The AP-1-like transcription factor ChAP1 balances tolerance and cell death in the response of the maize pathogen Cochliobolus heterostrophus to a plant phenolic.

Fungal pathogens need to contend with stresses including oxidants and antimicrobial chemicals resulting from host defenses. ChAP1 of Cochliobolus heterostrophus, agent of Southern corn leaf blight, encodes an ortholog of yeast YAP1. ChAP1 is retained in the nucleus in response to plant-derived phenolic acids, in addition to its well-studied activation by oxidants. Here, we used transcriptome profiling to ask which genes are regulated in response to ChAP1 activation by ferulic acid (FA), a phenolic abundant in the maize host. Nuclearization of ChAP1 in response to phenolics is not followed by strong expression of genes needed for oxidative stress tolerance. We, therefore, compared the transcriptomes of the wild-type pathogen and a ChAP1 deletion mutant, to study the function of ChAP1 in response to FA. We hypothesized that if ChAP1 is retained in the nucleus under plant-related stress conditions yet in the absence of obvious oxidant stress, it should have additional regulatory functions. The transcriptional signature in response to FA in the wild type compared to the mutant sheds light on the signaling mechanisms and response pathways by which ChAP1 can mediate tolerance to ferulic acid, distinct from its previously known role in the antioxidant response. The ChAP1-dependent FA regulon consists mainly of two large clusters. The enrichment of transport and metabolism-related genes in cluster 1 indicates that C. heterostrophus degrades FA and removes it from the cell. When this fails at increasing stress levels, FA provides a signal for cell death, indicated by the enrichment of cell death-related genes in cluster 2. By quantitation of survival and by TUNEL assays, we show that ChAP1 promotes survival and mitigates cell death. Growth rate data show a time window in which the mutant colony expands faster than the wild type. The results delineate a transcriptional regulatory pattern in which ChAP1 helps balance a survival response for tolerance to FA, against a pathway promoting cell death in the pathogen. A general model for the transition from a phase where the return to homeostasis dominates to a phase leading to the onset of cell death provides a context for understanding these findings.

Secretome of Trichoderma interacting with maize roots: role in induced systemic resistance.

Trichoderma virens is a biocontrol agent used in agriculture to antagonize pathogens of crop plants. In addition to direct mycoparasitism of soil-borne fungal pathogens, T. virens interacts with roots. This interaction induces systemic resistance (ISR), which reduces disease in above-ground parts of the plant. In the molecular dialog between fungus and plant leading to ISR, proteins secreted by T. virens provide signals. Only a few such proteins have been characterized previously. To study the secretome, proteins were characterized from hydroponic culture systems with T. virens alone or with maize seedlings, and combined with a bioassay for ISR in maize leaves infected by the pathogen Cochliobolus heterostrophus. The secreted protein fraction from coculture of maize roots and T. virens (Tv+M) was found to have a higher ISR activity than from T. virens grown alone (Tv). A total of 280 fungal proteins were identified, 66 showing significant differences in abundance between the two conditions: 32 were higher in Tv+M and 34 were higher in Tv. Among the 34 found in higher abundance in Tv and negatively regulated by roots were 13 SSCPs (small, secreted, cysteine rich proteins), known to be important in the molecular dialog between plants and fungi. The role of four SSCPs in ISR was studied by gene knockout. All four knockout lines showed better ISR activity than WT without affecting colonization of maize roots. Furthermore, the secreted protein fraction from each of the mutant lines showed improved ISR activity compared with WT. These SSCPs, apparently, act as negative effectors reducing the defense levels in the plant and may be important for the fine tuning of ISR by Trichoderma. The down-regulation of SSCPs in interaction with plant roots implies a revision of the current model for the Trichoderma-plant symbiosis and its induction of resistance to pathogens.

Plant phenolic acids induce programmed cell death of a fungal pathogen: MAPK signaling and survival of Cochliobolus heterostrophus.

Plant aromatic compounds provide signals and a nutrient source to pathogens, and also act as stressors. Structure-activity relationships suggest two pathways sensing these compounds in the maize pathogen Cochliobolus heterostrophus, one triggering a stress response, and one inducing enzymes for their degradation. Focusing on the stress pathway, we found that ferulic acid causes rapid appearance of TUNEL-positive nuclei, dispersion of histone H1:GFP, hyphal shrinkage, and eventually membrane damage. These hallmarks of programmed cell death (PCD) were not seen upon exposure to caffeic acid, a very similar compound. Exposure to ferulic acid dephosphorylated two MAP kinases: Hog1 (stress activated) and Chk1 (pathogenicity related), while increasing phosphorylation of Mps1 (cell integrity related). Mutants lacking Hog1 or Chk1 are hypersensitive to ferulic acid while Mps1 mutants are not. These results implicate three MAPK pathways in the stress response. Ferulic acid and the antifungal fludioxonil have opposite additive effects on survival and on dephosphorylation of Hog1, which is thus implicated in survival. The results may explain why some fungal pathogens of plants undergo cell death early in host invasion, when phenolics are released from plant tissue.

Plant phenolic compounds and oxidative stress: integrated signals in fungal-plant interactions.

Upon invasion of a host, fungal pathogens are exposed to a variety of stresses. Plants release reactive oxygen species, and mount a variety of preformed and induced chemical defenses. Phenolic compounds are one example: they are ubiquitous in plants, and an invading pathogen encounters them already at the leaf surface, or for soil-borne pathogens, in the rhizosphere. Phenolic and related aromatic compounds show varying degrees of toxicity to cells. Some compounds are quite readily metabolized, and others less so. It was known already from classical studies that phenolic substrates induce the expression of the enzymes for their degradation. Recently, the ability to degrade phenolics was shown to be a virulence factor. Conversely, phenolic compounds can increase the effectiveness of antifungals. Phenolics are known antioxidants, yet they have been shown to elicit cellular responses that would usually be triggered to counter oxidant stress. Here, we review the evidence for a connection between the fungal response to phenolics as small-molecule signals, and the response to oxidants. The connections proposed here should enable genetic screens to identify specific fungal receptors for plant phenolics. Furthermore, understanding how the pathogen detects plant phenolic compounds as a stress signal may facilitate new antifungal strategies.

G-quadruplexes are a source of vulnerability in BRCA2 deficient granule cell progenitors and medulloblastoma.

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 causes Fanconi anemia, complementation group FA-D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under SHH activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2 Δex3-4 ;Trp53 -/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping G-quadruplexes (G4s). Brca2-deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary MB tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2-deficient medulloblastomas.

Structure-activity relationships delineate how the maize pathogen Cochliobolus heterostrophus uses aromatic compounds as signals and metabolites.

The necrotrophic maize pathogen Cochliobolus heterostrophus senses plant-derived phenolic compounds, which promote nuclear retention of the redox-sensitive transcription factor ChAP1 and alter gene expression. The intradiol dioxygenase gene CCHD1 is strongly upregulated by coumaric and caffeic acids. Plant phenolics are potential nutrients but some of them are damaging compounds that need to be detoxified. Using coumaric acid as an inducer (16 to 160 µM), we demonstrated the rapid and simultaneous upregulation of most of the ß-ketoadipate pathway genes in C. heterostrophus. A cchd1 deletion mutant provided genetic evidence that protocatechuic acid is an intermediate in catabolism of a wide range of aromatic acids. Aromatics catabolism was slowed for compounds showing toxicity, and this was strongly correlated with nuclear retention of GFP-ChAP1. The activity of a structure series of compounds showed complementary requirements for upregulation of CCHD1 and for ChAP1 nuclear retention. Thus, there is an inverse correlation between the ability to metabolize a compound and the stress response (ChAP1 nuclear retention) that it causes. The ability to metabolize phenolics and to respond to them as signals should be an advantage to plant pathogens and may explain the presence of at least two response pathways detecting these compounds.

Adhesion as a Focus in Trichoderma-Root Interactions.

Fungal spores, germlings, and mycelia adhere to substrates, including host tissues. The adhesive forces depend on the substrate and on the adhesins, the fungal cell surface proteins. Attachment is often a prerequisite for the invasion of the host, hence its importance. Adhesion visibly precedes colonization of root surfaces and outer cortex layers, but little is known about the molecular details. We propose that by starting from what is already known from other fungi, including yeast and other filamentous pathogens and symbionts, the mechanism and function of Trichoderma adhesion will become accessible. There is a sequence, and perhaps functional, homology to other rhizosphere-competent Sordariomycetes. Specifically, Verticillium dahliae is a soil-borne pathogen that establishes itself in the xylem and causes destructive wilt disease. Metarhizium species are best-known as insect pathogens with biocontrol potential, but they also colonize roots. Verticillium orthologs of the yeast Flo8 transcription factor, Som1, and several other relevant genes are already under study for their roles in adhesion. Metarhizium encodes relevant adhesins. Trichoderma virens encodes homologs of Som1, as well as adhesin candidates. These genes should provide exciting leads toward the first step in the establishment of beneficial interactions with roots in the rhizosphere.

The fungal pathogen Cochliobolus heterostrophus responds to maize phenolics: novel small molecule signals in a plant-fungal interaction.

The transcription factor ChAP1 of the fungal pathogen of maize, Cochliobolus heterostrophus, responds to oxidative stress by migration to the nucleus and activation of antioxidant genes. Phenolic and related compounds found naturally in the host also trigger nuclear localization of ChAP1, but only slight upregulation of some antioxidant genes. ChAP1 thus senses phenolic compounds without triggering a strong antioxidant response. We therefore searched for genes whose expression is regulated by phenolic compounds and/or ChAP1. The C. heterostrophus genome contains a cluster of genes for metabolism of phenolics. One such gene, catechol dioxygenase CCHD1, was induced at least 10-fold by caffeic and coumaric acids. At high phenolic concentrations (≥ 1.6 mM), ChAP1 is needed for maximum CCHD1 expression. At micromolar levels of phenolics CCHD1 is as strongly induced in chap1 mutants as in the wild type. The pathogen thus detects phenolics by at least two signalling pathways: one causing nuclear retention of ChAP1, and another triggering induction of CCHD1 expression. The low concentrations required for induction of CCHD1 indicate fungal receptors for plant phenolics. Symbiotic and pathogenic bacteria are known to detect phenolics, and our findings generalize this to a eukaryotic pathogen. Phenolics and related compounds thus provide a ubiquitous plant-derived signal.

Genetic interaction of the stress response factors ChAP1 and Skn7 in the maize pathogen Cochliobolus heterostrophus.

The transcription factors ChAP1 and Skn7 of the maize pathogen Cochliobolus heterostrophus are orthologs of Yap1 and Skn7 in yeast, where they are predicted to work together in a complex. Previous work showed that in C. heterostrophus, as in yeast, ChAP1 accumulates in the nucleus in response to reactive oxygen species (ROS). The expression of genes whose products counteract oxidative stress depends on ChAP1, as shown by impaired ability of a Δchap1 mutant to induce these 'antioxidant' genes. In this study, we found that under oxidative stress, antioxidant gene expression is also partially impaired in the Δskn7 mutant but to a milder extent than in the Δchap1 mutant, whereas in the double mutant - Δchap1-Δskn7 - none of the tested genes was induced, with the exception of one catalase gene, CAT2. Both single mutants are capable of infecting the plant, showing similar virulence to the WT. The double mutant, however, showed clearly decreased virulence, pointing to additive contributions of ChAP1 and Skn7. Possible mechanisms are discussed, including additive regulation of gene expression by oxidative stress.

Role of the transcription factor ChAP1 in cytoplasmic redox homeostasis: imaging with a genetically encoded sensor in the maize pathogen Cochliobolus heterostrophus.

The redox-sensitive transcription factor ChAP1 [Cochliobolus heterostrophus YAP1 (Yeast Activator Protein 1) orthologue] of C. heterostrophus is required for oxidative stress tolerance. It is not known, however, to what extent the intracellular redox state changes on exposure of the fungus to oxidants, and whether ChAP1 is involved in the return of the cell to redox homeostasis. In order to answer these questions, we expressed a ratiometric redox-sensitive fluorescent protein sensor, pHyper, in C. heterostrophus. The fluorescence ratio was sensitive to extracellular hydrogen peroxide (H2O2) concentrations that had been shown previously to inhibit the germination of conidia and growth of the pathogen in culture. chap1 mutants showed a slower return to redox homeostasis than the wild-type on exposure to H2O2. Plant extracts that mimic oxidants in their ability to promote nuclear retention of ChAP1 reduced, rather than oxidized, the fungal cells. This result is consistent with other data suggesting that ChAP1 responds to plant-derived signals other than oxidants. pHyper should be a useful reporter of the intracellular redox state in filamentous fungi.