The autophagy genes ChATG4 and ChATG8 are required for reproductive development, virulence, and septin assembly in Cochliobolus heterostrophus

Autophagy is a highly conserved degrading process and is crucial for cell growth and development in eukaryotes, especially when they face starvation and stressful conditions. To evaluate the functions of Atg4 and Atg8 in mycelial growth, asexual and sexual development, and virulence in Cochliobolus heterostrophus, ΔChatg4 and ΔChatg8 mutants were generated by gene replacement. Strains deleted for ChATG4 and ChATG8 genes showed significant changes in vegetative growth and in development of conidia and ascospores compared with the wild-type strain. The autophagy process was blocked and the virulence was reduced dramatically in ΔChatg4 and ΔChatg8 mutants. In addition, deletion of ChATG4 and ChATG8 disordered Cdc10 subcellular localization and formation of septin rings. The direct physical interaction between ChAtg4 and ChAtg8 was detected by Yeast-two-hybrid, and ChAtg4-GFP was dispersed throughout the cytoplasm, although GFP-ChAtg8 appeared as punctate structures. All phenotypes were restored in complemented strains. Taken together, these findings indicated that ChATG4 and ChATG8 were crucial for autophagy to regulate fungal growth, development, virulence, and localization of septin in C. heterostrophus.

Developmental Roles of the Hog1 Protein Phosphatases of the Maize Pathogen Cochliobolus heterostrophus

Protein phosphorylation cascades are universal in cell signaling. While kinome diversity allows specific phosphorylation events, relatively few phosphatases dephosphorylate key signaling proteins. Fungal mitogen activated protein kinases (MAPK), in contrast to their mammalian counterparts, often show detectable basal phosphorylation levels. Dephosphorylation, therefore, could act as a signal. In Cochliobolus heterostrophus, the Dothideomycete causing Southern corn leaf blight, ferulic acid (FA)—an abundant phenolic found in plant host cell walls—acts as a signal to rapidly dephosphorylate the stress-activated MAP kinase Hog1 (High Osmolarity Glycerol 1). In order to identify the protein phosphatases responsible, we constructed mutants in Hog1 phosphatases predicted from the genome by homology to yeast and other species. We found that Cochliobolus heterostrophus mutants lacking PtcB, a member of the PP2C family, exhibited altered growth, sporulation, and attenuated dephosphorylation in response to FA. The loss of the dual-specificity phosphatase CDC14 led to slow growth, decreased virulence, and attenuated dephosphorylation. Mutants in two predicted tyrosine phosphatase genes PTP1 and PTP2 showed normal development and virulence. Our results suggest that a network of phosphatases modulate Hog1’s dual phosphorylation levels. The mutants we constructed in this work provide a starting point to further unravel the signaling hierarchy by which exposure to FA leads to stress responses in the pathogen.

Desarrollo de la mancha foliar por Bipolaris maydis (teleomorfo: Cochliobolus heterostrophus) en maíz dulce, en función de nitrógeno, potasio y silicio en invernadero

El objetivo del trabajo fue evaluar el desarrollo de la mancha foliar causada por Bipolaris maydis, enfermedad común e n maíz dulce, según la dosis y el acumulado de nitrógeno, potasio y silicio, en condiciones de invernadero. Los tratamientos consistieron en cinco dosis de nitrógeno (N) (0, 200, 400, 800 y 1.200 kg/ha), aplicación o no de potasio (K) (240 kg/ha) y aplicación o no de silicio (Si) (380 kg/ha). Se empleó un diseño experimental completamente aleatorizado con esquema factorial 5 x 2 x 2 y tres repeticiones. Se inocularon todos los tratamientos a los 30 días con una suspensión de 2 x 105 conidios por mL de B. maydis. A los 42 días se midió la acumulación de N, K y Si, así como el porcentaje de infección y el área bajo la curva de progreso de la enfermedad (ABCPE). Los tratamientos que recibieron fertilización con Si y K presentaron mayores acumulados de N, K y Si, y niveles más bajos de intensidad de ataque y ABCPE. Los acumulados de N, K y Si presentaron variaciones frente a las interacciones de la fertilización de K con N, y de Si con N; sin embargo, los niveles de las variables de la enfermedad fueron menores en la fertilización con K o Si, combinadas con las dosis de 0, 200 y 400 kg/ha de N.

Copper Ions are Required for Cochliobolus heterostrophus in Appressorium Formation and Virulence on Maize

Cochliobolus heterostrophus is the causal agent of southern corn leaf blight, a destructive disease on maize worldwide. However, how it regulates virulence on maize is still largely unknown. Here, we report that two copper transporter genes, ChCTR1 and ChCTR4, are required for its virulence. chctr1 and chctr4 mutants showed attenuated virulence on maize compared with the wild-type strain TM17 but development phenotypes of those mutants on media with or without infection-related stress agents were the same as the wild-type strain. Moreover, ChCTR1 and ChCTR4 play critical roles in appressorium formation and mutation of ChCTR1 or ChCTR4 suppresses the appressorium formation. Furthermore, copper-chelating agent ammonium tetrathiomolybdate suppressed the appressorium formation and virulence of C. heterostrophus on maize, whereas copper ions enhanced the appressorium formation and virulence on maize. The results indicate that copper ions are required for appressorium formation and virulence of C. heterostrophus on maize and are acquired from the environment by two copper transporters: ChCTR1 and ChCTR4.

Clues to an Evolutionary Mystery: The Genes for T-Toxin, Enabler of the Devastating 1970 Southern Corn Leaf Blight Epidemic, Are Present in Ancestral Species, Suggesting an Ancient Origin

The Southern corn leaf blight (SCLB) epidemic of 1970 devastated fields of T-cytoplasm corn planted in monoculture throughout the eastern United States. The epidemic was driven by race T, a previously unseen race of Cochliobolus heterostrophus. A second fungus, Phyllosticta zeae-maydis, with the same biological specificity, appeared coincidentally. Race T produces T-toxin, while Phyllosticta zeae-maydis produces PM-toxin, both host-selective polyketide toxins necessary for supervirulence. The present abundance of genome sequences offers an opportunity to tackle the evolutionary origins of T- and PM- toxin biosynthetic genes, previously thought unique to these species. Using the C. heterostrophus genes as probes, we identified orthologs in six additional Dothideomycete and three Eurotiomycete species. In stark contrast to the genetically fragmented race T Tox1 locus that encodes these genes, all newly found Tox1-like genes in other species reside at a single collinear locus. This compact arrangement, phylogenetic analyses, comparisons of Tox1 protein tree topology to a species tree, and Tox1 gene characteristics suggest that the locus is ancient and that some species, including C. heterostrophus, gained Tox1 by horizontal gene transfer. C. heterostrophus and Phyllosticta zeae-maydis did not exchange Tox1 DNA at the time of the SCLB epidemic, but how they acquired Tox1 remains uncertain. The presence of additional genes in Tox1-like clusters of other species, although not in C. heterostrophus and Phyllosticta zeae-maydis, suggests that the metabolites produced differ from T- and PM-toxin.