Duplication and Functional Divergence of Branched-Chain Amino Acid Biosynthesis Genes in Aspergillus nidulans

ABSTRACT Fungi, bacteria, and plants, but not animals, synthesize the branched-chain amino acids: leucine, isoleucine, and valine. While branched-chain amino acid (BCAA) biosynthesis has been well characterized in the yeast Saccharomyces cerevisiae, it is incompletely understood in filamentous fungi. The three BCAAs share several early biosynthesis steps before divergence into specific pathways. In Aspergillus nidulans, the genes for the first two dedicated steps in leucine biosynthesis have been characterized, but the final two have not. We used sequence searches of the A. nidulans genome to identify two genes encoding β-isopropylmalate dehydrogenase, which catalyzes the penultimate step of leucine biosynthesis, and six genes encoding BCAA aminotransferase, which catalyzes the final step in biosynthesis of all three BCAA. We have used combinations of gene knockouts to determine the relative contribution of each of these genes to BCAA biosynthesis. While both β-isopropylmalate dehydrogenase genes act in leucine biosynthesis, the two most highly expressed BCAA aminotransferases are responsible for BCAA biosynthesis. We have also characterized the expression of leucine biosynthesis genes using reverse transcriptase-quantitative PCR and found regulation in response to leucine availability is mediated through the Zn(II)2Cys6 transcription factor LeuB.

role (18). Two A. nidulans b-IPM dehydrogenase enzymes, encoded by AN0912 and AN2793, were identified in BLASTp searches with S. cerevisiae Leu2p as the query. AN0912 and AN2793 showed high levels of similarity and identity with Leu2p, Leu2A, and Leu2B, with AN0912 most similar to Leu2A and AN2793 most similar to Leu2B (Table 1). AN0912 and AN2793 showed 50.5% identity and 67.3% similarity with each other. Alignment of the five proteins revealed strong conservation throughout the protein, including in the substrate binding loop and NAD binding motif (see Fig. S1 in the supplemental material). We designated AN0912 leuD and AN2793 leuE. leuD is found on chromosome VIII in a region of highly conserved gene colinearity in all 27 Aspergillus species genomes available at FungiDB (Fig. S2A). In contrast, leuE is located on chromosome VI and lacks colinearity with its 24 orthologs in the 27 Aspergillus species (Fig. S2B).
We investigated the relationships of the two A. nidulans b-IPM dehydrogenase genes through construction of a phylogenetic tree (Fig. 2). LeuD and LeuE formed distinct clades with their respective Aspergillus orthologs. The LeuD clade is consistent with the position of A. nidulans in the fungal evolutionary tree (37), whereas the LeuE clade lies between the Ascomycota and the Basidiomycota clades.
leuD and leuE both function in leucine biosynthesis. To determine whether leuD and leuE are functional genes, we generated deletion mutants by gene replacement (Fig. S3A; see Materials and Methods). Deletion of genes required for leucine biosynthesis results in leucine auxotrophy (19,27), yet neither leuDD nor leuED strain conferred strict  , which generate a-ketoisocaproate (KIC), and two BCAA aminotransferases (BatA and BatB), which also function in isoleucine and valine biosynthesis and isoleucine, leucine, and valine (ILV) catabolism, were characterized in this work from eight candidate coding genes.
Leucine Biosynthesis in Aspergillus nidulans ® leucine auxotrophy (Fig. 3A). However, while the leuED strain grew similarly to the wild type in the absence of leucine, the leuDD mutant showed reduced growth compared with the wild type unless supplemented with exogenous leucine. Transformation of the leuD gene into the leuDD mutant restored leucine prototrophy (Fig. S4A). To determine whether the leaky nature of the leuDD leucine auxotrophy resulted from LeuE activity, we constructed a leuDD leuED double mutant by meiotic crossing and found that the double mutant was a strict auxotroph, showing growth only when supplemented with exogenous leucine (Fig. 3A). Leucine supplementation of a C. neoformans auxotroph lacking a-IPM isomerase is possible when glutamine or asparagine, but not ammonium, is the nitrogen source (6). In contrast, the leuCD mutant lacking a-IPM isomerase can be supplemented on ammonium (27). Likewise, the leuDD leuED leucine auxotrophy could be supplemented on the preferred nitrogen sources ammonium and glutamine and on the alternative nitrogen source nitrate (Fig. 3A). Therefore, regulation of leucine uptake in A. nidulans is not regulated by nitrogen metabolite repression.
To complement the tight leucine auxotrophy of the leuDD leuED double mutant, we introduced a plasmid carrying the wild-type leuE gene and directly selected transformants in the absence of leucine (Fig. S4B to D). Single-copy integration conferred partial leucine auxotrophy that resembled the leuDD single mutant, whereas multicopy transformants showed stronger growth, indicating that additional copies of the leuE gene partially suppress the leuDD phenotype. We next considered whether levels of expression were the source of the different degrees of effect of leuDD and leuED. We  found, using reverse transcription-quantitative PCR (RT-qPCR), that leuD had ;64-fold higher expression than leuE after 16 h of growth in 10 mM ammonium-minimal medium. In transcriptome sequencing (RNA-seq) data from wild-type mycelia, leuD showed higher expression than leuE when grown on ammonium (35-fold), alanine (12fold), and glutamine (13-fold) (Fig. 3B). As leucine production is regulated by feedback inhibition, we examined the effect of the leuDD mutation on expression of leuE and two other leucine biosynthesis genes, luA and leuC, by RT-qPCR, and gdhA, which is coregulated with leucine biosynthesis, using enzyme activity of LacZ expressed from the gdhA-lacZ translational fusion reporter gene (19,27). For all three leucine biosynthesis genes, and for gdhA-lacZ, we found that leuDD resulted in increased expression over wild-type levels ( Fig. 3C and D). Therefore, reduced leucine production as a result of leuDD results in compensation by upregulation of leuE and the other leucine biosynthesis genes as well as gdhA.
As leuED had no effect on growth and leuE upregulation in the leuDD deletion mutant is expected to be LeuB dependent, we constructed a leuBD leuDD double mutant (Fig. 3A). In contrast to the leuBD and leuDD single mutants, which are leaky leucine auxotrophs, the leuBD leuDD double mutant is a strict leucine auxotroph, suggesting , and leuBD leuDD (RT460) strains were grown at 37°C for 2 days on solid supplemented ANM with or without 2 mM leucine (Leu) and with 10 mM ammonium (NH 4 ), glutamine (Gln), and nitrate (NO 3 ) as the nitrogen source. Note that the yellow colony color of RT460 is due to the yA1 conidial color mutation and is unrelated to the leuBD leuDD phenotype. (B) Mean reads per kilobase per million mapped reads (RPKM) from RNA-seq of MH1 grown at 37°C for 16 h in supplemented liquid ANM with 10 mM ammonium (NH 4 ), glutamine (Gln), and alanine (Ala). (C) RT-qPCR quantification of mean fold change in transcript expression in leuDD (RT411) strain compared to the wild type (MH1) grown at 37°C for 16 h in supplemented liquid ANM-10 mM ammonium and 2 mM leucine. Bars indicate the mean fold change from three independent biological replicates (circles). *, P # 0.05. NS, not significant using two-tailed Student's t test with equal distribution. (D) LacZ specific activity for wild-type (MH12101), leuBD (MH12181), leuDD (RT458), and leuBD leuDD (RT460) strains, which contain the 2753 bp gdhA-lacZ reporter construct. Strains were grown at 37°C for 16 h in supplemented liquid ANM with 10 mM ammonium and 2 mM leucine (n = 3). *, P # 0.05; **, P # 0.001; ***, P # 0.0001; NS, not significant; using one-way ANOVA. For panels B to D, error bars depict standard error of the mean (N = 3).
Leucine Biosynthesis in Aspergillus nidulans ® that LeuB regulation of leuE is required for leucine biosynthesis in the absence of leuD. We assayed gdhA-lacZ reporter gene expression in the double mutant (Fig. 3D). Unlike the single leuDD mutant, there was no increase in expression above leuBD levels in the double mutant, consistent with the leuDD-induced upregulation of leucine biosynthesis genes occurring through LeuB.
Identification of six branched-chain amino acid aminotransferase genes. The final step in leucine biosynthesis, catalyzed by the BCAA aminotransferase (BAT), is common to isoleucine and valine biosynthesis (Fig. 1). In S. cerevisiae, BAT enzymes are encoded by two genes, BAT1 and BAT2 (38,39). Six BAT enzymes predicted to catalyze this step have been previously identified in A. nidulans (20). We confirmed the identity of these six BATs, and their coding genes, using BLASTP analysis and designated them BatA (AN4323), BatB (AN5957), BatC (AN7878), BatD (AN7876), BatE (AN0385), and BatF (AN8511). Pairwise protein sequence comparisons with Bat1p and Bat2p revealed .21% identity and .31% similarity to both proteins ( Table 2). Alignment of these eight proteins showed strong conservation of NAD cofactor binding residues and absolute conservation of the catalytic lysine residue (Fig. S5). The two S. cerevisiae BATs function in different subcellular compartments. Bat1p is primarily targeted to mitochondria, whereas Bat2p is cytoplasmic (39). To predict the subcellular location of the six A. nidulans BAT enzymes, we used DeepLoc-1.0, TargetP v1.1, and Predotar targeting signal predictions (40)(41)(42)(43). For all three algorithms, BatA and BatC, like Bat1p, were predicted to be predominantly mitochondrial, and the remaining BAT enzymes were predicted by DeepLoc-1.0 to localize in the cytoplasm (Data Set S1). The BAT protein alignment revealed that Bat1p, BatA, and BatC have extended N termini containing a predicted mitochondrial targeting signal (Fig. S5).
We examined the colinearity of genes surrounding each of the six A. nidulans BATencoding genes to identify orthologous genes (Fig. S6). batA and batB orthologs are conserved in regions of high colinearity in all 27 species. batE orthologs are found in a region of moderate colinearity in 13 species. In contrast, batC, batD, and batF were located in regions lacking colinearity. batD only had orthologs in A. niger and A. oryzae, whereas batC and batF have no predicted ortholog. Interestingly, two of the BATencoding genes, batC and batD, are separated by just 2 kbp within the aspercryptins secondary metabolite gene cluster (44)(45)(46)(47). The tight physical linkage of these two genes suggests that they arose from gene duplication by unequal crossover and, therefore, would show high sequence homology. However, the proteins encoded by these genes are highly diverged, showing only 28.9% protein sequence identity.
To determine the relationship of the six A. nidulans BATs, we performed phylogenetic analysis (Fig. 4). The BATs formed two distinct groups within the fungi. Group I, the larger group containing 37 out of 52 of the fungal BATs, included BatA, BatB, BatC, and S. cerevisiae Bat1p and Bat2p, as well as at least one protein from every other fungus examined. Group II was a smaller group, with only 15 of the 52 proteins, and was almost entirely composed of BAT enzymes from Pezizomycotina genera (Aspergillus, Penicillium, Fusarium, Neurospora, Magnaporthe) and lacked any Saccharomycotina genera (Saccharomyces, Candida). Notably, BatC is in group I and BatD is in group II, consistent with separate recruitment to the aspercryptins cluster. Genetic analysis of six A. nidulans BATs. The expansion of the number of BATencoding genes in A. nidulans indicates specialization for the production of isoleucine, leucine, or valine by specific BATs or the evolution of completely new roles. To determine which BAT-encoding genes were required for BCAA biosynthesis, we constructed individual knockout mutants of each of the six BATs ( Fig. S3B; see Materials and Methods). Growth tests of the six individual bat knockout mutants showed none were BCAA auxotrophs (Fig. 5A). Therefore, each of the six BATs is dispensable for BCAA biosynthesis. During this study, the two BAT genes found in the aspercryptins gene cluster batC (AN7878) and batD (AN7876) were published by others as atnH and atnJ, respectively, and are thought to be involved in biosynthesis of 2-aminocaprylic acid, 2-aminododecanoic acid, and 2-aminodecanoic acid, three unusual BCAAs that are components of aspercryptins (46,47).
Analysis of RNA-seq expression data from wild-type mycelia grown on ammonium, alanine, or glutamine (Fig. 6A) showed that batA has the highest expression under all three conditions. batB was the next most highly expressed and showed increased expression on alanine and glutamine compared to ammonium. batC, batD, and batE all showed intermediate expression levels, whereas batF was not expressed under these conditions. As batC and batD are involved in biosynthesis of unusual BCAAs (46,47), we focused on the other four BAT genes. We measured expression of batA, batB, batE, and batF using RT-qPCR of RNA prepared from samples grown on ammonium, alanine, or nitrate. batA, batB, and batE expression did not substantially change under these conditions (Fig. 6B).  batF was not expressed under these conditions, consistent with it being undetectable by RNA-seq. We constructed double, triple, and quadruple mutants combining batAD, batBD, batED, and batFD by meiotic crossing. The batAD batBD double mutant, which combined deletions of the two most related and highly expressed genes, was a strict BCAA auxotroph and could only grow if supplemented with all three BCAAs (Fig. 5B). Therefore, BatA and BatB are the major BAT enzymes for isoleucine, leucine, and valine (ILV) biosynthesis. The batAD batBD batED and batAD batBD batFD triple mutants and the batAD batBD batED batFD quadruple mutant showed BCAA auxotrophy identical to that of the batAD batBD double mutant. In contrast, all of the other double and triple mutants constructed, which contained a wild-type copy of either batA or batB, were BCAA prototrophs. We confirmed that introduction of either the batA or batB gene into the batAD batBD mutant restored BCAA prototrophy (Fig. S4E). We investigated whether Leucine Biosynthesis in Aspergillus nidulans ® loss of either batA or batB would cause a compensatory increase in expression of batB or batA, respectively. However, on ammonium, batA expression was not upregulated in the batBD mutant and batB expression was not upregulated in the batAD mutant (Fig. 6C). This indicates that the expression levels of either one of the major bat genes for BCAA biosynthesis is sufficient for prototrophy. We constructed leuBD batAD and leuBD batBD double mutants. These two double mutants showed leaky leucine auxotrophy similar to that of the leuBD single mutant, indicating that leuBD is epistatic to batAD and batBD (Fig. 6D).
In addition to their role in BCAA biosynthesis, BATs also form the first step in ILV catabolism (28). We examined expression of batA, batB, batE, and batF with ILV as the sole nitrogen source to determine their expression pattern during catabolic conditions (Fig. 6B). For both batA and batE, expression levels were similar under anabolic and catabolic conditions. However, batB levels were elevated substantially during ILV catabolism compared with biosynthetic growth conditions, suggesting that BatB is the predominant catabolic enzyme. batF expression was undetectable. During BCAA catabolic growth, neither batA nor batB expression showed compensatory upregulation in the batBD or batAD strain, respectively (Fig. 7A). We assessed whether mutants carrying single or multiple BAT gene deletions could utilize each BCAA as the predominant nitrogen source in the presence of lower levels of the other two BCAAs to supplement the auxotrophy (Fig. 7B). All six single BAT mutants could utilize the three BCAAs. Mutants lacking batB but not batA showed slightly reduced colony morphology compared with batB 1 strains. Notably, mutants lacking both batA and batB showed severely reduced growth on each of the BCAAs as a predominant nitrogen source, and the reduction in growth was greater on isoleucine and valine than on leucine. We also examined growth of the batAD and batBD single and double mutants on increasing concentrations of equimolar ILV and found that batBD shows reduced colony morphology compared with both wild-type and batAD strains but stronger growth than the batAD batBD double mutant (Fig. 7C). Therefore, BatA and BatB are the major BAT enzymes in A. nidulans for both BCAA biosynthesis and utilization. We did not observe a phenotype for batED or batFD mutant in BCAA catabolism. Transformation analysis of the batA or batB gene into the batAD batBD recipient repaired BCAA utilization to the wild-type phenotype (Fig. S4F).
Regulation of leucine biosynthesis pathway gene expression by LeuB. The transcription factor LeuB is thought to regulate leucine biosynthesis genes because the leuBD mutant is a leaky leucine auxotroph (19). To determine whether LeuB regulates these genes in response to leucine levels, we performed RT-qPCR on RNA isolated from mycelia grown with exogenous leucine, which represses LeuB activation (27), and in a leuBD strain (Fig. 8). leuB expression was not altered in response to leucine. The six genes we demonstrated to function in leucine biosynthesis, leuC, luA, leuD, leuE, batA, and batB, as well as batE, showed decreased expression in response to exogenous leucine and/or in the leuBD mutant compared to the wild type.

DISCUSSION
We have completed annotation of the A. nidulans leucine biosynthesis pathway and characterized the genes encoding enzymes for the final two steps. Our analysis has revealed divergence between aspergilli and yeast in the number of genes encoding the enzymes for each step. In S. cerevisiae, ketoisovalerate is converted to a-IPM by two a-IPM synthetases, Leu4p and Leu9p, which form homodimers and heterodimers that show differential sensitivity to leucine feedback inhibition (23)(24)(25)(26)31). In contrast, a single a-IPM synthetase gene exists in A. nidulans (27). a-IPM is converted to b-IPM by the isopropylmalate isomerase, which is encoded by a single gene in both S. cerevisiae (LEU1) and A. nidulans (luA) (19,21). b-IPM is then converted to ketoisocaproate by a single b-IPM dehydrogenase in S. cerevisiae, Leu2p (35, 36), but two enzymes, LeuD and LeuE, in A. nidulans. The final step in BCAA biosynthesis is catalyzed by BCAA aminotransferase (BAT). S. cerevisiae has two BAT genes (38,39). A. nidulans carries six BAT genes; however, primarily two, BatA and BatB, play major roles in ILV biosynthesis. Interestingly, the genes encoding the dimeric enzymes in the pathway, a-IPM synthetase (31), b-IPM dehydrogenase (48,49), and BAT (50), differ in copy number, whereas the nonduplicated gene for a-IPM isomerase is monomeric (51). The LEU4/LEU9 and BAT1/BAT2 gene duplications resulted from the ancestral whole-genome duplication (WGD) and exhibit functional diversification associated with the acquisition of fermentative metabolism (52).
The Aspergillus lineage did not experience an ancestral WGD, but alternative mechanisms have mediated gene duplication within the leucine biosynthesis pathway. The acquisition of additional copies of genes often leads to robustness via the evolution of new functions but in some cases confers fragility (52)(53)(54). We found that both leuD and leuE function in leucine biosynthesis, although leuE plays a lesser role based upon its low expression, the prototrophy of the leuED mutant, and the leaky leucine auxotrophy conferred by deletion of leuD. This gene duplication provides robustness in the form of redundancy, as perturbation of leucine biosynthesis by deletion of leuD resulted in LeuB-dependent upregulation of leuE and partial compensation of the leucine auxotrophy. Our functional analysis showed Leucine Biosynthesis in Aspergillus nidulans ® that each of the six A. nidulans BATs are dispensable. Combining BAT gene deletions, however, revealed that BatA and BatB are the major enzymes in both BCAA biosynthesis and utilization. BatA contains a mitochondrial targeting signal and shows higher biosynthetic expression, while the likely cytoplasmic BatB shows higher catabolic expression. Therefore, BatA and BatB are equivalent to mitochondrial and predominantly biosynthetic Bat1p and cytoplasmic and predominantly catabolic Bat2p in S. cerevisiae (50,55,56). BatA and BatB show redundancy in both biosynthesis and catabolism. BAT function is also distributed between two paralogs in Lachancea kluyveri, with one major biosynthetic BAT and both involved in aerobic metabolism (57). In contrast, Kluyveromyces lactis has just one BAT gene, which encodes a bifunctional enzyme for BCAA biosynthesis and degradation, and this is thought to be the ancestral type prior to the WGD and subfunctionalization of Bat1p and Bat2p in S. cerevisiae (50).
The dispensability of batC, batD, batE, and batF for BCAA biosynthesis and catabolism suggests evolution of novel roles. We showed that batE is regulated by leucine and LeuB, similar to other leucine biosynthesis genes, but expression levels are low and we did not observe a phenotype for the batED mutant. However, batE expression is induced during hypoxia in the absence of glucose-to-ethanol fermentation, in association with elevated BCAA biosynthesis that occurs as a mechanism to generate NAD 1 and survive anaerobic stress (20,58). BatE does not appear to contribute to BCAA metabolism under our normoxic growth conditions but may play a role during anaerobic stress. batC (atnH) and batD (atnJ) are members of the aspercryptin biosynthetic gene cluster, with presumed roles in transamination of the unusual BCAAs 2-aminocaprylic acid, 2-aminodecanoic acid, and 2-aminododecanoic acid (46,47). Aspercryptins contain three BCAAs (isoleucine or valine, 2-aminocaprylic acid, and 2-aminododecanoic or 2-aminodecanoic acid). Expression of batF was undetectable under our growth conditions or growth conditions used for RNA-seq by others (59). batF is adjacent to the terriquinone A (tdi) biosynthetic gene cluster and may also be associated with secondary metabolism (45,(60)(61)(62).
Regulation of leucine biosynthesis is best understood in S. cerevisiae where both activation and repression are mediated by the Zn(II) 2 Cys 6 transcription factor Leu3p (16). When leucine is abundant, it interacts with a-IPM synthetase, inhibiting its function, which decreases cellular a-IPM levels and leads to Leu3p acting as a repressor (16,24). When leucine levels decrease, a-IPM synthetase is not inhibited, and a-IPM interacts with Leu3p, causing a conformational change and resulting in Leu3p switching to an activator (16,30). We observed repression by exogenous leucine in wild-type cells of all six genes that function in leucine biosynthesis, luA, leuC, leuD, leuE, batA, and batB, as well as batE, indicating this feedback mechanism operates in A. nidulans. Deletion of the A. nidulans LEU3 ortholog leuB confers leaky leucine auxotrophy (19), which we have now shown is due to decreased expression of the leucine biosynthesis  genes. The leuDD mutant shows leaky leucine auxotrophy and increased expression of other leucine biosynthesis genes, which likely results from reduced cellular levels of the negative feedback mediator leucine and increased a-IPM inducer levels due to increased b-IPM levels increasing the reverse reaction rate carried out by the bidirectional a-IPM isomerase encoded by luA.
The absence of the leucine biosynthesis pathway in animals and the reduced virulence of leucine auxotrophs (4-6, 9, 33, 97) render leucine biosynthesis enzymes strong candidate targets for antifungals. Our studies of the genes in this pathway indicate that the feedback mechanisms and gene duplications present in the aspergilli must be considered in target selection to avoid increased LeuB-dependent expression of other leucine biosynthesis genes in response to an antifungal agent targeting this pathway. The strongest target would be a-IPM synthetase (LeuC), as reduced activity of this enzyme leads to decreased a-IPM and repression of leucine biosynthesis genes by LeuB (27). The benefit of targeting this step would be in cross regulation of nitrogen assimilation by reduced expression of gdhA and potentially reduced cellular glutamate and glutamine levels.
Overall, this study has completed the annotation of the genes required for leucine biosynthesis in A. nidulans and demonstrated regulation of the pathway genes by LeuB. We have found roles for leuD and leuE in leucine biosynthesis and for batA and batB in BCAA biosynthesis and catabolism. Roles for batC (atnH) and batD (atnJ) in aspercryptins production have now been reported (46,47), but the roles of batE and batF remain to be determined.

MATERIALS AND METHODS
A. nidulans strains, media, and genetic analysis. A. nidulans strains and genotypes are listed in Table 3 using conventional nomenclature (98). A. nidulans growth conditions and media were as described previously (63,64). Aspergillus nitrogen-free minimal medium (ANM), pH 6.5, containing 1% (wt/vol) glucose as the sole carbon source, was supplemented for auxotrophies and nitrogen sources (10 mM final concentration), unless otherwise stated. A. nidulans growth testing and genetic analysis were as described previously (64).
Standard molecular techniques. Escherichia coli NM522 cells [F9 proA 1 B 1 lacI q D(lacZ)M15/D(lac-proAB) glnV thi-1 D(hsdS-mcrB)5] (65) were employed for molecular cloning (66). Plasmid DNA was isolated using the Wizard Plus SV miniprep DNA purification kit (Promega). A. nidulans genomic DNA was isolated according to reference 67. PCR products and DNA fragments isolated from agarose gels were cleaned with the Wizard SV gel and PCR clean-up system (Promega). Restriction enzyme digestions (Promega, New England Biolabs), dephosphorylation with Arctic shrimp alkaline phosphatase (Promega), and ligations using T4 DNA ligase (Promega) followed the manufacturers' instructions. DNA was separated on 1 to 2% agarose gels by electrophoresis in 1Â Tris-acetate-EDTA (TAE) buffer. PCRs used Ex Taq (TaKaRa), Phusion (Finnzymes), or AccuStart II Geltrack PCR supermix (Quanta Biosciences) DNA polymerases according to the manufacturers' instructions, with 1 ng plasmid or 100 ng A. nidulans genomic DNA templates. All reactions followed recommended denaturing and annealing conditions with 33 to 36 amplification cycles. Oligonucleotide PCR primers (Integrated DNA Technologies) are described in Table S1 in the supplemental material. DNA sequencing to confirm correct amplifications and cloning was performed at the Kansas State University DNA Sequencing and Genotyping Facility. Southern hybridizations used either Hybond N1 or Hybond XL membranes (GE Healthcare) and the DIG (digoxigenin) high prime DNA labeling and detection starter kit II (Roche) by following the manufacturer's instructions.
Strain construction. A. nidulans transformation was performed as described previously (27) using the nkuAD nonhomologous integration-defective mutant for targeted integration (68). Knockout constructs, generated by the A. nidulans whole-genome gene deletion constructs program (69), were sourced from the Fungal Genetics Stock Center, Manhattan, KS (70), and were transformed into MH11068 (pyrG89 nkuAD::Bar) and selected for uracil and uridine prototrophy to generate leuDD (AN0912D; RT411, D27 to 11,431 bp), leuED (AN2793D; RT413, D26 to 11,233 bp), batAD (AN4323; RT415, D165 to 11,722 bp), batBD (AN5957D; RT440, D125 to 11,395 bp), batCD (AN7878D; RT475, D210 to 11,222 bp), batDD (AN7876D; RT419, D27 to 11,297 bp), batED (AN0385D; RT417, D127 bp to 1,302 bp), and batFD (AN8511; RT441, D29 to 11,230 bp) strains. Selection media for leuDD and leuED transformants were supplemented with 2 mM leucine, and BAT gene deletion transformants were supplemented with ILV (2 mM each). The Aspergillus fumigatus pyrG (AfpyrG) marker showed position effect in the batCD mutant, incompletely complementing the pyrimidine auxotrophy of the pyrG89 mutation. Full complementation of pyrG89 by AfpyrG was observed in the other deletion mutants generated in this study. Pyrimidine supplementation was used in all growth tests. All deletion mutants were confirmed by Southern blotting as a single homologous double-crossover integration at the correct locus by probing with the 982-bp KpnI-SspI fragment of AfpyrG 1 (data not shown). Meiotic crossing was used to generate double, triple, and quadruple mutants. The presence of each deletion in the progeny of crosses was confirmed by diagnostic Southern blotting or diagnostic PCR. The leuDD mutant was repaired by introduction of a wild-type leuD PCR product (2960 to 13216) amplified from MH1, with direct selection for simultaneous resistance to 1 mg ml 21  complemented with the plasmid pJS249, which carries leuE (2913 to 12877) PCR amplified from MH1 and cloned into pGEMTeasy by transformation with direct selection for leucine prototrophy. The batAD batBD double mutant was complemented with the wild-type batA (2717 to 12558) or batB (2725 to 12187) gene using plasmids (pJS244 and pJS255, respectively) containing PCR-amplified DNA from MH1 cloned into pGEMTeasy. Transformants were directly selected for growth in the absence of exogenous ILV. b-Galactosidase assays. b-Galactosidase assays were performed as described previously (71) using soluble protein extracts. b-Galactosidase specific activity is defined as A 420 Â 10 3 min 21 mg 21 of soluble protein. Protein concentrations were determined using Bio-Rad assay reagent (Bio-Rad).
RNA preparation. Total RNA was isolated by grinding mycelia under liquid nitrogen and subsequent addition to RNA extraction buffer (7.0 M urea, 100 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1.0% sodium dodecyl sulfate) followed by two phenol-chloroform-isoamyl alcohol extractions and one chloroform extraction (66). RNA was precipitated in 3 M ammonium acetate and 50% isopropanol, resuspended in diethyl pyrocarbonate-H 2 O, and reprecipitated overnight in 4 M lithium chloride at 220°C. RNA quality was determined by visualization after electrophoretic separation in a 1.2% agarose gel containing 1.1% formaldehyde run in 1Â morpholinepropanesulfonic acid (MOPS) buffer (20 mM MOPS, pH 7.0, 5 mM sodium acetate, 1 mM EDTA). RQ1 DNase (Promega) treatment of RNA followed the manufacturer's instructions.
RT-qPCR. For reverse transcriptase-quantitative PCR (RT-qPCR), cDNA was produced using the reverse transcriptase system (Promega) or qScript cDNA supermix (Quanta Biosciences). RT-qPCR used a MyiQ thermocycler (Bio-Rad) with iTAQ universal SYBR green supermix (Bio-Rad), and results were analyzed with iQ5 v2.1 (Bio-Rad). Fold change was calculated using the DDC T method with b-tubulin-encoding benA as the reference gene (72)(73)(74). Primers (IDT) were designed to specifically amplify cDNA by overlapping a splice junction. Primer sequences used for RT-qPCR, target regions, and efficiencies are listed in Table S2.