and AF genes that have not been
characterized.
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REGULATION OF MYCOTOXIN PRODUCTION 439
transcription factor regulating transcription of the aflatoxin biosynthetic genes (33,
126). Another cluster gene, aflJ, also seems to have a role in regulating aflatoxin
production in A. flavus (83). In A. nidulans the 60-kb sterigmatocystin cluster con-
sists of circa 25 genes also regulated by aflR (16, 44, 130). The functions of mostof the sterigmatocystin cluster genes have been determined and are orthologs of
aflatoxin cluster genes (55).
Trichothecenes
Trichothecenes comprise a large family of sesquiterpenoid metabolites produced
by a number of fungal genera, including Fusarium, Myrothecium, Stachybotrys,
Cephalosporium, Trichoderma, and Trichothecium (62, 104, 120). These com-
pounds not only exhibit toxicity to vertebrates and plants, but also are associated
with virulence in specific plant-pathogen interactions (38, 53, 95).
Biochemical and genetic analyses of the T-2 toxin producer F. sporotrichioides
ledto the identification of the first trichothecene biosynthetic gene cluster. The gene
cluster for deoxynivalenol production has also been identified in F. graminearum.
The two clusters contain 10 to 12 ORFs and span circa 29 kb (17, 18, 57). The
functions of ten genes have been determined. Seven of them encode biosynthetic
enzymes (6, 18, 19, 81). Tri6 and Tri10 are regulatory proteins and Tri12 is the
efflux pump that is implicated to play a self-protection role (7, 110). Recently, a
second mini-cluster has been found in F. graminearum that contains two more
enzymatic genes required for deoxynivalenol production (17). A trichothecene
cluster has also been described in Myrothecium roridum (113).
Fumonisins
Fumonisins are a group of polyketide mycotoxins that are produced primarily
by the economically important maize and sorghum pathogens Fusarium verticil-
lioides and Fusarium proliferatum (87). Fumonisin B1, the most toxic fumonisin,
promotes cancer and causes equine leukoencephalomalacia. The genes involved
in fumonisin biosynthesis are clustered in a 45-kb stretch of DNA. Expressionanalysis of F. verticillioides indicated that 15 genes (ORF1 and ORF6-19) are
coregulated and exhibited patterns of expression that were correlated with fumon-
isin production. These ORFs are designated as FUM genes (92). FUM5 encodes
the polyketide synthase gene that was shown to be required for fumonisin biosyn-
thesis (93). Disruption of FUM6 and FUM8 blocked production but did not lead
to accumulation of detectable intermediates (103); FUM9 and FUM13 both are
involved in side chain decoration of the carbon backbone (22, 23). Most recently,
a transcription factor, ZFR1, important in fumonisin regulation has been identified
(45). However, in contrast to the regulatory genes of the AF, ST, and trichothecenepathways, ZFR1 is not located in the fumonisin cluster.
Other identified gene clusters include those involved in production of other
mycotoxins and phytotoxins (HC toxin, 4; dothiostromin, 13; sirodesmin, 47;
gibberellin, 118; ergot alkaloids, 119; paxillin, 128; aflatrem, 135), antibiotics
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440 YU KELLER
(cephalosporin, 48; penicillin, 79), melanins (71, 115), and pharmaceuticals (com-
pactin, 2; lovastatin, 69).
TRANSCRIPTIONAL REGULATION
The coregulation of the cluster genes can be in part explained by transcriptional
control of structural genes by two classes of transcription factors, one class briefly
mentioned in the previous section that are specific to a particular metabolic path-
way (i.e., aflR) and a second class that mediate environmental signals including
pH, carbon, and nitrogen sources. This multilevel regulation by both specific and
broad-domain transcription factors ensures that secondary metabolite pathways
can respond to the demands of general cellular metabolism and the presence ofspecific pathway inducers.
Pathway-Specific Transcription Factors
Many, but not all, clusters contain genes encoding transcription factors that pos-
itively regulate gene expression. Perhaps the archetypal protein in this group is
AflR, the Zn(II)2Cys6 domain protein required for AF and ST biosynthetic gene
activation (33, 44, 126). Typical for this group of DNA binding proteins, AflR
recognizes and binds to a palindromic sequence, 5-TCG(N5)GCA, found in the
promoters of the AF/ST biosynthetic genes (41, 44, 90). A second binding site,5-TTAGGCCTAA, has also been reported for A. flavus and A. parasiticus and
is considered important in autoregulation of aflR transcript in these spp. (34, 35,
40, 90). Disruption ofaflR eliminates the expression of structural genes (130) and
modifications of its promoter region alter not only its own but subsequent clus-
ter gene expression (40). How AflR is negatively regulated by protein kinase A
signaling is described below.
Othercluster transcription factors include additionalZn(II)2Cys6 proteins (MlcR
for compactin biosynthesis, 1), Cys2His2 zinc finger proteins (Tri6 and MRTRI6
for trichothecene production, 94), an ankyrin repeat protein (ToxE for HC-toxinproduction, 91), a two-peptide forkhead complex (AcFKH1 and CPCR1 for cepha-
losporin production, 99, 100), and a HAP-like transcriptional complex (PENR1
for penicillin, 78). Additionally, PENR1 has also been shown to be important in
taka-amylase, xylanase, and cellobiohydrolase production (14).
Pathway-specific regulatory genes not obviously encoding transcription factors
include aflJrequired for AF/ST biosynthesis and Tri10 involved in trichothecene
biosynthesis (83, 110). Inactivation or mutation ofaflJgives a phenotype similar to
an aflR deletion, i.e., a great reduction in AF or ST production (83; R.A. Butchko &
N.P Keller, unpublished data). Although AflJ has not been studied in A. nidulans,several studies have partially defined its function inA. parasiticus (32, 83). Despite
lack of AF production in aflJ deletion strains, structural genes are still expressed
at a reduced level. This complex phenotype suggests that AflJ is not directly
responsible for AF/ST gene transcription or for any particular enzymatic step in
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REGULATION OF MYCOTOXIN PRODUCTION 441
the pathway, but is in some way enhancing transcription. Chang (32) demonstrated
an interaction of AflJ with AflR using a yeast two-hybrid system. Two regions of
AflR are required for this interaction, one located between aa 230238, the other
at the C terminus. However, it appears that the full-length AflJ protein is likelyrequired for activity as deletion of the first 9 amino acids reduces activity of the
protein by 85%90%, anddeletion of the final 11 amino acids eliminates its activity.
Global Regulatory Factors
CreA, AreA, PacC Secondary metabolite biosynthesis is responsive to environmen-
tal cues including carbon and nitrogen source, ambient temperature, light, and
pH. Several studies (42, 84) indicate that these environmental signals are mediated
through Cys2His2 zinc finger global transcription factors conveying carbon (CreA,
39), nitrogen (AreA, 59), and pH (PacC, 79, 112) signaling. Gene expression inseveral gene clusters including AF, ST, penicillin, and gibberellin clusters is reg-
ulated, either positively or negatively, by these zinc finger proteins. For example,
disruption of the positive-acting nitrogen regulatory areA-GF gene in Gibberella
fujikuroi led to a 10%20% reduction of gibberellin production in gibberellin
induction medium. In addition, the loss-of-function areA-GFstrains were insensi-
tive to ammonium-mediated gibberellin repression, supporting the conclusion that
gibberellin biosynthesis is under the control of AreA-GF (84).
The involvement of CreA in secondary metabolism may reflect the differences
seen in metabolite production when fungi are grown in different carbon sources(43). Another related factor in metabolite production may be the availability of
precursor units. For example, each secondary metabolite ultimately depends on
the available pools of a limited number of primary precursors for peak produc-
tion. Many secondary metabolites are classified as polyketides (ST, AF, fumonisin,
lovastatin, compactins, and melanins). This chemical class is derived from reit-
erative condensations of acetyl-CoA and malonyl-CoA moieties, malonyl-CoA
itself being derived from acetyl-CoA. Availability of acetyl-CoA might be ex-
pected to impact polyketide formation. Such a prediction has been recently borne
out in genetic and biochemical studies of A. nidulans where mutants in path-ways affecting either acetyl-CoA concentration (-oxidation mutants; 78a) or
availability via altered acyl-CoA ratios (methyl citrate mutants; 133, 134) can
reduce or even eliminate polyketide production despite an otherwise wild-type
phenotype.
LaeA A novel mechanism of gene cluster regulation was uncovered by com-
plementation of an A. nidulans ST mutant that was unable to express aflR. The
complementing gene, termed laeA for loss ofaflR expression, encodes a nuclear
protein with closest identity to arginine and histone methyltransferases (11). Lossof LaeA function silences not only ST cluster expression but also a multitude of
other metabolites including penicillin and numerous mycelial pigments inA. nidu-
lans and gliotoxin in A. fumigatus, whereas overexpression of laeA upregulates
cluster gene expression. Furthermore, microarray examination of the A. nidulans
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laeA deletion and laeA overexpression strains clearly shows that LaeA transcrip-
tionally regulates multiple novel secondary metabolite clusters, several of which
are currently being examined in our lab (J.W. Bok, L. Maggio-Hall, D. Hoffmeister
& N.P Keller, unpublished data). The findings that LaeA regulates multiple clus-ters may support a coregulation model for clustering (135), possibly via chromatin
remodeling of cluster loci. Putative LaeA orthologs are found in all filamentous
and dimorphic fungi examined to date, and it will be interesting to see if these
LaeA homologs play a role in secondary metabolism in other genera.
UPSTREAM SIGNALING MECHANISMS
All cells have the capacity to sense and respond to various external signals, such
as nutrients, hormones, as well as physical and chemical stimuli including envi-ronmental stress. Among various signaling elements, heterotrimeric G proteins
(G proteins) are conserved in all eukaryotes and play a central role in relaying
external cues into the cells to elicit appropriate physiological and biochemical
responses (reviewed in 86). In the past decade, G proteinmediated signaling has
been intensively studied in various filamentous fungal species, and outcomes of the
studies provided an important clue to understand the upstream regulation of fungal
secondary metabolite biosynthesis, which was hypothesized to be intimately asso-
ciated with sporulation (reviewed in 27). Progress has been made with the model
fungus A. nidulans, and this section primarily discusses signaling mechanismsgoverning development and ST production in this fungus with a few additional
examples.
The basic unit of heterotrimeric G protein is comprised of a seven-transmem-
brane-spanning domain G proteincoupled receptor (GPCR), a G protein consist-
ing of, , and subunits, and an intracellular effector that produces a second
messenger. G protein signaling is activated when ligand-bound GPCRs catalyze
GDP/GTP exchange of the G subunit, which provokes subsequent dissociation
of G-GTP and G. It is turned off when the intrinsic GTPase activity of the
G subunit hydrolyzes GTP to GDP, causing the formation of the inactive het-erotrimer GGDP:G. Dissociated (activated) G-GTP and/or Gcan trigger
the production or release of a large variety of second messengers including cAMP,
inositol 1,4,5-trisphosphate (IP3), diacylglycerol, cGMP, Ca2+, and nitric oxide.
These second messengers in turn initiate amplified cellular responses (reviewed in
86). In fungi, GPCR-G proteininitiated signaling is primarily transmitted to two
downstream signaling branches defined by adenylyl cyclase cAMP protein
kinase (PKA) and/or mitogen-activated protein kinase (MAPKKKMPAKK
MAPK) cascades, which eventually elicit cellular responses such as growth, mat-
ing, cell division, cell-cell fusion, morphogenesis, toxicogenesis, chemotaxis, andpathogenic development (12, 76, 122).
An important aspect of achieving pertinent cellular response to a (or multiple)
signal is to properly control the intensity of G protein signaling. Among various
controlling elements, regulators of G protein signaling (RGS proteins) play a key
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REGULATION OF MYCOTOXIN PRODUCTION 443
role in tightly controlling G protein signaling upstream or at the same level of
G proteins. RGS proteins contain a conserved 130 amino acid core domain
(RGS box) that functions in enhancing the intrinsic GTPase activity of the G
subunit, which results in increased GTP to GDP hydrolysis (inactivation) ratesof G subunits (Figure 2). Through activities of multiple RGS proteins, cells
can coordinate diverse incoming signals and fine-tune their cellular responses
(reviewed in 36). G proteins and RGS proteins function in global coordination of
fundamental biological processesin filamentous fungi including vegetative growth,
sporulation, mycotoxin/pigment production, pathogenicity, and mating.
AspergillusSpecies
ST/AF PRODUCTION REGULATION BY FadA (G) AND FlbA (RGS PROTEIN) G pro-
tein and its proper regulation play a central role coordinating hyphal growth,asexual/sexual development, and secondary metabolite production in A. nidulans.
Hyphal growth signaling is mediated by both the (FadA) and (SfaD) subunits of
a heterotrimeric G protein (96, 132). When FadA (G) is in its active-GTP bound-
state, it is dissociated with its cognate Gdimer (SfaD:GpgA) and the free FadA-
GTP and SfaD:GpgA can both activate downstream effectors for proliferation
(Figure 2), which inhibits both sexual and asexual development (96, 132; J.-A. Seo
& J.-H. Yu, unpublished data). In addition, activated FadA signaling also blocks
production of the mycotoxin sterigmatocystin (ST) and mRNA expression ofaflR,
indicating that FadA-GTP signaling negatively controls ST biosynthesis (56, 131,132) (Figure 3). For asexual/sexual development as well as ST biosynthesis to oc-
cur, FadA-GTP/SfaD:GpgA signaling needs to be at least partially inactivated. In
this attenuation of growth signaling, FlbA (an RGS protein) plays a key role and is
presumed to rapidly convert FadA-GTP to FadA-GDP by increasing the intrinsic
GTPase activity of FadA (Figure 3). Loss of flbA function results in the fluffy-
autolytic colony (Figure 2) that lacks sexual/asexual sporulation and ST production
due to the absence of proper down-regulation of FadA signaling (56, 73, 132).
While the primary role of FlbA in development and ST production is inactivat-
ing FadA, FlbA is found to have additional roles in conidiation and ST biosynthesis(56, 132). This was further supported by the observation that whereas overexpres-
sion of the conidiation activator brlA rescued conidiation in loss-of-function flbA
mutants (J. Hicks & N.P. Keller, unpublished data), forced expression of aflR or
site-directed mutagenized hyperactive AflR could not restore ST production in the
absence of flbA function (106). These results suggest that FlbA is necessary for
activity of the AflR protein via unknown mechanisms. While loss of SfaD (G)
or GpgA (G) function has clear effects on vegetative growth, conidiation, and
sexual development, their roles in ST production remain to be examined (96; J.-A.
Seo & J.-H. Yu, unpublished data).Importantly, G proteins are found to be functionally conserved in Aspergillus
species, in that the FadA-homologous pathway negatively controls AF biosynthesis
in both A. parasiticus and A. flavus. A study by Hicks et al. (56) showed that intro-
duction of the A. nidulans constitutively active FadA allele into an A. parasiticus
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Figure 3 Antagonistic pathways coordinate vegetative growth, conidiation, and pro-
duction of secondary metabolites in A. nidulans. Vegetative growth signaling by
(FadA) and (SfaD with a presumed G, GpgA) subunits of a heterotrimeric G pro-tein inhibits asexual/sexual sporulation and ST biosynthesis, but stimulates penicillin
production (56, 96, 111, 132). Activation of asexual development requires at least
partial inhibition of FadA-mediated signaling, which requires two genes, fluG and
flbA (7375). FluG is proposed to activate conidiation via removing repressive effects
imposed by multiple negative regulators (101). FlbA is an RGS protein that inhibits
FadA-GTP/SfaD::GpgA-mediated vegetative growth signaling (96, 132; J.-A. Seo &
J.-H. Yu, unpublished data).
strain inhibited production of AF (and/or intermediate products) as well as coni-diation in a dominant manner. Moreover, the similar results were observed in
A. flavus (82).
ProteinkinaseAandAflR FadA growth signaling is transduced in part via protein
kinase A (PKA; 107). Deletion of the pkaA gene encoding the primary PKA
catalytic subunit inA. nidulans resulted in elevated conidiationand highlyrestricted
vegetative growth. Analyses of epistatic interactions between fadA-flbA and pkaA
revealed that PkaA functions downstream of FlbA/FadA, i.e., deletion of pkaA
suppressed both developmental and ST biosynthesis defects caused by the absenceofflbA function. Conversely, overexpression ofpkaA caused reduced conidiation,
increased vegetative growth as well as inhibition ofaflR expression necessary for
ST biosynthesis (107). This study clarified that FadA-mediated signaling is (at
least in part) transmitted to a cAMP PKA signaling cascade and PkaA plays
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REGULATION OF MYCOTOXIN PRODUCTION 445
a major role in activation of vegetative growth and repression of both conidiation
and ST production (Figure 3).
The role of PkaA in negatively controlling ST production was further sup-
ported by a recent study by Shimizu et al. (106), where it was demonstrated thatAflR is phosphorylated by PkaA in vitro. Furthermore, probable posttranscrip-
tional negative regulation of AflR activity by PkaA-dependent phosphorylation
in vivo was also shown by substitution of the putative phosphorylation target
amino acid Ser to Ala in AflR. Such site-directed mutations abolished inhibitory
effects of overexpression ofpkaA on AflR activity. Moreover, the authors showed
that the requirement of FlbA for AflR-mediated ST production is PkaA indepen-
dent. This is consistent with the previous hypothesis that, in addition to inhibiting
FadA signaling, FlbA has an additional role in activating conidiation as well as
ST production (56, 132). Later, Bok & Keller (11) showed that PkaA negativelycontrols LaeA, which is required for the expression of clustered genes for ST,
penicillin, and lovastatin biosynthesis, respectively (see section on LaeA).
Recently, the roles of FadA, cAMP and PKA in regulation of AF biosynthesis
and conidiation in A. parasiticus were examined by Roze et al. (97). Interestingly,
while introduction of cAMP or dibutyryl-cAMP (DcAMP) onto solid medium
resulted in a 100-fold increase in intracellular cAMP/DcAMP, total cellular PKA
activity was lowered by 40- to 80-fold in the tested strain. These findings explained
why cAMP or DcAMP stimulated AF synthesis and conidiation in A. parasiticus
despite the functionally conserved FadA-mediated signaling mechanisms. Theauthors concluded: (a) the FadA/PKA signaling cascade negatively regulates AF
biosynthesis and conidiation via similar mechanisms in Aspergillus species; and
(b) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory
influence on conidiation and AF synthesis.
GanBandRgsA,thesecondG-RGSpair As in most filamentous fungal genomes
(12), the A. nidulans genome contains three G subunits, FadA, GanA, and GanB.
Recent studies identified three additional RGS proteins (RgsA, RgsB, and RgsC)
in A. nidulans (52). Han et al. (52) revealed that RgsA down-regulates pigmentproduction and conidial germination, but stimulates conidiation (and ST produc-
tion) via inhibiting GanB signaling. This study showed that deletion ofrgsA caused
reduced colony size with increased aerial hyphae, elevated accumulation of brown
(mycelial) pigments, but reduced ST production (Figure 4). The fact that dele-
tion of both flbA and rgsA resulted in an additive phenotype led the authors to
speculate that the G protein pathways controlled by FlbA and RgsA are differ-
ent. Morphological alterations, increased pigment but reduced ST production, as
well as restricted colony growth caused by deletion of rgsA were suppressed by
deletion of ganB, indicating that the primary role of RgsA is to negatively con-trol GanB-mediated signaling (Figure 4). The observations that overexpression
ofrgsA as well as deletion or dominant interfering mutations of ganB caused in-
appropriate hyperactive conidiation in liquid-submerged culture further support
that RgsA and GanB function in opposite manner and GanB-mediated signaling
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represses conidiation (and ST production; 31, 52). This second RGS-G pair in
A. nidulans may govern upstream regulation of fungal cellular responses to envi-
ronmental changes such as carbon sources and stresses. While the precise mech-
anism of GanB-mediated reduction of ST production remains to be determined,it was speculated that elevated accumulation of brown pigment(s) might partially
contribute to this phenotype (52). Taken together, a genetic model incorporating
the activities of two G protein signaling pathways and the cognate RGS proteins
in governing growth, development and ST production is presented (Figure 5).
Conidiation-specific functions and ST production A close relationship between
fungal development and secondary metabolite production has been observed in
Aspergillus species. Bennett and colleagues (9, 10) first observed that A. para-
siticus morphological mutants also lost the ability to produce wild-type levels of
Figure 5 G protein-RGS mediated regulation of development and ST production in
A. nidulans (adapted from Reference 52). Two independent G-RGS signaling path-
ways coordinately control cellular responses to various signals. FlbA-FadA primarily
governs vegetative growth versus development and ST production, and RgsA-GanB
controls stress response (pigmentation), carbon sensing, and germination. Conidiationoccurs through activation of brlA, which requires multiple upstream genes including
fluG (74). GanB and presumed SfaD:GpgA-mediated signaling is proposed to repress
asexual sporulation (31, 96). Possible direct activation of conidiation (and ST produc-
tion) by FlbA and RgsA is presented as dotted arrows.
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REGULATION OF MYCOTOXIN PRODUCTION 447
AF. Later, these sporulation defective mutants were found to fail to accumulate
mRNA ofaflR and genes encoding enzymes for AF biosynthesis (64, 65). Genetic
mechanisms interconnecting conidiation and AF/ST production were uncovered
by a series of studies that identified and characterized genes (fluG, flbE, flbD,flbB, flbC, and brlA) required for conidiation in A. nidulans (reviewed in 3, 7375,
124). BrlA is a key transcription factor that activates conidiophore (asexual spore-
bearing structure) formation, andfluG, flbE, flbD, flbB, andflbCare required for the
expression of brlA. Mutations in these upstream regulatory genes resulted in the
absence or delay of conidiation and overproliferation of hyphae, termed fluffy
phenotypes. Accordingly, a genetic model proposed that conidiation occurs via
activities of multiple positive regulators (reviewed in 3). Among these, the fluG
gene that functions at the most upstream of this genetic cascade was also found to
be necessary for ST production.Hicks et al. (56) demonstrated that loss of fluG function resulted in lack of ST
production. Importantly, the role of FluG in ST biosynthesis was found to be indi-
rectly inhibiting FadA-mediated vegetative growth signaling. They demonstrated
that although mutational inactivation offadA did not overcome the sporulation de-
fects caused by deletion offluG, it restored ST biosynthesis in the absence offluG
function (56, 132). This finding and discovery of the interdependent relationship
offluG and flbA in conidiation (75) strongly suggested that the role of fluG in ST
production is indirect, via activating FlbA, which in turn inhibits FadA signaling
(Figure 3). Later, Seo et al. (101) isolated and characterized suppressors offluG lossof function and proposed that the primary role of FluG in activating conidiation and
ST production is to remove repressive effects imposed by multiple negative regu-
lators of conidiation. These studies provided partial understanding of the genetic
mechanism for intimate correlation between conidiation and ST production as well
as upstream regulation of secondary metabolite biosynthesis in A. nidulans (see
Figure 3). Although a probable FluG homolog has been found in all Aspergilli ex-
amined (J.-H. Yu, unpublished data), precise mechanisms coordinating conidiation
and toxin biosynthesis in individual Aspergillus species remain to be uncovered.
Sexual developmental genes and ST/AF production An important characteristic
ofA. nidulans distinguishing it from many Aspergilli is that A. nidulans has both
sexual and asexual reproductive cycles (see 29, 63). Recent studies identified
a number of genes required for and/or associated with sexual development in
A. nidulans such as GPCRs (51, 102), G proteins (96), MAPKKK (123), various
transcription factors (50, 121), and novel genes such as veA with unclear functions
(70). Among these, the veA gene encoding a novel protein coordinating balanced
sexual/asexual development, particularly in response to light, is also found to
be required for ST production as well as aflR expression in A. nidulans (67).Furthermore,Calvoetal.(24)showedthata veA ortholog in the aflatoxin-producing
fungus A. parasiticus is essential for formation of sclerotia (protective spherical
structures) as well as production of AF (and expression of necessary genes). It
appears that deletion ofveA resulted in pleiotrophic effects in Aspergilli.
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448 YU KELLER
Role of small GTP-binding protein RasA in ST production Recently, Shimizu
et al. (106) reported that RasA (a small GTP-binding protein), a homolog of the
yeast Ras proteins, is negatively associated with ST production in A. nidulans.
High levels of activated RasA also inhibited production of ST via repression ofaflR expression (106). While this RasA-mediated transcriptional control of aflR
was independent of PkaA, posttranscriptional regulation of AflR by RasA was
found to be partially mediated by PkaA (106).
G PROTEIN SIGNALING AND PENICILLIN/CYCLOPIAZONIC ACID PRODUCTION Tag
et al. (111) showed that while the constitutively active FadA allele (G42R) in-
hibited conidiation and production of ST, it also caused elevated mRNA levels of
the isopenicillin synthetase gene (ipnA) in the penicillin biosynthetic gene cluster
as well as enhanced production of the well-known antibiotic penicillin in A. nidu-lans. This result implies that FadA-mediated activation of vegetative growth has
opposite roles in regulating the biosynthesis of two major secondary metabolites,
penicillin and ST, in A. nidulans (see Figure 3). This same allele, when expressed
in A. flavus, repressed both AF biosynthesis as well as cyclopiazonic acid produc-
tion (82). The veA gene was also shown to be necessary for penicillin production.
Kato et al. (67) demonstrated that although VeA repressed transcription of ipnA,
it was found to be required for expression of acvA, a gene encoding the delta-
(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase that acts at the first step of
penicillin biosynthesis.
FusariumSpecies
TRICHOTHECENES Tag et al. (111) also examined the morphological and physi-
ological consequences of a constitutively active FadA allele in Fusarium sporotri-
chioides. They demonstrated that the introduced mutant FadA allele reduced
Fusarium spore production by 50% to 95%, restricted colony growth, but ele-
vated production of the mycotoxin trichothecene. This result indicates that the
cellular responses to a given G protein signal can be different between fungalgenera. Jain et al. (60, 61) identified and characterized G protein and sub-
units in Fusarium oxysporum and found that these G proteins are necessary for
normal development and pathogenicity. However, roles of these G proteins in
controlling production of secondary metabolites in F. oxysporum remain to be
examined.
FUMONISINS Fumonisins are a group of mycotoxins produced by the maize
pathogen Fusarium verticillioides. Shim & Woloshuck (105) isolated a mutant
that was unable to produce fumonisins on cracked corn and identified the mu-tant locus named FCC1 for Fusarium cyclin C1. The fcc1 mutant produced a
dark purple substance when grown on cracked-corn medium. This study sug-
gests a possible role of cell-cycle regulators in coordinately controlling biosynthe-
sis of various metabolites including fumonisins. However, detailed mechanisms
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REGULATION OF MYCOTOXIN PRODUCTION 449
of FCC1-mediated regulation of secondary metabolite production remain to be
studied.
Pigment Production in Other FungiZuber et al. (136) identified the gasCgene encoding a G protein subunit (GanB-
homolog) in the opportunistic human pathogen Penicillium marneffei. Analyses
of the deletion, dominant-interfering, and constitutively active GasC mutations re-
vealed that GasC-mediated signaling is positively associated with germination as
well as production of an unknown secondary metabolite. In appropriate medium,
the gasCdeletion mutant and strains carrying a dominant-interfering GasC allele
exhibited reduced production of red pigment, whereas strains carrying a constitu-
tively active GasC allele overproduced red pigment and appeared dark red. This is
somewhat consistent with physiological outcomes (i.e., increased brown pigment
production) of GanB-mediated signaling in A. nidulans (52; see above).
In Cryphonectria parasitica, the chestnut-blight fungus, deletion of cpg1 and
cpgb-1 encoding a G protein subunit and subunit, respectively, resulted in
reduced hyphal growth, lowered spore formation, a loss of virulence as well as de-
creased pigment production (46, 66), indicating that these G proteins are necessary
for normal level pigment production.
LigandsThe demonstration of how important G protein signaling is in secondary meta-
bolism, sporulation, and virulence indicated that various environmental ligands
must also be important in initiating these cascades, presumably through GPCRs or
similar transmembrane proteins. One of the first extracellular signals described to
regulate both asexual and sexual spore development is psi factor, the collective term
for a series of oleic, linoleic, and linolenic acid-derived oxylipins, produced by
A. nidulans (25, 28, 30, 80) and other fungal genera (15, 49, 109). The proportion
of these three compounds to each other was reported to regulate asexual to sexual
spore development in A. nidulans (28).These fungal sporogenic lipids bear structural and biosynthetic similarities to
plant defense oxylipins, particularly the lipoxygenase products 9S-HPODE (9S-
hydroperoxy-10E,12Z-octadecadienoic acid) and 13S-HPODE (13S-hydroperoxy-
9Z,11E-octadecdienoic acid). Detailed studies of Aspergillus spp. showed that
purified linoleic acid and hydroperoxy linoleic acids derived from seed exhibit
sporogenic activities towards several Aspergillus spp. including A. nidulans,
A. flavus, and A. parasiticus (26) and, furthermore, that Aspergillus infection of
seed induces expression of seed lipoxygenases responsible for synthesis of 9S-
HPODE (20, 125). Additionally, the seed oxylipins also had a profound effect onAF and ST production in these species where 9S-HPODE had a stimulatory ef-
fect and 13S-HPODE an inhibitory effect on toxin biosynthesis (21). These results,
coupled with the studies from other fungal research groups (88, 89, 108), suggested
that linoleic acid and its derivatives are conserved signal molecules modulating
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450 YU KELLER
mycotoxin biosynthesis, fungal sporulation, and other aspects of fungal differen-
tiation processes.
The fungal oxylipin biosynthetic pathway has been partially elucidated in A.
nidulans. Two oxygenases, bearing similarity to mammalian prostaglandin syn-thetases and, to a lesser degree, plant lipoxygenases have been recently charac-
terized (116, 117). Eliminination of ppoA (psi-producing oxygenase) from the
genome results in a strain with an increased asexual to sexual spore ratio and re-
duced levels of the linoleic acid-derived psiB (117), whereas the deletion ofppoC
reduces levels of oleic acid-derived psiB and decreases the asexual to sexual spore
ratio (116). Moreover, the PpoA::gfp fusion protein located the oxygenase to both
asexual and sexual spore-bearing structures (117). These results complemented
previous physiological and biochemical studies that pointed out an important role
for oxylipins in integrating mitotic and meiotic spore development (30). Currentstudies have revealed a third oxygenase, ppoB, deletion of which greatly increases
asexual spore production (116a).
ST biosynthesis is also affected in ppo mutants. Deletion of both ppoA and
ppoC in the same strain eliminated ST production, whereas deletion of ppoB
greatly stimulated its synthesis (D.I. Tsitsigiannis & N.P. Keller, unpublished
data). These opposite effects on ST are reminiscent of the differential effects
of 9S- and 13S-HPODE on toxin biosynthesis (21). The myriad effects of Ppo
activity were reflected at a transcriptional level where expression of transcription
factors required for ST (aflR), asexual sporulation (brlA), and sexual sporulation(nsdA) were upregulated or down-regulated, respectively, with concomitant toxin
and spore production (116, 116a, 117; D.I. Tsitsigiannis & N.P. Keller, unpub-
lished data). Ppo orthologs have been found in all filamentous fungi examined by
genome database, and deletion of a ppo gene in F. sporotrichiodes generated a
strain impaired in both conidiation and T toxin production (82). Current studies
in our laboratories suggest a model where the different oxylipin products gener-
ated by Ppo oxygenases are secreted and function as ligands activating specific
GPCR signaling cascades inAspergillus and other fungi (Figure 5). The conserved
presence of ppo genes in fungal genomes coupled with conserved lipid stimula-tion of sporulation in several filamentous fungi suggests a putative global oxylipin
signaling cascade in the fungal kingdom.
CONCLUSION
Although still not complete, our knowledge of the molecular genetics of fungal
secondary metabolism has soared in the past decade. The establishment of the
secondary metabolite cluster motif and identification of both pathway-specific
and global regulators of these clusters lend themselves well to identification and
manipulation of additional clusters. Signaling cascades link sporulation processes
with metabolite synthesis. Coupling of secondary metabolism with morphologi-
cal development of the fungus appears to be a universal constant in filamentous
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REGULATION OF MYCOTOXIN PRODUCTION 451
fungi and may indicate an underlying evolutionary mechanism important in fungal
survival and possibly aspects of pathogenesis.
ACKNOWLEDGMENTSThe authors are thankful to those who made contributions to the subject areas.
This work was supported by National Science Foundation grant MCB-04,21863
to J.H.Y. and MCB-02,36393 to N.P.K.
The Annual Review of Phytopathology is online at
http://phyto.annualreviews.org
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REGULATION OF MYCOTOXIN PRODUCTION C-1
Figure 2 RGS proteins enhance the intrinsic GTPase activity of G subunits.
When a GPCR is sensitized by ligand binding, it catalyzes GDP to GTP exchange of
a G subunit, which subsequently provokes dissociation of G-GTP and G. Once
freed, either G-GTP or G, or both, can mediate signaling. Hydrolysis of G-
GTP to G-GDP causes the formation of the inactive heterotrimer G-GDP:G,
thereby turning off the signal. InA. nidulans, FadA-GTP and the cognate G het-
erodimer mediate signaling for vegetative growth, which involves PkaA (107) and
presumed transcription factors (TFs). FlbA is proposed to rapidly turn off FadA-
mediated vegetative growth signaling by acting as a GTPase Activating Protein
(132). Colony photographs are wild type (WT) and thef lbA deletion mutant (f lbA).Note that the f lbA mutant does not produce asexual spores and center of the f lbA
colony autolyzed (arrow).
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C-2 YU KELLER
Figure 4 GanB-RgsA signaling and production of pigment and ST (modified from
Reference 52) The ganBrgsA mutant exhibits colony growth and pigmentation
phenotypes identical to the ganB mutant. A TLC chromatograph shows that the
rgsA mutant produced a high level of fast migrating brown pigments (top two
arrows) while accumulating hardly detectable level of ST. Note that ST is readily
detectable in wild-type or ganB strains, and that the ganBrgsA mutant restored
the production of ST to the level of the ganB mutant.
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Annual Review of Phytopathology
Volume 43, 2005
CONTENTS
FRONTISPIECE, Robert K. Webster xii
BEING AT THE RIGHT PLACE, AT THE RIGHT TIME, FOR THE RIGHT
REASONSPLANT PATHOLOGY, Robert K. Webster 1
FRONTISPIECE, Kenneth Frank Baker
KENNETH FRANK BAKERPIONEER LEADER IN PLANT PATHOLOGY,
R. James Cook 25
REPLICATION OF ALFAMO- AND ILARVIRUSES: ROLE OF THE COAT PROTEIN,
John F. Bol 39
RESISTANCE OF COTTON TOWARDS XANTHOMONAS CAMPESTRISpv.
MALVACEARUM, E. Delannoy, B.R. Lyon, P. Marmey, A. Jalloul, J.F. Daniel,
J.L. Montillet, M. Essenberg, and M. Nicole 63
PLANT DISEASE: A THREAT TO GLOBAL FOOD SECURITY, Richard N. Strange
and Peter R. Scott 83
VIROIDS AND VIROID-HOST INTERACTIONS, Ricardo Flores,Carmen Hernandez, A. Emilio Mart nez de Alba, Jose-Antonio Daros,
and Francesco Di Serio 117
PRINCIPLES OF PLANT HEALTH MANAGEMENT FOR ORNAMENTAL PLANTS,
Margery L. Daughtrey and D. Michael Benson 141
THE BIOLOGY OF PHYTOPHTHORA INFESTANS AT ITS CENTER OF ORIGIN,
Niklaus J. Gr unwald and Wilbert G. Flier 171
PLANT PATHOLOGY AND RNAi: A BRIEF HISTORY, John A. Lindbo
and William G. Doughtery 191
CONTRASTING MECHANISMS OF DEFENSE AGAINST BIOTROPHIC AND
NECROTROPHIC PATHOGENS, Jane Glazebrook 205
LIPIDS, LIPASES, AND LIPID-MODIFYING ENZYMES IN PLANT DISEASE
RESISTANCE, Jyoti Shah 229
PATHOGEN TESTING AND CERTIFICATION OF VITIS AND PRUNUSSPECIES,
Adib Rowhani, Jerry K. Uyemoto, Deborah A. Golino,
and Giovanni P. Martelli 261
MECHANISMS OF FUNGAL SPECIATION, Linda M. Kohn 279
vii
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viii CONTENTS
PHYTOPHTHORA RAMORUM: INTEGRATIVE RESEARCH AND MANAGEMENT
OF AN EMERGING PATHOGEN IN CALIFORNIA AND OREGON FORESTS,
David M. Rizzo, Matteo Garbelotto, and Everett M. Hansen 309
COMMERCIALIZATION AND IMPLEMENTATION OF BIOCONTROL, D.R. Fravel 337
EXPLOITING CHINKS IN THE PLANTS ARMOR: EVOLUTION AND EMERGENCE
OF GEMINIVIRUSES, Maria R. Rojas, Charles Hagen, William J. Lucas,
and Robert L. Gilbertson 361
MOLECULAR INTERACTIONS BETWEEN TOMATO AND THE LEAF MOLD
PATHOGEN CLADOSPORIUM FULVUM, Susana Rivas
and Colwyn M. Thomas 395
REGULATION OF SECONDARY METABOLISM IN FILAMENTOUS FUNGI,
Jae-Hyuk Yu and Nancy Keller 437
TOSPOVIRUS-THRIPS INTERACTIONS, Anna E. Whitfield, Diane E. Ullman,and Thomas L. German 459
HEMIPTERANS AS PLANT PATHOGENS, Isgouhi Kaloshian
and Linda L. Walling 491
RNA SILENCING IN PRODUCTIVE VIRUS INFECTIONS, Robin MacDiarmid 523
SIGNAL CROSSTALK AND INDUCED RESISTANCE: STRADDLING THE LINE
BETWEEN COST AND BENEFIT, Richard M. Bostock 545
GENETICS OF PLANT VIRUS RESISTANCE, Byoung-Cheorl Kang, Inhwa Yeam,
and Molly M. Jahn 581
BIOLOGY OF PLANT RHABDOVIRUSES, Andrew O. Jackson, Ralf G. Dietzgen,
Michael M. Goodin, Jennifer N. Bragg, and Min Deng 623
INDEX
Subject Index 661
ERRATA
An online log of corrections to Annual Review of Phytopathology chapters
may be found at http://phyto.annualreviews.org/
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