Pathogenicity Assay for <i>Cochliobolus heterostrophus</i> G-Protein and MAPK Signaling Deficiency Strains

Abstract

Cochliobolus heterostrophus is an agriculturally important and emerging model pathogen for studying the signaling hierarchies' role during the maize host colonization. In particular, G-protein and MAPK-linked pathways are playing a major role during pathogenesis. Although gene disruption studies are an efficient way of identifying the role of these cascades, differentiating between the mutant strains' virulence ability may become an intricate task. For example, in C. heterostrophus, mutants in a G-protein α subunit gene, cga1, are defective in mating and appressorium formation, but unlike mutants in homologous genes in other fungal pathogens, the cga1 mutants remained highly virulent to corn under some host physiological conditions. Here, we used the cga1 strain as a model for developing an in vivo sensitive and accurate pathogenicity assay. A detailed and well controlled analysis of wild type (WT) and cga1 pathogenic behavior revealed that detached leaves are significantly more vulnerable to the disease than intact ones. In intact leaves, cga1 mutants were less infective of maize under most conditions. This difference was maximized when the first seedling leaf was chosen for inoculation and when the infected leaves, with spores or mycelia fragments droplets, were incubated for a period of four days. This optimal condition set enabled us to classify the C. heterostrophus G-protein signaling mutants deficient in α, β or both subunits in order of decreasing virulence: WT > cga1> cgb1> cga1 cgb1. The method presented proved to be accurate and sensitive enough to identify even slight variations in virulence. Moreover, it could be modified for use in studies of other foliar phytoparasitic fungi.

Characterization of Botrytis–plant interactions using PathTrack©—an automated system for dynamic analysis of disease development

“…, Table S1, see Supporting Information). Because there is no structural change in detached relative to attached leaves, the differences in kinetics of infection on living detached leaves might be attributed, at least in part, to the stimulation of the defence response caused by wounding of the leaf by the detachment process, and hence higher levels of resistance in the detached leaves (Degani, ; Degani et al ., ).…”

Plant growth hormones suppress the development of Harpophora maydis, the cause of late wilt in maize

“…Moreover, detaching the roots may accelerate their senescence and susceptibility to the pathogen (as shown in leaves for the foliar pathogen Cochliobolus heterostrophus (Degani 2014)). The roots themselves may be subject to the hormone influence (CK and IAA inhibit senescence while the ET precursor ACC or ACPC accelerates senescence) that can alter their susceptibility to pathogen invasion (Degani et al 2004).…”

Methods for Studying Magnaporthiopsis maydis, the Maize Late Wilt Causal Agent

“…The effect of environmental suppressors, enhancers, or regulators on the ability of M. maydis to infect detached roots can be assessed efficiently using the detached root assay when the fungus grows on its natural food source. Detaching the roots may accelerate their senescence and susceptibility to the pathogen (as shown in leaves for the foliar pathogen Cochliobolus heterostrophus [51]). This is important for increasing the sensitivity of this test.…”