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

Abstract

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.

Detection of Cochliobolus heterostrophus races in South China

“…The most important research progress on C. heterostrophus focuses on genome sequencing of races T and O Turgeon & Baker, 2007) and determination of essential genes required for synthesizing T-toxin in race T (Baker et al, 2006;Inderbitzin, Asvarak, & Turgeon, 2010). Many transcription factors are involved in stress response, toxin production or virulence regulation (Lev et al, 2005;Ronen, Shalaby, & Horwitz, 2013;Shalaby, Larkov, Lamdan, & Horwitz, 2013;Zhang et al, 2013;Bi, Wu, Zhu, & Gillian Turgeon, 2013;Ganem et al, 2004;Horwitz et al, 1999;Igbaria et al, 2008;Izumitsu et al, 2009;Lev, Sharon, Hadar, Ma, & Horwitz, 1999;Oide et al, 2010;Wu, Oide, Zhang, Choi, & Turgeon, 2012). Among race types, race O is the predominant agent of maize disease in China; maize resistance against race O is determined by quantitative trait locus (Balint-Kurti et al, 2007;Carson, Stuber, & Senior, 2004).…”

Reactive oxygen species metabolism and plant-fungal interactions

“…A genetically encoded HyPer (hydrogen peroxide) sensor used in filamentous fungi-originally developed to detect intracellular ROS in mammalian cells using a circularly permuted yellow fluorescent protein (cpYFP) bound to the regulatory domain of the H 2 O 2 -sensing OxyR protein in Escherichia coli (Belousov et al, 2006)-has been optimized for several fungal phytopathogens (Huang et al, 2016;Ronen et al, 2013). The HyPer sensor allows for intracellular measurement of ROS and is specific and sensitive to H 2 O 2 (Belousov et al, 2006).…”

“…The HyPer sensor allows for intracellular measurement of ROS and is specific and sensitive to H 2 O 2 (Belousov et al, 2006). The necrotrophic maize pathogen, C. heterostrophus, redox sensor pHyPer was initially used to measure the role of the transcription regulator ChAP1 in mediating redox homeostasis (Ronen et al, 2013). Ronen et al (2013) used the pHyPer probe to monitor the time it took to reestablish redox homeostasis after challenge with oxidative stress in a Δchap1 mutant by measuring fluorescent excitation ratios.…”

“…The necrotrophic maize pathogen, C. heterostrophus, redox sensor pHyPer was initially used to measure the role of the transcription regulator ChAP1 in mediating redox homeostasis (Ronen et al, 2013). Ronen et al (2013) used the pHyPer probe to monitor the time it took to reestablish redox homeostasis after challenge with oxidative stress in a Δchap1 mutant by measuring fluorescent excitation ratios. Subsequently, a more sensitive HyPer2 sensor was developed to measure redox balance in mutants impaired for oxidative stress regulation in F. graminearum (Mentges and Bormann, 2015).…”

Optimization of the HyPer sensor for robust real‐time detection of hydrogen peroxide in the rice blast fungus

“…Ronen et al . () used the HyPer construct to examine H 2 O 2 levels in the necrotrophic corn pathogen Cochliobolus heterostrophus after exposure to plant phenolics, and a comparison was made between the wild‐type and a fungal line mutant in the AP1 homologue, CHAP1 . Both endogenous sensors demonstrated that, on exposure to ROS, the mutants took longer to recover redox homeostasis.…”