The Barley Mutant emr1 was Identified in a Mutational Screen for Resistance Against Magnaporthe oryzae

Abstract

Barley plants can be colonized by the fungus Magnaporthe oryzae, a pathogen initially known from rice plant cultivation. A mutational screen was performed in the barley mlo-genetic background which is, in comparison to wild-type MLO-genotypes, hypersusceptible against this fungus. This led to the identification of a mutant, referred to as emr1 (enhanced Magnaporthe resistance), that showed partially restored resistance. Disease symptoms on leaves of emr1 were significantly less severe than on mlo5-genotypes but still more than on wt MLO-barley plants.

Segregation analysis showed that emr1 was inherited as a single recessive trait. Insight into the mode of action of emr1-dependent resistance against M. oryzae was gained by microscopic analysis. The results of these experiments revealed that mutant emr1 blocked penetration by M. oryzae by the formation of effective papillae in approximately half of all incidences. At about 30% of the interaction sites fungal growth was arrested effectively by an HR in the epidermal cell. Only a low frequency of fungal infection sites proceed into the mesophyll where fungal invasion resulted in the onset of a hypersensitive response (HR)-like cell death. Here, we report further evidence that barley shows a mesophyll HR in response to colonisation by M. oryzae. The possibility that the fungus turns this ostensible defence reaction to its own advantage and profits from the dead host tissue by switching to a necrotrophic lifestyle is discussed.

Plant pathogenic fungi from the genus Magnaporthe (Ascomycota) cause disease on monocots in the Poaceae. The most prevalent disease in rice plant cultivation world-wide is referred to as “rice blast,” caused by M. oryzae.¹ Breeding for resistance against M. oryzae has resulted in the release of a large number of new rice varieties with major resistance genes against M. oryzae.² However, in the so-called boom and bust cycle (Hammond-Kosack and Parker 2003), these resistances are rapidly overcome by a shift in the pathogen population to new races. Searching for novel sources for durable resistance, we investigated whether race-non-specific resistance mechanisms known to be highly effective against other fungal plant pathogens might be transferable to the rice/M. oryzae pathosystem. Since barley is also a host for M. oryzae, the survey was started with the barley mlo-resistance trait. Recessive alleles of the mlo-locus confer a durable race-non-specific resistance against the barley powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh). Importantly, mlo-conditioned resistance has proven stable under field conditions.³ In contrast, barley plants with a wild-type MLO-allele are susceptible to Bgh. Performing infections on these two barley genotypes with M. oryzae revealed the amazing phenomenon that alleles of the barley MLO-locus behave reciprocally in response to these two pathogens. More precisely, plants homozygous for mutant alleles (mlo) are resistant to powdery mildew but hypersusceptible to M. oryzae and vice versa for plants with at least one wild-type MLO-allele.^4,5 This clearly showed that the transfer of the mlo-scenario to rice would probably result in more devastating blast epidemics.

It is apparent that M. oryzae blast epidemics on barley are becoming more prevalent in the field.⁶ Therefore, we adopted the long-term goal of improving the resistance of barley against M. oryzae. Moreover, it would be desireable to keep the mlo-dependent resistance of barley against Bgh and to add a trait that confers resistance against the blast fungus in addition. In this context a mutational screen was set up in the barley mlo-genetic background and resulted in the identification of mutant emr1.⁵ Resistance in this mutant did not result from a back-mutation to wild-type MLO-alleles as evidenced by retained resistance against Bgh. Infection experiments with the obligate-biotropic rust fungus Puccinia hordei or the necrotrophic fungus Drechslera teres revealed no altered resistance phenotype with these pathogens. At the cellular level resistance against M. oryzae was manifested in most incidences by a block of penetration. This resembles the most frequent resistance response observed against M. oryzae in moderately susceptible wild-type MLO-plants and this penetration resistance is compromised in mlo-genotypes.^4,7 It could be speculated that wild-type EMR1 encodes for a suppressor that knocks down defence against M. oryzae only in mlo-but not in MLO-genotypes.

At this stage it must be stated clearly that all barley cultivars under investigation were hosts for M. oryzae, however they differed in their degree of susceptibility.^4,5,9 On question aroused from this observation targeted the mechanisms by which the pathogen establish compatibility on each genotype. On the microscopic level enhanced susceptibility correlated strictly with cell collapse and the accumulation of autofluorescent material in the mesophyll.^4,5 These histological criteria were reminiscent of a typical HR as described for resistant barley cultivars attacked by Bgh.¹⁰ However, since the term HR is normally used to describe a rapid form of programmed cell death associated with resistance,¹¹ we refer to the observed phenomenon of cell death in M. oryzae attacked mesophyll of barley as “HR-like cell death”. In this way we hope to avoid misleading interpretations. However, the phenomenon of HR is still enigmatic^12,13 and we looked for additional criteria, apart from microscopy, to distinguish HR from necrosis, e.g., molecular markers like harpin-induced 1 (HIN1) or HSR203J, HMGR, HSR201 and HSR515.¹⁴ Accordingly, we performed northern analysis using a barley HIN1 cDNA as probe (Fig. 1). Transcripts specific for this probe did not accumulate in any of the non-inoculated plants. HIN1 transcripts accumulated most strongly in mlo-genotypes at 72 to 96 h post inoculation. In contrast, only a weak accumulation of transcripts were detectable in MLO genotypes and an intermediate intensity was observed in plants of the emr1 mutant genotype. This pattern of HIN1 accumulation across the genotypes perfectly matched the disease severity on leaves and—even more important—with the observed indications for a HR-like cell death in the mesophyll. This result strongly supported our interpretation that this is a form of pathogen-associated programmed host cell death rather than the simple killing of attacked cells as would be expected in the case of a necrosis. Finally, it could be concluded that in the barley/M. oryzae interaction the timing and/or qualitative composition of the HR-like cell death in the mesophyll is not capable of arresting fungal invasion, most properly due to the ability of the facultative biotrophic fungus to switch in these incidences to a necrotrophic life-style.

Figure 1.

Accumulation of HIN1-specific transcripts in different barley genotypes (MLO, mlo5, and emr1). Inoculation with spores of M. oryzae race 031 (200000 spores/mL) was done eight days after sowings (8 d p.s.). Samples were harvested from 0 to 96 hours after inoculation (h p.i.). Equal loading of the gel was monitored by ethidium-bromide (EtBr) staining. Membranes were hybridized according to Peterhänsel et al⁸ with a probe labelled with Digoxigenin by in-vitro transcription of a plasmid containing a fragment of the barley HIN1 cDNA (clone ID HV08H24, CR-EST, IPK Gatersleben).

In summary, it was demonstrated that emr1 plants combine pathogen resistance against two diseases in barley crop cultivation, namely powdery mildew and blast. With respect to M. oryzae infections emr1 exhibited an intermediate resistance phenotype between wild-type MLO-and mlo-genotypes, respectively. Further investigations aimed at identifying the mutated gene(s) will show whether the emr1 locus could be utilised for traditional breeding programs or transgenic approaches.

Addendum to: Jansen M, Jarosch B, Schaffrath U. The Barley Mutant emr1 Exhibits Restored Resistance against Magnaporthe oryzae in the Hypersusceptible mlo-Genetic Background. Planta. 2007;225:1381–1391. doi: 10.1007/s00425-006-0447-1.