Regulation of Secondary Metabolism in Filamentous Fungi

26 Jul 2005 11:51 AR AR250-PY43-18.tex XMLPublishSM(2004/02/24) P1: KUV10.1146/annurev.phyto.43.040204.140214

Annu. Rev. Phytopathol. 2005. 43:437–58doi: 10.1146/annurev.phyto.43.040204.140214

Copyright c© 2005 by Annual Reviews. All rights reserved

REGULATION OF SECONDARY METABOLISM

IN FILAMENTOUS FUNGI

Jae-Hyuk Yu1 and Nancy Keller1,2

1Department 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 the current understanding of the genetic regula-tion of secondary metabolism from clustering of biosynthetic genes to global regulatorsbalancing 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 haveobscure or unknown functions in the producing organism but have tremendousimportance to humankind in that they display a broad range of useful antibioticand pharmaceutical activities as well as less desirable immunosuppressant andtoxic activities.

In most cases, the function of secondary metabolites for the producing fungus isunknown but is inferred from a few studies using mutants or enzyme inhibitors. Themost obvious fungal natural products are the pigments—typically brown and blackpigments referred to as melanins—giving color to spores, appressoria, sclerotia,sexual bodies, and other developmental structures. Studies of pigment functionin 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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protectants (72, 77), antigrowth deterrents (98), or ROS scavengers (37). Fungalphytotoxins are proven pathogenicity or virulence factors that cause significantdisease on agricultural crops (5, 54, 127). Another notorious group of agriculturallyimportant secondary metabolites are the mycotoxins, which are excreted by fungias they grow in various commodities (8). The primary aim of this review is topresent our current understanding of how secondary metabolites are regulated infungi, 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 anoriginal premise that fungal metabolic genes would be scattered throughout thegenome. Rather, the hallmark of secondary metabolite genes—in contrast to genesinvolved in primary metabolism—is that they are clustered in fungal genomes(reviewed in 68, 135). As described below, the contiguous clustering of metabolicgenes specific to one product has considerable bearing on the regulation of thesegenes.

Aflatoxin and Sterigmatocystin

With the possible exception of the penicillin metabolic cluster, the most thor-oughly examined fungal secondary metabolite gene clusters are those involvedin 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 isthe penultimate precursor of AF (16, 129). The AF cluster in A. parasiticus andA. flavus contains genes that constitute a cluster spanning more than 70 kb. Amongthese 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 indicatedby the same bar pattern. Solid black bars represent ST and AF genes that have not beencharacterized.

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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 aflatoxinproduction 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 ofaflatoxin cluster genes (55).

Trichothecenes

Trichothecenes comprise a large family of sesquiterpenoid metabolites producedby 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 associatedwith virulence in specific plant-pathogen interactions (38, 53, 95).

Biochemical and genetic analyses of the T-2 toxin producer F. sporotrichioidesled to the identification of the first trichothecene biosynthetic gene cluster. The genecluster 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). Thefunctions of ten genes have been determined. Seven of them encode biosyntheticenzymes (6, 18, 19, 81). Tri6 and Tri10 are regulatory proteins and Tri12 is theefflux pump that is implicated to play a self-protection role (7, 110). Recently, asecond “mini-cluster” has been found in F. graminearum that contains two moreenzymatic genes required for deoxynivalenol production (17). A trichothecenecluster has also been described in Myrothecium roridum (113).

Fumonisins

Fumonisins are a group of polyketide mycotoxins that are produced primarilyby 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 involvedin fumonisin biosynthesis are clustered in a ∼45-kb stretch of DNA. Expressionanalysis of F. verticillioides indicated that 15 genes (ORF1 and ORF6-19) arecoregulated and exhibited patterns of expression that were correlated with fumon-isin production. These ORFs are designated as FUM genes (92). FUM5 encodesthe polyketide synthase gene that was shown to be required for fumonisin biosyn-thesis (93). Disruption of FUM6 and FUM8 blocked production but did not leadto accumulation of detectable intermediates (103); FUM9 and FUM13 both areinvolved 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 othermycotoxins and phytotoxins (HC toxin, 4; dothiostromin, 13; sirodesmin, 47;gibberellin, 118; ergot alkaloids, 119; paxillin, 128; aflatrem, 135), antibiotics

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(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 transcriptionalcontrol of structural genes by two classes of transcription factors, one class brieflymentioned in the previous section that are specific to a particular metabolic path-way (i.e., aflR) and a second class that mediate environmental signals includingpH, carbon, and nitrogen sources. This multilevel regulation by both specific andbroad-domain transcription factors ensures that secondary metabolite pathwayscan 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 isAflR, the Zn(II)2Cys6 domain protein required for AF and ST biosynthetic geneactivation (33, 44, 126). Typical for this group of DNA binding proteins, AflRrecognizes and binds to a palindromic sequence, 5′-TCG(N5)GCA, found in thepromoters 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 andis considered important in autoregulation of aflR transcript in these spp. (34, 35,40, 90). Disruption of aflR eliminates the expression of structural genes (130) andmodifications of its promoter region alter not only its own but subsequent clus-ter gene expression (40). How AflR is negatively regulated by protein kinase Asignaling is described below.

Other cluster transcription factors include additional Zn(II)2Cys6 proteins (MlcRfor compactin biosynthesis, 1), Cys2His2 zinc finger proteins (Tri6 and MRTRI6for 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 (PENR1for penicillin, 78). Additionally, PENR1 has also been shown to be important intaka-amylase, xylanase, and cellobiohydrolase production (14).

Pathway-specific regulatory genes not obviously encoding transcription factorsinclude aflJ required for AF/ST biosynthesis and Tri10 involved in trichothecenebiosynthesis (83, 110). Inactivation or mutation of aflJ gives a phenotype similar toan 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 in A. parasiticus (32, 83). Despitelack of AF production in aflJ deletion strains, structural genes are still expressedat a reduced level. This complex phenotype suggests that AflJ is not directlyresponsible for AF/ST gene transcription or for any particular enzymatic step in

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the pathway, but is in some way enhancing transcription. Chang (32) demonstratedan interaction of AflJ with AflR using a yeast two-hybrid system. Two regions ofAflR are required for this interaction, one located between aa 230–238, the otherat 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 theprotein by 85%–90%, and deletion 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, andpH. Several studies (42, 84) indicate that these environmental signals are mediatedthrough 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 Gibberellafujikuroi led to a 10%–20% reduction of gibberellin production in gibberellininduction medium. In addition, the loss-of-function areA-GF strains were insensi-tive to ammonium-mediated gibberellin repression, supporting the conclusion thatgibberellin biosynthesis is under the control of AreA-GF (84).

The involvement of CreA in secondary metabolism may reflect the differencesseen in metabolite production when fungi are grown in different carbon sources(43). Another related factor in metabolite production may be the availability ofprecursor units. For example, each secondary metabolite ultimately depends onthe 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-CoAitself being derived from acetyl-CoA. Availability of acetyl-CoA might be ex-pected to impact polyketide formation. Such a prediction has been recently borneout in genetic and biochemical studies of A. nidulans where mutants in path-ways affecting either acetyl-CoA concentration (β-oxidation mutants; 78a) oravailability via altered acyl-CoA ratios (methyl citrate mutants; 133, 134) canreduce 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. Thecomplementing gene, termed laeA for loss of aflR expression, encodes a nuclearprotein with closest identity to arginine and histone methyltransferases (11). Lossof LaeA function silences not only ST cluster expression but also a multitude ofother metabolites including penicillin and numerous mycelial pigments in A. nidu-lans and gliotoxin in A. fumigatus, whereas overexpression of laeA upregulatescluster 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 whichare 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 chromatinremodeling of cluster loci. Putative LaeA orthologs are found in all filamentousand dimorphic fungi examined to date, and it will be interesting to see if theseLaeA 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, suchas 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 relayingexternal cues into the cells to elicit appropriate physiological and biochemicalresponses (reviewed in 86). In the past decade, G protein–mediated signaling hasbeen intensively studied in various filamentous fungal species, and outcomes of thestudies provided an important clue to understand the upstream regulation of fungalsecondary metabolite biosynthesis, which was hypothesized to be intimately asso-ciated with sporulation (reviewed in 27). Progress has been made with the modelfungus A. nidulans, and this section primarily discusses signaling mechanismsgoverning development and ST production in this fungus with a few additionalexamples.

The basic unit of heterotrimeric G protein is comprised of a seven-transmem-brane-spanning domain G protein–coupled receptor (GPCR), a G protein consist-ing of α, β, and γ subunits, and an intracellular effector that produces a secondmessenger. G protein signaling is activated when ligand-bound GPCRs catalyzeGDP/GTP exchange of the Gα subunit, which provokes subsequent dissociationof Gα-GTP and Gβγ . It is turned off when the intrinsic GTPase activity of theGα subunit hydrolyzes GTP to GDP, causing the formation of the inactive het-erotrimer Gα–GDP:Gβγ . Dissociated (activated) Gα-GTP and/or Gβγ can triggerthe 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 in86). In fungi, GPCR-G protein–initiated signaling is primarily transmitted to twodownstream signaling branches defined by adenylyl cyclase → cAMP → proteinkinase (PKA) and/or mitogen-activated protein kinase (MAPKKK → MPAKK →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 variouscontrolling elements, regulators of G protein signaling (RGS proteins) play a key

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role in tightly controlling G protein signaling upstream or at the same level ofG proteins. RGS proteins contain a conserved ∼130 amino acid core domain(RGS box) that functions in enhancing the intrinsic GTPase activity of the Gα

subunit, which results in increased GTP to GDP hydrolysis (inactivation) ratesof Gα subunits (Figure 2). Through activities of multiple RGS proteins, cellscan coordinate diverse incoming signals and fine-tune their cellular responses(reviewed in 36). G proteins and RGS proteins function in global coordination offundamental biological processes in filamentous fungi including vegetative growth,sporulation, mycotoxin/pigment production, pathogenicity, and mating.

Aspergillus Species

ST/AF PRODUCTION REGULATION BY FadA (Gα) 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 α (FadA) and β (SfaD) subunits ofa heterotrimeric G protein (96, 132). When FadA (Gα) is in its active-GTP bound-state, it is dissociated with its cognate Gβγ dimer (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 blocksproduction of the mycotoxin sterigmatocystin (ST) and mRNA expression of aflR,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. Inthis attenuation of growth signaling, FlbA (an RGS protein) plays a key role and ispresumed to rapidly convert FadA-GTP to FadA-GDP by increasing the intrinsicGTPase activity of FadA (Figure 3). Loss of flbA function results in the fluffy-autolytic colony (Figure 2) that lacks sexual/asexual sporulation and ST productiondue 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 flbAmutants (J. Hicks & N.P. Keller, unpublished data), forced expression of aflR orsite-directed mutagenized hyperactive AflR could not restore ST production in theabsence of flbA function (106). These results suggest that FlbA is necessary foractivity of the AflR protein via unknown mechanisms. While loss of SfaD (Gβ)or GpgA (Gγ ) function has clear effects on vegetative growth, conidiation, andsexual 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 Aspergillusspecies, in that the FadA-homologous pathway negatively controls AF biosynthesisin 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 α

(FadA) and β (SfaD with a presumed Gγ , GpgA) subunits of a heterotrimeric G pro-tein inhibits asexual/sexual sporulation and ST biosynthesis, but stimulates penicillinproduction (56, 96, 111, 132). Activation of asexual development requires at leastpartial inhibition of FadA-mediated signaling, which requires two genes, fluG andflbA (73–75). FluG is proposed to activate conidiation via removing repressive effectsimposed by multiple negative regulators (101). FlbA is an RGS protein that inhibitsFadA-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 inA. flavus (82).

Protein kinase A and AflR FadA growth signaling is transduced in part via proteinkinase A (PKA; 107). Deletion of the pkaA gene encoding the primary PKAcatalytic subunit in A. nidulans resulted in elevated conidiation and highly restrictedvegetative growth. Analyses of epistatic interactions between fadA-flbA and pkaArevealed that PkaA functions downstream of FlbA/FadA, i.e., deletion of pkaAsuppressed both developmental and ST biosynthesis defects caused by the absenceof flbA function. Conversely, overexpression of pkaA caused reduced conidiation,increased vegetative growth as well as inhibition of aflR expression necessary forST biosynthesis (107). This study clarified that FadA-mediated signaling is (atleast in part) transmitted to a cAMP → PKA signaling cascade and PkaA plays

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a major role in activation of vegetative growth and repression of both conidiationand 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 phosphorylationin vivo was also shown by substitution of the putative phosphorylation targetamino acid Ser to Ala in AflR. Such site-directed mutations abolished inhibitoryeffects of overexpression of pkaA on AflR activity. Moreover, the authors showedthat the requirement of FlbA for AflR-mediated ST production is PkaA indepen-dent. This is consistent with the previous hypothesis that, in addition to inhibitingFadA signaling, FlbA has an additional role in activating conidiation as well asST 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 biosynthesisand conidiation in A. parasiticus were examined by Roze et al. (97). Interestingly,while introduction of cAMP or dibutyryl-cAMP (DcAMP) onto solid mediumresulted in a 100-fold increase in intracellular cAMP/DcAMP, total cellular PKAactivity was lowered by 40- to 80-fold in the tested strain. These findings explainedwhy cAMP or DcAMP stimulated AF synthesis and conidiation in A. parasiticusdespite the functionally conserved FadA-mediated signaling mechanisms. Theauthors concluded: (a) the FadA/PKA signaling cascade negatively regulates AFbiosynthesis and conidiation via similar mechanisms in Aspergillus species; and(b) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatoryinfluence on conidiation and AF synthesis.

GanB and RgsA, the second Gα-RGS pair As in most filamentous fungal genomes(12), the A. nidulans genome contains three Gα 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 of rgsA causedreduced 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 tospeculate that the G protein pathways controlled by FlbA and RgsA are differ-ent. Morphological alterations, increased pigment but reduced ST production, aswell as restricted colony growth caused by deletion of rgsA were suppressed bydeletion of ganB, indicating that the primary role of RgsA is to negatively con-trol GanB-mediated signaling (Figure 4). The observations that overexpressionof rgsA as well as deletion or dominant interfering mutations of ganB caused in-appropriate hyperactive conidiation in liquid-submerged culture further supportthat 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-Gα pair inA. 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 partiallycontribute to this phenotype (52). Taken together, a genetic model incorporatingthe activities of two G protein signaling pathways and the cognate RGS proteinsin governing growth, development and ST production is presented (Figure 5).

Conidiation-specific functions and ST production A close relationship betweenfungal development and secondary metabolite production has been observed inAspergillus 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 inA. nidulans (adapted from Reference 52). Two independent Gα-RGS signaling path-ways coordinately control cellular responses to various signals. FlbA-FadA primarilygoverns vegetative growth versus development and ST production, and RgsA-GanBcontrols stress response (pigmentation), carbon sensing, and germination. Conidiationoccurs through activation of brlA, which requires multiple upstream genes includingfluG (74). GanB and presumed SfaD:GpgA-mediated signaling is proposed to repressasexual 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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AF. Later, these sporulation defective mutants were found to fail to accumulatemRNA of aflR and genes encoding enzymes for AF biosynthesis (64, 65). Geneticmechanisms interconnecting conidiation and AF/ST production were uncoveredby 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, 73–75,124). BrlA is a key transcription factor that activates conidiophore (asexual spore-bearing structure) formation, and fluG, flbE, flbD, flbB, and flbC are required for theexpression of brlA. Mutations in these upstream regulatory genes resulted in theabsence or delay of conidiation and overproliferation of hyphae, termed “fluffy”phenotypes. Accordingly, a genetic model proposed that conidiation occurs viaactivities of multiple positive regulators (reviewed in 3). Among these, the fluGgene that functions at the most upstream of this genetic cascade was also found tobe necessary for ST production.

Hicks et al. (56) demonstrated that loss of fluG function resulted in lack of STproduction. Importantly, the role of FluG in ST biosynthesis was found to be indi-rectly inhibiting FadA-mediated vegetative growth signaling. They demonstratedthat although mutational inactivation of fadA did not overcome the sporulation de-fects caused by deletion of fluG, it restored ST biosynthesis in the absence of fluGfunction (56, 132). This finding and discovery of the interdependent relationshipof fluG and flbA in conidiation (75) strongly suggested that the role of fluG in STproduction is indirect, via activating FlbA, which in turn inhibits FadA signaling(Figure 3). Later, Seo et al. (101) isolated and characterized suppressors of fluG lossof function and proposed that the primary role of FluG in activating conidiation andST production is to remove repressive effects imposed by multiple negative regu-lators of conidiation. These studies provided partial understanding of the geneticmechanism for intimate correlation between conidiation and ST production as wellas upstream regulation of secondary metabolite biosynthesis in A. nidulans (seeFigure 3). Although a probable FluG homolog has been found in all Aspergilli ex-amined (J.-H. Yu, unpublished data), precise mechanisms coordinating conidiationand toxin biosynthesis in individual Aspergillus species remain to be uncovered.

Sexual developmental genes and ST/AF production An important characteristicof A. nidulans distinguishing it from many Aspergilli is that A. nidulans has bothsexual and asexual reproductive cycles (see 29, 63). Recent studies identifieda number of genes required for and/or associated with sexual development inA. nidulans such as GPCRs (51, 102), G proteins (96), MAPKKK (123), varioustranscription factors (50, 121), and novel genes such as veA with unclear functions(70). Among these, the veA gene encoding a novel protein coordinating balancedsexual/asexual development, particularly in response to light, is also found tobe required for ST production as well as aflR expression in A. nidulans (67).Furthermore, Calvo et al. (24) showed that a veA ortholog in the aflatoxin-producingfungus A. parasiticus is essential for formation of sclerotia (protective sphericalstructures) as well as production of AF (and expression of necessary genes). Itappears that deletion of veA resulted in pleiotrophic effects in Aspergilli.

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Role of small GTP-binding protein RasA in ST production Recently, Shimizuet al. (106) reported that RasA (a small GTP-binding protein), a homolog of theyeast 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 aflRwas independent of PkaA, posttranscriptional regulation of AflR by RasA wasfound to be partially mediated by PkaA (106).

G PROTEIN SIGNALING AND PENICILLIN/CYCLOPIAZONIC ACID PRODUCTION Taget al. (111) showed that while the constitutively active FadA allele (G42R) in-hibited conidiation and production of ST, it also caused elevated mRNA levels ofthe isopenicillin synthetase gene (ipnA) in the penicillin biosynthetic gene clusteras well as enhanced production of the well-known antibiotic penicillin in A. nidu-lans. This result implies that FadA-mediated activation of vegetative growth hasopposite roles in regulating the biosynthesis of two major secondary metabolites,penicillin and ST, in A. nidulans (see Figure 3). This same allele, when expressedin 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 ofpenicillin biosynthesis.

Fusarium Species

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 reducedFusarium spore production by 50% to 95%, restricted colony growth, but ele-vated production of the mycotoxin trichothecene. This result indicates that thecellular responses to a given G protein signal can be different between fungalgenera. Jain et al. (60, 61) identified and characterized G protein α and β sub-units in Fusarium oxysporum and found that these G proteins are necessary fornormal development and pathogenicity. However, roles of these G proteins incontrolling production of secondary metabolites in F. oxysporum remain to beexamined.

FUMONISINS Fumonisins are a group of mycotoxins produced by the maizepathogen Fusarium verticillioides. Shim & Woloshuck (105) isolated a mutantthat was unable to produce fumonisins on cracked corn and identified the mu-tant locus named FCC1 for Fusarium cyclin C1. The fcc1 mutant produced adark 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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of FCC1-mediated regulation of secondary metabolite production remain to bestudied.

Pigment Production in Other Fungi

Zuber et al. (136) identified the gasC gene encoding a G protein α subunit (GanB-homolog) in the opportunistic human pathogen Penicillium marneffei. Analysesof the deletion, dominant-interfering, and constitutively active GasC mutations re-vealed that GasC-mediated signaling is positively associated with germination aswell as production of an unknown secondary metabolite. In appropriate medium,the gasC deletion mutant and strains carrying a dominant-interfering GasC alleleexhibited reduced production of red pigment, whereas strains carrying a constitu-tively active GasC allele overproduced red pigment and appeared dark red. This issomewhat consistent with physiological outcomes (i.e., increased brown pigmentproduction) of GanB-mediated signaling in A. nidulans (52; see above).

In Cryphonectria parasitica, the chestnut-blight fungus, deletion of cpg1 andcpgb-1 encoding a G protein α subunit and β subunit, respectively, resulted inreduced hyphal growth, lowered spore formation, a loss of virulence as well as de-creased pigment production (46, 66), indicating that these G proteins are necessaryfor normal level pigment production.

Ligands

The demonstration of how important G protein signaling is in secondary meta-bolism, sporulation, and virulence indicated that various environmental ligandsmust also be important in initiating these cascades, presumably through GPCRs orsimilar transmembrane proteins. One of the first extracellular signals described toregulate both asexual and sexual spore development is psi factor, the collective termfor a series of oleic, linoleic, and linolenic acid-derived oxylipins, produced byA. nidulans (25, 28, 30, 80) and other fungal genera (15, 49, 109). The proportionof these three compounds to each other was reported to regulate asexual to sexualspore development in A. nidulans (28).

These fungal sporogenic lipids bear structural and biosynthetic similarities toplant 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 thatpurified linoleic acid and hydroperoxy linoleic acids derived from seed exhibitsporogenic activities towards several Aspergillus spp. including A. nidulans,A. flavus, and A. parasiticus (26) and, furthermore, that Aspergillus infection ofseed 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), suggestedthat linoleic acid and its derivatives are conserved signal molecules modulating

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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 thegenome results in a strain with an increased asexual to sexual spore ratio and re-duced levels of the linoleic acid-derived psiBα (117), whereas the deletion of ppoCreduces levels of oleic acid-derived psiBα and decreases the asexual to sexual sporeratio (116). Moreover, the PpoA::gfp fusion protein located the oxygenase to bothasexual and sexual spore-bearing structures (117). These results complementedprevious physiological and biochemical studies that pointed out an important rolefor oxylipins in integrating mitotic and meiotic spore development (30). Currentstudies have revealed a third oxygenase, ppoB, deletion of which greatly increasesasexual spore production (116a).

ST biosynthesis is also affected in ppo mutants. Deletion of both ppoA andppoC in the same strain eliminated ST production, whereas deletion of ppoBgreatly stimulated its synthesis (D.I. Tsitsigiannis & N.P. Keller, unpublisheddata). These opposite effects on ST are reminiscent of the differential effectsof 9S- and 13S-HPODE on toxin biosynthesis (21). The myriad effects of Ppoactivity were reflected at a transcriptional level where expression of transcriptionfactors required for ST (aflR), asexual sporulation (brlA), and sexual sporulation(nsdA) were upregulated or down-regulated, respectively, with concomitant toxinand spore production (116, 116a, 117; D.I. Tsitsigiannis & N.P. Keller, unpub-lished data). Ppo orthologs have been found in all filamentous fungi examined bygenome database, and deletion of a ppo gene in F. sporotrichiodes generated astrain impaired in both conidiation and T toxin production (82). Current studiesin our laboratories suggest a model where the different oxylipin products gener-ated by Ppo oxygenases are secreted and function as ligands activating specificGPCR signaling cascades in Aspergillus and other fungi (Figure 5). The conservedpresence of ppo genes in fungal genomes coupled with conserved lipid stimula-tion of sporulation in several filamentous fungi suggests a putative global oxylipinsignaling cascade in the fungal kingdom.

CONCLUSION

Although still not complete, our knowledge of the molecular genetics of fungalsecondary metabolism has soared in the past decade. The establishment of thesecondary metabolite cluster motif and identification of both pathway-specificand global regulators of these clusters lend themselves well to identification andmanipulation of additional clusters. Signaling cascades link sporulation processeswith 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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fungi and may indicate an underlying evolutionary mechanism important in fungalsurvival and possibly aspects of pathogenesis.

ACKNOWLEDGMENTS

The authors are thankful to those who made contributions to the subject areas.This work was supported by National Science Foundation grant MCB-04,21863to J.H.Y. and MCB-02,36393 to N.P.K.

The Annual Review of Phytopathology is online athttp://phyto.annualreviews.org

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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 ofa G� subunit, which subsequently provokes dissociation of G�-GTP and G��. Oncefreed, 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. In A. nidulans, FadA-GTP and the cognate G�� het-erodimer mediate signaling for vegetative growth, which involves PkaA (107) andpresumed 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 the f lbA deletion mutant (�f lbA).Note that the �f lbA mutant does not produce asexual spores and center of the �f lbAcolony autolyzed (arrow).

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Figure 4 GanB-RgsA signaling and production of pigment and ST (modified fromReference 52) The �ganB�rgsA mutant exhibits colony growth and pigmentationphenotypes identical to the �ganB mutant. A TLC chromatograph shows that the�rgsA mutant produced a high level of fast migrating brown pigments (top twoarrows) while accumulating hardly detectable level of ST. Note that ST is readilydetectable in wild-type or �ganB strains, and that the �ganB�rgsA mutant restoredthe production of ST to the level of the �ganB mutant.

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P1: KUV

July 14, 2005 11:17 Annual Reviews AR250-FM

Annual Review of PhytopathologyVolume 43, 2005

CONTENTS

FRONTISPIECE, Robert K. Webster xii

BEING AT THE RIGHT PLACE, AT THE RIGHT TIME, FOR THE RIGHT

REASONS—PLANT PATHOLOGY, Robert K. Webster 1

FRONTISPIECE, Kenneth Frank Baker

KENNETH FRANK BAKER—PIONEER 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 CAMPESTRIS pv.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. Strangeand Peter R. Scott 83

VIROIDS AND VIROID-HOST INTERACTIONS, Ricardo Flores,Carmen Hernandez, A. Emilio Martınez 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. Grunwald and Wilbert G. Flier 171

PLANT PATHOLOGY AND RNAi: A BRIEF HISTORY, John A. Lindboand 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 PRUNUS SPECIES,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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July 14, 2005 11:17 Annual Reviews AR250-FM

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 PLANT’S 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 Rivasand 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 Kaloshianand 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 chaptersmay be found at http://phyto.annualreviews.org/

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Regulation of Secondary Metabolism in Filamentous Fungi

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