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Background Controlled turnover of proteins as mediated by the ubiquitin proteasome system (UPS) is an important element in plant defense against environmental and pathogen stresses. E3 ligases play a central role in subjecting proteins to hydrolysis by the UPS. Recently, it has been demonstrated that a specific class of E3 ligases termed the U-box ligases are directly associated with the defense mechanisms against abiotic and biotic stresses in several plants. However, no studies on U-box E3 ligases have been performed in one of the important staple crops, barley. Results In this study, we identified 67 putative U-box E3 ligases from the barley genome and expressed sequence tags (ESTs). Similar to Arabidopsis and rice U-box E3 ligases, most of barley U-box E3 ligases possess evolutionary well-conserved domain organizations. Based on the domain compositions and arrangements, the barley U-box proteins were classified into eight different classes. Along with this new classification, we refined the previously reported classifications of U-box E3 ligase genes in Arabidopsis and rice. Furthermore, we investigated the expression profile of 67 U-box E3 ligase genes in response to drought stress and pathogen infection. We observed that many U-box E3 ligase genes were specifically up-and-down regulated by drought stress or by fungal infection, implying their possible roles of some U-box E3 ligase genes in the stress responses. Conclusion This study reports the classification of U-box E3 ligases in barley and their expression profiles against drought stress and pathogen infection. Therefore, the classification and expression profiling of barley U-box genes can be used as a platform to functionally define the stress-related E3 ligases in barley. Electronic supplementary material The online version of this article (10.1186/s12864-019-5696-z) contains supplementary material, which is available to authorized users.

Barley (Hordeum vulgare L.) produces five leucine-derived hydroxynitrile glucosides, potentially involved in alleviating pathogen and environmental stresses. These compounds include the cyanogenic glucoside epiheterodendrin. The biosynthetic genes are clustered. Total hydroxynitrile glucoside contents were previously shown to vary from zero to more than 10,000 nmoles g−1 in different barley lines. To elucidate the cause of this variation, the biosynthetic genes from the high-level producer cv. Mentor, the medium-level producer cv. Pallas, and the zero-level producer cv. Emir were investigated. In cv. Emir, a major deletion in the genome spanning most of the hydroxynitrile glucoside biosynthetic gene cluster was identified and explains the complete absence of hydroxynitrile glucosides in this cultivar. The transcript levels of the biosynthetic genes were significantly higher in the high-level producer cv. Mentor compared to the medium-level producer cv. Pallas, indicating transcriptional regulation as a contributor to the variation in hydroxynitrile glucoside levels. A correlation between distinct single nucleotide polymorphism (SNP) patterns in the biosynthetic gene cluster and the hydroxynitrile glucoside levels in 227 barley lines was identified. It is remarkable that in spite of the demonstrated presence of a multitude of SNPs and differences in transcript levels, the ratio between the five hydroxynitrile glucosides is maintained across all the analysed barley lines. This implies the involvement of a stably assembled multienzyme complex.

Barley (Hordeum vulgare L.) produces five leucine-derived hydroxynitrile glucosides (HNGs), of which only epiheterodendrin is a cyanogenic glucoside. The four non-cyanogenic HNGs are the β-HNG epidermin and the γ-HNGs osmaronin, dihydroosmaronin and sutherlandin. By analyzing 247 spring barley lines including landraces and old and modern cultivars, we demonstrated that the HNG level varies notably between lines whereas the overall ratio between the compounds is constant. Based on sequence similarity to the sorghum (Sorghum bicolor) genes involved in dhurrin biosynthesis, we identified a gene cluster on barley chromosome 1 putatively harboring genes that encode enzymes in HNG biosynthesis. Candidate genes were functionally characterized by transient expression in Nicotiana benthamiana. Five multifunctional P450s, including two CYP79 family enzymes and three CYP71 family enzymes, and a single UDP-glucosyltransferase were found to catalyze the reactions required for biosynthesis of all five barley HNGs. Two of the CYP71 enzymes needed to be co-expressed for the last hydroxylation step in sutherlandin synthesis to proceed. This observation, together with the constant ratio between the different HNGs, suggested that HNG synthesis in barley is organized within a single multi-enzyme complex.

Ramularia leaf spot disease (RLS), caused by the ascomycete fungus Ramularia collo-cygni, has emerged as a major economic disease of barley. No substantial resistance has been identified, so far, among barley genotypes and, based on the epidemiology of the disease, a quantitative genetic determinacy of RLS has been suggested. The relative contributions of barley and R. collo-cygni genetics to disease infection and epidemiology are practically unknown. Here, we present an integrated genome-wide analysis of host and pathogen transcriptome landscapes identified in a sensitive barley cultivar following infection by an aggressive R. collo-cygni isolate. We compared transcriptional responses in the infected and noninfected leaf samples in order to identify which molecular events are associated with RLS symptom development. We found a large proportion of R. collo-cygni genes to be expressed in planta and that many were also closely associated with the infection stage. The transition from surface to apoplastic colonization was associated with downregulation of cell wall–degrading genes and upregulation of nutrient uptake and resistance to oxidative stresses. Interestingly, the production of secondary metabolites was dynamically regulated within the fungus, indicating that R. collo-cygni produces a diverse panel of toxic compounds according to the infection stage. A defense response against R. collo-cygni was identified in barley at the early, asymptomatic infection and colonization stages. We found activation of ethylene signaling, jasmonic acid signaling, and phenylpropanoid and flavonoid pathways to be highly induced, indicative of a classical response to necrotrophic pathogens. Disease development was found to be associated with gene expression patterns similar to those found at the onset of leaf senescence, when nutrients, possibly, are used by the infecting fungus. These analyses, combining both barley and R. collo-cygni transcript profiles, demonstrate the activation of complex transcriptional programs in both organisms.

Plants produce secondary metabolites promoting adaptation to changes in the environment and challenges by pathogenic microorganisms. A future climate with increased temperature and CO2 and ozone levels will likely alter the chemical composition of plants and thereby plant-pathogen interactions. To investigate this, barley was grown at elevated CO2, temperature and ozone levels as single factors or in combination resembling future climatic conditions. Increased basal resistance to the powdery mildew fungus was observed when barley was grown under elevated CO2, temperature and ozone as single factors. However, this effect was neutralized in the combination treatments. Twenty-five secondary metabolites were putatively identified in healthy and diseased barley leaves, including phenylpropanoids, phenolamides and hydroxynitrile glucosides. Accumulation of the compounds was affected by the climatic growth conditions. Especially elevated temperature, but also ozone, showed a strong impact on accumulation of many compounds, suggesting that these metabolites play a role in adaptation to unfavorable growth conditions. Many compounds were found to increase in powdery mildew diseased leaves, in correlation with a strong and specific influence of the climatic growth conditions. The observed disease phenotypes could not be explained by accumulation of single compounds. However, decreased accumulation of the powdery mildew associated defense compound p-coumaroylhydroxyagmatine could be implicated in the increased disease susceptibility observed when barley was grown under combination of elevated CO2, temperature and ozone. The accumulation pattern of the compounds in both healthy and diseased leaves from barley grown in the combination treatments could not be deduced from the individual single factor treatments. This highlights the complex role and regulation of secondary metabolites in plants' adaptation to unfavorable growth conditions.

The response in production parameters to projected future levels of temperature, atmospheric carbon dioxide ([CO2]), and ozone ([O3]) was investigated in 138 spring barley accessions. The comprehensive set of landraces, cultivars, and breeder-lines, were during their entire life cycle exposed to a two-factor treatment of combined elevated temperature (+5 °C day/night) and [CO2] (700 ppm), as well as single-factor treatments of elevated temperature (+5 °C day/night), [CO2] (700 ppm), and [O3] (100–150 ppb). The control treatment was equivalent to present average South Scandinavian climate (temperature: 19/12 °C (day/night), [CO2]: 385 ppm). Overall grain yield was found to decrease 29% in the two-factor treatment with concurrent elevation of [CO2] and temperature, and this response could not be predicted from the results of treatments with elevated [CO2] and temperature as single factors, where grain yield increased 16% and decreased 56%, respectively. Elevated [O3] was found to decrease grain yield by 15%. Substantial variation in response to the applied climate treatments was found between the accessions. The results revealed landraces, cultivars, and breeder-lines with phenotypes applicable for breeding towards stable and high yield under future climate conditions. Further, we suggest identifying resources for breeding under multifactor climate conditions, as single-factor treatments did not accurately forecast the response, when factors were combined.

Barley (Hordeum vulgare) was grown in different climatic environments with elevated [CO2] (700 vs 385 ppm), [O3] (60/90 vs 20 ppb) and temperature (24/19 vs 19/12°C day/night) as single factors and in combinations, to evaluate the impact of these climatic factors on photosynthesis and susceptibility to powdery mildew and spot blotch disease. No significant increase in net CO2 assimilation rate was observed in barley grown under elevated [CO2] at ambient temperature.However, this rate was positively stimulated under elevated temperature together with a slightly higher potential quantum efficiency of PSII, both at ambient and elevated [CO2], suggesting that photosynthesis was not limited by [CO2] at ambient temperature. When growing under elevated temperature or [O3], infection by the biotrophic powdery mildew fungus decreased, whereas disease symptoms and growth of the toxin-secreting hemibiotrophic spot blotch fungus increased compared to ambient conditions, implying that climate-induced changes in disease severity could be linked to the trophic lifestyle of the pathogens. Elevated [CO2] decreased powdery mildew infection but had no effect on spot blotch disease compared to ambient condition. However, the effect of elevated [CO2], [O3] and temperature did not act in an additive manner when combined. This led to a surprising disease development in the combination treatments, where powdery mildew infection increased despite the individual reducing effect of the climatic factors, and spot blotch disease decreased despite the individual promoting effect of temperature and ozone, emphasizing the importance of conducting multifactorial experiments when evaluating the potential effects of climate change.

Pea powdery mildew incited by Erysiphe pisi represents a major constraint for pea crop worldwide. Only three resistance genes (er1, er2 and Er3) have been identified in pea germplasm so far. Cellular and molecular aspects involved in their interaction with the pathogen are still unknown. In the present study, we selected three well-characterised powdery mildew pea accessions carrying each of these resistance genes JI2302 (er1), JI2480 (er2) and IFPI3260 (Er3) for a combined cDNA array and histological analyses. Transcripts of twenty pea genes, including three housekeeping genes, were generated at 24, 48 and 72 h after fungal inoculation. The 17 genes analysed encoded for phenylpropanoids, structurally related genes, disease relative response genes, or homologues genes to characterised pathogenesis-related family (PR) genes from barley known to be up-regulated after powdery mildew infection. A total of 16 genes out of the 20 studied were differentially expressed between genotypes and/or E. pisi infection, as well as between the time points considered. The transcript inductions observed are expressed as reinforcement of cell walls, activation of pathogenesis-related proteins and the activation of the phenylpropanoid pathway. Leaves of genotype JI2302 (er1) showed mainly Pschitin and Chi2 (encoding for endochitinases) accumulation after E. pisi inoculation, as well as genes encoding for pea defensins. Leaves of IFPI3260 (Er3) showed the overall highest expression of DRR230a, DRR230b and DRR230c (encoding pea defensins) and Prx7 (encoding an elicitor-inducible peroxidase) after pathogen inoculation. Genotype JI2480 (er2) showed Pschitin and Chi2 accumulation after E. pisi inoculation, as well as reduced activation of pea defensins, compared to er1 and Er3 genotypes, after pathogen attack. Up-regulation of genes involved in these mechanisms combined with high constitutive expression determines a more effective defence against E. pisi. © 2014 Springer Science+Business Media Dordrecht., Financial support by AGL2011-22524 Project co-financed by FEDER is acknowledged. E. Barilli was financed by a JAEdoc Grant.

The receptor-like protein kinases (RLKs) constitute a large and diverse group of proteins controlling numerous plant physiological processes, including development, hormone perception and stress responses. The cysteine-rich RLKs (CRKs) represent a prominent subfamily of transmembrane-anchored RLKs. We have identified a putative barley (Hordeum vulgare) CRK gene family member, designated HvCRK1. The mature putative protein comprises 645 amino acids, and includes a putative receptor domain containing two characteristic 'domain 26 of unknown function' (duf26) domains in the N-terminal region, followed by a rather short 17-amino-acid transmembrane domain, which includes an AAA motif, two features characteristic of endoplasmic reticulum (ER)-targeted proteins and, finally, a characteristic putative protein kinase domain in the C-terminus. The HvCRK1 transcript was isolated from leaves inoculated with the biotrophic fungal pathogen Blumeria graminis f.sp. hordei (Bgh). HvCRK1 transcripts were observed to accumulate transiently following Bgh inoculation of susceptible barley. Transient silencing of HvCRK1 expression in bombarded epidermal cells led to enhanced resistance to Bgh, but did not affect R-gene-mediated resistance. Silencing of HvCRK1 phenocopied the effective penetration resistance found in mlo-resistant barley plants, and the possible link between HvCRK1 and MLO was substantiated by the fact that HvCRK1 induction on Bgh inoculation was dependent on Mlo. Finally, using both experimental and in silico approaches, we demonstrated that HvCRK1 localizes to the ER of barley cells. The negative effect on basal resistance against Bgh and the functional aspects of MLO- and ER-localized HvCRK1 signalling on Bgh inoculation are discussed.

Transgenic crops are now grown commercially in 25 countries worldwide. Although pathogens represent major constraints for the growth of many crops, only a tiny proportion of these transgenic crops carry disease resistance traits. Nevertheless, transgenic disease-resistant plants represent approximately 10% of the total number of approved field trials in North America, a proportion that has remained constant for 15 years. In this review, we explore the socioeconomic and biological reasons for the paradox that although technically useful solutions now exist for providing transgenic disease resistance, very few new crops have been introduced to the global market. For bacteria and fungi, the majority of transgenic crops in trials express antimicrobial proteins. For viruses, three-quarters of the transgenics express coat protein (CP) genes. There is a notable trend toward more biologically sophisticated solutions involving components of signal transduction pathways regulating plant defenses. For viruses, RNA interference is increasingly being used.

Hypersensitive response (HR) against Blumeria graminis f. sp. hordei infection in barley (Hordeum vulgare) was associated with stomata "lockup" leading to increased leaf water conductance (gl). Unique spatiotemporal patterns of HR formation occurred in barley with Mla1, Mla3, or MlLa R genes challenged with B. graminis f. sp. hordei. With Mla1, a rapid HR, limited to epidermal cells, arrested fungal growth before colonies initiated secondary attacks. With Mla3, mesophyll HR preceded that in epidermal cells whose initial survival supported secondary infections. With MlLa, mesophyll survived and not all attacked epidermal cells died immediately, allowing colony growth and secondary infection until arrested. Isolines with Mla1, Mla3, or MlLa genes inoculated with B. graminis f. sp. hordei ranging from 1 to 100 conidia mm2 showed abnormally high gl during dark periods whose timing and extent correlated with those of each HR. Each isoline showed increased dark gl with the nonpathogen B. graminis f. sp. avenae which caused a single epidermal cell HR. Guard cell autofluorescence was seen only after drying of epidermal strips and closure of stomata suggesting that locked open stomata were viable. The data link stomatal lock-up to HR associated cell death and has implications for strategies for selecting disease resistant genotypes. © 2010 The American Phytopathological Society, E. Prats was supported by a Marie Curie European Reintegration Grant and by the project AGL2007-65031/AGR, T. Carver and B. Thomas by Defra Project AR0712, and A. P. Gay by ERDF interreg. IIIB Atlantic Area Project 190 (PIMHAI).

The concept ‘functional genomics’ refers to the methods used for the functional characterisation of genomes. The methods utilised provide new opportunities for studying the nature and role of defence mechanisms in plants. Unlike Arabidopsis, poplar and rice, the full genomic sequence of barley is not available. In this case, the analysis of barley gene expression data plays a pivotal role for obtaining insight into the functional characterisation of individual gene products. Many genes are activated transcriptionally following attack by pathogens and these often contribute to the defence mechanisms which underlie disease resistance. The use of large-scale complementary DNA library constructions and genome-wide transcript profiles of plants exposed to biotic stress provide the data required to drive hypotheses concerning the function of newly identified genes. In this paper, we illustrate how publicly available gene expression data has proved valid for studies of plant defence responses; enabling a cost-effective workflow starting from isolated gene transcripts to elucidation of biological function upon biotic stress.

Pathogens induce the expression of many genes encoding plant transcription factors, though specific knowledge of the biological function of individual transcription factors remains scarce. NAC transcription factors are encoded in plants by a gene family with proposed functions in both abiotic and biotic stress adaptation, as well as in developmental processes. In this paper, we provide convincing evidence that a barley NAC transcription factor has a direct role in regulating basal defence. The gene transcript was isolated by differential display from barley leaves infected with the biotrophic powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh). The full-length cDNA clone was obtained using 5'-RACE and termed HvNAC6, due to its high similarity to the rice homologue, OsNAC6. Gene silencing of HvNAC6 during Bgh inoculation compromises penetration resistance in barley epidermal cells towards virulent Bgh. Complementing the effect of HvNAC6 gene silencing, transient overexpression of HvNAC6 increases the occurrence of penetration resistant cells towards Bgh attack. Quantitative RT-PCR shows the early and transient induction of HvNAC6 in barley epidermis upon Bgh infection. Additionally, our results show that the Arabidopsis HvNAC6 homologue ATAF1 is also induced by Bgh and the ataf1-1 mutant line shows decreased penetration resistance to this non-host pathogen. Collectively, these data suggest a conserved role of HvNAC6 and ATAF1 in the regulation of penetration resistance in monocots and dicots, respectively.

In many plant-pathogen interactions, there are several possible outcomes for simultaneous attacks on the same leaf. For instance, an attack by the powdery mildew fungus on one barley leaf epidermal cell may succeed in infection and formation of a functional haustorium, whereas a neighboring cell attacked at the same time may resist fungal penetration. To date, the mixed cellular responses seen even in susceptible host leaves have made it difficult to relate induced changes in gene expression to resistance or susceptibility in bulk leaf samples. By microextraction of cell-specific mRNA and subsequent cDNA array analysis, we have successfully obtained separate gene expression profiles for specific mildew-resistant and -infected barley cells. Thus, for the first time, it is possible to identify genes that are specifically regulated in infected cells and, presumably, involved in fungal establishment. Further, although much is understood about the genetic basis of effective papilla resistance associated with mutant mlo barley, we provide here the first evidence for gene regulation associated with effective papilla-based nonspecific resistance expressed in nominally “susceptible” wild-type barley.

Resistance and susceptibility in barley to the powdery mildew fungus (Blumeria graminis f. sp. hordei) is determined at the single-cell level. Even in genetically compatible interactions, attacked plant epidermal cells defend themselves against attempted fungal penetration by localized responses leading to papilla deposition and reinforcement of their cell wall. This conveys a race-nonspecific form of resistance. However, this defense is not complete, and a proportion of penetration attempts succeed in infection. The resultant mixture of infected and uninfected leaf cells makes it impossible to relate powdery mildew-induced gene expression in whole leaves or even dissected epidermal tissues to resistance or susceptibility. A method for generating transcript profiles from individual barley epidermal cells was established and proven useful for analyzing resistant and successfully infected cells separately. Contents of single epidermal cells (resistant, infected, and unattacked controls) were collected, and after cDNA synthesis and PCR amplification, the resulting sample was hybridized to dot-blots spotted with genes, including some previously reported to be induced upon pathogen attack. Transcripts of several genes, (e.g., PR1a, encoding a pathogenesis related protein, and GLP4, encoding a germin-like protein) accumulated specifically in resistant cells, while GRP94, encoding a molecular chaperone, accumulated in infected cells. Thus, the single-cell method allows discrimination of transcript profiles from resistant and infected cells. The method will be useful for microarray expression profiling for simultaneous analysis of many genes.

When barley, wheat or oat leaf epidermal cells were attacked by their appropriate forma specialis (f.sp.) of Blumeria graminis DC. Speer (f.sp. hordei, tritici and avenae, respectively), many attempted penetrations succeeded, functional haustoria were formed and very few plant cells died. When attacked by either of the two possible inappropriate ff.spp., penetration attempts failed in association with papilla deposition by epidermal cells, attacked cells died, or if visible haustoria were formed the plant cell died very soon afterwards. Double inoculation experiments were performed where each cereal species was first attacked by its appropriate f.sp., as inducer, and later by the different ff.spp. as challenger. Infection by the appropriate inducer profoundly affected cellular responses to challenger attack. Suppression of defensive responses was dramatic within epidermal cells containing the inducer haustorium, evident to some extent in adjacent cells, but undetectable at two cells distance. Suppression of penetration resistance allowed most challenger attacks, even by inappropriate ff.spp., to form a haustorium. Furthermore, death of penetrated epidermal cells was also suppressed so that haustoria of the inappropriate ff.spp. functioned to support colony development. In oat, delayed epidermal cell death prevented full colony development by inappropriate ff.spp., but in barley and wheat, no cell death was apparent by four days after inoculation and colonies of the inappropriate ff.spp. produced extensive hyphae, secondary haustoria and conidial chains.

Future barley cultivars will have to produce under the constraints of higher temperature in combination with increased concentrations of atmospheric carbon dioxide and ozone as a consequence of climate change. A diverse set of 167 spring barley genotypes was cultivated under elevated levels of temperature (+5 °C) and [CO2] (700 ppm) as single factors and in combination as well as under elevated [O3] (100–150 ppb) as single factor. The setting in general resembled changes projected by IPCC (AR5) to take place at the end of this century. A genome-wide association study (GWAS) was performed to identify markers for increased primary production under climate change conditions and reveal possible genes of interest. Phenotyped traits included grain yield, number of grains, number of ears per plant, aboveground vegetative biomass, harvest index and stability of the production parameters over the five applied treatments. The GWAS encompassed 7864 SNP markers (Illumina iselect), a compressed mixed linear model with the GAPIT package, and conservative validation of markers. A total of 60 marker-trait associations [−log10(P value) 2.97–5.58] were identified, e.g. grain yield under elevated temperature on barley chromosome 2H, static stability of grain yield on 7H, sites for exploitation of elevated [CO2] on 4H and 7H and associations under the two-factor treatment. Marker-trait associations identified from single-factor treatments were not retrieved, when elevated [CO2] and temperature were combined emphasizing the need for multifactor experiments. This GWA study identified markers and chromosome regions to be targeted in breeding for development of climate resilient cultivars.

Zeyen, R. J., Kruger, W. M., Lyngkjaer, M. F., Carver, T. L. W. (2002). Differential effects of D-mannose and 2-deoxy-D-glucose on attempted powdery mildew fungal infection of inappropriate and appropriate Gramineae. Physiological and Molecular Plant Pathology, 61, (6), 315-323.

Treatment with mycelial extracts, prepared from liquid cultures of Bipolaris oryzae, Pythium ultimum and Rhizopus stolonifer, protected barley (Hordeum vulgare) against powdery mildew disease caused by the fungus Blumeria graminis f.sp. hordei. The mechanisms of this protection were studied using histopathological methods and molecular analysis. Germination and appressorial formation of B. graminis were generally reduced after treatment with mycelial extracts. Although this reduction (between 12 and 62% depending on treatment and experiment) was inconsistent and only occasionally significantly different from the water-treated control, it indicated a direct antifungal effect of the extracts. In situations where the fungus succeeded in forming an appressorium, penetration efficiency and haustorium formation from these appressoria was not affected – no enhanced penetration resistance associated with papilla formation was detected. However, a post-penetration effect was observed, as B. graminis colonies on mycelial extract-treated leaves produced 50% fewer hyphae than on controls. Northern blot analyses showed earlier accumulation of defence-related gene transcripts following treatment with B. oryzae and P. ultimum mycelial extracts, and to a lesser extent R. stolonifer mycelial extract, compared with water-treated leaves. It is suggested that the protection mechanism of the mycelial extracts involves direct antifungal effects and possible induced resistance for the B. oryzae and P. ultimum mycelial extracts.

Leaves of near isogenic barley lines containing the race-specific resistance alleles Mla1 or mla1 were subjected to double inoculation treatments with virulent and avirulent Blumeria graminis isolates. Attack by the avirulent isolate alone frequently caused hypersensitive death of individual Mla1 epidermal cells, but underlying mesophyll cells survived. Virulent and avirulent B. graminis isolates were inoculated sequentially (inducer followed by challenger) in all possible combinations. As shown previously, where a papilla formed due to inducer attacks, the attacked epidermal cell, and to some extent its neighbours, showed highly increased resistance to challenger penetration. When epidermal cells died in response to avirulent inducer attack, adjacent cells also showed increased penetration resistance. By contrast, where the virulent inducer formed a haustorium, invaded epidermal cells, and to some extent their neighbours, showed induced penetration susceptibility to later challenger attack. Furthermore, when virulent inducer haustoria formed in Mla1 epidermal cells, these cells became highly susceptible to successful infection by the avirulent isolate and vigorous colonies developed. It appears that suppressive factors released during virulent inducer infection prevented the hypersensitive epidermal cell death that normally is induced by the race-specific avirulence elicitor. However, suppression of Mla1 race-specific resistance was confined to the epidermis. The presence of successful avirulent fungus infection of the epidermis often led to the death of the underlying mesophyll cells, thus generating a novel cellular response phenotype in B. graminis attacked Mla1 barley.

High levels of protection against infection from Blumeria graminis f.sp. hordei in the susceptible barley cv. Pallas were obtained by pretreatment with mycelial extracts or culture filtrates from seven different fungi (Bipolaris oryzae, B. sorokiniana, Drechslera teres f. maculata, Fusarium culmorum, Trichoderma harzianum, Pythium ultimum and Rhizopus stolonifer). Three mycelial extracts from the taxonomically different fungi B. oryzae, P. ultimum and R. stolonifer were selected for detailed study. In general the number of colonies formed was reduced by 70–98% compared with controls. Furthermore, the few colonies that developed on treated leaves were generally smaller and showed reduced spore production. Protection was limited to the area of the leaves treated with mycelial extracts and a systemic effect could not be detected. No differences in the protection level were observed when treatment was conducted between 2 h and 3 days before inoculation. It is suggested that components in the mycelial extracts interacted directly with B. graminis and that this antifungal effect was responsible for the observed protection. However, indirect effects of the mycelial extracts mediated by locally induced resistance cannot be precluded.

This review briefly summarises recently generated knowledge about mlo powdery mildew resistance in barley. Barley mlo resistance has remained highly effective since commercial spring barley varieties with the resistance were first released in 1979. Currently, this resistance is the most used resistance in spring barley grown throughout Europe. Barley mlo resistance confers nearly total resistance against fungal penetration attempts. However, the efficiency of the resistance depends on several factors including epidermal cell type, host genetic background, environmental conditions and fungal genotype. Recently, the barley Mlo-gene has been cloned, but the exact function of the gene is not known. The Mlo-gene most likely regulates several mechanisms involved in penetration resistance against powdery mildew, and mlo mutations cause disfunction of the wild type Mlo-protein leading to increased resistance. The resistance mechanisms involved probably include earlier deposition and increased size of the host papilla response, callose deposition, production of phenolic compounds and cell wall strengthening by cross binding.

Field-grown plants sequentially encounter many different fungal pathogens and nonpathogens that are capable of triggering an array of responses that may affect the subsequent level of disease they develop following later pathogen attack. These changes, which are induced by prior encounters, may be manifest as increased susceptibility or enhanced resistance to later pathogen attack; they may be expressed systemically or their effects may be localized within a few cells distance of the original encounter site. Here, we review our recent investigations of cellular changes effected by sequential inoculations of cereal leaves with the powdery mildew fungus Blumeria graminis DC. In susceptible barley and oats, a successful B. graminis attack followed by haustorium formation, renders the attacked cell, and to some extent its adjacent cells, highly accessible to later B. graminis attacks. By contrast, a failed attack due to papilla formation by the attacked host cells, renders the attacked cell and its adjacent cells highly inaccessible to later B. graminis attacks. Importantly, barley carrying the mlo5 allele for powdery mildew resistance is also conditioned to accessibility if prior attacks by an mlo-virulent isolate penetrates successfully. In the partial resistant oat cultivar Maldwyn B. graminis attacks either succeeded, failed due to papilla deposition, or failed because the attacked host cells died in response to the attacks. Sequential inoculation of Maldwyn demonstrated the induction of accessibility and inaccessibility, as well as a complete suppression of cell death response to attack where the cells had survived an earlier attack. Furthermore, when a prior attack induced cell death, a later attack on adjacent cells caused greatly increased rate of cell death, demonstrating potentiation of cell death. The importance of the induced cellular changes for plant resistance in the field is discussed.

First-formed (seedling) and later-formed leaves of oat cvs Selma (susceptible) and Maldwyn (adult plant resistance under complex genetic control) were subjected to a double inoculation procedure (‘inducer’ followed by ‘challenger’) with conidia of Blumeria graminis (DC.) Speer (Syn. Erysiphe graminis DC.). Successful penetration and haustorium formation by the inducer rendered living epidermal cells highly accessible to later challenge attack as judged by increased frequency of challenge penetration success compared to controls. Conversely, where failure of inducer attack on living epidermal cells was associated with papilla deposition, cells were rendered highly inaccessible to challenge attack. These effects were localized, being expressed most strongly in the single cell subject to direct inducer attack (D0 cell), to a lesser degree in immediately adjacent cells, and relatively weakly, if at all, at two cells distance. The degree of induced (in)accessibility in D0 cells appeared correlated to localized autofluorescent host cell responses to challenge appressoria, but independent of the inherent resistance of leaf tissues. These results agree with earlier observations from barley, suggesting that induced changes in (in)accessibility may be a common consequence of B. graminis attack in cereals. As expected, in Maldwyn, cell death was a consistent but infrequent response to attack (5–20% of attacks caused cell death in controls). Here, the successful formation of an inducer haustorium, or of a papilla due to failed attack, totally suppressed the cell death response to later challenge attack on D0 cells, but had less effect on neighbouring cells. Conversely, death of a Maldwyn epidermal cell due to inducer attack potentiated cell death in adjacent cells where up to 80% of challenge attacks caused death. This effect was transmitted to some extent to two cells distance.

First leaves of barley line Riso 5678R, with the recessive mlo5 allele conditioning highly efficient, papilla-based penetration resistance to field isolates of Blumeria graminis (DC.) Speer (Syn. Erysiphe graminis DC.) were used. Leaves were subjected to a double inoculation procedure (“inducer” followed by “challenger”) using two fungal isolates. Isolate GE3 is a wild-type, unable to infect Riso-R, and is termed “ mlo -avirulent”. Isolate HL3/5, selected from GE3, is capable of infecting Riso-R, and is termed “ mlo -virulent”. When HL3/5 was used as inducer, some attacks penetrated successfully and formed haustoria within epidermal cells. When either isolate was inoculated onto the same leaves as challenger 48 h later, attacks on cells containing an inducer haustorium were almost invariably successful. Thus, cells containing an inducer haustorium showed almost complete induced accessibility to challenge attacks, even by the avirulent isolate. Accessibility was also induced to some extent in adjacent cells, but the effect was localized. By contrast, where inducer attacks failed, and a papilla was formed in the attacked cell, later challenge attacks on the same cells always failed. These, and adjacent cells, showed almost complete induced inaccessibility. Induced inaccessibility, therefore, was more effective in preventing penetration than inherent resistance due to mlo . Papillae formed in inaccessible cells were larger than in controls, which might be related to their efficient penetration resistance. Autofluorogens accumulated in papillae and locally in surrounding host cell wall areas. Some evidence suggested that accessibility may be related to suppression of localized autofluorescent host cell responses, and, conversely that inaccessibility may be related to their enhancement.

Powdery mildew infection of barley with the mlo5 barley powdery mildew resistance gene was examined, using near-isogenic barley lines, with and without mlo5 resistance, and two near-isogenic powdery mildew isolates, HL3/5 and GE3 with high (virulent) or low (avirulent) penetration efficiency on the resistant barley line. In all isolate–host combinations (except GE3 on the resistant barley line), frequency of haustorium formation increased significantly from zero at 11 h after inoculation to a maximum by 13 h, and there was no subsequent increase up to 24 h. In the susceptible barley line, 27% of appressoria from both isolates formed haustoria. Although this was significantly higher than the frequency of haustorium formation (18%) of HL3/5 on the resistant barley line, HL3/5 was much more successful than GE3 (frequency of haustorium formation less than 1%). The fact that HL3/5 did not possess a generally higher ability to penetrate successfully to form haustoria on the susceptible barley line, indicates that HL3/5 did not overcome the mlo5 resistance by being generally more vigorous. In the resistant barley line, papillae were larger than in the susceptible line; however, both isolates were associated with papillae of the same diameter at the time of penetration. We suggest that the mlo5 resistant barley line confers two different forms of resistance: isolate-specific and isolate-nonspecific.

Suppression of resistance against penetration byErysiphe graminisf.sp.hordeiconferred by themlo5barley powdery mildew resistance gene was accomplished by using the glucose analogue, 2-deoxy-D-glucose (DDG) or mannose applied to true leaf tissue of themlo5containing Riso-R/barley isoline. Additional suppression ofmlo5penetration resistance against the avirulentE. graminisisolate was achieved by using DDG, mannose, or glucose in combination with the phenylalanine ammonia lyase inhibitor α-aminooxy-β-phenylpropionic acid (AOPP). Amlovirulent isolate ofE. graminiswas also tested against Riso-R and the isoline Riso-S. The penetration efficiency of themlovirulent isolate against both isolines was also enhanced by treatment of leaf tissues with DDG or mannose alone or in combination with AOPP. It was concluded that Riso-R penetration resistance utilizes a backup defence involving phenolic compound synthesis, but that phenolics are not responsible for the primary mechanism ofmlo5penetration resistance. Sequestration of phosphate ions caused by complexing with DDG or mannose may lower the energy available for penetration resistance in these barley lines, obviating both inherent andmlo5based penetration resistance. Barley epidermal cell papillae were associated with penetration resistance in both barley isolines, and all papillae contained callose. However, papillae were significantly smaller and less frequent when tissues were treated with DDG or mannose. The potential inhibitory effects of glucose, DDG and mannose on activity of the enzyme callose synthasein vitrowere tested and proved negative.

Barley HvNAC6 is a member of the plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factor family and we have shown previously that it acts as a positive regulator of basal resistance in barley against the biotrophic pathogen Blumeria graminis f. sp. hordei (Bgh). In this study, we use a transgenic approach to constitutively silence HvNAC6 expression, using RNA interference (RNAi), to investigate the in vivo functions of HvNAC6 in basal resistance responses in barley in relation to the phytohormone ABA. The HvNAC6 RNAi plants displayed reduced HvNAC6 transcript levels and were more susceptible to Bgh than wild-type plants. Application of exogenous ABA increased basal resistance against Bgh in wild-type plants, but not in HvNAC6 RNAi plants, suggesting that ABA is a positive regulator of basal resistance which depends on HvNAC6. Silencing of HvNAC6 expression altered the light/dark rhythm of ABA levels which were, however, not influenced by Bgh inoculation. The expression of the two ABA biosynthetic genes HvNCED1 and HvNCED2 was compromised, and transcript levels of the ABA conjugating HvBG7 enzyme were elevated in the HvNAC6 RNAi lines, but this effect was not clearly associated with transgene-mediated resistance. Together, these data support a function of HvNAC6 as a regulator of ABA-mediated defence responses for maintenance of effective basal resistance against Bgh.

A Japanese field isolate (Race I) of Erysiphe graminis f.sp, hordei was tested on 17 barley lines carrying the mlo powdery mildew resistance gene. Race I produced many successful infections with infection type larger than or equal to 2 on six lines (M66, MC20, SRI, SR7, A tem and Totem), On the remaining 11 lines it reacted with infection type 0. Colony numbers on the Mlo-lines were between 26% and 12 9% of the numbers on a susceptible cultivar Manchuria, These numbers were larger than, or similar to those produced by isolate HL3/5, which has the highest recorded infection efficiency on Mlo-resistant cultivars. The interaction between isolates and lines was highly significant. The isolate GE3, from which HL3/5 was derived by selection, gave rise to occasional colonies corresponding to less than 03° o of the number produced on cv. Manchuria.

Climate change is likely to decrease crop yields worldwide. Developing climate resilient cultivars is one way to combat this production scarcity, however, little is known of crop response to future climate conditions and in particular the variability within crops. In Scandinavia, barley is widely cultivated, but yields have stagnated since the start of this century. In this study we cultivated 138 spring barley accessions in a climate phytotron under four treatments mimicking forecasted levels of temperature, carbon dioxide concentration ([CO2]) and ozone ([O3]) at the end of the 21st century1. The ambient control had 19/12 °C (day/night) and [CO2] at 385 ppm. Three single-factor treatments had elevated temperature +5 °C day/night, [CO2] at 700 ppm or [O3] at 120 ppb, and in a two-factor treatment the combination of elevated temperature and [CO2] was applied. Treatment effects were assessed on grain yield, grain protein concentration, grain protein harvested, number of grains, number of ears, aboveground vegetative biomass and harvest index. In addition, stability of the production was calculated over the applied treatments for the assessed parameters. In the climate scenario of elevated temperature and [CO2] the grain yield of barley decreased 29% and harvested grain protein declined 22%. Vast variation was identified among the individual barley accessions, which should be exploited by plant breeders in the development of climate resilient cultivars. A genome-wide association study (GWAS) of recorded phenotypes and 3967 SNP-markers identified 60 marker-trait associations (-logp>2.95)2. Markers were found associated with grain yield under all three single factor treatments temperature, [CO2] and [O3], as well as with stability over treatments. To our knowledge, this is the first study that evaluates numerous barley accessions under future climate conditions and identifies candidate markers for abiotic stress tolerance - markers that could be used in the development of cultivars to secure future primary production.

SUMMARY Fungal-induced inaccessibility in oat to Blumeria graminis requires active cell processes. These are reiterative de novo cell processes involved in inherent penetration resistance. Therefore, induced inaccessibility may well involve cellular memory of the initial attack. Phenylpropanoid biosynthesis inhibitors (AOPP and OH-PAS) and phosphate scavengers (DDG and d-mannose) strongly suppressed induced inaccessibility, but silicon nutrition had no effect. Induced accessibility was modulated by the presence of fungal haustoria inside cells. Haustoria actively suppress or reprogram infected plant cells toward a constant state of penetration susceptibility. Neither inhibitor treatments nor silicon nutrition affected fungal-induced accessibility.

ATAF1 is a member of a largely uncharacterized plant-specific gene family encoding NAC transcription factors, and is induced in response to various abiotic and biotic stimuli in Arabidopsis thaliana. Previously, we showed that a mutant allele of ATAF1 compromises penetration resistance in Arabidopsis with respect to the non-host biotrophic pathogen Blumeria graminis f. sp. hordei (Bgh). In this study, we have used genome-wide transcript profiling to characterize signalling perturbations in ataf1 plants following Bgh inoculation. Comparative transcriptomic analyses identified an over-representation of abscisic acid (ABA)-responsive genes, including the ABA biosynthesis gene AAO3, which is significantly induced in ataf1 plants compared to wild-type plants following inoculation with Bgh. Additionally, we show that Bgh inoculation results in decreased endogenous ABA levels in an ATAF1-dependent manner, and that the ABA biosynthetic mutant aao3 showed increased penetration resistance to Bgh compared to wild-type plants. Furthermore, we show that ataf1 plants show ABA-hyposensitive phenotypes during seedling development and germination. Our data support a negative correlation between ABA levels and penetration resistance, and identify ATAF1 as a new stimuli-dependent attenuator of ABA signalling for the mediation of efficient penetration resistance in Arabidopsis upon Bgh attack.

Extreme climate events as heatwaves, floods and storms cause acute changes in season variability influencing primary production and are very likely to increase in magnitude and/or frequency (IPCC, AR5, WGI).In the present study 22 primarily Nordic barley accessions were grown in four basic climate treatments of 1) 19/12°C (day/night) and 400 ppm carbon dioxide concentration [CO2] mimicking ambient South Scandinavian summer conditions, 2) elevated temperature (+5°C day/night), 3) elevated [CO2] at 700 ppm and 4) the combination of elevated temperature and [CO2]. Temperature and [CO2] were at levels representing a worst case scenario (~RCP8.5, IPCC) at the end of the 21st century. A 10 day-heatwave of 33/22°C (day/night) was superimposed around the time of flowering on the basic climate treatments.The superimposed heatwave decreased overall grain yield in all combinations, however, vast variation in response was identified among accessions. In the two-factor treatment the decrease in grain yield varied from 2-80%. The heatwave caused the strongest overall effect in the treatment of elevated [CO2] decreasing grain yield by 48% and the least effect (35%) was observed under elevated temperature suggesting elevated temperature to have a priming effect. In all heatwave treatments allocation of biomass was changed, increasing aboveground vegetative biomass and decreasing grain yield as previously reported.The treatment with the combination of elevated temperature, [CO2] and the superimposed heatwave may best represent a future climate scenario since more than one climate factor most likely will change at a time. From the basic ambient treatment to the two-factor treatment including heatwave, grain yield decreased 52%.Our study emphasizes the need for assessing the effects of extreme events under climate change conditions on numerous accessions in order to select appropriate genotypes for breeding future cultivars that can secure the primary production.