T-toxin Production Research Articles

T-Toxin Virulence Genes: Unconnected Dots in a Sea of Repeats.

In 1970, the Southern Corn Leaf Blight epidemic ravaged U.S. fields to great economic loss. The outbreak was caused by never-before-seen, supervirulent, Race T of the fungus Cochliobolus heterostrophus. The functional difference between Race T and O, the previously known, far less aggressive strain, is production of T-toxin, a host-selective polyketide. Supervirulence is associated with ~1 Mb of Race T-specific DNA; only a fraction encodes T-toxin biosynthetic genes (Tox1). Tox1 is genetically and physically complex, with unlinked loci (Tox1A, Tox1B) genetically inseparable from breakpoints of a Race O reciprocal translocation that generated hybrid Race T chromosomes. Previously, we identified 10 genes for T-toxin biosynthesis. Unfortunately, high-depth, short-read sequencing placed these genes on four small, unconnected scaffolds surrounded by repeated A+T rich sequence, concealing context. To sort out Tox1 topology and pinpoint the hypothetical Race O translocation breakpoints corresponding to Race T-specific insertions, we undertook PacBio long-read sequencing which revealed Tox1 gene arrangement and the breakpoints. Six Tox1A genes are arranged as three small islands in a Race T-specific sea (~634 kb) of repeats. Four Tox1B genes are linked, on a large loop of Race T-specific DNA (~210 kb). The race O breakpoints are short sequences of race O-specific DNA; corresponding positions in race T are large insertions of race T-specific, A+T rich DNA, often with similarity to transposable (predominantly Gypsy) elements. Nearby, are 'Voyager Starship' elements and DUF proteins. These elements may have facilitated Tox1 integration into progenitor Race O and promoted large scale recombination resulting in race T. IMPORTANCE In 1970 a corn disease epidemic ravaged fields in the United States to great economic loss. The outbreak was caused by a never-before seen, supervirulent strain of the fungal pathogen Cochliobolus heterostrophus. This was a plant disease epidemic, however, the current COVID-19 pandemic of humans is a stark reminder that novel, highly virulent, pathogens evolve with devastating consequences, no matter what the host-animal, plant, or other organism. Long read DNA sequencing technology allowed in depth structural comparisons between the sole, previously known, much less aggressive, version of the pathogen and the supervirulent version and revealed, in meticulous detail, the structure of the unique virulence-causing DNA. These data are foundational for future analysis of mechanisms of DNA acquisition from a foreign source.

Characterization and potential evolutionary impact of transposable elements in the genome of Cochliobolus heterostrophus.

BackgroundCochliobolus heterostrophus is a dothideomycete that causes Southern Corn Leaf Blight disease. There are two races, race O and race T that differ by the absence (race O) and presence (race T) of ~ 1.2-Mb of DNA encoding genes responsible for the production of T-toxin, which makes race T much more virulent than race O. The presence of repetitive elements in fungal genomes is considered to be an important source of genetic variability between different species.ResultsA detailed analysis of class I and II TEs identified in the near complete genome sequence of race O was performed. In total in race O, 12 new families of transposons were identified. In silico evidence of recent activity was found for many of the transposons and analyses of expressed sequence tags (ESTs) demonstrated that these elements were actively transcribed. Various potentially active TEs were found near coding regions and may modify the expression and structure of these genes by acting as ectopic recombination sites. Transposons were found on scaffolds carrying polyketide synthase encoding genes, responsible for production of T-toxin in race T. Strong evidence of ectopic recombination was found, demonstrating that TEs can play an important role in the modulation of genome architecture of this species. The Repeat Induced Point mutation (RIP) silencing mechanism was shown to have high specificity in C. heterostrophus, acting only on transposons near coding regions.ConclusionsNew families of transposons were identified. In C. heterostrophus, the RIP silencing mechanism is efficient and selective. The co-localization of effector genes and TEs, therefore, exposes those genes to high rates of point mutations. This may accelerate the rate of evolution of these genes, providing a potential advantage for the host. Additionally, it was shown that ectopic recombination promoted by TEs appears to be the major event in the genome reorganization of this species and that a large number of elements are still potentially active. So, this study provides information about the potential impact of TEs on the evolution of C. heterostrophus.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-536) contains supplementary material, which is available to authorized users.

Cochliobolus heterostrophus Llm1 – A Lae1-like methyltransferase regulates T-toxin production, virulence, and development

A Lae1-like methyltransferase, Llm1, was identified in maize pathogen Cochliobolus heterostrophus which is renowned for production of the secondary metabolite host-selective toxin, T-toxin, and is a model for mechanisms of reproduction of heterothallic Dothideomycetes. Previously, we determined that C. heterostrophus mutants lacking Lae1 and Vel1 proteins were decreased in ability to produce T-toxin when the fungus was grown in the dark, demonstrating that these proteins are positive regulators of toxin production. We showed also that Lae1 and Vel1 regulate resistance to oxidative stress and both sexual and asexual reproduction. Here, it is demonstrated that Llm1, one of nine Lae1-like methyltransferases in the C. heterostrophus genome, acts as a negative regulator of T-toxin production and thus impacts virulence to the host. In vitro, in the dark, and in planta, llm1 mutants make more T-toxin than do wild-type (WT) strains, while overexpressing strains make less than WT. Virulence (amount of chlorosis) to maize, due to T-toxin, follows accordingly. Expression of nine genes involved in T-toxin production is elevated in llm1 mutants and reduced in overexpressing strains. llm1 mutations cannot rescue deficiencies in T-toxin production of lae1 or vel1 mutants indicating that Llm1 represses T-toxin biosynthesis, and that vel1 and lae1 mutations are epistatic to llm1 mutations. Thus, increased T-toxin production, and presumably gene expression, in the llm1 mutant is dependent on the presence of Vel1 and Lae1 proteins. There is no evidence that Llm1 has an effect on oxidative stress tolerance. llm1 mutants are fully fertile in crosses to WT mating testers, while LLM1 overexpressing strains and llm1lae1 and llm1vel1 double mutants are unable to act as females. Overexpression of LLM1 leads to de-repression of asexual sporulation during sexual development, and of asexual sporulation in the light and the dark during vegetative growth, as is the case for vel1, llm1vel1, and llm1lae1-deletion strains. llm1vel1 and llm1lae1 double mutants are similar to lae1 single mutants and accumulate more hyphal melanin in liquid medium than do llm1 or vel1 single mutants, implying Llm1 plays a redundant role in regulating pigmentation with Vel1, while Lae1 plays a major role.

ChLae1 and ChVel1 Regulate T-toxin Production, Virulence, Oxidative Stress Response, and Development of the Maize Pathogen Cochliobolus heterostrophus

LaeA and VeA coordinate secondary metabolism and differentiation in response to light signals in Aspergillus spp. Their orthologs, ChLae1 and ChVel1, were identified in the maize pathogen Cochliobolus heterostrophus, known to produce a wealth of secondary metabolites, including the host selective toxin, T-toxin. Produced by race T, T-toxin promotes high virulence to maize carrying Texas male sterile cytoplasm (T-cms). T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT. Correspondingly, expression of T-toxin biosynthetic genes (Tox1) is up-regulated in the dark in WT, while dark-induced expression is much reduced/minimal in Chvel1 and Chlae1 mutants. Toxin production and Tox1 gene expression are increased in ChVEL1 overexpression (OE) strains grown in the dark and in ChLAE1 strains grown in either light or dark, compared to WT. These observations establish ChLae1 and ChVel1 as the first factors known to regulate host selective toxin production. Virulence of Chlae1 and Chvel1 mutants and OE strains is altered on both T-cms and normal cytoplasm maize, indicating that both T-toxin mediated super virulence and basic pathogenic ability are affected. Deletion of ChLAE1 or ChVEL1 reduces tolerance to H2O2. Expression of CAT3, one of the three catalase genes, is reduced in the Chvel1 mutant. Chlae1 and Chvel1 mutants also show decreased aerial hyphal growth, increased asexual sporulation and female sterility. ChLAE1 OE strains are female sterile, while ChVEL1 OE strains are more fertile than WT. ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains. Thus, ChLae1 and ChVel1 positively regulate T-toxin biosynthesis, pathogenicity and super virulence, oxidative stress responses, sexual development, and aerial hyphal growth, and negatively control melanin biosynthesis and asexual differentiation.

Six New Genes Required for Production of T-Toxin, a Polyketide Determinant of High Virulence of Cochliobolus heterostrophus to Maize

Southern Corn Leaf Blight, one of the worst plant disease epidemics in modern history, was caused by Cochliobolus heterostrophus race T, which produces T-toxin, a determinant of high virulence to maize carrying Texas male sterile cytoplasm. The genetics of T-toxin production is complex and the evolutionary origin of associated genes is uncertain. It is known that ability to produce T-toxin requires three genes encoded at two unlinked loci, Tox1A and Tox1B, which map to the breakpoints of a reciprocal translocation. DNA associated with Tox1A and Tox1B sums to about 1.2 Mb of A+T rich, repeated DNA that is not found in less virulent race O or other Cochliobolus species. Here, we describe identification and targeted deletion of six additional genes, three mapping to Tox1A and three to Tox1B. Mutant screens indicate that all six genes are involved in T-toxin production and high virulence to maize. The nine known Tox1 genes encode two polyketide synthases (PKS), one decarboxylase, five dehydrogenases, and one unknown protein. Only two have a similar phylogenetic profile. To trace evolutionary history of one of the core PKS, DNA from more than 100 Dothideomycete species were screened for homologs. An ortholog (60% identity) was confirmed in Didymella zeae-maydis, which produces PM-toxin, a polyketide of similar structure and biological specificity as T-toxin. Only one additional Dothideomycete species, the dung ascomycete Delitschia winteri harbored a paralog. The unresolved evolutionary history and distinctive gene signature of the PKS (fast-evolving, discontinuous taxonomic distribution) leaves open the question of lateral or vertical transmission.

Two Polyketide Synthase-Encoding Genes Are Required for Biosynthesis of the Polyketide Virulence Factor, T-toxin, by Cochliobolus heterostrophus

Cochliobolus heterostrophus race T, causal agent of southern corn leaf blight, requires T-toxin (a family of C35 to C49 polyketides) for high virulence on T-cytoplasm maize. Production of T-toxin is controlled by two unlinked loci, Tox1A and Tox1B, carried on 1.2 Mb of DNA not found in race O, a mildly virulent form of the fungus that does not produce T-toxin, or in any other Cochliobolus spp. or closely related fungus. PKS1, a polyketide synthase (PKS)-encoding gene at Tox1A, and DEC1, a decarboxylase-encoding gene at Tox1B, are necessary for T-toxin production. Although there is evidence that additional genes are required for T-toxin production, efforts to clone them have been frustrated because the genes are located in highly repeated, A+T-rich DNA. To overcome this difficulty, ligation specificity-based expression analysis display (LEAD), a comparative amplified fragment length polymorphism/gel fractionation/capillary sequencing procedure, was applied to cDNAs from a near-isogenic pair of race T (Tox1+) and race O (Tox1-) strains. This led to discovery of PKS2, a second PKS-encoding gene that maps at Tox1A and is required for both T-toxin biosynthesis and high virulence to maize. Thus, the carbon chain of each T-toxin family member likely is assembled by action of two PKSs, which produce two polyketides, one of which may act as the starter unit for biosynthesis of the mature T-toxin molecule.

A decarboxylase encoded at the Cochliobolus heterostrophus translocation-associated Tox1B locus is required for polyketide (T-toxin) biosynthesis and high virulence on T-cytoplasm maize.

Genes at two unlinked loci (Tox1A and Tox1B) are required for production of the polyketide T-toxin by Cochliobolus heterostrophus race T, a pathogenic fungus that requires T-toxin for high virulence to maize with T-cytoplasm. Previous work indicated that Tox1A encodes a polyketide synthase (PKS1) required for T-toxin biosynthesis and for high virulence. To identify genes at Tox1B, a wild-type race T cDNA library was screened for genes missing in the genome of a Tox1B deletion mutant. The library was probed, first with a 415-kb NotI restriction fragment from the genome of the Tox1B mutant, then with the corresponding 560-kb fragment from the genome of wild type. Two genes, DEC1 (similar to acetoacetate decarboxylase-encoding genes) and RED1 (similar to genes encoding members of the medium-chain dehydrogenase/reductase superfamily), were recovered. Targeted disruption of DEC1 drastically reduced both T-toxin production and virulence of race T to T-cytoplasm maize, whereas specific inactivation of RED1 had no apparent effect on T-toxin production (as determined by bioassay) or on virulence. DEC1 and RED1 map within 1.5 kb of each other on Tox1B chromosome 6;12 and are unique to the genome of race T, an observation consistent with the hypothesis that these genes were acquired by C. heterostrophus via a horizontal transfer event.

The translocation-associated tox1 locus of Cochliobolus heterostrophus is two genetic elements on two different chromosomes.

Previously, Tox1 was defined as a single genetic element controlling the difference between races of Cochliobolus heterostrophus: race T is highly virulent on T-cytoplasm corn and produces the polyketide T-toxin; race O is weakly virulent and does not produce T-toxin. Here we report that Tox1 is two loci, Tox1A and Tox1B, on two different chromosomes. Evidence for two loci derives from: (1) the appearance of 25% Tox+ progeny in crosses between induced Tox1(-) mutants, one defective at Tox1A, the other at Tox1B; (2) the ability of Tox1A- + Tox1B- heterokaryons to complement for T-toxin production; and (3) electrophoretic karyotypes proving that Tox1(-) mutations are physically located on two different chromosomes. Data showing Tox1 as a single genetic element are reconciled with those proving it is two loci by the fact that Tox1 is inseparably linked to the breakpoints of a reciprocal translocation; the translocation results in a four-armed linkage group. In crosses where the translocation is heterozygous (i.e., race T by race O), all markers linked to the four-armed intersection appear linked to each other; in crosses between induced Tox1(-) mutants, complications due to the translocation are eliminated and the two loci segregate independently.

A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin.

Race T of the fungal pathogen Cochliobolus heterostrophus is highly virulent toward Texas male sterile (T) maize and differs from its relative, race O, at a locus (Tox1) that is responsible for the production of T-toxin, a family of linear long-chain (C35 to E41) polyketides. In a previous study, the restriction enzyme-mediated integration procedure was used to mutagenize and tag Tox1. Here, we report that the DNA recovered from the insertion site of one mutant encodes a 7.6-kb open reading frame (2530 amino acids) that identifies a multifunctional polyketide synthase (PKS)-encoding gene (PKS1) with six catalytic domains arranged in the following order, starting at the N terminus: beta-ketoacyl synthase, acyltransferase, dehydratase, enoyl reductase, beta-ketoacyl reductase, and acyl carrier protein. PKS1 is interrupted by four apparent introns (74, 57, 49, and 41 bp) and exists in the genome as a single copy surrounded by highly repetitive, A + T-rich DNA. When PKS1 in race T was inactivated by targeted gene disruption, T-toxin production and high virulence were eliminated, indicating that this PKS is required for fungal virulence. Race O strains, which do not produce T-toxin, lack a detectable homolog of PKS1, suggesting that race T may have acquired PKS1 by horizontal transfer of DNA rather than by vertical inheritance from an ancestral strain.

A reciprocal translocation and possible insertion(s) tightly associated with host-specific virulence inCochliobolus heterostrophus

A reciprocal translocation and one or more apparent insertions are shown to be tightly associated with Tox1, a locus controlling T-toxin production and host-selective virulence in race T of the maize pathogen Cochliobolus heterostrophus. Chromosome arrangements were examined by separating chromosomal DNAs of a variety of Tox+ and Tox− strains by pulsed-field gel electrophoresis and hybridizing with probes known to detect RFLPs genetically linked to Tox1. The existence of the translocation was demonstrated by chromosome hybridization patterns; the existence of the putative insertion(s) was deduced from chromosome migration rates. Both differences in chromosome arrangement were detected between 8 Tox+ and 8 Tox− near-isogenic laboratory strains, suggesting that the differences are tightly linked to Tox1. The reciprocal translocation was also detected between all 7 Tox+ and 8 Tox− field isolates examined, suggesting that the translocation is common in natural populations. The field isolates may also differ by the insertion(s); however, numerous additional chromosome size polymorphisms in the field isolates prevented a firm conclusion. The tight association of the translocation and insertion(s) with T-toxin production suggests that chromosome rearrangements may have been involved in the evolution of race T and Tox1. These genomic differences may be causally related to the previously reported reduced fitness of race T relative to race O on N-cytoplasm maize. Key words : chromosome rearrangement, southern corn leaf blight, pulsed-field electrophoresis, T-toxin, evolution.

Function and chromosomal location of theCochliobolus heterostrophus TOX1locus

Conventional genetic analyses have firmly established that the difference in virulence between race T and O of the corn pathogen Cochliobolus heterostrophus is determined by a single genetic locus called Tox1, which also controls production of T-toxin, a polyketide specifically toxic to corn with Texas male sterile (T) cytoplasm. More recently, molecular genetic analyses have revealed that Tox1 is not a single gene but rather at least two genetic loci situated on two different chromosomes. DNA at both of these loci is required for the biosynthesis of T-toxin and for the high virulence of race T to corn carrying T-cytoplasm. One of the loci encodes a polyketide synthase that is necessary for the assembly of the T-toxin molecule. Key words: polyketide, restriction enzyme mediated integration (REMI), host-specific toxin, corn, fungus, virulence.

Toxin-deficient mutants from a toxin-sensitive transformant of Cochliobolus heterostrophus.

Tox1 is the only genetic element identified which controls production of T-toxin, a linear polyketide involved in the virulence of Cochliobolus heterostrophus to its host plant, corn. Previous attempts to induce toxin-deficient (Tox-) mutants, using conventional mutagenesis and screening procedures, have been unsuccessful. As a strategy to enrich for Tox- mutants, we constructed a Tox1+ strain that carried the corn T-urf13 gene (which confers T-toxin sensitivity) fused to a fungal mitochondrial signal sequence; the fusion was under control of the inducible Aspergillus nidulans pelA promoter which, in both A. nidulans and C. heterostrophus, is repressed by glucose and induced by polygalacturonic acid (PGA). We expected that a transformant carrying this construction would be sensitive to its own toxin when the T-urf13 gene was expressed. Indeed, the strain grew normally on medium containing glucose but was inhibited on medium containing PGA. Conidia of this strain were treated with ethylmethanesulfonate and plated on PGA medium. Among 362 survivors, 9 were defective in T-toxin production. Authenticity of each mutant was established by the presence of the transformation vector, proper mating type, and a restriction fragment length polymorphism tightly linked to the Tox1+ locus. Progeny of each mutant crossed to a Tox1+ tester segregated 1:1 (for wild type toxin production vs. no or reduced toxin production), indicating a single gene mutation in each case. Progeny of each mutant crossed to a Tox1- tester segregated 1:1 (for no toxin production vs. no or reduced toxin production) indicating that each mutation mapped at the Tox1 locus. Availability of Tox- mutants will permit mapping in the Tox1 region without interference from a known Tox1 linked translocation breakpoint.

Genetic Control and Distorted Segregation of T-Toxin Production in Field Isolates ofCochliobolus heterostrophus

Genetic Control and Distorted Segregation of T-Toxin Production in Field Isolates of<i>Cochliobolus heterostrophus</i>

Nonrandom abortion of ascospores containing alternate alleles at the Tox-1 locus of the fungal plant pathogen Cochliobolus heterostrophus

Crosses involving lab strains of C. heterostrophus that were previously selected for improved fertility yielded high frequencies of four-spored asci and only occasional asci with the full set of eight spores. Fertility in crosses between field isolates was low. Most asci contained two haploid spores; no asci had more than four spores. We investigated the distribution of aborted spores with respect to the three defined genetic loci Mat (mating type), Alb-1 (albinism), and Tox-1 (T-toxin production) and determined the frequency of mitotic twins in four-spored asci. Nonrandom spore abortion was evident from the observations that 80–100% of four-spored asci contained two sets of twins (9% expected for random abortion of mitotic products) and up to 95% of four-spored asci segregated 2:2 for alleles at the Tox-1 locus (67% expected for random abortion of meiotic products), suggesting preferential recovery of nonsister meiotic products. Segregation in crosses between field isolates tended to be skewed in favor of the tox-1a allele (specifies no toxin production). Alleles at Mat and Alb-1 segregated as expected for random abortion of meiotic products in both types of crosses. Low fertility and aberrant segregation among progeny segregating for ability to produce T toxin are likely due in part to abortion-inducing factors linked to Tox-1.Key words: fertility, ascomycetes, T toxin, Helminthosporium maydis, Bipolaris maydis.

Dominance at the Tox1 locus controlling T-toxin production by Cochliobolus heterostrophus

Heterokaryons of Cochliobolus heterostrophus were constructed which contained approximately equal numbers of nuclei bearing either the tox1+ allele for presence of T-toxin or the tox1− allele for absence of T-toxin in culture filtrates. The heterokaryons were grown in a liquid medium known to support production of T-toxin; the resulting culture filtrates were assayed for T-toxin activity using 2 different bioassays: seedling root growth and dark CO2 fixation. Both assay procedures detected the presence of T-toxin in culture filtrates of tox1+tox1− heterokaryons; levels of activity were slightly less than those found in filtrates from tox1+tox1+ heterokaryons. Since these heterokaryons are known to function as diploids, the simplest interpretation of the results is that the tox1+ allele, which determines high virulence as well as T-toxin production, is either dominant or semidominant.

T-Toxin Production by Near-Isogenic Isolates ofCochliobolus heterostrophusRaces T and O

T-Toxin Production by Near-Isogenic Isolates of<i>Cochliobolus heterostrophus</i>Races T and O