Integrative transcript to proteome analysis of barley during Ramularia collo-cygni leaf spot development identified several proteins that are related to fungal recognition and infection responses.

Introduction: Ramularia leaf spot (RLS) disease is a growing threat to barley cultivation, but with no substantial resistance identified to date. Similarly, the understanding of the lifestyle of Ramularia collo-cygni (Rcc) and the prediction of RLS outbreak severity remain challenging, with Rcc displaying a rather untypical long endophytic phase and a sudden change to a necrotrophic lifestyle. The aim of this study was to provide further insights into the defense dynamics during the different stages of colonization and infection in barley in order to identify potential targets for resistance breeding. Methods: Utilizing the strength of proteomics in understanding plant-pathogen interactions, we performed an integrative analysis of a published transcriptome dataset with a parallel generated proteome dataset. Therefore, we included two spring barley cultivars with contrasting susceptibilities to Rcc and two fungal isolates causing different levels of RLS symptoms. Results: Interestingly, early responses in the pathogen recognition phase of the host were driven by strong responses differing between isolates. An important enzyme in this process is a xylanase inhibitor, which protected the plant from cell wall degradation by the fungal xylanase. At later time points, the differences were driven by cultivar-specific responses, affecting mostly features contributing to the pathogenesis- and senescence-related pathways or photosynthesis. Discussion: This supports the hypothesis of a hemibiotrophic lifestyle of Rcc, with slight differences in trophism of the two analyzed isolates. The integration of these data modalities highlights a strength of protein-level analysis in understanding plant-pathogen interactions and reveals new features involved in fungal recognition and susceptibility in barley cultivars.

Deciphering Molecular Host-Pathogen Interactions During Ramularia Collo-Cygni Infection on Barley.

Ramularia collo-cygni is the causal agent of Ramularia leaf spot disease (RLS) on barley and became, during the recent decades, an increasing threat for farmers across the world. Here, we analyze morphological, transcriptional, and metabolic responses of two barley cultivars having contrasting tolerance to RLS, when infected by an aggressive or mild R. collo-cygni isolate. We found that fungal biomass in leaves of the two cultivars does not correlate with their tolerance to RLS, and both cultivars displayed cell wall reinforcement at the point of contact with the fungal hyphae. Comparative transcriptome analysis identified that the largest transcriptional differences between cultivars are at the early stages of fungal colonization with differential expression of kinases, calmodulins, and defense proteins. Weighted gene co-expression network analysis identified modules of co-expressed genes, and hub genes important for cultivar responses to the two R. collo-cygni isolates. Metabolite analyses of the same leaves identified defense compounds such as p-CHDA and serotonin, correlating with responses observed at transcriptome and morphological level. Together these all-round responses of barley to R. collo-cygni provide molecular tools for further development of genetic and physiological markers that may be tested for improving tolerance of barley to this fungal pathogen.

Dissection of Ramularia Leaf Spot Disease by Integrated Analysis of Barley and Ramularia collo-cygni Transcriptome Responses.

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.

Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

The barley HvNAC6 transcription factor affects ABA accumulation and promotes basal resistance against powdery mildew.

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.

Regulation of basal resistance by a powdery mildew-induced cysteine-rich receptor-like protein kinase in barley.

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.

Engineering pathogen resistance in crop plants: current trends and future prospects.

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.

Blumeria graminis interactions with barley conditioned by different single R genes demonstrate a temporal and spatial relationship between stomatal dysfunction and cell death.

Hypersensitive response (HR) against Blumeria graminis f. sp. hordei infection in barley (Hordeum vulgare) was associated with stomata "lock-up" leading to increased leaf water conductance (g(l)). Unique spatio-temporal 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 mm(2) showed abnormally high g(l) during dark periods whose timing and extent correlated with those of each HR. Each isoline showed increased dark g(l) 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.

Transcriptional regulation by an NAC (NAM-ATAF1,2-CUC2) transcription factor attenuates ABA signalling for efficient basal defence towards Blumeria graminis f. sp. hordei in Arabidopsis.

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.

Single-cell transcript profiling of barley attacked by the powdery mildew fungus.

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.

Induced inaccessibility and accessibility in the oat powdery mildew system: insights gained from use of metabolic inhibitors and silicon nutrition.

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.

Differential gene expression in individual papilla-resistant and powdery mildew-infected barley epidermal cells.

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.

The germinlike protein GLP4 exhibits superoxide dismutase activity and is an important component of quantitative resistance in wheat and barley.

Germinlike proteins (GLP) are encoded in plants by a gene family with proposed functions in plant development and defense. Genes of GLP subfamily 4 of barley (HvGLP4, formerly referred to as HvOxOLP) and the wheat orthologue TaGLP4 (formerly referred to as TaGLP2a) were previously found to be expressed in pathogen-attacked epidermal tissue of barley and wheat leaves, and the corresponding proteins are proposed to accumulate in the apoplast. Here, the role of HvGLP4 and TaGLP4 in the defense of barley and wheat against Blumeria graminis (DC.) E. O. Speer, the cereal powdery mildew fungus, was examined in an epidermal transient expression system and in transgenic Arabidopsis thaliana plants overexpressing His-tagged HvGLP4. Leaf extracts of transgenic Arabidopsis overexpressing HvGLP4 contained a novel His-tagged protein with superoxide dismutase activity and HvGLP4 epitopes. Transient overexpression of TaGLP4 and HvGLP4 enhanced resistance against B. graminis in wheat and barley, whereas transient silencing by RNA interference reduced basal resistance in both cereals. The effect of GLP4 overexpression or silencing was strongly influenced by the genotype of the plant. The data suggest that members of GLP subfamily 4 are components of quantitative resistance in both barley and wheat, acting together with other, as yet unknown, plant components.