Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens

Journal of Experimental Botany Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens Graham R. D. McGrann 1 2 Andrew Steed 2 Christopher Burt 0 2 Paul Nicholson 2 James K. M. Brown 2 0 Present address: RAGT Seeds Ltd. , Grange Road, Ickleton, Essex, CB10 1TA , UK 1 Present address: Crop Protection Team, Crop and Soil Systems Group, SRUC , West Mains Road, Edinburgh, EH9 3JG , UK 2 Department of Crop Genetics, John Innes Centre , Norwich Research Park, Norwich, NR4 7UH , UK Lesion mimic mutants display spontaneous necrotic spots and chlorotic leaves as a result of mis-regulated cell death programmes. Typically these mutants have increased resistance to biotrophic pathogens but their response to facultative fungi that cause necrotrophic diseases is less well studied. The effect of altered cell death regulation on the development of disease caused by Ramularia collo-cygni, Fusarium culmorum and Oculimacula yallundae was explored using a collection of barley necrotic (nec) lesion mimic mutants. nec8 mutants displayed lower levels of all three diseases compared to nec9 mutants, which had increased R. collo-cygni but decreased F. culmorum disease symptoms. nec1 mutants reduced disease development caused by both R. collo-cygni and F. culmorum. The severity of the nec1-induced lesion mimic phenotype and F. culmorum symptom development was reduced by mutation of the negative cell death regulator MLO. The significant reduction in R. collo-cygni symptoms caused by nec1 was completely abolished in the presence of the mlo-5 allele and both symptoms and fungal biomass were greater than in the wild-type. These results indicate that physiological pathways involved in regulation of cell death interact with one another in their effects on different fungal pathogens. Cell death; disease resistance; endophyte; hemibiotroph; hypersensitive response; mlo; necrotroph; plant-microbe interactions - Programmed cell death is essential for many plant developmental processes such as leaf senescence and plays a critical role in defence against pathogens (Jones, 2001). Localized cell death at the sites of pathogen infection is termed the hypersensitive response (HR). HR forms part of the defence response referred to as effector triggered immunity (ETI), which is associated with the production of antimicrobial compounds, cell wall cross-linking, deposition of callose, and a prolonged reactive oxygen species (ROS) burst (Nurnberger et al., 2004; Jones and Dangl, 2006). ETI is particularly effective against pathogens that have a biotrophic lifestyle, requiring living host tissue on which to feed (Glazebrook, 2005; Jones and Dangl, 2006). However, the role of cell death in defence against facultative pathogens that may benefit from, or actively induce host cell death is not as clear. Cell death can operate against some hemibiotrophic pathogens that require a period of biotrophic development before becoming necrotrophic but is not effective against pathogens during the necrotrophic phase (Glazebrook, 2005; Mengiste, 2012). Mutagenesis of plants resulting in altered disease resistance has proved valuable in dissecting the defence response to different pathogens (Hammond-Kosack and Parker 2003). Lesion mimic mutants develop spontaneous necrotic lesions in the absence of pathogen infection. This phenotype is caused © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. by altered regulation of cell death processes such as HR and senescence or by perturbation of metabolic pathways resulting in cell death (Dangl et al., 1996). Mutations in genes involved in processes such as cellular signalling, chlorophyll biosynthesis, redox homeostasis, and disease resistance can result in lesion mimic phenotypes and these have advanced our understanding of the programmed cell death and HR pathways (Dangl et al., 1996; Lorrain et  al., 2003; Moeder & Yoshioka, 2008). As a consequence of the association with cell death, lesion mimic mutants often exhibit accelerated leaf senescence and altered ROS homeostasis (Lorrain et al., 2003). Lesion mimic mutants have been extensively studied in relation to plant defence responses and typically show enhanced resistance against biotrophic pathogens such as rusts and mildews (Kamlofski et al., 2007; Zhang et al., 2009). More variable responses have been reported between lesion mimics and facultative fungi, ranging from enhanced resistance (Persson et al., 2008; 2009) to supersusceptibility (Wright et al., 2013). One gene of agronomic significance which when mutated in barley causes necrotic lesions is MLO. Recessive mlo mutations confer broad-spectrum durable resistance to the obligate biotrophic powdery mildew fungus Blumeria graminis f.  sp. hordei and cause developmentally controlled lesion mimic phenotypes in the absence of disease (Wolter et  al., 1993). Mutant MLO alleles have associated deleterious agronomic effects including reduced yield (Kjaer et al., 1990) and increased susceptibility to some facultative pathogens such as Fusarium graminearum (Jansen et al., 2005), Magnaporthe oryzae (Jarrosch et  al., 1999), Bipolaris sorokiniana (Kumar et  al., 2001) and Ramularia collo-cygni (McGrann et  al., 2014). MLO encodes a seven-transmembrane domain protein that has been proposed to act as a negative regulator of cell death and disease resistance (Peterhänsel et al., 1997; Piffanelli et al., 2002), but the exact biochemical function of this protein remains undetermined (Buschges et al., 1997). Necrotic (nec) mutants from a fast-neutron exposed barley collection show varying degrees of leaf spotting, chlorosis and in most cases increased expression of HR-induced genes (Rostoks et al., 2003). Genetic analyses of some of these mutants have identified the genes responsible for the lesion mimic phenotype. nec1 mutants which show reduced basal resistance against powdery mildew fungi and enhanced nonhost resistance against Pseudomonas syringae pv. tomato (Keisa et  al., 2011) have mutations in a cyclic nucleotide-gated ion channel 4 protein (CNGC4; Rostoks et  al., 2006). CNGCs are cation channel proteins involved in regulating intracellular fluxes of ions such as Ca2+ (Ma and Berkowitz, 2011). These non-selective cation channels have been well studied in the model plant Arabidopsis thaliana and function in biological processes including ion homeostasis, development, plant defence and programmed cell death (Ma and Berkowitz, 2011; Moeder et  al., 2011). Barley nec8 mutants show elevated resistance against stem rust (Puccinia graminis) but not stripe rust (P. striiformis f. sp. hordei; Zhang et al., 2009). (...truncated)