Monothiol glutaredoxin DaGrxB couples iron homeostasis with redox balance to orchestrate development and virulence in the peach shoot blight fungus Diaporthe amygdali

Abstract Peach shoot blight (PSB), caused by Diaporthe amygdali , is one of the most devastating fungal diseases affecting peach production worldwide. Glutaredoxins (Grxs) are ubiquitous redox-regulatory proteins involved in antioxidant defense and iron metabolism, yet their functions in phytopathogenic fungi remain largely unknown. Here, we identified four Grxs (DaGrxA-DaGrxD) in D. amygdali . Among them, DaGrxB, a monothiol glutaredoxin, is the sole member essential for vegetative growth, asexual reproduction and virulence. DaGrxB interacts with iron-regulatory transcription factors DaHapX and DaSreA and modulates their activities via iron-dependent nucleocytoplasmic shuttling, coordinating iron acquisition and storage gene expression. During infection, D. amygdali encounters a biphasic iron environment: an inherently iron-limited host environment at early stages (1-2 dpi) followed by host-mediated iron excess (3-4 dpi), and DaGrxB is essential for navigating this transition. Consistently, deletion of either DaHAPX or DaSREA significantly reduced virulence, confirming that iron homeostasis is a critical determinant of D. amygdali virulence. Notably, deletion of DaGRXB disrupts Fe-S cluster biosynthesis, causing mitochondrial Fe²⁺ accumulation, impaired aconitase activity, and reduced ATP production, which triggers constitutive activation of the GSH-dependent antioxidant system, with elevated GSH/GSSG ratios, increased GR and GST activities, and reduced H₂O₂ levels. Exogenous GSH and H₂O₂ depletion both reduced virulence, suggesting that disturbed redox balance directly contributes to attenuated virulence. Additionally, the Trx domain assists the Grx domain in nucleocytoplasmic translocation through intramolecular interactions. Finally, LDH-delivered dsRNA targeting DaGRXB effectively controls PSB. Collectively, these findings reveal how a monothiol glutaredoxin integrates iron homeostasis, redox balance and virulence in a plant pathogen, providing a foundation for RNAi-based disease management. Author summary Peach shoot blight is a devastating disease that causes substantial yield losses in peach production worldwide. The causal fungus, Diaporthe amygdali , also infects other economically important fruit crops. During infection, plants use iron as a weapon: either by starving the pathogen of iron or by releasing toxic levels of it. Glutaredoxins (Grxs) are ubiquitous redox-regulatory proteins involved in antioxidant defense and iron metabolism, and they are emerging as key players in this process. We studied how this fungus fights back. We found that it carries four glutaredoxin (Grx) proteins, but only DaGrxB is essential for its development and virulence. DaGrxB acts as a molecular switch that helps the fungus sense and respond to the changing iron levels inside the host. It does this by moving between the nucleus and cytoplasm, where it works together with two iron-regulatory proteins DaHapX and DaSreA to control iron uptake and storage genes expression. When we deleted DaGrxB , the fungus could no longer manage iron properly, leading to disrupted energy production and an overactive antioxidant system. Importantly, we also developed a spray that uses RNA interference to target DaGRXB , which effectively controlled the disease. Our findings reveal how a single protein helps a fungal pathogen survive the host’s iron-based defenses, and offer a promising strategy for disease management.