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Oat ( Avena sativa ) is susceptible to Fusarium head blight (FHB). The quality of oat grain is threatened by the accumulation of mycotoxins, particularly the trichothecene deoxynivalenol (DON), which also acts as a virulence factor for the main pathogen Fusarium graminearum . The plant can defend itself, e.g., by DON detoxification by UGT-glycosyltransferases (UTGs) and accumulation of PR-proteins, even though these mechanisms do not deliver effective levels of resistance. We studied the ability of the fungal biocontrol agent (BCA) Clonostachys rosea to reduce FHB and mycotoxin accumulation. Greenhouse trials showed that C. rosea -inoculation of oat spikelets at anthesis 3 days prior to F. graminearum inoculation reduced both the amount of Fusarium DNA (79%) and DON level (80%) in mature oat kernels substantially. DON applied to C. rosea -treated spikelets resulted in higher conversion of DON to DON-3-Glc than in mock treated plants. Moreover, there was a significant enhancement of expression of two oat UGT-glycosyltransferase genes in C. rosea -treated oat. In addition, C. rosea treatment activated expression of genes encoding four PR-proteins and a WRKY23-like transcription factor, suggesting that C. rosea may induce resistance in oat. Thus, C. rosea IK726 has strong potential to be used as a BCA against FHB in oat as it inhibits F. graminearum infection effectively, whilst detoxifying DON mycotoxin rapidly.

The enigmatic endophytic fungi are beginning to reveal their secrets. Like pathogens, they can manipulate the host for their own benefit to create their own optimal habitat. Some endophytic manipulations induce resistance or otherwise outcompete pathogens and can thus be exploited for biological control. Like pathogens and other symbionts, endophytes produce effector proteins and other molecules, ranging from specialised metabolites, phytohormones and microRNAs, to manipulate their hosts and other microorganisms they meet. There is a continuum from endophyte to pathogen: some organisms can infest or cause disease in some hosts, but not in others. Molecular genetics approaches coupled with functional characterisation have demonstrated their worth for understanding the biological phenomena underlying endophytic fungal interactions., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusarium graminearum . Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichia coli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.

The fungal endophyte Penicillium olsonii ML37 is a biocontrol agent of Fusarium head blight in wheat (caused by Fusarium graminearum ), which has shown a limited direct inhibition of fungal growth in vitro. We used RNA-seq and LC-MS/MS analyses to elucidate metabolic interactions of the three-way system Penicillium-wheat-Fusarium in greenhouse experiments. We demonstrated that P. olsonii ML37 colonises wheat spikes and transiently activates plant defence mechanisms, as pretreated spikes show a faster and stronger expression of the defence metabolism during the first 24 h after pathogen inoculation. This effect was transient and the expression of the same genes was lower in the pathogen-infected spikes than in those infected by P. olsonii alone. This response to the endophyte includes the transcriptional activation of several WRKY transcription factors. This early activation is associated with a reduction in FHB symptoms and significantly lower levels of the F. graminearum metabolites 15-acetyl-DON and culmorin. An increase in the Penicillium-associated metabolite asperphanamate confirms colonisation by the endophyte. Our results suggest that the mode of action used by P. olsonii ML37 is via a local defence activation in wheat spikes, and that this fungus has potential as a novel biological alternative in wheat disease control.

Specialised metabolites produced during plant-fungal associations often define how symbiosis between the plant and the fungus proceeds. They also play a role in the establishment of additional interactions between the symbionts and other organisms present in the niche. However, specialised metabolism and its products are sometimes overlooked when studying plant-microbe interactions. This limits our understanding of the specific symbiotic associations and potentially future perspectives of their application in agriculture. In this study, we used the interaction between the root endophyte Serendipita indica and tomato ( Solanum lycopersicum ) plants to explore how specialised metabolism of the host plant is regulated upon a mutualistic symbiotic association. To do so, tomato seedlings were inoculated with S. indica chlamydospores and subjected to RNAseq analysis. Gene expression of the main tomato specialised metabolism pathways was compared between roots and leaves of endophyte-colonised plants and tissues of endophyte-free plants. S. indica colonisation resulted in a strong transcriptional response in the leaves of colonised plants. Furthermore, the presence of the fungus in plant roots appears to induce expression of genes involved in the biosynthesis of lignin-derived compounds, polyacetylenes, and specific terpenes in both roots and leaves, whereas pathways producing glycoalkaloids and flavonoids were expressed in lower or basal levels.

Ustilaginoidea virens , causing rice false smut (RFS) is an economically important ascomycetous fungal pathogen distributed in rice-growing regions worldwide. Here, we identified a novel transcription factor UvCGBP1 ( C utinase G -box b inding p rotein) from this fungus, which is unique to ascomycetes. Deletion of UvCGBP1 affected development and virulence of U. virens . A total of 865 downstream target genes of UvCGBP1 was identified using ChIP-seq and the most significant KEGG enriched functional pathway was the MAPK signaling pathway. Approximately 36% of target genes contain the AGGGG (G-box) motif in their promoter. Among the targets, deletion of UvCGBP1 affected transcriptional and translational levels of UvPmk1 and UvSlt2 , both of which were important in virulence. ChIP-qPCR, yeast one-hybrid and EMSA confirmed that UvCGBP1 can bind the promoter of UvPmk1 or UvSlt2 . Overexpression of UvPmk1 in the ∆UvCGBP1-33 mutant restored partially its virulence and hyphae growth, indicating that UvCGBP1 could function via the MAPK pathway to regulate fungal virulence. Taken together, this study uncovered a novel regulatory mechanism of fungal virulence linking the MAPK pathway mediated by a G-box binding transcription factor, UvCGBP1.

Interactions between plant-associated fungi and their hosts are characterized by a continuous crosstalk of chemical molecules. Specialized metabolites are often produced during these associations and play important roles in the symbiosis between the plant and the fungus, as well as in the establishment of additional interactions between the symbionts and other organisms present in the niche. Serendipita indica , a root endophytic fungus from the phylum Basidiomycota, is able to colonize a wide range of plant species, conferring many benefits to its hosts. The genome of S. indica possesses only few genes predicted to be involved in specialized metabolite biosynthesis, including a putative terpenoid synthase gene ( SiTPS ). In our experimental setup, SiTPS expression was upregulated when the fungus colonized tomato roots compared to its expression in fungal biomass growing on synthetic medium. Heterologous expression of SiTPS in Escherichia coli showed that the produced protein catalyzes the synthesis of a few sesquiterpenoids, with the alcohol viridiflorol being the main product. To investigate the role of SiTPS in the plant-endophyte interaction, an SiTPS -over-expressing mutant line was created and assessed for its ability to colonize tomato roots. Although overexpression of SiTPS did not lead to improved fungal colonization ability, an in vitro growth-inhibition assay showed that viridiflorol has antifungal properties. Addition of viridiflorol to the culture medium inhibited the germination of spores from a phytopathogenic fungus, indicating that SiTPS and its products could provide S. indica with a competitive advantage over other plant-associated fungi during root colonization.

Fungi living inside plants affect many aspects of plant health, but little is known about how plant genotype influences the fungal endophytic microbiome. However, a deeper understanding of interactions between plant genotype and biotic and abiotic environment in shaping the plant microbiome is of significance for modern agriculture, with implications for disease management, breeding and the development of biocontrol agents. For this purpose, we analysed the fungal wheat microbiome from seed to plant to seeds and studied how different potential sources of inoculum contributed to shaping of the microbiome. We conducted a large-scale pot experiment with related wheat cultivars over one growth-season in two environments (indoors and outdoors) to disentangle the effects of host genotype, abiotic environment (temperature, humidity, precipitation) and fungi present in the seed stock, air and soil on the succession of the endophytic fungal communities in roots, flag leaves and seeds at harvest. The communities were studied with ITS1 metabarcoding and environmental climate factors were monitored during the experimental period. Host genotype, tissue type and abiotic factors influenced fungal communities significantly. The effect of host genotype was mostly limited to leaves and roots, and was location-independent. While there was a clear effect of plant genotype, the relatedness between cultivars was not reflected in the microbiome. For the phyllosphere microbiome, location-dependent weather conditions factors largely explained differences in abundance, diversity, and presence of genera containing pathogens, whereas the root communities were less affected by abiotic factors. Our findings suggest that airborne fungi are the primary inoculum source for fungal communities in aerial plant parts whereas vertical transmission is likely to be insignificant. In summary, our study demonstrates that host genotype, environment and presence of fungi in the environment shape the endophytic fungal community in wheat over a growing season., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Mycoviruses are viruses that infect fungi, and hypovirulence-associated mycoviruses have the potential to control fungal diseases. However, it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field, and no mycovirus has been applied for field crop protection. In this study, we found that a previously identified small DNA mycovirus (SsHADV-1) can convert its host, Sclerotinia sclerotiorum, from a typical necrotrophic pathogen to a beneficial endophytic fungus. SsHADV-1 downregulates the expression of key pathogenicity factor genes in S. sclerotiorum during infection. When growing in rapeseed, the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense, hormone signaling, and circadian rhythm pathways. As a result, plant growth is promoted and disease resistance is enhanced. Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6% and improve yield by 14.9%. Moreover, we discovered that SsHADV-1 could also infect other S. sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants. These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism. Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Little is known about the influence of host genotype and phytohormones on the composition of fungal endophytic communities. We investigated the influence of host genotype and phytohormones on the structure of the fungal endophytic communities of tomato roots by amplicon sequencing of the ITS1 region and combined this approach with isolation and functional characterization of the isolates. A significant effect of the host genotype on the dominant fungal species was found by comparing the cultivars Castlemart and UC82B and, surprisingly, root pathogens were among the most abundant taxa. In contrast, smaller changes in the relative abundance of the dominant species were found in mutants impaired in jasmonic acid biosynthesis (def1) and ethylene biosynthesis (8338) compared to the respective wild types. However, def1 showed significantly higher species richness compared to the wild type. Analysis of the phytohormone profiles of these genotypes indicates that changes in the phytohormone balance may contribute to this difference in species richness. Assessing the lifestyle of isolated fungi on tomato seedlings revealed the presence of both beneficial endophytes and latent pathogens in roots of asymptomatic plants, suggesting that the interactions between members of the microbiome maintain the equilibrium in the community preventing pathogens from causing disease., (© FEMS 2020.)

Fusarium head blight (FHB) is a devastating disease of wheat heads. It is caused by several species from the genus Fusarium. Several endophytic fungi also colonize wheat spikes asymptomatically. Pathogenic and commensal fungi share and compete for the same niche and thereby influence plant performance. Understanding the natural dynamics of the fungal community and how the pre-established species react to pathogen attack can provide useful information on the disease biology and the potential use of some of these endophytic organisms in disease control strategies. Fungal community composition was assessed during anthesis as well as during FHB attack in wheat spikes during 2016 and 2017 in two locations. Community metabarcoding revealed that endophyte communities are dominated by basidiomycete yeasts before anthesis and shift towards a more opportunistic ascomycete-rich community during kernel development. These dynamics are interrupted when Fusarium spp. colonize wheat spikes. The Fusarium pathogens appear to exclude other fungi from floral tissues as they are associated with a reduction in community diversity, especially in the kernel which they colonize rapidly. Similarly, the presence of several endophytes was negatively correlated with Fusarium spp. and linked with spikes that stayed healthy despite exposure to the pathogen. These endophytes belonged to the genera Cladosporium, Itersonillia and Holtermanniella. These findings support the hypothesis that some naturally occurring endophytes could outcompete or prevent FHB and represent a source of potential biological control agents in wheat.

Ash dieback, caused by the fungus Hymenoscyphus fraxineus, has threatened ash trees in Europe for more than two decades. However, little is known of how endophytic communities affect the pathogen, and no effective disease management tools are available. While European ash (Fraxinus excelsior) is severely affected by the disease, other more distantly related ash species do not seem to be affected. We hypothesise that fungal endophytic communities of tolerant ash species can protect the species against ash dieback, and that selected endophytes have potential as biocontrol agents. These hypotheses were tested by isolating members of the fungal communities of five tolerant ash species, and identifying them using ITS regions. Candidate endophytes were tested by an in vitro antagonistic assay with H.fraxineus. From a total of 196 isolates we identified 9 fungal orders, 15 families, and 40 species. Fungi in orders Pleosporales, such as Boeremia exigua and Diaporthe spp., and Hypocreales (e.g., Fusarium sp.), were recovered in most communities, suggesting they are common taxa. The in vitro antagonistic assay revealed five species with high antagonistic activity against H. fraxineus. These endophytes were identified based on ITS region as Sclerostagonospora sp., Setomelanomma holmii, Epicoccum nigrum, B. exigua and Fusarium sp. Three of these taxa have been described previously as antagonists of plant pathogenic microbes, and are of interest for future studies of their potential as biological control agents against ash dieback, especially for valuable ash trees in parks and urban areas., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Cerato-platanin proteins (CPs), which are secreted by filamentous fungi, are phytotoxic to host plants, but their functions have not been well defined to date. Here we characterized a CP (SsCP1) from the necrotrophic phytopathogen Sclerotinia sclerotiorum. Sscp1 transcripts accumulated during plant infection, and deletion of Sscp1 significantly reduced virulence. SsCP1 could induce significant cell death when expressed in Nicotiana benthamiana. Using yeast two-hybrid, GST pull-down, co-immunoprecipitation and bimolecular florescence complementation, we found that SsCP1 interacts with PR1 in the apoplast to facilitate infection by S. sclerotiorum. Overexpressing PR1 enhanced resistance to the wild-type strain, but not to the Sscp1 knockout strain of S. sclerotiorum. Sscp1-expressing transgenic plants showed increased concentrations of salicylic acid (SA) and higher levels of resistance to several plant pathogens (namely Botrytis cinerea, Alternaria brassicicola and Golovinomyces orontii). Our results suggest that SsCP1 is important for virulence of S. sclerotiorum and that it can be recognized by plants to trigger plant defense responses. Our results also suggest that the SA signaling pathway is involved in CP-mediated plant defense ., (© 2017 State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University New Phytologist © 2017 New Phytologist Trust.)

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.

Clonostachys rosea is a mycoparasitic fungus that can control several important plant diseases. Here, we report on the genome sequencing of C. rosea and a comparative genome analysis, in order to resolve the phylogenetic placement of C. rosea and to study the evolution of mycoparasitism as a fungal lifestyle. The genome of C. rosea is estimated to 58.3 Mb, and contains 14,268 predicted genes. A phylogenomic analysis shows that C. rosea clusters as sister taxon to plant pathogenic Fusarium species, with mycoparasitic/saprotrophic Trichoderma species in an ancestral position. A comparative analysis of gene family evolution reveals several distinct differences between the included mycoparasites. Clonostachys rosea contains significantly more ATP-binding cassette (ABC) transporters, polyketide synthases, cytochrome P450 monooxygenases, pectin lyases, glucose-methanol-choline oxidoreductases, and lytic polysaccharide monooxygenases compared with other fungi in the Hypocreales. Interestingly, the increase of ABC transporter gene number in C. rosea is associated with phylogenetic subgroups B (multidrug resistance proteins) and G (pleiotropic drug resistance transporters), whereas an increase in subgroup C (multidrug resistance-associated proteins) is evident in Trichoderma virens. In contrast with mycoparasitic Trichoderma species, C. rosea contains very few chitinases. Expression of six group B and group G ABC transporter genes was induced in C. rosea during exposure to the Fusarium mycotoxin zearalenone, the fungicide Boscalid or metabolites from the biocontrol bacterium Pseudomonas chlororaphis. The data suggest that tolerance toward secondary metabolites is a prominent feature in the biology of C. rosea., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The fungus Clonostachys rosea is antagonistic against plant pathogens, including Fusarium graminearum, which produces the oestrogenic mycotoxin zearalenone (ZEA). ZEA inhibits other fungi, and C. rosea can detoxify ZEA through the enzyme zearalenone lactonohydrolase (ZHD101). As the relevance of ZEA detoxification for biocontrol is unknown, we studied regulation and function of ZHD101 in C. rosea. Quantitative reverse-transcription PCR revealed zhd101 gene expression in all conditions studied and demonstrated dose-dependent induction by ZEA. Known inducers of the Polyketide Synthase pathway did not induce zhd101 expression, suggesting specificity of the enzyme towards ZEA. To assess the role of ZHD101 during biocontrol interactions, we generated two Δzhd101 mutants incapable of ZEA-detoxification and confirmed their defect in degrading ZEA by HPLC. The Δzhd101 mutants displayed a lower in vitro ability to inhibit growth of the ZEA-producing F. graminearum (strain 1104-14) compared to the wild type. In contrast, all three C. rosea strains equally inhibited growth of the F. graminearum mutant (ΔPKS4), which is impaired in ZEA-production. Furthermore, the Δzhd101 mutants failed to protect wheat seedlings against foot rot caused by the ZEA-producing F. graminearum. These data show that ZEA detoxification by ZHD101 is important for the biocontrol ability of C. rosea against F. graminearum., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Background: Clonostachys rosea strain IK726 is a mycoparasitic fungus capable of controlling mycotoxin-producing Fusarium species, including F. graminearum and F. culmorum, known to produce Zearalenone (ZEA) and Deoxynivalenol (DON). DON is a type B trichothecene known to interfere with protein synthesis in eukaryotes. ZEA is a estrogenic-mimicing mycotoxin that exhibits antifungal growth. C. rosea produces the enzyme zearalenone hydrolase (ZHD101), which degrades ZEA. However, the molecular basis of resistance to DON in C. rosea is not understood. We have exploited a genome-wide transcriptomic approach to identify genes induced by DON and ZEA in order to investigate the molecular basis of mycotoxin resistance C. rosea., Results: We generated DON- and ZEA-induced cDNA libraries based on suppression subtractive hybridization. A total of 443 and 446 sequenced clones (corresponding to 58 and 65 genes) from the DON- and ZEA-induced library, respectively, were analysed. DON-induced transcripts represented genes encoding metabolic enzymes such as cytochrome P450, cytochrome c oxidase and stress response proteins. In contrast, transcripts encoding the ZEA-detoxifying enzyme ZHD101 and those encoding a number of ATP-Binding Cassette (ABC) transporter transcripts were highly frequent in the ZEA-induced library. Subsequent bioinformatics analysis predicted that all transcripts with similarity to ABC transporters could be ascribed to only 2 ABC transporters genes, and phylogenetic analysis of the predicted ABC transporters suggested that they belong to group G (pleiotropic drug transporters) of the fungal ABC transporter gene family. This is the first report suggesting involvement of ABC transporters in ZEA tolerance. Expression patterns of a selected set of DON- and ZEA-induced genes were validated by the use of quantitative RT-PCR after exposure to the toxins. The qRT-PCR results obtained confirm the expression patterns suggested from the EST redundancy data., Conclusion: The present study identifies a number of transcripts encoding proteins that are potentially involved in conferring resistance to DON and ZEA in the mycoparasitic fungus C. rosea. Whilst metabolic readjustment is potentially the key to withstanding DON, the fungus produces ZHD101 to detoxify ZEA and ABC transporters to transport ZEA or its degradation products out from the fungal cell.

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.

The ascomycete fungal pathogen Fusarium graminearum (teleomorph stage: Gibberella zeae) is the causal agent of Fusarium head blight in wheat and barley. This disease leads to significant losses of crop yield, and especially quality through the contamination by diverse fungal mycotoxins, which constitute a significant threat to the health of humans and animals. In recent years, high-throughput proteomics, aiming at identifying a broad spectrum of proteins with a potential role in the pathogenicity and host resistance, has become a very useful tool in plant-fungus interaction research. In this review, we describe the progress in proteomics applications toward a better understanding of F. graminearum pathogenesis, virulence, and host defense mechanisms. The contribution of proteomics to the development of crop protection strategies against this pathogen is also discussed briefly.

A large number of effector candidates have been identified recently in powdery mildew fungi. However, their roles and how they perform their functions remain unresolved. In this study, we made use of host-induced gene silencing and confirmed that the secreted barley powdery mildew effector candidate, CSEP0055, contributes to the aggressiveness of the fungus. This result suggests that CSEP0055 is involved in the suppression of plant defence. A yeast two-hybrid screen indicated that CSEP0055 interacts with members of the barley pathogenesis-related protein families, PR1 and PR17. Interaction with PR17c was confirmed by bimolecular fluorescence complementation analyses. Down-regulation and over-expression of PR17c in epidermal cells of barley confirmed that this protein is important for penetration resistance against the powdery mildew fungus. In line with this, PR17c was found to be apoplastic, localizing to the papillae formed in response to this fungus. The CSEP0055 transcript did not start to accumulate until 24 h after inoculation. This suggests that this gene is expressed too late to influence primary penetration events, but rather sustains the fungus at sites of secondary penetration, where PR17c appears to be able to accumulate., (© 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.)

Fusarium graminearum is a phytopathogenic fungus primarily infecting small grain cereals, including barley and wheat. Secreted enzymes play important roles in the pathogenicity of many fungi. In order to access the secretome of F. graminearum, the fungus was grown in liquid culture with barley or wheat flour as the sole nutrient source to mimic the host-pathogen interaction. A gel-based proteomics approach was employed to identify the proteins secreted into the culture medium. Sixty-nine unique fungal proteins were identified in 154 protein spots, including enzymes involved in the degradation of cell walls, starch and proteins. Of these proteins, 35% had not been identified in previous in planta or in vitro studies, 70% were predicted to contain signal peptides and a further 16% may be secreted in a nonclassical manner. Proteins identified in the 72 spots showing differential appearance between wheat and barley flour medium were mainly involved in fungal cell wall remodelling and the degradation of plant cell walls, starch and proteins. The in planta expression of corresponding F. graminearum genes was confirmed by quantitative reverse transcriptase-polymerase chain reaction in barley and wheat spikelets harvested at 2-6 days after inoculation. In addition, a clear difference in the accumulation of fungal biomass and the extent of fungal-induced proteolysis of plant β-amylase was observed in barley and wheat. The present study considerably expands the current database of F. graminearum secreted proteins which may be involved in Fusarium head blight., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

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., (© 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.)

Many carrots are discarded during post harvest cold storage due to development of fungal infections, caused by, e.g., Mycocentrospora acerina (liquorice rot). We compared the susceptibility of carrots grown under conventional and organic agricultural practices. In one year, organically cultivated carrots showed 3× to 7× more symptoms than conventionally cultivated, when studying naturally occurring disease at 4 and 6 months, respectively. On the other hand, we have developed a bioassay for infection studies of M. acerina on carrots and observed that organic roots were more susceptible after one month of storage than conventional ones, but no differences were apparent after four or six months storage. Levels of polyacetylenes (falcarinol, falcarindiol and falcarindiol-3-acetate) did not change, whereas the isocoumarin phytoalexin (6-methoxymellein) accumulated in infected tissue as well as in healthy tissue opposite the infection. The proteomes of carrot and M. acerina were characterized, the intensity of 33 plant protein spots was significantly changed in infected roots including up regulation of defence and stress response proteins but also a decrease of proteins involved in energy metabolism. This combined metabolic and proteomic study indicates that roots respond to fungal infection through altered metabolism: simultaneous induction of 6-methoxymellein and synthesis of defence related proteins., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Quantitative real-time polymerase chain reaction differentiating 10 Fusarium spp. and Microdochium nivale or M. majus was applied to a total of 396 grain samples of wheat, barley, triticale, oat, and rye sampled across Denmark from 2003 to 2007, along with selected samples of wheat and barley from 1957 to 2000, to determine incidence and abundance of individual Fusarium spp. The mycotoxins deoxynivalenol (DON), nivalenol, zearalenone, T-2, and HT-2 were quantified using liquid chromatography-double mass spectrometry. Major differences in the Fusarium species complex among the five cereals as well as great yearly variation were seen. Fusarium graminearum, F. culmorum, and F. avenaceum were dominant in wheat, with DON as the dominant mycotoxin. F. langsethiae, F. culmorum, and F. avenaceum were dominant in barley and oat, leading to relatively high levels of the mycotoxins T-2 and HT-2. F. graminearum, F. culmorum, and F. avenaceum dominated in triticale and rye. The nontoxigenic M. nivale/majus were present in significant amounts in all cereal species. Wheat and barley samples from 1957 to 1996 exhibited no or very low amounts of F. graminearum, indicating a recent increase of this pathogen. Biomass and mycotoxin data exhibited good correlations between Fusarium spp. and their corresponding mycotoxins under field conditions.

Sheath blight caused by Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is a major cause of crop loss in intensive rice production systems. No economically viable control methods have been developed. We screened aqueous extracts of common herbal plants that could reduce sheath blight lesions and found that foliar spraying and seed soaking application of extracts of either fresh or dried leaves of Chromolaena odorata gave up to 68% reduction in sheath blight lesion lengths under controlled and semi-field conditions. The observed reductions were not dependent on growth conditions of C. odorata and rice cultivar. The effect was observed until 21 days after inoculation and was not dependent on microbial activity. Under semi-field conditions, extracts also reduced severity of other important rice diseases, i.e., blast (Pyricularia oryzae) using foliar spray (up to 45%), brown spot (Bipolaris oryzae) using seed treatment (up to 57%), and bacterial blight (Xanthomonas oryzae pv. oryzae) using both application methods (up to 50%).

A proteomic analysis was conducted to map the events during the initial stages of the interaction between the fungal pathogen Fusarium graminearum and the susceptible barley cultivar Scarlett. Quantification of fungal DNA demonstrated a sharp increase in fungal biomass in barley spikelets at 3 days after inoculation. This coincided with the appearance of discrete F. graminearum-induced proteolytic fragments of β-amylase. Based on these results, analysis of grain proteome changes prior to extensive proteolysis enabled identification of barley proteins responding early to infection by the fungus. In total, the intensity of 51 protein spots was significantly changed in F. graminearum-infected spikelets and all but one were identified. These included pathogenesis-related proteins, proteins involved in energy metabolism, secondary metabolism and protein synthesis. A single fungal protein of unknown function was identified. Quantitative real-time RT-PCR analysis of selected genes showed a correlation between high gene expression and detection of the corresponding proteins. Fungal genes encoding alkaline protease and endothiapepsin were expressed during 1-3 days after inoculation, making them candidates for generation of the observed β-amylase fragments. These fragments have potential to be developed as proteome-level markers for fungal infection that are also informative about grain protein quality.

The effect of nitrogen on Fusarium Head Blight (FHB) in a susceptible barley cultivar was investigated using gel-based proteomics. Barley grown with either 15 or 100kgha(-1)N fertilizer was inoculated with Fusarium graminearum (Fg). The storage protein fraction did not change significantly in response either to N level or Fg, whereas eighty protein spots in the water-soluble albumin fraction increased and 108 spots decreased more than two-fold in intensity in response to Fg. Spots with greater intensity in infected plants contained fungal proteins (9 spots) and proteolytic fragments of plant proteins (65 spots). Identified fungal proteins included two superoxide dismutases, L-xylulose reductase in two spots, peptidyl prolyl cis-trans isomerase and triosephosphate isomerase, and proteins of unknown function. Spots decreasing in intensity in response to Fg contained plant proteins possibly degraded by fungal proteases. Greater spot volume changes occurred in response to Fg in plants grown with low nitrogen, although proteomes of uninfected plants were similar for both treatments. Correlation of proteome changes with measurement of Fusarium-damaged kernels, fungal biomass and mycotoxin levels indicated that increased Fusarium infection occurred in barley with low N and suggests control of N fertilization as a possible way to minimise FHB in barley.

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.

RNA-directed RNA polymerases (RDRs) play crucial roles in the RNA silencing response of plants by enhancing and maintaining silencing signals. At least two members of the RDR group, namely RDR1 and RDR6, are implicated in defence against plant viruses. RDRs have so far only been characterized in dicot species. In this report, we identified and characterized HvRDR1, HvRDR2 and HvRDR6 genes in the monocot plant barley (Hordeum vulgare). We analysed their expression under various biotic and abiotic stresses including fungal and viral infections, salicylic acid treatment as well as during plant development. The different classes and subclasses of barley RDRs displayed contrasting expression patterns during pathogen challenge and development suggesting their involvement in specific regulatory pathways. Their response to heat and salicylic acid treatment suggests a conserved pattern of expression of these genes between monocot and dicot plant species. The existence of two HvRDR1 and two HvRDR6 genes suggests an evolutionary selection for specialization in response to biotic and abiotic stresses after gene duplication.

The accumulation of the pathogenesis-related (PR) proteins beta-1,3-glucanase and chitinase and structural defence responses were studied in leaves of wheat either resistant or susceptible to the hemibiotrophic pathogen Septoria tritici. Resistance was associated with an early accumulation of beta-1,3-glucanase and chitinase transcripts followed by a subsequent reduction in level. Resistance was also associated with high activity of beta-1,3-glucanase, especially in the apoplastic fluid, in accordance with the biotrophic/endophytic lifestyle of the pathogen in the apoplastic spaces, thus showing the highly localized accumulation of defence proteins in the vicinity of the pathogen. Isoform analysis of beta-1,3-glucanase from the apoplastic fluid revealed that resistance was associated with the accumulation of an endo-beta-1,3-glucanase, previously implicated in defence against pathogens, and a protein with identity to ADPG pyrophosphatase (92%) and germin-like proteins (93%), which may be involved in cell wall reinforcement. In accordance with this, glycoproteins like extensin were released into the apoplast and callose accumulated to a greater extent in cell walls, whereas lignin and polyphenolics were not found to correlate with defence. Treatment of a susceptible wheat cultivar with purified beta-1,3-glucan fragments from cell walls of S. tritici gave complete protection against disease and this was accompanied by increased gene expression of beta-1,3-glucanase and the deposition of callose. Collectively, these data indicate that resistance is dependent on a fast, initial recognition of the pathogen, probably due to beta-1,3-glucan in the fungal cell walls, and this results in the accumulation of beta-1,3-glucanase and structural defence responses, which may directly inhibit the pathogen and protect the host against fungal enzymes and toxins.

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.

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.

Hydrogen peroxide (H(2)O(2)) is reported to inhibit biotrophic but benefit necrotrophic pathogens. Infection by necrotrophs can result in a massive accumulation of H(2)O(2) in hosts. Little is known of how pathogens with both growth types are affected (hemibiotrophs). The hemibiotroph, Septoria tritici, infecting wheat (Triticum aestivum) is inhibited by H(2)O(2) during the biotrophic phase, but a large H(2)O(2) accumulation occurs in the host during reproduction. Here, we infiltrated catalase, H(2)O(2) or water into wheat during the biotrophic or the necrotrophic phase of S. tritici and studied the effect of infection on host physiology to get an understanding of the survival strategy of the pathogen. H(2)O(2) removal by catalase at both early and late stages made plants more susceptible, whereas H(2)O(2) made them more resistant. H(2)O(2) is harmful to S. tritici throughout its life cycle, but it can be tolerated. The late accumulation of H(2)O(2) is unlikely to result from down-regulation of photosynthesis, but probably originates from damage to the peroxisomes during the general tissue collapse, which is accompanied by release of soluble sugars in a susceptible cultivar.

To study the mechanisms of inducible disease resistance in conifers, changes in transcript accumulation in roots of Norway spruce (Picea abies (L.) Karst.) seedlings exposed to the root rot pathogen Ceratobasidium bicorne Erikss. and Ryv. (anamorph: Rhizoctonia sp.) were monitored by differential display (DD). Because C. bicorne attacks root tips, a desiccation treatment was added to exclude genes induced by pathogen-related desiccation stress. The DD analysis was defined by the use of 11 sets of primers, covering about 5% of the transcriptome. A comparison of gene expression in control, desiccation- and pathogen-stressed roots revealed 36 pathogen-induced gene transcripts. Based on database searches, these transcripts were assigned to four groups originating from spruce mRNA (25 transcripts), rRNA (five transcripts), fungal mRNA (two transcripts) and currently unknown cDNAs (four transcripts). Real-time PCR was applied to verify and quantify pathogen-induced changes in transcript accumulation. Of the 18 transcripts tested, nine were verified to be Norway spruce gene transcripts up-regulated from 1.3- to 66-fold in the infected roots. Four germin-like protein isoforms, a peroxidase and a glutathione S-transferase, all implicated in oxidative processes, including the oxidative burst, were predicted from sequence similarity searches. Seven class IV chitinase isoforms implicated in fungal cell wall degradation and a nucleotide binding site-leucine rich repeat (NBS-LRR) disease resistance protein homologue related to pathogen recognition were identified. Several transcript species, such as the NBS-LRR homologue and the germin-like protein homologues, have not previously been identified as pathogen-inducible genes in gymnosperms.

14-3-3 proteins function as regulators of a wide range of target proteins in all eukaryotes by effecting direct protein-protein interactions. Primarily, interactions between 14-3-3 proteins and their targets are mediated by phosphorylation at specific sites on the target protein. Hence, interactions with 14-3-3s are subject to environmental control through signalling pathways which impact on 14-3-3 binding sites. Because 14-3-3 proteins regulate the activities of many proteins involved in signal transduction, there are multiple levels at which 14-3-3 proteins may play roles in stress responses in higher plants. In this article, we review evidence which implicates 14-3-3 proteins in responses to environmental, metabolic and nutritional stresses, as well as in defence responses to wounding and pathogen attack. This evidence includes stress-inducible changes in 14-3-3 gene expression, interactions between 14-3-3 proteins and signalling proteins and interactions between 14-3-3 proteins and proteins with defensive functions.

The 14-3-3 protein family is a family of regulatory proteins involved in diverse cellular processes. In a previous study of regulation of individual 14-3-3 isoforms in the germinating barley embryo, we found that a post-translationally modified, 28 kDa form of 14-3-3A was present in specific cell fractions of the germinated embryo. In the present study, we identify the nature of the modification of 14-3-3A, and show that the 28 kDa doublet is the result of cleavage of the C-terminus. The 28 kDa forms of 14-3-3A lack ten or twelve amino acid residues at the non-conserved C-terminus of the protein, respectively. Barley 14-3-3B and 14-3-3C are not modified in a similar way. Like the 30 kDa form, in vitro produced 28 kDa 14-3-3A is still capable of binding AHA2 H+-ATPase in an overlay assay. Our results show a novel isoform-specific post-translational modification of 14-3-3 proteins that is regulated in a tissue-specific and developmental way.

Summary Two barley (Hordeum vulgare L.) cDNA clones (pBH6-12 and pBH6-17) were isolated from a cDNA library prepared from leaves 6 h after inoculation with Blumeria graminis f.sp. hordei (Bgh). The two transcripts accumulate strongly in response to Bgh, peaking around 6, 15-24 and 48-96 h after inoculation, concomitant with fungal penetration attempts, hypersensitive response and fungal growth. The encoded proteins, HvPR-17a and HvPR-17b, belong to a new family of plant pathogenesis-related proteins, designated 'PR-17'. The family also include NtPRp27 from tobacco (Okushima et al., 2000, Plant Mol. Biol.42, 479-488) and WCI-5 from wheat (Görlach et al., 1996, Plant Cell8, 629-643), responsive to viral and fungal infection, respectively. Antisera were raised to HvPR-17a and HvPR-17b, and the proteins exhibit apparent molecular weights of 26 and 24 kDa, respectively. They accumulate in the mesophyll apoplast following Bgh-inoculation, as well as in the leaf epidermis, the only tissue to be invaded by the fungus. Several homologous plant proteins exist, and a highly conserved part of the members of this new protein family show similarity to the active site and to the peptide-binding groove of the exopeptidase 'aminopeptidase N' from eukaryotes and the endopeptidase 'thermolysin' from bacteria.

14-3-3 proteins form a family of highly conserved proteins with central roles in many eukaryotic signalling networks. In plants, they bind to and activate the plasma membrane H+-ATPase, creating a binding site for the phytotoxin fusicoccin. Barley 14-3-3 transcripts accumulate in the epidermis upon inoculation with the powdery mildew fungus. We have isolated a cDNA encoding a plasma membrane H+-ATPase (HvHAI), which is also induced by powdery mildew attack. The C-terminal domain of this H+-ATPase interacts with 14-3-3 proteins in the yeast two-hybrid system. Inoculation with the powdery mildew fungus, or treatment with fusicoccin, results in an increase in fusicoccin binding ability of barley leaf membranes. Overlay assays show a fungus-induced increase in binding of digoxygenin-labelled 14-3-3 protein to several proteins including a 100 kDa membrane protein, probably the plasma membrane H+-ATPase. These effects are seen specifically in the inoculated epidermis and not in the whole leaf. We propose that 14-3-3 proteins are involved in an epidermis-specific response to the powdery mildew fungus, possibly via an activation of the plasma membrane H+-ATPase.

We propose a model for activation of the epidermal cell hypersensitive response (HR) in the barley/powdery mildew interaction. The model suggests that the plasma membrane proton pump (H+-ATPase) of epidermal cells is activated following penetration by an avirulent powdery mildew fungus. This will cause an acidification of the apoplast towards the mesophyll cells, thereby activating generation of H2O2 from the mesophyll, which subsequently triggers the epidermal cell to undergo HR. The model is supported by the following data: (1) the earliest HR-related H2O2 is found in the attachment zones between the epidermal cell and underlying mesophyll cells; (2) scavenger treatment reduces HR; (3) treatment of leaves with low-pH (3.5) citrate and succinate buffers causes more cells to undergo HR in the compatible interaction, while treatment with the same buffers at pH 5.5 reduces the number of HR-cells in the incompatible interaction; (4) race-specific proton extrusion is observed underneath epidermal tissue detached from leaves inoculated 15 h earlier; and (5) treatment of leaves with fusicoccin, an activator of the plasma membrane H+-ATPase, increases the number of HR-cells in the compatible interaction.

The enigmatically named 14-3-3 proteins have been the subject of considerable attention in recent years since they have been implicated in the regulation of diverse physiological processes, in eukaryotes ranging from slime moulds to higher plants. In plants they have roles in the regulation of the plasma membrane H+-ATPase and nitrate reductase, among others. Regulation of target proteins is achieved through binding of 14-3-3 to short, often phosphorylated motifs in the target, resulting either in its activation (e.g. H+-ATPase), inactivation (e.g. nitrate reductase) or translocation (although this function of 14-3-3 proteins has yet to be demonstrated in plants). The native 14-3-3 proteins are homo- or heterodimers and, as each monomer has a binding site, a dimer can potentially bind two targets, promoting their association. Alternatively, target proteins may have more than one 14-3-3-binding site. In this mini review, we present a synthesis of recent results from plant 14-3-3 research and, with reference to known 14-3-3-binding motifs, suggest further subjects for research.

A cDNA clone was isolated by differential hybridization from a library prepared from barley leaves inoculated with the fungus Blumeria graminis f.sp. hordei (Bgh). The open reading frame of the insert (designated HvCHS2) encoded a polypeptide with 72-79% identity to chalcone synthases (CHS) and 65-68% identity to stilbene synthases. Alignments of the amino acid sequence of HvCHS2 with the consensus sequence of naringenin-CHS (EC 2.3.1.74) reveals significant differences between HvCHS2 and naringenin-CHS. HvCHS2 transcripts accumulate strongly in barley leaves in response to inoculation with Bgh, whereas only insignificant accumulation of barley naringenin-CHS (CHS1) transcripts is seen upon the inoculation. The accumulation of HvCHS2 transcripts is also elicited by UV light. To compare the activity of HvCHS2 with the activity of CHS1, the two enzymes were expressed in Escherichia coli. Both HvCHS2 and CHS1 catalyse the formation of chalcones. However, HvCHS2 and CHS1 differ in their substrate requirements. CHS1 uses cinnamoyl-CoA and 4-coumaroyl-CoA at comparable rates whereas feruloyl-CoA is a poor substrate for this enzyme. In contrast, HvCHS2 converts feruloyl-CoA and caffeoyl-CoA at the highest rate whereas cinnamoyl-CoA is a poor substrate. Thus, HvCHS2 is a novel pathogen and UV light induces homoeriodictyol/eriodictyol CHS involved in the direct production of flavonoids possessing multi-substituted B-rings.

Previously we reported that oxalate oxidase activity increases in extracts of barley (Hordeum vulgare) leaves in response to the powdery mildew fungus (Blumeria [syn. Erysiphe] graminis f.sp. hordei) and proposed this as a source of H2O2 during plant-pathogen interactions. In this paper we show that the N terminus of the major pathogen-response oxalate oxidase has a high degree of sequence identity to previously characterized germin-like oxalate oxidases. Two cDNAs were isolated, pHvOxOa, which represents this major enzyme, and pHvOxOb', representing a closely related enzyme. Our data suggest the presence of only two oxalate oxidase genes in the barley genome, i.e. a gene encoding HvOxOa, which possibly exists in several copies, and a single-copy gene encoding HvOxOb. The use of 3' end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in response to the powdery mildew fungus. The transcripts were detected in both compatible and incompatible interactions with a similar accumulation pattern. The oxalate oxidase is found exclusively in the leaf mesophyll, where it is cell wall located. A model for a signal transduction pathway in which oxalate oxidase plays a central role is proposed for the regulation of the hypersensitive response.

We have shown previously that transcripts corresponding to the cDNA clone pBH72-F1, with similarities to O-methyltransferases (OMT), accumulated in barley leaves in response to attack by the pathogenic fungus Blumeria graminis (Plant Mol Biol 26 (1994) 1797). To investigate the accumulation pattern in the defence response and the organ localization of the pBH72-F1-encoded polypeptide (F1-OMT), an antiserum was raised against Escherichia coli expressed F1-OMT. The 43 kDa protein was absent in normal leaves but accumulated strongly in response to pathogen attack. The F1-OMT protein accumulated faster in barley lines inoculated with an avirulent B. graminis isolates compared to a virulent isolate. Additionally, F1-OMT related proteins were detected in developing kernels. F1-OMT was expressed as a functional enzyme in E. coli and the substrate specificity was investigated. The enzyme exhibited OMT activity towards flavonoid aglycones with the highest activity against apigenin (4',5,7-trihydroxyflavone). In contrast, caffeic acid did not serve as substrate for F1-OMT. The product of F1-OMT was analyzed by HPLC and GC-MS and found to be genkwanin (4',5-dihydroxy-7-methoxyflavone). Initial velocity data were best represented by a sequential bi-bi mechanism, and kinetic parameters of KSAM = 10.9 microM, Kapigenin = 4.6 microM and a specific activity of 0.45 mukat/g were obtained. Barley F1-OMT, apigenin 7-O-methyltransferase, is suggested to be involved in the production of a methylated flavonoid phytoalexin.