An extended physical map of the TOX2 locus of Cochliobolus carbonum required for biosynthesis of HC-toxin

In genetic crosses, HC-toxin production in the filamentous fungus Cochliobolus carbonum appears to be controlled by a single locus, TOX2. At the molecular level, TOX2 is composed of at least seven duplicated and coregulated genes involved in HC-toxin biosynthesis, export, and regulation. All copies of four of the TOX2 genes were previously mapped within a 540-kb stretch of DNA in strain SB111. Subsequently, an additional three TOX2 genes, TOXE, TOXF, and TOXG, have been discovered. In this paper we have mapped all copies of the new genes, a total of seven, and show that except for one of the two copies of TOXE, which was previously shown to be on a chromosome of 0.7 Mb in strain SB111, they are all linked to the previously known TOX2 genes within approximately 600 kb of each other on a chromosome of 3.5 Mb. We show here that this chromosome also contains at least one non-TOX2 gene, EXG2, which encodes an exo-beta1,3-glucanase. EXG2 is still present in strains that have undergone spontaneous deletion of up to approximately 1.4 Mb of the 3.5-Mb chromosome. The results contribute to our understanding of the complex organization of the genes involved in HC-toxin biosynthesis and are consistent with the hypothesis that a reciprocal chromosomal translocation accounts for the pattern of distribution of the TOX2 genes in different C. carbonum isolates.     

Murine Toxicity of Cochliobolus carbonum

Seventeen wild-type strains of the phytopathogenic fungus Cochliobolus carbonum, tested by intraperitoneal injection into mice, were lethal within 48 hr. The lethal effect appeared to be a toxic rather than an infectious process, because death occurred within 3 hr after injection of two of the isolates and heat-killed cultures were lethal. Assays of ascospore progeny from two crosses involving three isolates indicated that the toxic metabolites were under genetic control and quantitative regulation. Studies of the toxicological, cultural, and chemical characteristics of these three strains indicated that more than one murine toxin was present.     

Molecular Features Affecting the Biological Activity of the Host-Selective Toxins from Cochliobolus victoriae

The structures of the toxins produced by Cochliobolus victoriae, victorin B, C, D, E, and victoricine, have recently been established. These toxins and modified forms of victorin C were tested for their effect on dark CO₂ fixation in susceptible oat (Avena sativa) leaf slices. Half-maximal inhibition of dark CO₂ fixation occurred with the native toxins in the range of 0.004 to 0.546 micromolar. An essential component for the inhibitory activity of victorin is the glyoxylic acid residue, particularly its hydrated aldehyde group. Removal of glyoxylic acid completely abolished the inhibitory activity of victorin, and the reduction of the aldehydo group transformed the toxin into a protectant. Conversion of victorin to its methyl ester resulted in diminution of inhibitory activity to 10% of the original activity of the toxin, whereas derivatization of the ε-amino group of the β-hydroxylysine moiety resulted in a decrease of inhibitory activity to 1% of that of victorin C. However, the derivatized toxin retained its host selectivity. In addition, the opening of the macrocyclic ring of the toxin drastically reduced the inhibitory activity.     

Mutational analysis of beta-glucanase genes from the plant-pathogenic fungus Cochliobolus carbonum

Two new beta-glucanase-encoding genes, EXG2 and MLG2, were isolated from the plant-pathogenic fungus Cochliobolus carbonum using polymerase chain reaction based on amino acid sequences from the purified proteins. EXG2 encodes a 46.6-kDa exo-beta1,3-glucanase and is located on the same 3.5-Mb chromosome that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8-kDa mixed-linked (beta1,3-beta1,4) glucanase with low activity against beta1,4-glucan and no activity against beta1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multiple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg1/mlg2) were also constructed by sequential disruption and by crossing. Total mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and exg1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total beta1,3-glucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/exg2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a modest decrease in growth on beta1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.     

The cyclic peptide synthetase catalyzing HC-toxin production in the filamentous fungus Cochliobolus carbonum is encoded by a 15.7-kilobase open reading frame.

Race 1 of Cochliobolus carbonum, a fungal plant pathogen, owes its exceptional virulence on certain genotypes of maize to the production of HC-toxin, a cyclic tetrapeptide. Production of HC-toxin is controlled by a single known gene, TOX2. Race 1, but not races that do not make HC-toxin, contains two copies of a 22-kilobase (kb) region of chromosomal DNA that is required for HC-toxin biosynthesis and hence virulence. We have sequenced this 22-kb region and here show that it contains an open reading frame of 15.7 kb that encodes a multifunctional cyclic peptide synthetase of potential M(r)574,620. This gene, called HTS1, apparently contains no introns. The predicted gene product, HC-toxin synthetase (HTS), contains four amino acid-binding (adenylate-forming) domains that are highly similar to those found in other cyclic peptide synthetases and other adenylate-binding enzymes. The DNA sequence encodes tryptic peptides derived from two HC-toxin biosynthetic enzymes, HC-toxin synthetase 1 (HTS-1) and HC-toxin synthetase 2 (HTS-2), indicating that these two enzymes exist in vivo as part of a single polypeptide. Consistent with this, in some enzyme preparations antibodies against the enzyme HTS-2, which was originally purified as a protein with a subunit M(r) of 160,000, recognize a protein with an estimated subunit M(r) greater than 480,000.     

Morphogenic Regulation of Pathotoxin Synthesis in Cochliobolus carbonum

The fungus Cochliobolus carbonum causes leaf spot disease of maize. Highly virulent isolates of the pathogen produce a host-selective, peptide toxin that is active against susceptible genotypes of maize. Prior to infection, spores must germinate and differentiate appressoria, structures specialized for leaf penetration. Analysis of spore germination fluids by plasma desorption mass spectrometry, which allowed detection of as little as 0.5 ng toxin, revealed that spores induced to form appressoria in vitro synthesized and released the toxin at a time coincident with maturation of appressoria. Spores incubated under conditions that did not induce appressorium formation failed to produce toxin. These observations indicate that synthesis of the host-selective toxin, which is essential for successful pathogenesis of maize by C. carbonum, is regulated by infection-related morphogenesis.     

Targeted Mutants of Cochliobolus carbonum Lacking the Two Major Extracellular Polygalacturonases

The filamentous fungus Cochliobolus carbonum produces endo-α1,4-polygalacturonase (endoPG), exo-α1,4-polygalacturonase (exoPG), and pectin methylesterase when grown in culture on pectin. Residual activity in a pgn1 mutant (lacking endoPG) was due to exoPG activity, and the responsible protein has now been purified. After chemical deglycosylation, the molecular mass of the purified protein decreased from greater than 60 to 45 kDa. The gene that encodes exoPG, PGX1, was isolated with PCR primers based on peptide sequences from the protein. The product of PGX1, Pgx1p, has a predicted molecular mass of 48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an exoPG from the saprophytic fungus Aspergillus tubingensis. Strains of C. carbonum mutated in PGX1 were constructed by targeted gene disruption and by gene replacement. Growth of pgx1 mutant strains on pectin was reduced by ca. 20%, and they were still pathogenic on maize. A double pgn1/pgx1 mutant strain was constructed by crossing. The double mutant grew as well as the pgx1 single mutant on pectin and was still pathogenic despite having less than 1% of total wild-type PG activity. Double mutants retained a small amount of PG activity with the same cation-exchange retention time as Pgn1p and also pectin methylesterase and a PG activity associated with the mycelium. Continued growth of the pgn1/pgx1 mutant on pectin could be due to one or more of these residual activities.     

An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum.

We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.     

Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize.

A gene (PGN1) encoding extracellular endopolygalacturonase was isolated from the fungal maize pathogen Cochliobolus carbonum race 1. A probe was synthesized by polymerase chain reaction using oligonucleotides based on the endopolygalacturonase amino acid sequence. Genomic and cDNA copies of the gene were isolated and sequenced. The corresponding mRNA was present in C. carbonum grown on pectin but not on sucrose as carbon source. The single copy of PGN1 in C. carbonum was disrupted by homologous integration of a plasmid containing an internal fragment of the gene. Polygalacturonase activity in one transformant chosen for further analysis was 10% or 35% of the wild-type activity based on viscometric or reducing sugar assays, respectively. End product analysis indicated that the residual activity in the mutant was due to an exopolygalacturonase. Pathogenicity on maize of the mutant lacking endopolygalacturonase activity was qualitatively indistinguishable from the wild-type strain, indicating that in this disease interaction endopolygalacturonase is not required. Either pectin degradation is not critical to this interaction or exopolygalacturonase alone is sufficient.     

Inhibition of maize histone deacetylases by HC toxin, the host-selective toxin of Cochliobolus carbonum.

HC toxin, the host-selective toxin of the maize pathogen Cochliobolus carbonum, inhibited maize histone deacetylase (HD) at 2 microM. Chlamydocin, a related cyclic tetrapeptide, also inhibited HD activity. The toxins did not affect histone acetyltransferases. After partial purification of histone deacetylases HD1-A, HD1-B, and HD2 from germinating maize embryos, we demonstrated that the different enzymes were similarly inhibited by the toxins. Inhibitory activities were reversibly eliminated by treating toxins with 2-mercaptoethanol, presumably by modifying the carbonyl group of the epoxide-containing amino acid Aeo (2-amino-9,10-epoxy-8-oxodecanoic acid). Kinetic studies revealed that inhibition of HD was of the uncompetitive type and reversible. HC toxin, in which the epoxide group had been hydrolyzed, completely lost its inhibitory activity; when the carbonyl group of Aeo had been reduced to the corresponding alcohol, the modified toxin was less active than native toxin. In vivo treatment of embryos with HC toxin caused the accumulation of highly acetylated histone H4 subspecies and elevated acetate incorporation into H4 in susceptible-genotype embryos but not in the resistant genotype. HDs from chicken and the myxomycete Physarum polycephalum were also inhibited, indicating that the host selectivity of HC toxin is not determined by its inhibitory effect on HD. Consistent with these results, we propose a model in which HC toxin promotes the establishment of pathogenic compatibility between C. carbonum and maize by interfering with reversible histone acetylation, which is implicated in the control of fundamental cellular processes, such as chromatin structure, cell cycle progression, and gene expression.     

The effect of thiram concentration on the spore germination of isolates of Cochliobolus carbonum

Inhibition of spore germination of selected isolates ofCochliobolus carbonum was studied with increasing concentrations of thiram. Isolate differences were detected for the TMTD polymodal curve. This difference was shown to be heritable and independent of tolerance to thiram as indicated by vegetative growth. The polymodal TMTD curve of spore germination inhibition inC. carbonum may result from two (or more) thiram sensitive loci each genetically conditioned independently.A joint contribution of the Department of Plant Pathology and the Pesticides Research Laboratory. The Pennsylvania State University, University Park 16802.     

Identification of Races and Mating Types of Cochliobolus carbonum from Corn in the Yunnan Province in China

Northern corn leaf spot, a foliar disease caused by Cochliobolus carbonum, has become prevalent in southwestern China, especially in the Yunnan Province. Races and mating types were identified for 169 isolates collected from 13 prefectures of Yunnan by artificial inoculation using six hybrid corns as differential hosts and by crossing with three standard mating strains: CC092 (MAT1‐2), CC120 (MAT1‐1) and CC026 (MAT1‐1). Results showed the existence of three races: CCR1 (one isolate), CCR2 (43 isolates) and CCR3 (125 isolates). Most isolates were moderately or weakly virulent with only five being highly virulent. CCR3 was widely distributed and significantly more virulent than CCR2 that coexisted with CCR3 in many locations. On Sach's nutrient agar, 20.71% of the Yunnan isolates self‐mated, forming sterile perithecia. Fully developed perithecia could be formed between isolates of different geographic origins, but only 15.98% strains mated successfully with CC092 and 5.33% formed mature perithecia with 4–6 ascospores per asus. Similar results were obtained in crossing with CC026 or CC120. Mating could also occur between CCR3 and CCR2. Both mating types were found in Yunnan with 84 MAT1‐1 strains (one CCR1, 10 CCR2 and 73 CCR3) and 85 MAT1‐2 strains (33 CCR2 and 52 CCR3) and they coexisted in most areas. To identify the mating type rapidly, three specific primers were successfully developed and employed to amplify the mating‐type genes, with stable patterns of 1627 and 876 bp fragments obtained from MAT1‐1 and MAT1‐2 isolates, respectively. The ratio between MAT1‐1 and MAT1‐2 was 1 : 1, indicating that the mating‐type genes segregated randomly in the field naturally.     

Polymorphic Chromosomes Bearing the Tox2 Locus in Cochliobolus carbonum Behave as Homologs during Meiosis

The HTS1 gene in the Tox2 locus of the fungal pathogen Cochliobolus carbonum race 1 is required for synthesis of a host-selective phytotoxin and for increased virulence on susceptible genotypes of maize. The locus is present in race 1 isolates but absent from isolates of the other races, which do not produce the toxin. By pulsed-field gel electrophoresis and Southern analysis with HTS1 sequences and chromosome-specific markers, the HTS1 gene was detected on a 4-Mb chromosome in one group of isolates and on a 2.3-Mb chromosome in another group, which lacked the 4-Mb chromosome. A chromosome-specific marker from C. heterostrophus hybridized to a 2.3-Mb chromosome in non-toxin-producing isolates and in toxin-producing isolates, including those with a 4-Mb chromosome. A marker from C. carbonum hybridized to the 4-Mb chromosome, but in isolates lacking the 4-Mb chromosome, this marker hybridized to a smaller, 2.0-Mb chromosome. Thus, the Tox2 locus is on different chromosomes in different groups of race 1 isolates. Single ascospore progeny from crosses between isolates having HTS1 on different chromosomes were analyzed for toxin-producing ability, virulence, and the presence and chromosomal location of HTS1. All progeny produced HC toxin in culture, incited race 1-type lesions on susceptible maize genotypes, and contained HTS1 sequences, as determined by PCR amplification with gene-specific primers. Analysis of the chromosomal complements of several progeny indicated that they all had only one Tox2-containing chromosome. Thus, despite their differences in size, these chromosomes behave as homologs during meiosis and may have arisen by a translocation.     

Cloning, disruption, and expression of two endo-beta 1, 4-xylanase genes, XYL2 and XYL3, from Cochliobolus carbonum.

In culture, the filamentous fungus Cochliobolus carbonum, a pathogen of maize, makes three cationic xylanases, XYL1, which encodes the major endoxylanase (Xyl1), was earlier cloned and shown by gene disruption to encode the first and second peaks of xylanase activity (P. C. Apel, D. G. Panaccione, F. R. Holden, and J. D. Walton, Mol. Plant-Microbe Interact. 6:467-473, 1993). Two additional xylanase genes, XYL2 and XYL3, have now been cloned from C. carbonum. XYL2 and XYL3 are predicted to encode 22-kDa family G xylanases similar to Xyl1. Xyl2 and Xyl3 are 60% and 42% identical, respectively, to Xyl1, and Xyl2 and Xyl3 are 39% identical. XYL1 and XYL2 but not XYL3 mRNAs are present in C. carbonum grown in culture, and XYL1 and XYL3 but not XYL2 mRNAs are present in infected plants. Transformation-mediated gene disruption was used to construct strains mutated in XYL1, XYL2, and XYL3. Xyl1 accounts for most of the total xylanase activity in culture, and disruption of XYL2 or XYL3 does not result in the further loss of any xylanase activity. In particular, the third peak of cationic xylanase activity is still present in a xyl1 xyl2 xyl3 triple mutant, and therefore this xylanase must be encoded by yet a fourth xylanase gene. A minor protein of 22 kDa that can be detected immunologically in the xyl1 mutant disappears in the xyl2 mutant and is therefore proposed to be the product of XYL2. The single xylanase mutants were crossed with each other to obtain multiple xylanase disruptions within the same strain. Strains disrupted in combinations of two and in all three xylanases were obtained. The triple mutant grows at the same rate as the wild type on xylan and on maize cell walls. The triple mutant is still fully pathogenic on maize with regard to lesion size, morphology, and rate of lesion development.     

Reduced virulence caused by meiotic instability of the TOX2 chromosome of the maize pathogen Cochliobolus carbonum

The mechanisms by which pathogenic fungi evolve are poorly understood. Production of the host-selective cyclic peptide HC-toxin is controlled by a complex locus, TOX2, in the plant pathogen Cochliobolus carbonum. Crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, as well as crosses between isolates in which the TOX2 genes were on chromosomes of different size, yielded progeny that had lost one or more copies of one or more of the TOX2 genes. Of approximately 200 progeny analyzed, eight (4%) had lost at least one TOX2 gene. All of them still had at least one functional copy of all of the known genes required for HC-toxin production (HTS1, TOXA, TOXC, and TOXE). Most deletion strains could be explained by simple chromosome breaks resulting in the loss of major contiguous portions (0.8 to 1.4 Mb) of the 3.5-Mb TOX2 chromosome, whereas others had more complicated patterns. All deletion strains had normal growth and were fertile, indicating that the 1.4 Mb of DNA contained no essential housekeeping genes. Most strains were also still virulent (Tox2+), but two had a novel phenotype of reduced virulence (RV), characterized by smaller lesions that expanded at a reduced rate and an inability to colonize plants systemically. Although the RV strains made no detectable HC-toxin in culture, the RV phenotype was dependent on the presence of a functional copy of HTS1, which encodes the central enzyme in HC-toxin biosynthesis. We propose that the RV strains still make a low level of HC-toxin, at least in planta, and that this is due to the loss of one or more genes that contribute to, but are not absolutely required for, HC-toxin synthesis.     

Chromosomal organization of TOX2, a complex locus controlling host-selective toxin biosynthesis in Cochliobolus carbonum.

Race 1 isolates of the filamentous fungus Cochliobolus carbonum are exceptionally virulent on certain genotypes of maize due to production of a cyclic tetrapeptide, HC-toxin. In crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, toxin production segregates in a simple 1:1 pattern, suggesting the involvement of a single genetic locus, which has been named TOX2. Earlier work had shown that in isolate SB111, TOX2 consists in part of two copies of a gene, HTS1, that encodes a 570-kD cyclic peptide synthetase and is lacking in Tox2- isolates. The genomic structure of TOX2 and the relationship between the two copies of HTS1 have now been clarified by using pulsedfield gel electrophoresis and physical mapping. In isolate SB111, both copies of HTS1 are on the largest chromosome (3.5 Mb), which is not present in the related Tox2- strain SB114. Two other genes known or thought to be important for HC-toxin biosynthesis, TOXA and TOXC, are also on the same chromosome in multiple copies. Other independent Tox2+ isolates also have two linked copies of HTS1, but in some isolates the size of the chromosome containing HTS1 is 2.2 Mb. Evidence obtained with Tox2+ -unique and with random probes is consistent with a reciprocal translocation as the cause of the difference in the size of the HTS1-containing chromosome among the Tox2+ isolates studied here. Physical mapping of the 3.5-Mb chromosome of SB111 that contains HTS1 using rare-cutting restriction enzymes and engineered restriction sites was used to map the chromosome location of the two copies of HTS1 and the three copies of TOXC. The results indicate that TOX2 is a complex locus that extends over more than 500 kb. The capacity to produce HC-toxin did not evolve by any single, simple mechanism.     

Novel Cyclic Peptide,Epichlicin, from the Endophytic Fungus,Epichloe typhina

The novel cyclic peptide, epichlicin, was isolated from Epichloe typhina, an endophytic fungus of the timothy plant (Phleum pretense L.). Its structure was determined by NMR studies and by mass spectrometry. Enantiomers of 3-amino tetradecanoic acid, a constituent amino acid of epichlicin, were synthesized as authentic standards. The stereochemistry of each amino acid was elucidated through a combination of the advanced Marfey method and chemical manipulation. Epichlicin showed inhibitory activity toward the spore germination of Cladosporium phlei, a pathogenic fungus of the timothy plant at an IC₅₀ value of 22 nM.     

Cloning and targeted gene disruption of EXG1, encoding exo-beta 1, 3-glucanase, in the phytopathogenic fungus Cochliobolus carbonum.

The phytopathogenic fungus Cochliobolus carbonum produces an extracellular enzyme capable of degrading beta 1,3-glucan in an exolytic manner. On the basis of partial amino acid sequences of the purified enzyme, two degenerate oligonucleotides were synthesized and used as PCR primers to amplify a 1.1-kb fragment of corresponding genomic DNA. The PCR product was used to isolate the genomic copy of the gene, called EXG1. Partial sequencing of the genomic DNA confirmed that the PCR product corresponded to EXG1. A strain of the fungus specifically mutated in the EXG1 gene was constructed by homologous integration of an internal fragment of EXG1. In the mutant, enzymatic activity and the corresponding peak of UV absorption during high-pressure liquid chromatography purification were reduced by at least 98%. However, crude culture filtrates of the mutant retained 44% of the wild-type beta 1,3-glucanase activity. This residual activity was due to two additional activities which were chromatographically separable from the product of EXG1 and which were coeluted with beta 1,3-beta 1,4-glucanase activity. Growth of the EXG1 mutant was normal on sucrose and oat bran but was reduced by 65% on pure beta 1,3-glucan. The EXG1 mutant was still pathogenic to maize.     

Cloning and Targeted Disruption of MLG1, a Gene Encoding Two of Three Extracellular Mixed-Linked Glucanases of Cochliobolus carbonum

Mixed-linked glucanases (MLGases), which are extracellular enzymes able to hydrolyze β1,3-1,4-glucans (also known as mixed-linked glucans or cereal β-glucans), were identified in culture filtrates of the plant-pathogenic fungus Cochliobolus carbonum. Three peaks of MLGase activity, designated Mlg1a, Mlg1b, and Mlg2, were resolved by cation-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Mlg1a and Mlg1b also hydrolyze β1,3-glucan (laminarin), whereas Mlg2 does not degrade β1,3-glucan but does degrade β1,4-glucan to a slight extent. Mlg1a, Mlg1b, and Mlg2 have monomer molecular masses of 33.5, 31, and 29.5 kDa, respectively. The N-terminal amino acid sequences of Mlg1a and Mlg1b are identical (AAYNLI). Mlg1a is glycosylated, whereas Mlg1b is not. The gene encoding Mlg1b, MLG1, was isolated by using PCR primers based on amino acid sequences of Mlg1b. The product of MLG1 has no close similarity to any known protein but does contain a motif (EIDI) that occurs at the active site of MLGases from several prokaryotes. An internal fragment of MLG1 was used to create mlg1 mutants by transformation-mediated gene disruption. The total MLGase and β1,3-glucanase activities in culture filtrates of the mutants were reduced by approximately 50 and 40%, respectively. When analyzed by cation-exchange HPLC, the mutants were missing the two peaks of MLGase activity corresponding to Mlg1a and Mlg1b. Together, the data indicate that Mlg1a and Mlg1b are products of the same gene, MLG1. The growth of mlg1 mutants in culture medium supplemented with macerated maize cell walls or maize bran and the disease symptoms on maize were identical to the growth and disease symptoms of the wild type.