Principles of Carbon Catabolite Repression in the Rice Blast Fungus: Tps1, Nmr1-3, and a MATE–Family Pump Regulate Glucose Metabolism during Infection

Abstract

Understanding the genetic pathways that regulate how pathogenic fungi respond to their environment is paramount to developing effective mitigation strategies against disease. Carbon catabolite repression (CCR) is a global regulatory mechanism found in a wide range of microbial organisms that ensures the preferential utilization of glucose over less favourable carbon sources, but little is known about the components of CCR in filamentous fungi. Here we report three new mediators of CCR in the devastating rice blast fungus Magnaporthe oryzae: the sugar sensor Tps1, the Nmr1-3 inhibitor proteins, and the multidrug and toxin extrusion (MATE)–family pump, Mdt1. Using simple plate tests coupled with transcriptional analysis, we show that Tps1, in response to glucose-6-phosphate sensing, triggers CCR via the inactivation of Nmr1-3. In addition, by dissecting the CCR pathway using Agrobacterium tumefaciens-mediated mutagenesis, we also show that Mdt1 is an additional and previously unknown regulator of glucose metabolism. Mdt1 regulates glucose assimilation downstream of Tps1 and is necessary for nutrient utilization, sporulation, and pathogenicity. This is the first functional characterization of a MATE–family protein in filamentous fungi and the first description of a MATE protein in genetic regulation or plant pathogenicity. Perturbing CCR in Δtps1 and MDT1 disruption strains thus results in physiological defects that impact pathogenesis, possibly through the early expression of cell wall–degrading enzymes. Taken together, the importance of discovering three new regulators of carbon metabolism lies in understanding how M. oryzae and other pathogenic fungi respond to nutrient availability and control development during infection.

Regulation of Superoxide Dismutase Synthesis in Humcola lutea Cells under Cu2+ Stress Conditions

“…Moreover, when glucose was replaced with G6P, a marked increase in SOD activity was immediately produced in Cutreated cultures. The influence of G6P as an effector of catabolite repression has also been shown in bacteria and fungi [ 19 ]. Graham et al [ 20 ] proposed that the signal giving rise to a change in gene expression of glucose repressed enzymes originates from the intracellular concentration of hexose sugars or the flux of hexose sugars into glycolysis.…”

Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein � DNA Interactions in Magnaporthe oryzae

“…This is achieved by transforming the plasmid DNA isolated in step A6 into M. oryzae protoplasts and selecting for hygromycin resistance. Protoplast generation and transformation, as well as screening for fungal transformants, are performed as described previously (Fernandez et al, 2012;Wilson et al, 2010) and summarized as follows:…”

Molecular mechanisms regulating plant infection in the rice blast fungus Magnaporthe oryzae

“…M. oryzae is considered a hemibiotrophic fungus based on its nutritional mode during host invasion. However, genes regulating the switch in life style and acquisition of nutrients during plant infection are largely unknown (Kankanala, et al, 2007, Fernandez & Wilson, 2012. The duration of the biotrophic versus the necrotrophic phase in M. oryzae is also unknown.…”

“…CCR is a genetic mechanism that ensures the preferential use of glucose over other, less-preferred carbon sources, and it is also present in M. oryzae (Fernandez & Wilson, 2012). M. oryzae has the ability to use a wide range of mono-and disaccharides as sole carbon source but has a strong preference for glucose (Tanzer, et al, 2003, Wilson, et al, 2007, Fernandez & Wilson, 2012.…”

“…M. oryzae has the ability to use a wide range of mono-and disaccharides as sole carbon source but has a strong preference for glucose (Tanzer, et al, 2003, Wilson, et al, 2007, Fernandez & Wilson, 2012. In A. nidulans, CCR is mediated at DNA level by the global transcriptional repressor CreA.…”