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The establishment of host-microbe interactions requires molecular communication between both partners, which may involve the mutual transfer of noncoding small RNAs. Previous evidence suggests that this is also true for powdery mildew disease in barley, which is caused by the fungal pathogen Blumeria hordei. However, previous studies lacked spatial resolution regarding the accumulation of small RNAs upon host infection by B. hordei. Here, we analysed site-specific small RNA repertoires in the context of the barley-B. hordei interaction. To this end, we dissected infected leaves into separate fractions representing different sites that are key to the pathogenic process: epiphytic fungal mycelium, infected plant epidermis, isolated haustoria, a vesicle-enriched fraction from infected epidermis, and extracellular vesicles. Unexpectedly, we discovered enrichment of specific 31-33-base 5'-terminal fragments of barley 5.8S ribosomal RNA in extracellular vesicles and infected epidermis, as well as particular B. hordei transfer RNA fragments in haustoria. We describe canonical small RNAs from both the plant host and the fungal pathogen that may confer cross-kingdom RNA interference activity. Interestingly, we found first evidence of phased small interfering RNAs in B. hordei, a feature usually attributed to plants, which may be associated with the posttranscriptional control of fungal coding genes, pseudogenes, and transposable elements. Our data suggest a key and possibly site-specific role for cross-kingdom RNA interference and noncoding RNA fragments in the host-pathogen communication between B. hordei and its host barley., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Powdery mildew is a common and widespread plant disease of considerable agronomic relevance. It is caused by obligate biotrophic fungal pathogens which, in most cases, epiphytically colonize aboveground plant tissues. The disease has been typically studied as a binary interaction of the fungal pathogen with its plant hosts, neglecting, for the most part, the mutual interplay with the wealth of other microorganisms residing in the phyllo- and/or rhizosphere and roots. However, the establishment of powdery mildew disease can be impacted by the presence/absence of host-associated microbiota (epi- and endophytes) and, conversely, plant colonization by powdery mildew fungi might disturb indigenous microbial community structures. In addition, other (foliar) phytopathogens could interact with powdery mildews, and mycoparasites may affect the outcome of plant-powdery mildew interactions. In this review, we discuss the current knowledge regarding the intricate and multifaceted interplay of powdery mildew fungi, host plants and other microorganisms, and outline current gaps in our knowledge, thereby setting the basis for potential future research directions., (© 2018 The Authors. Molecular Plant Pathology published by BSPP and John Wiley & Sons Ltd.)

Filamentous phytopathogens, such as fungi and oomycetes, secrete effector proteins to establish successful interactions with their plant hosts. In contrast with oomycetes, little is known about effector functions in true fungi. We used a bioinformatics pipeline to identify Blumeria effector candidates (BECs) from the obligate biotrophic barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). BEC1-BEC5 are expressed at different time points during barley infection. BEC1, BEC2 and BEC4 have orthologues in the Arabidopsis thaliana-infecting powdery mildew fungus Golovinomyces orontii. Arabidopsis lines stably expressing the G. orontii BEC2 orthologue, GoEC2, are more susceptible to infection with the non-adapted fungus Erysiphe pisi, suggesting that GoEC2 contributes to powdery mildew virulence. For BEC3 and BEC4, we identified thiopurine methyltransferase, a ubiquitin-conjugating enzyme, and an ADP ribosylation factor-GTPase-activating protein (ARF-GAP) as potential host targets. Arabidopsis knockout lines of the respective HvARF-GAP orthologue (AtAGD5) allowed higher entry levels of E. pisi, but exhibited elevated resistance to the oomycete Hyaloperonospora arabidopsidis. We hypothesize that ARF-GAP proteins are conserved targets of powdery and downy mildew effectors, and we speculate that BEC4 might interfere with defence-associated host vesicle trafficking., (© 2013 BSPP AND JOHN WILEY & SONS LTD.)

Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea., (© 2011 The Authors. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd.)

Conidiospores are the asexual propagation units of many plant-pathogenic fungi. In this article, we report an annotated proteome map of ungerminated conidiospores of the ascomycete barley powdery mildew pathogen, Blumeria graminis f.sp. hordei. Using a combination of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, we have identified the proteins in 180 spots, which probably represent at least 123 distinct fungal gene products. Most of the identified proteins have a predicted function in carbohydrate, lipid or protein metabolism, indicating that the spore is equipped for the catabolism of storage compounds as well as for protein biosynthesis and folding on germination.

SUMMARY Durability and effectiveness against all genetic variants of a microbial species are hallmarks of so-called plant 'non-host' resistance. Highly effective immunity of monocotyledonous barley against the fungal powdery mildew pathogen, which is conferred by loss-of-function mutant alleles of the barley Mlo locus, likewise is a durable and broad-spectrum type of resistance. Although this was long considered as being a barley-specific phenomenon, recent findings indicate that mlo resistance can also occur in the distantly related dicotyledonous species Arabidopsis thaliana. Shared histological and phytopathological characteristics plus a conserved requirement for a set of genes in Arabidopsis mlo and non-host powdery mildew resistance indicate a potential common mechanism for these two seemingly distinct types of immunity.

SUMMARY Barley lines PRU1, URS1 and URS2 represent three candidate mlo mutants induced in either the two-rowed cultivar Prudentia or the six-rowed cultivar Ursula. Both Prudentia and Ursula are North American malting barley varieties with specific malting properties. Here, we analysed the three candidate mutants at the molecular level. We identified lesions in the Mlo gene of all three lines, causing either a premature stop codon (PRU1), a shift in the reading frame (URS1) or a single amino acid replacement (URS2). In a transient gene expression assay, the URS2 mlo allele fails to complement a barley null mutant genotype, indicating that URS2 is a genuine mlo mutant (here designated as mlo-33). The MLO-33 mutant variant accumulates to similar levels as the wild-type MLO protein in Arabidopsis protoplasts, suggesting that MLO-33 is stable in planta. We show that the mlo-33 allele can be readily detected in barley genomic DNA by a cleaved amplified polymorphic sequence marker, rendering this allele particularly suited for marker-assisted breeding.

SUMMARY Despite considerable technical progress in past years, genetic manipulation of cereals remains a tedious task. Thus, transgenic approaches in monocot species to study plant-microbe interactions are limited to date. Transient gene expression in single epidermal cells mediated by particle bombardment has emerged recently as an attractive alternative for testing the impact of (over-)expressing or silencing single host genes in the context of cereal-powdery mildew interactions. The ease and pace of this assay enables the analysis of candidate genes within a fraction of the time needed to generate stable transgenic lines. Genetically encoded fluorescent sensors expressed in single cells are ideally suited to monitor gene expression, subcellular protein localization and changes of physiological parameters at the single cell level. Likewise, single cell gene expression can be employed to study protein-protein interactions of fluorophore-tagged polypeptides by fluorescence resonance energy transfer or fluorescence (cross) correlation spectroscopy. An integrated approach, combining single cell gene expression technology with modern cell biological tools and single cell sampling via laser capture microdissection, may provide in-depth insights into the molecular events in epidermal host cells in the course of cereal-mildew interactions.

SUMMARY Powdery mildew is a common fungal disease of many monocotyledonous and dicotyledonous plant species. In a moderately temperate and humid climate, these ascomycete fungi cause severe yield losses in a wide range of crops. In recent years, several plant genes encoding proteins that control disease resistance against powdery mildew were isolated from the model organism Arabidopsis and the crop barley. Here we review the presumptive biochemical functions of the respective proteins and discuss potential mechanisms which mediate pathogen recognition, resistance signalling, and the termination of host colonization. Perhaps not surprisingly, these advances also promise to shed light on the molecular basis of pathogenesis and biotrophic lifestyle.