- Top of page
- Materials and Methods
- Results and Discussion
- Supporting Information
Rice blast disease caused by Magnaporthe oryzae is a major global emerging infectious disease (EID) causing rice losses sufficient to feed 3–10.6% of the world's population 2000 calories a day for 1 yr (Fisher et al., 2012). Infection starts with the germination of a three-celled spore on the host surface, producing a short germ tube that develops a domed infection structure, termed the appressorium (Wilson & Talbot, 2009). Development requires progression through a single cell cycle in the apical cell, followed by autophagy (Veneault-Fourrey et al., 2006) and mobilization of storage reserves to fuel appressorium development (Thines et al., 2000; Wang et al., 2007; Patkar et al., 2012a). The appressorium becomes melanized and builds up sufficient turgor pressure to drive an infection peg into the host epidermal cell (Howard et al., 1991; Money & Howard, 1996; Wilson & Talbot, 2009). In a susceptible host, the infection hyphae ramify through the infected cell and then colonize adjacent cells through plasmodesmata (Kankanala et al., 2007). In resistant varieties, infection is halted at the penetration stage or during early colonization, and is associated with a hypersensitive response (HR) (Torres, 2010; Heller & Tudzynski, 2011).
Plants respond to infection by the rapid production of reactive oxygen species (ROS) using membrane-bound NADPH oxidases (Marino et al., 2012), or secreted peroxidases and amine oxidases (Bolwell et al., 2002), as part of the general pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) or more specific effector-triggered immunity (ETI) responses (Yoshioka et al., 2009; Torres, 2010; Heller & Tudzynski, 2011; Thomma et al., 2011; Tudzynski et al., 2012). ROS produce cross-linked plant wall polymers to form a barrier to penetration, attack the pathogen directly or act as diffusible signals in the plant to up-regulate pathogenesis-related proteins (Lamb & Dixon, 1997; Shetty et al., 2008; Heller & Tudzynski, 2011). In a resistant host challenged by an avirulent pathogen, the initial ROS burst is followed by a longer lasting second phase, culminating in an HR (Mur et al., 2008) and programmed cell death (Levine et al., 1994). In biotrophic and hemibiotrophic fungi, such as M. oryzae, which require living hosts, the first phase of ROS production still occurs, but the second phase is suppressed in susceptible hosts, probably through the secretion of effectors through the biotrophic interfacial complex (Valent & Khang, 2010), which re-programme the metabolic pathways involved in host ROS production (Parker et al., 2009).
Fungi require effective anti-oxidant defence systems to operate in such an environment in which oxidative stress is endemic. An abundant array of anti-oxidant genes exists in the M. grisea genome (Dean et al., 2005; Egan & Talbot, 2008; Morel et al., 2008), although less is known about the low-molecular-weight anti-oxidants in fungi more generally (Georgiou & Petropoulou, 2001; Patsoukis & Georgiou, 2004). Nevertheless, the need to detoxify host ROS can be inferred from fungal mutants that lack critical anti-oxidant enzymes, or from treatments that manipulate ROS levels during infection. Thus, Magnaporthe mutants in glutathione peroxidase, Hyr1, are less tolerant to ROS and produce smaller lesions on susceptible plants (Huang et al., 2011). Likewise, several redox-sensitive transcription factors, such as MoAP1 (Guo et al., 2011) and MoSwi6 (Qi et al., 2012), and the defence suppressor Des1 (Chi et al., 2009) increase resistance to external H2O2, with mutants showing reduced pathogenicity and pleiotropic changes in gene expression, including decreases in extracellular peroxidases. Conversely, the reduction of external ROS levels during infection through the addition of exogenous catalase (Tanabe et al., 2009) or the NADPH oxidase inhibitor diphenylene iodonium (DPI) (Chi et al., 2009) promotes increased infection of compatible and incompatible strains, or particular H2O2-sensitive mutants.
Nevertheless, the significance of other putative anti-oxidant defences is less clear. Thus, the deletion of the major secreted M. oryzae catalase-peroxidase, CPXB, increases sensitivity to exogenous H2O2, but does not affect overall pathogenicity (Tanabe et al., 2011). Likewise, mutants lacking the large subunit catalase, catB, are less pathogenic, but through changes in normal fungal wall strengthening rather than by detoxification of host-derived H2O2 (Skamnioti et al., 2007). Furthermore, expression data from transcriptome profiling show that the genes most highly up-regulated in M. oryzae during infection are related to nutrient limitation rather than oxidative stress (Mathioni et al., 2011). This echoes results for the necrotrophic fungus Botrytis cinerea, in which the redox-sensitive AP1 transcription factor homologue, Bap1, is critical for ROS resistance in vitro, but deletion mutants do not show reduced virulence, and the suite of downstream target genes regulated by Bap1 is not highly expressed in planta (Temme & Tudzynski, 2009). The authors concluded that B. cinerea does not suffer H2O2 stress in planta, in contrast with conventional expectations of the role of the oxidative burst in restricting infection (Temme & Tudzynski, 2009), but actually exploits the host HR as part of its necrotrophic habit (Govrin & Levine, 2000).
The interplay between ROS and anti-oxidant defences is further complicated, as ROS are produced by normal cell metabolism and act as signalling intermediates associated with key transitions in microbial development, including differentiation, sexual reproduction, conidiation, spore germination, secondary metabolism and apoptosis (Hansberg & Aguirre, 1990; Aguirre et al., 2005; D'Autreaux & Toledano, 2007; Gessler et al., 2007; Takemoto et al., 2007; Egan & Talbot, 2008; Scott & Eaton, 2008; Shetty et al., 2008; Aguirre & Lambeth, 2010; Heller & Tudzynski, 2011; Tudzynski et al., 2012). Indeed, Hansberg and Aguirre originally proposed that microbial cell differentiation might be triggered by transient oxidation that initiates a shift between developmental states (Hansberg & Aguirre, 1990; Aguirre et al., 2005). ROS production is well documented during pre-penetration in M. oryzae, whereas scavenging external ROS reduces infection rates, all consistent with a role for ROS in the developmental programme (Egan et al., 2007; Ryder et al., 2013).
The major cytoplasmic anti-oxidant that mitigates oxidative stress in eukaryotes is glutathione (Belozerskaya & Gessler, 2007; Gessler et al., 2007; Meyer, 2008). However, little is known about the glutathione concentrations and dynamics in filamentous fungi. Tools are now available to quantify both the amount of glutathione in vivo (Fricker et al., 2000; Meyer & Fricker, 2000, 2008; Fricker & Meyer, 2001; Meyer et al., 2001) and the electrochemical potential of the reduced glutathione:oxidized glutathione (GSH:GSSG) redox couple (EGSH) using transgenic redox green fluorescent protein (GFP)-based reporters (Dooley et al., 2004; Schwarzlander et al., 2008; Meyer & Dick, 2010). In particular, Grx1-roGFP2 includes a glutaredoxin (Grx) subunit to improve the response kinetics (Gutscher et al., 2008) and is known to function correctly in fungi (Heller et al., 2012). Notably, the difference in the mid-point potential between the Grx1-roGFP redox couple and the GSH:GSSG redox couple makes Grx1-roGFP exquisitely sensitive to small changes in the degree of glutathione oxidation from the highly reduced level typically found in vivo (Meyer & Dick, 2010). Thus, in this article, we use multi-parameter live-cell confocal imaging and a range of fluorescent reporters to determine, first, whether there is physiological evidence for the redox control of early development in M. oryzae mediated by changes in EGSH; second, what is the relative level of endogenous ROS production during development; third, what is the capacity of the glutathione anti-oxidant system to deal with an imposed oxidative burst, as might be encountered during host infection; and fourth, what impact is exerted by the actual host oxidative burst on EGSH in vivo during susceptible and resistant interactions.