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A key role for vesicles in fungal secondary metabolism
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  A key role for vesicles in fungalsecondary metabolism Anindya Chanda a , Ludmila V. Roze a , Suil Kang a,1 , Katherine A. Artymovich a , Glenn R. Hicks b , Natasha V. Raikhel b ,Ana M. Calvo c , and John E. Linz a,d,e,2 a Department of Food Science and Human Nutrition,  d National Food Safety and Toxicology Center, and  e Department of Microbiology and MolecularGenetics, Michigan State University, East Lansing, MI 48824;  b Center for Plant Cell Biology and Department of Botany and Plant Sciences, University ofCalifornia, Riverside, CA 92521; and  c Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115Edited by Joan Wennstrom Bennett, Rutgers University, New Brunswick, NJ, and approved September 28, 2009 (received for review July 6, 2009) Eukaryotes have evolved highly conserved vesicle transport ma-chinery to deliver proteins to the vacuole. In this study we showthat the filamentous fungus  Aspergillus parasiticus  employs thisdelivery system to perform new cellular functions, the synthesis,compartmentalization, and export of aflatoxin; this secondarymetabolite is one of the most potent naturally occurring carcino-gens known. Here we show that a highly pure vesicle-vacuolefraction isolated from  A. parasiticus  under aflatoxin-inducing con-ditions converts sterigmatocystin, a late intermediate in aflatoxinsynthesis, to aflatoxin B 1 ; these organelles also compartmentalizeaflatoxin. The role of vesicles in aflatoxin biosynthesis and exportwas confirmed by blocking vesicle-vacuole fusion using 2 indepen-dent approaches. Disruption of  A. parasiticus vb1  (encodes aprotein homolog of AvaA, a small GTPase known to regulatevesicle fusion in  A. nidulans ) or treatment with Sortin3 (blocksVps16 function, one protein in the class C tethering complex)increased aflatoxin synthesis and export but did not affect afla-toxin gene expression, demonstrating that vesicles and not vacu-oles are primarily involved in toxin synthesis and export. We alsoobserved that development of aflatoxigenic vesicles (aflatoxi-somes) is strongly enhanced under aflatoxin-inducing growthconditions. Coordination of aflatoxisome development with afla-toxin gene expression is at least in part mediated by  Velvet   (VeA),a global regulator of  Aspergillus  secondary metabolism. We pro-pose a unique 2-branch model to illustrate the proposed role forVeAinregulationofaflatoxisomedevelopmentandaflatoxingeneexpression. aflatoxin biosynthesis    aflatoxisomes    compartmentalization    VeA    vb1 S econdary metabolites, natural products generated by fila-mentous fungi, plants, bacteria, algae, and animals, have anenormous impact on humans due to their application in health,medicine, and agriculture. Many secondary metabolites arebeneficial (antibiotics, statins, morphine, etc.), though phyto-toxins (e.g., ricin, crotin, amygdalin) and fungal poisons calledmycotoxins (e.g., aflatoxin, sterigmatocystin, fumonisin) aredetrimental to humans and animals. To control or customizebiosynthesis of these natural products we must understand howand where secondary metabolism is orchestrated within the cell.Vacuoles and vesicles are known to sequester secondarymetabolites to protect host cells from self-toxicity (1). Enzymesinvolvedinsecondarymetabolismareoftenfoundinvesiclesand vacuoles, including those for biosynthesis of alkaloids (e.g.,berberine, sanguinarine, camptotecin, and morphine; reviewedin refs. 1 and 2) and flavonoids (e.g., aurone) (reviewed in refs.1 and 3) in plants and the nonribosomal peptide cyclosporin (4),the   -lactam antibiotic penicillin (5) (localization of ACVS isstill controversial), and the polyketide aflatoxin (6–8) in fungi.However, the functional role of these compartments in second-ary metabolism was unclear because these organelles potentiallycould be involved in synthesis, storage, protein turnover, trans-port, or export of the end product or biosynthetic enzymes. Aflatoxins are polyketide-derived furanocoumarins synthe-sized primarily by the filamentous fungi  Aspergillus parasiticus and  A. flavus  (9, 10). Aflatoxin biosynthesis is one of the mosthighlycharacterizedsecondarymetabolicpathwaysandprovidesa useful system to understand secondary metabolism in eu-karyotes. Aflatoxin B 1  (AFB 1 ) is the most potent naturallyoccurring carcinogen known (10) and has significant health andeconomic impacts worldwide (10). Aflatoxin biosynthesis in- volves at least 17 enzyme activities encoded by 25 or more genesthat are clustered in a 75-Kb region on one chromosome. Themolecular mechanisms that regulate aflatoxin biosynthesis havebeen studied extensively by us and others (reviewed in refs. 10and 11).Theintracellularsiteforsynthesisoffungalpolyketide-derivedsecondary metabolites was not known before our current work.Previous studies from our laboratory demonstrated that asaflatoxin synthesis increases, early (Nor-1), middle (Ver-1), andlate pathway enzymes (OmtA) localize to 2 primary subcellularlocations: ( i ) the cytoplasm (6–8) and ( ii ) vesicle and vacuole-like organelles (6–8) (we define organelles   2.5   m in size as vesicles; we define organelles   2.5   m as vacuoles). As a first step to determine the functional role of theseorganelles in  Aspergillus  secondary metabolism, we developed aunique ‘‘high-density sucrose cushion’’ method for purificationof a vesicle-vacuole fraction from  A. parasiticus  during aflatoxinsynthesis (12). In the current study, we show the functional roleof this fraction in aflatoxin synthesis, storage, and export. Thisfraction could convert sterigmatocystin (ST), a late intermediatein aflatoxin biosynthesis, to aflatoxin B 1  and could compart-mentalize aflatoxin. To differentiate the roles of vesicles and vacuoles, we blocked vesicle-vacuole fusion using 2 independentapproaches; the data show that predominantly, vesicles catalyzethe final 2 steps in aflatoxin biosynthesis and compartmentalizeand export aflatoxin to the cell exterior. An increase in vesiclenumber [high vesicle number (HVN) phenotype] was positivelycorrelatedtoaflatoxin-inducinggrowthconditionsandaflatoxinaccumulation/export; the HVN phenotype was inversely corre-lated with down-regulation of   A. parasiticus avaA  and  vps16  geneexpression. Finally we show that  Velvet  (13–15), a key globalregulator of   Aspergillus  secondary metabolism, helps to mediate Authorcontributions:A.C.,L.V.R.,andJ.E.L.designedresearch;A.C.,L.V.R.,S.K.,andK.A.A.performed research; G.R.H., N.V.R., and A.M.C. contributed new reagents/analytic tools;A.C., L.V.R., and J.E.L. analyzed data; and A.C., L.V.R., and J.E.L. wrote the paper.The authors declare no conflict of interest.This article is a PNAS Direct Submission.Freely available online through the PNAS open access option.Data deposition: The nucleotide sequence for  Aspergillus parasiticus vb1  ( avaA ) has beendeposited in GenBank (accession no. AY52045). 1 Present address: International Environmental Research Center, Gwangju Institute ofScience and Technology, Gwangju, South Korea. 2 To whom correspondence should be addressed. E-mail: jlinz@msu.edu.This article contains supporting information online at www.pnas.org/cgi/content/full/ 0907416106/DCSupplemental. www.pnas.org  cgi  doi  10.1073  pnas.0907416106 PNAS    November 17, 2009    vol. 106    no. 46    19533–19538      M     I     C     R     O     B     I     O     L     O     G     Y  the observed changes in aflatoxin gene expression and vesiclenumber. Results High Vesicle Number Phenotype Is Positively Correlated with Afla-toxin Synthesis.  Yeast extract sucrose (YES) is an aflatoxin-inducing growth medium, and yeast extract peptone (YEP) is anoninducing medium; these media have the same compositionexcept that peptone in YEP replaces sucrose as carbon source.Under standard growth conditions (100 mL YES liquid shakeculture, batch fermentation)  A. parasiticus  initiates aflatoxinsynthesis between 24 and 30 h, and by 40 h, synthesis occurs atpeak levels (16). Aflatoxin enzymes and transcripts are alsodetected between 30 and 40 h (16) in roughly the same order asthe genes in the aflatoxin cluster. Under standard aflatoxin-inducing conditions (YES), we observed a 5-fold increase in vesicle number (HVN) from 24 to 40 h in  A. parasiticus  SU-1(Fig. 1 and Table 1) as aflatoxin increased up to 11-fold (16). Incontrast, no change in vesicle number [low vesicle number(LVN) phenotype] was evident under standard aflatoxin-noninducing conditions (YEP) (Fig. 1). The fungus was thengrown in YES or YEP for 36 h and transferred to the oppositemedium (nutritional shift). Within 6 h after transfer from YESto YEP, we observed a 2-fold decrease in vesicle number thatprecededadeclineinaflatoxinaccumulation;transferfromYEPto YES resulted in a 2-fold increase in vesicle number precedingan increase in aflatoxin accumulation (Fig. 1). Vesicles and Vacuoles Are One Primary Site for the Late Steps inAflatoxin Synthesis and Storage. Wepreviouslyobservedthatearly(Nor-1), middle (Ver-1), and late (OmtA) aflatoxin enzymeslocalize in vesicles and vacuoles in  A. parasiticus  (6–8). Todetermine the functional role of these organelles in aflatoxinsynthesis, a vesicle-vacuole fraction (V) was first isolated from  A. parasiticus  grown for 36 h in aflatoxin-inducing medium (YES)using a high-density sucrose cushion method.Next, we performed feeding experiments (using a protocoldescribed previously) (17) with protoplasts, the vesicle-vacuolefraction (V), and the nonvacuole fraction (NV) (contains theremaining cell materials) in vitro with a late pathway interme-diate sterigmatocystin (ST) to test whether these could convertST to aflatoxin B 1  (AFB 1 ).In protoplasts (Fig. 2  A ) fed ST, aflatoxin increased 6-fold; without added ST, aflatoxin increased 2-fold (Fig. 2 C ). Analo-gous feeding experiments were performed with protoplastsobtained from 2 strains impaired in aflatoxin synthesis, including AFS10 (gene disruption in a positive pathway regulator,  aflR ; noaflatoxin enzymes or aflatoxin are synthesized) and LW1432(mutations in 2 pathway genes  omtA  and  ver-1 ; accumulates thepathway intermediate versicolorin A). An increase in AFB 1  wasnot observed in these strains, strongly suggesting that incorpo-ration of ST into AFB 1  in SU-1 was dependent on enzymeactivities contained within the protoplast. The vesicle-vacuolefraction purified from protoplasts that had been fed ST carriedthe vast majority of the aflatoxin as compared with the non- vacuole fraction (Fig. 2  D ). ST fed directly to the vesicle-vacuolefraction increased aflatoxin more than 5-fold (Fig. 2  E ). Incontrast, ST fed to the nonvacuole fraction resulted in nearly Fig. 1.  Positive correlation between aflatoxin biosynthesis and high vesiclenumberphenotype.  A.parasiticus SU-1wasgrownfor24or40hinYESorYEPmedia; vesicle-vacuole morphology was analyzed by light microscopy andaflatoxin per flask was analyzed by ELISA (see  Methods ). (  A ) Bright-fieldmicroscopy of  A. parasiticus  grown in aflatoxin-inducing (  sucrose, YES) or-noninducingconditions(  sucrose,YEP).(Scalebar:5  m.)( B )Ratioofvesiclenumber to vacuole number (vesicle:vacuole ratio) calculated at 24 or 40 h inYEPorYESmedium.( C  )Nutritionalshift:SU-1wasgrowninYESorYEPgrowthmedium for 36 h. Mycelia were harvested and transferred to the oppositemedium, and vesicle:vacuole ratio was calculated before ( B ) and 6 h after (  A )media shift. Table 1. Fungal stains, genotypes, and vesicle number under aflatoxin-inducing and -noninducing conditions Fungal strains and genotype  Sucrose (YES)   Sucrose (YEP) v V v/V v V v/V  SU-1 (wild type; NRRL5862) 69  8 11  5 8.3  4.5 12  3 21  4 0.6  0.2SU-1 treated with Sortin3 70  5 12  5 7.5  3.5 56  6 10  3 6.4  2.5AFS10 ( aflR ) a 59  6 14  3 4.5  2.5 14  5 20  1 0.7  0.3ATCC 36595 ( ver-1 ) b 63  6 15  4 4.7  1.6 12  3 17  4 0.8  0.4  veA  ( ver-1 wh-1 veA ) c 19  4 18  1 1.1  0.3 15  3 21  5 0.8  0.3  veA  treated with Sortin3 62  5 9  2 9.6  2.2 65  6 11  2 6.2  1.7TJYP1–22 ( brn nor1 fadA G42R ) d 57  4 11  2 5.5  1.3 21  4 23  5 1.0  0.4NR1 ( niaD ) b 60  4 11  3 6.0  2.0 12  4 22  4 0.6  0.3  vb1 e (this work) f 73  10 10  4 9.1  4.7 70  8 7  1 10.0  2.6AC34 ( niaD   ) (this work)  66  7 14  5 5.6  2.5 13  3 21  5 0.7  0.3 Fungal strains were grown for 40 h in either YES or YEP medium and then analyzed by light microscopy (see  Methods ).  v  , number of vesicles per 50   m ofmycelial length;  V  , number of vacuoles per 50   m of mycelial length. Strains provided by  a Jeff Cary;  b our laboratory;  c Anna Calvo;  d Nancy Keller. e AC11 was used as a representative of the  vb1  disruptant strains AC11, 5, and 7; this strain was labeled  vb1  in the text and figures for convenience. f Sortin3 treatment of SU-1 produces a similar vesicle phenotype as observed in AC11, AC5, and AC7. 19534    www.pnas.org  cgi  doi  10.1073  pnas.0907416106 Chanda et al.  undetectable levels of aflatoxin. These data suggested that the vesicle-vacuole fraction carries functional aflatoxin enzymes andcompartmentalizes aflatoxin. Blocking Vesicle-Vacuole Fusion Increases Aflatoxin Synthesis/Exportand Aflatoxin Enzyme Accumulation but Does Not Affect AflatoxinGene Expression.  In  A. parasiticus , the delivery of OmtA to vacuoles appears to occur through fusion of vesicles containingOmtA to vacuoles (8); this fusion event represents an importantlate step in vacuole biogenesis (18). To clarify which compart-ments (vesicles or vacuoles) associate functionally with aflatoxinsynthesis, we targeted the class C Vps tethering complex (Fig.3  A ) that mediates fusion of vesicles and other prevacuolarcompartments to vacuoles (18). We disrupted  A. parasiticus vb1 that encodes a homolog of Ypt7 (one member of the tetheringcomplex) in yeast and AvaA in  Aspergillus nidulans  that arenecessary for vacuole biogenesis;  ypt7   and  avaA  disruptionresults in fragmented vacuoles (19, 20). Vb1 contains 205 aa andis 70% identical to yeast Ypt7, 74% identical to mammalianRab7 GTPases, and 94% identical to  A. nidulans  AvaA.Disruption of   vb1  (supporting information (SI) Fig. S1) in  A. parasiticus  strain AC11 and 2 other genetically identical isolates(AC5 and AC7) generated a fragmented vacuole morphologyanalogoustothatseenwithdisruptionof   avaA in  A.nidulans .Wealso observed a HVN phenotype even under aflatoxin-noninducing growth conditions (YEP; Fig. 3  B ); however, AC5, AC7, and AC11 did not accumulate aflatoxin in YEP. Weconfirmed the presence of the  vb1  gene disruption at the level of DNA (Fig. S1) and mRNA (Fig. 3 C iv ). Under aflatoxin-inducing growth conditions (YES), AC11 and AC5 synthesized7-fold more total aflatoxin and exported aflatoxin to the cellexterior at significantly higher levels than SU-1 (Fig. 3 C i  and  ii ). AC11 also accumulated higher quantities of aflatoxin enzymesthan SU-1 (Fig. 3 C iii ); Vbs and OmtA increased 2-fold; Ver-1,3-fold; and Nor-1, 2.5-fold as determined by densitometry. Theincrease in Ver-1 and OmtA proteins occurred in the absence of a significant change in gene expression at the mRNA level (Fig.3 C iv ). In contrast to these data, the quantities of aflatoxin and vesicles were not statistically different from SU-1 in AC34, acontrol transformant in which  vb1  was not disrupted [confirmedat the DNA (Fig. S1) and mRNA level (Fig. 3 C iv )]. These dataconfirm that the process of transformation was not responsiblefor the changes in phenotype observed in AC11, AC7, and AC5.Sortin3 blocks the activity of Vps16 (21), another protein inthe class C Vps tethering complex in yeast (18) (Fig. 3  A ). Sortin3generates a fragmented vacuole morphology reminiscent of Ypt7 disruption and causes enhanced export of cellular hydro-lases (like carboxypeptidase Y, CPY) to the cell exterior (21).TreatmentofSU-1withSortin3resultedinahighvesiclenumberphenotype under aflatoxin-noninducing growth conditions(YEP) in agreement with  vb1  disruption (Fig. 3  A ). Sortin3treatment of SU-1 grown for 40 h in YES increased aflatoxinaccumulation 5-fold (Fig. S2  A ) and also increased accumulationof the aflatoxin enzymes Vbs (1.4-fold), Ver-1 (3.5-fold), andOmtA (1.5-fold) as compared with untreated cells (Fig. S2  B ).Based on these observations, we conclude that vesicles representone primary site for the late steps in aflatoxin synthesis, com-partmentalization, and export to the cell exterior; in contrast, vacuoles appear to play only a minor role in these processes. We Fig. 2.  Feeding experiments.  A. parasiticus  SU-1 was grown for 36 h in YESmedium, and protoplasts (P), a vesicle-vacuole fraction (V), and a nonvesicle-vacuole (NV) fraction were purified (see  Methods ). ST was then fed to P, V, orNV overnight and the quantity of aflatoxin measured using ELISA (see  Meth-ods ). (  A ) Bright-field microscopy of P fraction. (Scale bar: 25   m.) ( B ) Trans-mission electron microscopy of V fraction. (Scale bar: 50 nm.) [Fig. 2  A  and  B reproduced with permission from Chanda et al. (12) (copyright 2009,Elsevier).]( C  )FeedingSTtoPfraction.P  STandP-ST,protoplastsfedandnotfedwithST,respectively.( D )FeedingSTtoPfollowedbydetectionofaflatoxinin V and NV fractions. V(P  ST) and NV(P  ST), V and NV fractions obtainedfrom P fed with ST; V(P–ST) and NV(P–ST), V and NV fractions obtained fromprotoplasts not fed with ST. ( E  ) Feeding ST to fractions V and NV. V  ST andNV  ST, V and NV fractions fed with ST; V–ST and NV–ST, V and NV fractionsnot fed with ST. Two-tailed  P   values used to determine statistical significancewere calculated using an unpaired  t   test with sample size of 3 (aflatoxinmeasurements in each experiment were conducted in triplicate). Feedingexperiments were repeated 3 times with similar trends. Fig. 3.  Effect of blocking vesicle-vacuole fusion in  A. parasiticus . (  A ) Sche-maticrepresentationoftheexperiment.Tc,theclassCVpstetheringcomplex.Fusion of prevacuolar vesicles was blocked either by disruption of  vb1  or byinhibition with Sortin3 treatment. ( B ) Fungal strains were grown for 40 h in  sucrose (YEP), and vesicles were analyzed by light microscopy. (Scale bar: 5  m.) ( C  ) Effect of  vb1  disruption on aflatoxin biosynthesis. ( i  ) Aflatoxin inmycelium and medium per 100 mL of culture. ( ii  ) Aflatoxin export (calculatedasratioofaflatoxininthemediumtoaflatoxininmediumplusmyceliumina100 mL culture). ( iii  ) Accumulation of early (Nor-1), middle (Vbs, Ver-1), andlate (OmtA) aflatoxin enzymes. ( iv  ) Relative expression (RE) of  ver-1 ,  omtA , vb1 ,and  -tubulin . Two-tailed P  valuesfor( i  )and( ii  )werecalculatedusinganunpaired t  testwithsamplesizeof3(aflatoxinmeasurementswereconductedintriplicate).gDNA,genomicDNAfromSU-1.SeeTable2fordescriptionofRE. Chanda et al. PNAS    November 17, 2009    vol. 106    no. 46    19535      M     I     C     R     O     B     I     O     L     O     G     Y  hypothesize that vacuoles predominantly participate in aflatoxinenzymeturnover;thisissupportedbytheobservationthatblocking vesicle-vacuole fusion increases the quantity of aflatoxin enzymes(in the absence of increased transcription), possibly by preventingthem from reaching the vacuole for degradation. A HVN Phenotype Is Inversely Correlated with Downregulation of  vb1 and  vps16   Expression.  What drives the shift to a HVN phenotypeduring aflatoxin biosynthesis on sucrose? We hypothesized thatone possible mechanism is the down-regulation of vesicle- vacuole fusion via decreased activity of the class C tetheringcomplex. We analyzed  vb1  and  vps16  expression in  A. parasiticus SU-1 grown for 24, 30, and 40 h under aflatoxin-inducing (YES)and -noninducing (YEP) growth conditions.  vb1  and  vps16 transcript levels remained nearly constant in YEP. However, inYES, expression of both genes declined more than 2-fold from24 to 40 h (Table 2 and Fig. S3) in parallel with the observed increase in aflatoxin accumulation (16) and in vesicle number(Fig. 1). We hypothesize therefore that the observed shift to aHVN phenotype is functionally linked to the down-regulation of   vb1  and  vps16  expression. VeA Coregulates the Onset of Aflatoxin Gene Expression and the Shiftto a HVN Phenotype.  What are the molecular factors that partic-ipate in coregulation of aflatoxin gene expression and the shiftto a HVN phenotype? AFS10 (   aflR , see above) does notsynthesize aflatoxin or aflatoxin enzymes. Constitutive activa-tion of FadA, an alpha subunit of a heterotrimeric G protein in  A. parasiticus  TJYP1–22, exerts a negative regulatory influenceon aflatoxin synthesis and conidiation (22).  Velvet  protein,encoded by  veA , is a global regulator of secondary metabolismand development in Aspergilli in response to light (13–15); thegene is expressed and its gene product is active predominantly inthe dark. Disruption of   veA  in  A. parasiticus  (   veA ) blockssynthesis of aflatoxin pathway intermediates and aflatoxin,blocks sclerotia development, and impairs conidiation in thelight and dark (15). Aflatoxin synthesis is also blocked in  A. parasiticus  ATCC36537 that carries a mutation in the pathwaygene,  ver- 1 (15) (accumulates the pathway intermediate versi-colorin A). Although each of the 4 strains— AFS10, TJYP1–22,   veA , and ATCC36537—is impaired in aflatoxin gene expres-sion and/or aflatoxin enzyme accumulation, only   veA  failed toshift to a HVN phenotype under aflatoxin-inducing conditions(YES) at 48 h in the dark (Table 1 and Fig. S4). Wild-type SU-1 and the 3 other mutant strains did undergo this shift. However,Sortin3 treatment restored the HVN shift in    veA  in thepresence or absence of sucrose (Table 1 and Fig. S4). Ourobservations strongly suggest that  veA  helps to coordinate theshift to the HVN phenotype and the synthesis of aflatoxinenzymes. The data also suggest that in addition to globalregulation by VeA, the LVN to HVN shift and induction of aflatoxin gene expression can be regulated independently.How does VeA mediate the LVN-to-HVN shift? We analyzedVb1 and Vps16 expression at the RNA level in    veA . Weobserved a decrease in  vb1  and  vps16  expression in controls(SU-1 and ATCC36537) from 24 to 72 h under aflatoxin-inducing conditions, whereas expression of these genes in   veA remained nearly constant (Table 3; and Fig. S5). These data suggest that VeA regulates class C Vps complex activity bymodulating expression of tethering complex proteins. Discussion Our studies provide direct demonstration of the functionalsignificance for vesicles and vacuoles in fungal polyketide bio-synthesis and export. The current study shows that at least thelast 2 enzymatic steps in aflatoxin biosynthesis are completed in vesicles, and these organelles also participate in compartmen-talization and export of the end product, aflatoxin, into thegrowth medium. We hypothesize that aflatoxin enzymes likelycontinue to make aflatoxin until they are eventually turned overin vacuoles.Our data strongly suggest that the development of aflatoxin-synthesizing vesicles (aflatoxisomes) is functionally linked todown-regulation of   vb1  and  vps16  genes. VeA plays a novel andcritical dual role in this process by helping to coordinateregulation of aflatoxin gene expression and the shift to Table 2. Relative expression (RE) of  vb1 ,  vps16  , and  ver-1  in  A. parasiticus  grown inaflatoxin-inducing (  sucrose) or -noninducing (  sucrose) growth media for 24, 30,or 40 h Gene  Sucrose (YES)   Sucrose (YEP)24 h 30 h 40 h 24 h 30 h 40 h vb1  1.0 0.6 0.3 0.7 0.7 0.6 vps16   0.8 0.5 0.3 1.0 0.9 0.9 ver-1  0.1 1.0 1.0 0.0 0.0 0.0 Relative expression (RE) was determined by RT-PCR (see  Methods ). RE data for  vb1 ,  vps16  , and  -tubulin are provided. RE measures the relative signal intensity (RI) of RT-PCR products resolved byagarosegelelectrophoresis(seeFigs.S3andS5foragarosegelimages).RIiscalculatedusingtheratioof absolute intensity for a RT-PCR product generated from one time point as compared with thehighest absolute intensity recorded for any time point in which that gene was analyzed. As RE valuesdecrease, gene expression declines (and vice versa). Absolute intensity values were measured bydensitometry using Adobe Photoshop software. Table 3. Relative expression (RE) of  vb1 ,  vps16  , and   -tubulin  in SU-1, 36537, and  veA  grown in YES for 24, 40, and 72 h Fungal strain vb1 vps16    - tubulin24 h 40 h 72 h 24 h 40 h 72 h 24 h 40 h 72 hSU-1 1.0 0.5 0.4 1.0 0.4 0.3 0.9 1.0 1.0ATCC36537 1.0 0.3 0.3 1.0 0.4 0.6 1.0 1.0 1.0  veA  0.9 1.0 1.0 1.0 0.9 1.0 1.0 1.0 0.9 See footnote for Table 2 for description of RE. 19536    www.pnas.org  cgi  doi  10.1073  pnas.0907416106 Chanda et al.  aflatoxisome formation. Based on previous and current work, we propose a 2-branch model for coordination of subcellularcompartmentalization, gene regulation, and carbon flow as-sociated with aflatoxin biosynthesis in  A. parasiticus  (Fig. 4; seedetailed description in figure legend). This model represents alogical expansion of a previous model (16) focused on carbonsource regulation of aflatoxin synthesis. Understanding themechanisms that coregulate the activation of aflatoxin syn-thesis and the LVN-to-HVN shift to form aflatoxisomes is akey to development of efficient strategies to manipulatesecondary metabolism in general and aflatoxin synthesis spe-cifically. Our studies also tend to support the idea that Aspergilli, like other organisms with small genomes, useexisting conserved cellular machinery (vesicle trafficking) toconduct new cellular functions (aflatoxin synthesis).We know that VeA activity is light sensitive; the protein isexpressed at highest levels and localizes to the nucleus in thedark (13, 14, 15). VeA is also reported to form a complex withseveral proteins and may play a role in chromatin remodeling(13). We therefore hypothesize that on the soil surface, littleVeA is expressed or active, and low levels of aflatoxin accu-mulate. However, deeper in the soil where light does notpenetrate, VeA mediates an increase in aflatoxin production.This in turn protects the colony and sclerotia from predationby worms and insects [building on recent studies by Rohlfs etal. (23)] and hence helps  A. parasiticus  to survive in itsecological niche.The use of an endomembrane system to compartmentalizeproteins, substrates, intermediates, and products appears to bea common feature in eukaryotic secondary metabolism (1–8, 24,25). Another illustration of compartmentalization of pathwayenzymes in fungal secondary metabolism is the biosynthesis of penicillin (an amino acid-derived secondary metabolite) in  Penicillium chrysogenum  [reviewed recently by Evers et al. (25)]and  A. nidulans  (28). Although it is clear that the penicillinpathway enzymes localize to unique subcellular compartments[Golgi-derived vesicles (26), vacuoles (5), cytosol (25), andperoxisomes (25, 27, 28)], the data do not directly demonstratethe functional role of these compartments in penicillin biosyn-thesis, storage, or export. An interesting feature of penicillinbiosynthesis is that carbon (  -aminoadipate) flows from vacu-oles to the cytoplasm to the peroxisome before exiting the cell.In contrast, carbon (acetyl-CoA) appears to flow in aflatoxinbiosynthesis from peroxisome to the vesicle and then to the cellexterior (Fig. 4). Maggio-Hall et al. (24) reported that per-oxisomes supply at least part of the acetyl-CoA required foraflatoxin synthesis and demonstrated that norsolorinic acid(NA) accumulates in peroxisomes in a  nor-1  mutant strain.These data suggest that the early steps in aflatoxin synthesis(before averantin) may occur in this location. The increasedaccumulation of Nor-1, Ver-1, Vbs, and OmtA levels thatresults from a block in the fusion of vesicles with vacuoles(current study) implies that all subsequent steps occur inaflatoxisomes. Previous data from our lab and others stronglysuggest that peroxisomes are not part of the purified vesicle- vacuole fraction (12).During the course of these studies, we generated  vb1  genedisruption mutant strains to analyze the role of this gene and theclass C tethering complex in vesicle fusion and aflatoxin syn-thesis.  A. parasiticus  Vb1 exhibits 94% identity with  A. nidulans  AvaA at the amino acid level, and the phenotypes generatedupon disruption of   avaA  and  vb1  are very similar. We hypoth-esize that  vb1  and  avaA  are homologous members of thetethering complex; we also propose to rename  vb1 ,  avaA. We recently conducted preliminary analysis of the vesicle- vacuole proteome (the same fraction used for feeding experi-ments in the current study) using multidimensional proteinidentification technology (MudPIT) and detected   200 pro-teins, including 9 aflatoxin enzymes, suggesting that a largeportion of the aflatoxin pathway is present in this organelle. Insupport of this notion, Fas1, which catalyzes the first step in thepathway (synthesis of hexanoyl CoA), was among these 9 en-zymes. Details of how vesicles and vacuoles undergo morpho-logical, chemical, and functional development as cells switch tosecondary metabolism will be elucidated in our future studiesusing proteome analysis. Methods Strains, Media, and Growth Conditions.  The strains used in this study are listedin Table 1 (see  SI Methods  for generation of  vb1  strains). YES liquid medium[contains 2% yeast extract and 6% sucrose (pH 5.8)] was used as an aflatoxin-inducing growth medium, and YEP liquid medium [contains 2% yeast extractand 6% peptone (pH 5.8)] was used as an aflatoxin-noninducing medium.Sterigmatocystin(ST)usedforfeedingexperimentswasobtainedfromSigma.Sortin3 was obtained from DIVERSetE (Chembridge). Sortin3 treatment wasconductedwithestablisheddosages(21).Fungalgrowthwasconductedinthedark. Feeding Experiments.  Feeding experiments (see  SI Text  ) were conducted 3times with similar trends. Aflatoxin was measured by ELISA using standard Fig.4.  Two-branchmodel:regulationofsubcellularcompartmentalization,aflatoxin gene expression, and carbon flow. Compartmentalization (branch1):themodelproposesthatNor-1(early),Ver-1(middle),andOmtA(late)aresynthesized on free ribosomes in the cytoplasm, packaged into transportvesicles (6–8), and transported to vacuoles via the cytoplasm-to-vacuole tar-geting (Cvt) pathway (6, 8). In contrast Vbs localizes in the cytoplasm and instructuresthoughttobeGolgi(29),suggestingitistransportedtovacuolesviathe classical secretory pathway; VBS is glycolsylated in support of this notion.Gene regulation (branch 2): sensing of glucose concentration or glucosemetabolism initiates a FadA/cAMP/PKA signaling pathway (16); details ofmechanismforsensingormetabolismofsucrosearenotknown.Carbonflowhighlighted in red. Mitochondria and peroxisomes supply acetyl-CoA (24) topolyketide synthesis. The occurrence of early steps in aflatoxin synthesis(acetyl-CoA 3  norsolorinicacid)inperoxisomeswasproposedbyothers(24).Inthe current study, we show the function of late pathway enzymes in aflatoxi-genic vesicles (aflatoxisomes) that also participate in aflatoxin export. Ourdata also suggest the presence and activity of early and middle pathwayenzymesinaflatoxisomes.Coordinationof2branches(currentstudy).Atleast2 separate signals (carbon source and light) trigger VeA activity that coordi-nates regulation of the 2 branches involved in aflatoxin synthesis/export(denoted by thick black arrows). One branch up-regulates gene transcriptionviaactivationofgeneralandspecifictranscriptionfactors(reviewedinref.10).The second branch down-regulates class C Vps tethering complex (Tc) activity(current study), resulting in accumulation of transport vesicles. When bothbranches are operational, aflatoxin enzymes accumulate in aflatoxisomeswhere they carry out aflatoxin synthesis and export the toxin to the cellexterior. Hypothesized pathways denoted by dashed lines; known pathwaysdenoted by solid lines. PM, plasma membrane. Chanda et al. PNAS    November 17, 2009    vol. 106    no. 46    19537      M     I     C     R     O     B     I     O     L     O     G     Y
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