Molecular Characterization of the Purine Degradation Pathway Genes ALA1 and URE1 of the Maize Anthracnose Fungus Colletotrichum graminicola Identified Urease as a Novel Target for Plant Disease Control

Fungal pathogenicity is governed by environmental factors, with nitrogen playing a key role in triggering pathogenic development. Spores germinating on the plant cuticle are exposed to a nitrogen-free environment, and reprograming of nitrogen metabolism is required for bridging the time needed to gain access to the nitrogen sources of the host. Although degradation of endogenous purine bases efficiently generates ammonium and may allow the fungus to bridge the preinvasion nitrogen gap, the roles of the purine degradation pathway and of the key genes encoding allantoicase and urease are largely unknown in plant pathogenic fungi. To investigate the roles of the allantoicase and urease genes ALA1 and URE1 of the maize anthracnose fungus Colletotrichum graminicola in pathogenic development, we generated ALA1:eGFP and URE1:eGFP fusion strains as well as allantoicase- and urease-deficient mutants. Virulence assays, live cell, and differential interference contrast imaging, chemical complementation and employment of a urease inhibitor showed that the purine degradation genes ALA1 and URE1 are required for bridging nitrogen deficiency at early phases of the infection process and for full virulence. Application of the urease inhibitor acetohydroxamic acid did not only protect maize from C. graminicola infection, but also interfered with the infection process of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici, the maize and broad bean rusts Puccinia sorghi and Uromyces viciae-fabae, and the potato late blight pathogen Phytophthora infestans. Our data strongly suggest that inhibition of the purine degradation pathway might represent a novel approach to control plant pathogenic fungi and oomycetes.

Excess Iron Enhances Purine Catabolism Through Activation of Xanthine Oxidase and Impairs Myelination in the Hippocampus of Nursing Piglets

Background Few studies have addressed the risk of nutritional iron overexposure in infancy. We previously found that excess dietary iron in nursing piglets resulted in iron overload in the liver and hippocampus and diminished socialization with novel conspecifics in a test for social novelty preference. Objectives This experiment aimed to identify metabolites and metabolic pathways affected by iron overload in the liver and hippocampus of nursing piglets. Methods Liver and hippocampal tissues collected from 22-d-old piglets (Hampshire × Yorkshire crossbreed; 5.28 ± 0.53 kg body weight; 50% male) that received orally 0 (NI group) or 50 mg iron/(d · kg body weight) (HI group) from postnatal day (PD) 2 to PD21 were analyzed for mRNA and protein expression and enzyme activity of xanthine oxidase (XO). Untargeted metabolomics was performed using GC-MS. Expression of myelin basic protein (MBP) in the hippocampus was determined using western blot. Results There were 108 and 126 metabolites identified in the hippocampus and liver, respectively. Compared with NI, HI altered 15 metabolites (P < 0.05, q < 0.2) in the hippocampus, including a reduction in myo-inositol (0.86-fold) and N-acetylaspartic acid (0.84-fold), 2 metabolites important for neuronal function and myelination. Seven metabolites involved in purine and pyrimidine metabolism (e.g., hypoxanthine, xanthine, and β-alanine) were coordinately changed in the hippocampus (P < 0.05, q < 0.2), suggesting that iron excess enhanced purine catabolism. The mRNA expression (2.3-fold) (P < 0.05) and activity of XO, a rate-limiting enzyme in purine degradation, was increased. Excess iron increased hippocampal lipid peroxidation by 74% (P < 0.05) and decreased MBP by 44% (P = 0.053). The hepatic metabolome was unaffected. Conclusions In nursing piglets, excess iron enhances hippocampal purine degradation through activation of XO, which may induce oxidative stress and alter energy metabolism in the developing brain.

Early Life Iron Excess Enhances Hippocampal Purine Catabolism Through Activation of Xanthine Oxidase in a Nursing Piglet Model (P11-072-19)

Objectives Our prior work demonstrated that dietary iron excess in early life results in iron overload in both liver and hippocampus in pre-weanling piglets. Herein, we aimed to identify metabolic processes altered by iron overload in liver and hippocampus. Methods Liver and hippocampal tissues collected from 21-day old nursing piglets receiving high (HI; 50 mg iron/d ${\cdot}$ kg body weight), n = 5) or no oral iron supplementation (NI, n = 5) from birth to PD 21 were analyzed for non-targeted metabolomics, using gas chromatography mass spectrometry. Based on profiled changes in hippocampal metabolites, we further analyzed xanthine oxidase (XO), a rate limiting enzyme for purine degradation, for its mRNA, protein and enzyme activity by RT-qPCR, western blot, and ELISA. Results 108 and 126 metabolites were identified in hippocampus and liver, respectively. In comparison with NI, HI altered abundance of 15 metabolites in hippocampus (P < 0.05, q < 0.2). Myo-inositol and N-acetylaspartic acid, two abundant metabolites in the CNS with broad implications in neuronal function and myelination, were decreased in response to hippocampal iron overload. Seven metabolites involved in purine and pyrimidine metabolism (e.g., hypoxanthine, xanthine and beta-alanine) in hippocampus were modulated in a coordinated pattern by HI, implicating a shift from purine salvage towards degradation, governed by XO. In support of these findings, up-regulation of XO mRNA expression (2.3-fold, P < 0.05) and activity (fold/stats? ) was found in hippocampus but not in liver (P > 0.05). Despite overt iron loading, the hepatic metabolome remained stable (q > 0.2). Conclusions Our findings suggest that iron overload increases hippocampal purine degradation via enhanced XO expression and activity, deleteriously altering tissue redox balance and ROS production. Purine salvage contributes to ATP production in the CNS, where a global shift from purine salvage to degradation due to HI may compromise energetics in the developing hippocampus. Funding Sources UC Davis; NIFA.