Iron, Oxidative Stress, and Virulence: Roles of Iron-Sensitive Transcription Factor Sre1 and the Redox Sensor ChAp1 in the Maize Pathogen Cochliobolus heterostrophus

Abstract

The gene SRE1, encoding the GATA transcription factor siderophore biosynthesis repressor (Sre1), was identified in the genome of the maize pathogen Cochliobolus heterostrophus and deleted. Mutants were altered in sensitivity to iron, oxidative stress, and virulence to the host. To gain insight into mechanisms of this combined regulation, genetic interactions among SRE1 (the nonribosomal peptide synthetase encoding gene NPS6, which is responsible for extracellular siderophore biosynthesis) and ChAP1 (encoding a transcription factor regulating redox homeostasis) were studied. To identify members of the Sre1 regulon, expression of candidate iron and oxidative stress-related genes was assessed in wild-type (WT) and sre1 mutants using quantitative reverse-transcription polymerase chain reaction. In sre1 mutants, NPS6 and NPS2 genes, responsible for siderophore biosynthesis, were derepressed under iron replete conditions, whereas the high-affinity reductive iron uptake pathway associated gene, FTR1, was not, in contrast to outcomes with other well-studied fungal models. C. heterostrophus L-ornithine-N(5)- monooxygenase (SIDA2), ATP-binding cassette (ABC6), catalase (CAT1), and superoxide dismutase (SOD1) genes were also derepressed under iron-replete conditions in sre1 mutants. Chap1nps6 double mutants were more sensitive to oxidative stress than either Chap1 or nps6 single mutants, while Chap1sre1 double mutants showed a modest increase in resistance compared with single Chap1 mutants but were much more sensitive than sre1 mutants. These findings suggest that the NPS6 siderophore indirectly contributes to redox homeostasis via iron sequestration, while Sre1 misregulation may render cells more sensitive to oxidative stress. The double-mutant phenotypes are consistent with a model in which iron sequestration by NPS6 defends the pathogen against oxidative stress. C. heterostrophus sre1, nps6, Chap1, Chap1nps6, and Chap1sre1 mutants are all reduced in virulence toward the host, compared with the WT.

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).…”

Intracellular siderophore but not extracellular siderophore is required for full virulence in Metarhizium robertsii

“…This is not unexpected since fungi rely on multiple iron acquisition pathways to adapt to different environments. In this study we attempted to shed some light on the contribution of one of these pathways, SIA, to M. robertsii fitness, by identifying mrsidC, mrsidD and mrsreA as the orthologs of the ferricrocin synthetase sidC/NPS2, the coprogen-type siderophore synthetase sidD/NPS6, and the transcription regulator sreA/SRE1 previously characterized in other fungal species (Oberegger et al, 2001;Oide et al, 2007;Oide et al, 2006;Schrettl et al, 2004;Zhang et al, 2013). Predicted proteins encoded by mrsidC, mrsidD and mrsreA display structural characteristics typical of their predicted function as NRPSs and GATA-type transcription factors and their genomic regions are largely conserved in other Hypocrealeans.…”

“…For instance, in A. fumigatus ΔsreA mutants were as virulent as the WT, while in C. neoformans the GATA factor cir1 not only controls iron homeostasis, but also several virulence factors and its loss attenuated virulence (Jung and Kronstad, 2008). Similarly, in the plant pathogen C. heterostrophus, SRE1 mutants were reduced in virulence (Zhang et al, 2013). Mutations examined in this study had no effect on growth on rich undefined media with the only exception of ΔmrsreA strains, which showed slight, but significant, inhibition (Fig.…”

Genetic interaction of the stress response factors ChAP1 and Skn7 in the maize pathogenCochliobolus heterostrophus

“…On commercial hybrid maize cultivars Jubilee (Lev et al, 2005) and Royalty (this study), loss of ChAP1 did not compromise virulence in droplet inoculation assays. On the maize cultivar W64A, spray inoculation with Dchap1 resulted in about twofold decreased lesion size as compared with WT (Zhang et al, 2013). Necrotrophs like C. heterostrophus are thought to thrive in an oxidant-rich environment (see Heller & Tudzynski, 2011).…”

“…Again, this suggests that the Skn7 regulon includes additional genes that are critical for tolerance to oxidants and other stresses. C. heterostrophus should be a good model necrotrophic pathogen in which to address these questions at the systems level, considering that the genome is being studied intensively (Ohm et al, 2012;Condon et al, 2013), as is the genetic basis for stress physiology (Lev et al, 2005;Igbaria et al, 2008;Oide et al, 2010;Wu et al, 2012;Zhang et al, 2013).…”