Benjamin A. Horwitz

Trichoderma: the genomics of opportunistic success

Trichoderma is a genus of common filamentous fungi that display a remarkable range of lifestyles and interactions with other fungi, animals and plants. Because of their ability to antagonize plant-pathogenic fungi and to stimulate plant growth and defence responses, some Trichoderma strains are used for biological control of plant diseases. In this Review, we discuss recent advances in molecular ecology and genomics which indicate that the interactions of Trichoderma spp. with animals and plants may have evolved as a result of saprotrophy on fungal biomass (mycotrophy) and various forms of parasitism on other fungi (mycoparasitism), combined with broad environmental opportunism.

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Diverse Lifestyles and Strategies of Plant Pathogenesis Encoded in the Genomes of Eighteen Dothideomycetes Fungi

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.

Use of green fluorescent protein (GFP) for studying development and fungal-plant interaction in Cochliobolus heterostrophus

The green fluorescent protein (GFP) has been widely used as an extremely useful vital marker in a large number of organisms, but good expression in filamentous ascomycetes has not been reported. To facilitate the research of fungal development and fungal-plant interaction, we constructed two plasmid vectors for the expression of the synthetic SGFP-TYG gene in ascomycete species, and used these vectors for transformation of the maize pathogen Cochliobolus heterostrophus. High level expression of GFP was obtained, as detected by anti-GFP antibodies and by fluorescence microscopy. The intense fluorescence was used as a highly efficient vital marker to detect cytoplasmic and developmental changes that occur in the fungus, and to follow phytopathogenic development of the fungus on and inside maize leaves. The hyphae within the leaf form a unique parallel growth pattern, closely associated with, and apparently determined by, the anatomy of the leaf. Fluorescence intensity was quantified by digital analysis of the green fluorescence image and was highly correlated with the amount of mycelium and levels of disease. Expression of GFP was obtained in additional ascomycetes that were transformed with the new constructs, indicating that SGFP-TYG can be used as a highly effective vital marker in ascomycetes.

Looking through the eyes of fungi: molecular genetics of photoreception

Filamentous fungi respond to a variety of environmental signals. One of them is light, providing critical information about orientation, or impending stress. Cells of filamentous fungi appear to sense blue light through a unique transcription factor that has a flavin chromophore and activates its targets in a light‐dependent manner, the white collar (WC) complex. Fungal photophysiology, though, predicted a greater complexity of responses to the whole visible spectrum. The rapidly growing fungal genome database provides candidates to explain how fungi see not only blue, but also near‐UV, green and red light. At the same time, there are surprises in the genomes, including photoreceptors for which there are no obvious photoresponses. Linking these genes and their functions will help understand how a list of only a few biological chromophores accounts for such a diversity of responses. At the same time, deeper mechanistic understanding of how the WC complex functions will lead to fundamental insights at the point where the environment impinges, in this case in the form of photons, on the transcriptional machinery of the cell.

Trichoderma atroviride G-Protein α-Subunit Gene tga1 Is Involved in Mycoparasitic Coiling and Conidiation

ABSTRACT The soil fungus Trichoderma atroviride, a mycoparasite, responds to a number of external stimuli. In the presence of a fungal host, T. atroviride produces hydrolytic enzymes and coils around the host hyphae. In response to light or nutrient depletion, asexual sporulation is induced. In a biomimetic assay, different lectins induce coiling around nylon fibers; coiling in the absence of lectins can be induced by applying cyclic AMP (cAMP) or the heterotrimeric G-protein activator mastoparan. We isolated a T. atroviride G-protein α-subunit (Gα) gene (tga1) belonging to the fungal subfamily with the highest similarity to the Gαi class. Generated transgenic lines that overexpress Gα show very delayed sporulation and coil at a higher frequency. Furthermore, transgenic lines that express an activated mutant protein with no GTPase activity do not sporulate and coil at a higher frequency. Lines that express an antisense version of the gene are hypersporulating and coil at a much lower frequency in the biomimetic assay. The loss of Tga1 in these mutants correlates with the loss of GTPase activity stimulated by the peptide toxin Mas-7. The application of Mas-7 to growing mycelial colonies raises intracellular cAMP levels, suggesting that Tga1 can activate adenylyl cyclase. In contrast, cAMP levels and cAMP-dependent protein kinase activity drop when diffusible host signals are encountered and the mycoparasitism-related genes ech42 and prb1 are highly expressed. Mycoparasitic signaling is unlikely to be a linear pathway from host signals to increased cAMP levels. Our results demonstrate that the product of the tga1 gene is involved in both coiling and conidiation.

Melanin biosynthesis in the maize pathogen Cochliobolus heterostrophus depends on two mitogen-activated protein kinases, Chk1 and Mps1, and the transcription factor Cmr1.

ABSTRACT The maize pathogen Cochliobolus heterostrophus requires two mitogen-activated protein kinases (MAPKs), Chk1 and Mps1, to produce normal pigmentation. Young colonies of mps1 and chk1 deletion mutants have a white and autolytic appearance, which was partially rescued by a hyperosmotic environment. We isolated the transcription factor Cmr1, an ortholog of Colletotrichum lagenarium Cmr1 and Magnaporthe grisea Pig1, which regulates melanin biosynthesis in C. heterostrophus. Deletion of CMR1 in C. heterostrophus resulted in mutants that lacked dark pigmentation and acquired an orange-pink color. In cmr1 deletion strains the expression of putative scytalone dehydratase (SCD1) and hydroxynaphthalene reductase (BRN1 and BRN2) genes involved in melanin biosynthesis was undetectable, whereas expression of PKS18, encoding a polyketide synthase, was only moderately reduced. In chk1 and mps1 mutants expression of PKS18, SCD1, BRN1, BRN2, and the transcription factor CMR1 itself was very low in young colonies, slightly up-regulated in aging colonies, and significantly induced in hyperosmotic conditions, compared to invariably high expression in the wild type. These findings indicate that two MAPKs, Chk1 and Mps1, affect Cmr1 at the transcriptional level and this influence is partially overridden in stress conditions including aging culture and hyperosmotic environment. Surprisingly, we found that the CMR1 gene was transcribed in both sense and antisense directions, apparently producing mRNA as well as a long noncoding RNA transcript. Expression of the antisense CMR1 was also Chk1 and Mps1 dependent. Analysis of chromosomal location of the melanin biosynthesis genes in C. heterostrophus resulted in identification of a small gene cluster comprising BRN1, CMR1, and PKS18. Since expression of all three genes depends on Chk1 and Mps1 MAPKs, we suggest their possible epigenetic regulation.

Activation of an AP1-Like Transcription Factor of the Maize Pathogen Cochliobolus heterostrophus in Response to Oxidative Stress and Plant Signals

ABSTRACT Redox sensing is a ubiquitous mechanism regulating cellular activity. Fungal pathogens face reactive oxygen species produced by the host plants oxidative burst in addition to endogenous reactive oxygen species produced during aerobic metabolism. An array of preformed and induced detoxifying enzymes, including superoxide dismutase, catalases, and peroxidases, could allow fungi to infect plants despite the oxidative burst. We isolated a gene (CHAP1) encoding a redox-regulated transcription factor in Cochliobolus heterostrophus, a fungal pathogen of maize. CHAP1 is a bZIP protein that possesses two cysteine-rich domains structurally and functionally related to Saccharomyces cerevisiae YAP1. Deletion of CHAP1 in C. heterostrophus resulted in decreased resistance to oxidative stress caused by hydrogen peroxide and menadione, but the virulence of chap1 mutants was unaffected. Upon activation by oxidizing agents or plant signals, a green fluorescent protein (GFP)-CHAP1 fusion protein became localized in the nucleus. Expression of genes encoding antioxidant proteins was induced in the wild type but not in chap1 mutants. Activation of CHAP1 occurred from the earliest stage of plant infection, in conidial germ tubes on the leaf surface, and persisted during infection. Late in the course of infection, after extensive necrotic lesions were formed, GFP-CHAP1 redistributed to the cytosol in hyphae growing on the leaf surface. Localization of CHAP1 to the nucleus may, through changes in the redox state of the cell, provide a mechanism linking extracellular cues to transcriptional regulation during the plant-pathogen interaction.

TmkA, a mitogen-activated protein kinase of Trichoderma virens, is involved in biocontrol properties and repression of conidiation in the dark.

ABSTRACT Trichoderma virens is a mycoparasitic fungus used in biocontrol of soilborne plant pathogens. It inhibits or kills plant-pathogenic fungi through production of antifungal antibiotics and parasitism of hyphae and sclerotia. Conidiation, or the production of asexual spores, an inducible process triggered by light or nutrient stress, is an important trait in survival and also development of formulation products. In many fungi, signaling pathways, including mitogen-activated protein kinase (MAPK) cascades, have been implicated in parasitism of host plants as well as in the production of asexual spores. Here, we have studied the role of a MAPK gene, that for TmkA, in conidiation and antagonistic properties of a biocontrol strain of T. virens. Through single- and double-crossover recombination, we obtained three tmkA loss-of-function mutants. The TmkA transcript was not detectable in these mutants. The mutants conidiated in the dark, although photoinduction was normal and the light sensitivities of the wild type and the mutant were the same. The mutants had, overall, normal colony morphology, but their radial growth rate was reduced by about 16%, with no decrease in biomass production. Against Rhizoctonia solani hyphae, the knockout mutants exhibited mycoparasitic coiling and lysis of host hyphae similar to that of the wild type. The mutants, however, were less effective in colonizing the sclerotia of R. solani. On Sclerotium rolfsii, the MAPK loss-of-function mutants had reduced antagonistic properties in confrontation assays and failed to parasitize the sclerotia. TmkA-dependent and -independent pathways are thus involved in antagonism against different hosts. Finally, in contrast to the case for other filamentous fungi studied so far, signaling through a MAPK represses, rather than induces, asexual sporulation.

A Mitogen-Activated Protein Kinase Pathway Modulates the Expression of Two Cellulase Genes in Cochliobolus heterostrophus during Plant Infection

Conserved eukaryotic signaling elements play an important role in the development of fungal pathogens on their hosts. Chk1, a mitogen-activated protein kinase (MAPK), functions in virulence, mating, and sporulation of the maize leaf pathogen Cochliobolus heterostrophus. Suppression subtractive hybridization was used to identify fungal genes whose expression on the host plant is affected in chk1 deletion mutants. Two of the genes isolated in this screen were predicted to encode cellulolytic enzymes: a cellobiohydrolase, CBH7, and an endoglucanase, EG6. Expression of EG6 and CBH7 was followed by the fusion of their upstream regulatory regions to the coding sequence of the green fluorescent protein. Induction of both genes began at the onset of invasive growth and reached its maximal extent during leaf necrosis. Furthermore, EG6 was induced preferentially within necrotic lesions. Disruption of MAPK CHK1 resulted in a delay in the penetration of hyphae into the leaf and a concomitant delay in the induction of expression of both cellulase genes. In saprophytic culture, the absence of Chk1 resulted in a marked delay in the induction of CBH7 expression by crystalline cellulose. EG6 was expressed at a basal level in culture, and this expression was found to depend strictly on Chk1. Thus, the Chk1 MAPK signaling pathway is involved in the regulation of two cellulase-encoding genes and is necessary for their timely induction by environmental signals.