Volatile-mediated signaling induces resistance of barley against infection with the biotrophic fungus Blumeria graminis f.sp. hordei

Plants have evolved a vast variety of secondary metabolites to counteract biotic stress. Volatile organic compounds (VOCs) are carbon-based molecules induced by herbivore attack or pathogen infection. A mixture of plant VOCs is released for direct or indirect plant defense, plant-plant or plant-insect communication. Recent studies suggest that VOCs can also induce biotic stress resistance in distant organs and neighboring plants. Among other VOCs, green leaf volatiles (GLVs) are quickly released by plant tissue after the onset of herbivory or wounding. We analysed VOCs emitted by 13-day old barley plants (Hordeum vulgare L.) after mechanical wounding using passive absorbers and TD-GC/MS detection. We investigated the influence of pure (Z)-3-hexenyl acetate (Z3HAC) as well as complex VOCs from wounded barley plants on the barley - powdery mildew interaction by pre-exposure in a static and a dynamic headspace connected to a powdery mildew susceptibility assay. GLVs dominated the volatile profile of wounded barley plants with Z3HAC as the most prominent compound. Pre-exposure with Z3HAC resulted in induced resistance of barley against fungal infection. Barley complex volatiles emitted after mechanical wounding, similarly, enhanced resistance in receiver plants. We found volatile-induced modification of the interaction towards an enhanced resistance against fungal infection. In addition, Z3HAC triggered a modulation of the alcohol dehydrogenase isoenzyme activity in receiver plants, a physiological response that possibly contributes to induced resistance. Plant-originated volatile metabolites could be a useful supplementation for future agronomic or horticultural practices.

The barley immune receptor Mla recognizes multiple pathogens and contributes to host range dynamics

Crop losses caused by plant pathogens are a primary threat to stable food production. Stripe rust (Puccinia striiformis) is a fungal pathogen of cereal crops that causes significant, persistent yield loss. Stripe rust exhibits host species specificity, with lineages that have adapted to infect wheat and barley. While wheat stripe rust and barley stripe rust are commonly restricted to their corresponding hosts, the genes underlying this host specificity remain unknown. Here, we show that three resistance genes, Rps6, Rps7, and Rps8, contribute to immunity in barley to wheat stripe rust. Rps7 cosegregates with barley powdery mildew resistance at the Mla locus. Using transgenic complementation of different Mla alleles, we confirm allele-specific recognition of wheat stripe rust by Mla. Our results show that major resistance genes contribute to the host species specificity of wheat stripe rust on barley and that a shared genetic architecture underlies resistance to the adapted pathogen barley powdery mildew and non-adapted pathogen wheat stripe rust.

Mlo Resistance to Powdery Mildew (Blumeria graminis f. sp. hordei) in Barley Landraces Collected in Yemen

Barley (Hordeumvulgare L.) is one of the most important cereal crops in the world. Powdery mildew on barley, which is caused by the pathogen Blumeria graminis f. sp. hordei, occurs world-wide and can result in severe yield loss. Thousands of barley accessions are stored in national gene banks, and their characterization for breeding purposes is needed. This study was conducted to determine the resistance to powdery mildew in 33 barley landraces from Yemen, which were obtained from the ICARDA gene bank. Twenty differential isolates of barley powdery mildew were used. Nine single plant lines were selected from five landraces, based on tests that were performed with 30 plants per landrace, after inoculation with the most avirulent isolate of barley powdery mildew available. Two of these landraces originated from the Al Bayda province in Yemen, and three others originated from Dhamar, Sanaa, and Taizz, respectively. Next, single plant lines were tested using a set of 20 differential isolates of powdery mildew. Two lines that were selected from landrace from the Al Bayda province in Yemen, showed disease reaction designated as 0(4), which is specific for the presence of Mlo resistance. The new source of highly effective Mlo powdery mildew resistance that is described in this study could be used in barley breeding programs.

The barley powdery mildew effectors CSEP0139 and CSEP0182 suppress cell death and promote B. graminis fungal virulence in plants

The powdery mildew fungi secrete numerous Candidate Secreted Effector Proteins (CSEPs) to manipulate host immunity during infection of host plants. However, the function of most of these CSEPs in cell death suppression has not yet been established. Here, we identified several CSEPs from Blumeria graminis f. sp. hordei (Bgh) that have the potential to suppress BAX- and NtMEK2DD-triggered cell death in Nicotiana benthamiana. We further characterized two effector candidates, CSEP0139 and CSEP0182, from family six and thirty-two, respectively. CSEP0139 and CSEP0182 contain a functional signal peptide and are likely secreted effectors. Expression of either CSEP0139 or CSEP0182 suppressed cell death triggered by BAX and NtMEK2DD but not by the AVRa13/MLA13 pair in N. benthamiana. Transient overexpression of CSEP0139 or CSEP0182 also inhibited BAX-induced cell death and collapse of cytoplasm in barley cells. Furthermore, overexpression of either CSEPs significantly increased Bgh haustorial formation in barley, whereas host-induced gene silencing (HIGS) of the CSEP genes reduced haustorial formation, suggesting both CSEPs promote Bgh virulence in barley. In addition, expression of CSEP0139 and CSEP0182 reduced size of the lesions caused by the necrotrophic Botrytis cinerea in N. benthamiana. Our findings suggest that CSEP0139 and CSEP0182 may target cell death components in plants to promote fungal virulence, which extends the current understanding of the functions of Bgh CSEPs and provides an opportunity for further investigation of fungal virulence in relation to cell death pathways in host plants.

Nicotinamide Mononucleotide Potentiates Resistance to Biotrophic Invasion of Fungal Pathogens in Barley

Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), induces disease resistance to the Fusarium head blight fungus Fusarium graminearum in Arabidopsis and barley, but it is unknown at which stage of the infection it acts. Since the rate of haustorial formation of an obligate biotrophic barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was significantly reduced in NMN-treated coleoptile epidermal cells, the possibility that NMN induces resistance to the biotrophic stage of F. graminearum was investigated. The results show that NMN treatment caused the wandering of hyphal growth and suppressed the formation of appressoria-like structures. Furthermore, we developed an experimental system to monitor the early stage of infection in real-time and analyzed the infection behavior. We observed that the hyphae elongated windingly by NMN treatment. These results suggest that NMN potentiates resistance to the biotrophic invasion of F. graminearum as well as Bgh.

Physiological Changes in Barley mlo-11 Powdery Mildew Resistance Conditioned by Tandem Repeat Copy Number

Wild barley accessions have evolved broad-spectrum defence against barley powdery mildew through recessive mlo mutations. However, the mlo defence response is associated with deleterious phenotypes with a cost to yield and fertility, with implications for natural fitness and agricultural productivity. This research elucidates the mechanism behind a novel mlo allele, designated mlo-11(cnv2), which has a milder phenotype compared to standard mlo-11. Bisulphite sequencing and histone ChIP-seq analyses using near-isogenic lines showed pronounced repression of the Mlo promoter in standard mlo-11 compared to mlo-11(cnv2), with repression governed by 24 nt heterochromatic small interfering RNAs. The mlo-11(cnv2) allele appears to largely reduce the physiological effects of mlo while still endorsing a high level of powdery mildew resistance. RNA sequencing showed that this is achieved through only partly restricted expression of Mlo, allowing adequate temporal induction of defence genes during infection and expression close to wild-type Mlo levels in the absence of infection. The two mlo-11 alleles showed copy number proportionate oxidase and peroxidase expression levels during infection, but lower amino acid and aromatic compound biosynthesis compared to the null allele mlo-5. Examination of highly expressed genes revealed a common WRKY W-box binding motif (consensus ACCCGGGACTAAAGG) and a transcription factor more highly expressed in mlo-11 resistance. In conclusion, mlo-11(cnv2) appears to significantly mitigate the trade-off between mlo defence and normal gene expression.

Y2H-SCORES: A statistical framework to infer protein-protein interactions from next-generation yeast-two-hybrid sequence data

Interactomes embody one of the most effective representations of cellular behavior by revealing function through protein associations. In order to build these models at the organism scale, high-throughput techniques are required to identify interacting pairs of proteins. Next-generation interaction screening (NGIS) protocols that combine yeast two-hybrid (Y2H) with deep sequencing are promising approaches to generate protein-protein interaction networks in any organism. However, challenges remain to mining reliable information from these screens and thus, limit its broader implementation. Here, we describe a statistical framework, designated Y2H-SCORES, for analyzing high-throughput Y2H screens that considers key aspects of experimental design, normalization, and controls. Three quantitative ranking scores were implemented to identify interacting partners, comprising: 1) significant enrichment under selection for positive interactions, 2) degree of interaction specificity among multi-bait comparisons, and 3) selection of in-frame interactors. Using simulation and an empirical dataset, we provide a quantitative assessment to predict interacting partners under a wide range of experimental scenarios, facilitating independent confirmation by one-to-one bait-prey tests. Simulation of Y2H-NGIS identified conditions that maximize detection of true interactors, which can be achieved with protocols such as prey library normalization, maintenance of larger culture volumes and replication of experimental treatments. Y2H-SCORES can be implemented in different yeast-based interaction screenings, accelerating the biological interpretation of experimental results. Proof-of-concept was demonstrated by discovery and validation of a novel interaction between the barley powdery mildew effector, AVRA13, with the vesicle-mediated thylakoid membrane biogenesis protein, HvTHF1.Author SummaryOrganisms respond to their environment through networks of interacting proteins and other biomolecules. In order to investigate these interacting proteins, many in vitro and in vivo techniques have been used. Among these, yeast two-hybrid (Y2H) has been integrated with next generation sequencing (NGS) to approach protein-protein interactions on a genome-wide scale. The fusion of these two methods has been termed next-generation-interaction screening, abbreviated as Y2H-NGIS. However, the massive and diverse data sets resulting from this technology have presented unique challenges to analysis. To address these challenges, we optimized the computational and statistical evaluation of Y2H-NGIS to provide metrics to identify high-confidence interacting proteins under a variety of dataset scenarios. Our proposed framework can be extended to different yeast-based interaction settings, utilizing the general principles of enrichment, specificity, and in-frame prey selection to accurately assemble protein-protein interaction networks. Lastly, we showed how the pipeline works experimentally, by identifying and validating a novel interaction between the barley powdery mildew effector AVRA13 and the barley vesicle-mediated thylakoid membrane biogenesis protein, HvTHF1. Y2H-SCORES software is available at GitHub repository https://github.com/Wiselab2/Y2H-SCORES.

High Humidity Causes Abnormalities in the Process of Appressorial Formation of Blumeria graminis f. sp. hordei

High humidity decreases the penetration rate of barley powdery mildew Blumeria graminis f. sp. hordei. However, the mechanism is not well understood. In this study, the morphological and cytochemical analyses revealed that substances containing proteins leaked from the tip of the appressorial germ tube of conidia without the formation of appressorium under a high humidity condition. In addition, exposure to high humidity prior to the formation of appressorium caused the aberrant formation of the appressorial germ tube without appressorium formation, resulting in failure to penetrate the host cell. These findings suggest that the formation and maturation of the appressorium requires a low humidity condition, and will be clues to improve the disease management by humidity control.