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The barley powdery mildew fungus, Blumeria hordei (Bh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley nucleotide-binding leucine-rich-repeat (NLR) receptor MLA1 and therefore is designated AVR A1 . Here, we show that AVR A1 and the sequence-unrelated Bh effector BEC1016 (CSEP0491) suppress immunity in barley. We used yeast two-hybrid next-generation interaction screens (Y2H-NGIS), followed by binary Y2H and in planta protein-protein interactions studies, and identified a common barley target of AVR A1 and BEC1016, the endoplasmic reticulum (ER)-localized J-domain protein HvERdj3B. Silencing of this ER quality control (ERQC) protein increased Bh penetration. HvERdj3B is ER luminal, and we showed using split GFP that AVR A1 and BEC1016 translocate into the ER signal peptide-independently. Overexpression of the two effectors impeded trafficking of a vacuolar marker through the ER; silencing of HvERdj3B also exhibited this same cellular phenotype, coinciding with the effectors targeting this ERQC component. Together, these results suggest that the barley innate immunity, preventing Bh entry into epidermal cells, requires ERQC. Here, the J-domain protein HvERdj3B appears to be essential and can be regulated by AVR A1 and BEC1016. Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host NLR receptors. Previous work has shown that AVR A1 is directly recognized in the cytosol by the immune receptor MLA1. We speculate that the AVR A1 J-domain target being inside the ER, where it is inapproachable by NLRs, has forced the plant to evolve this challenging direct recognition., (© 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

A crucial step in functional genomics is identifying actively translated ORFs and linking them to biological functions. The challenge lies in identifying short ORFs, as their identification is greatly influenced by data quality and depth. Here, we improved the coverage of super-resolution Ribo-seq in Arabidopsis (Arabidopsis thaliana), revealing uncharacterized translation events for nuclear, chloroplastic, and mitochondrial genes. Assisted by a transcriptome assembly, we identified 7,751 unconventional translation events, comprising 6,996 upstream ORFs (uORFs) and 209 downstream ORFs on annotated protein-coding genes, as well as 546 ORFs in presumed noncoding RNAs. Proteomic data confirmed the production of stable proteins from some of these unannotated translation events. We present evidence of active translation from primary transcripts of trans-acting small interfering RNAs (TAS1-4) and microRNAs (pri-MIR163 and pri-MIR169) and periodic ribosome stalling supporting cotranslational decay. Additionally, we developed a method for identifying extremely short uORFs, including 370 minimum uORFs (AUG-stop), and 2,921 tiny uORFs (2 to 10 amino acids) and 681 uORFs that overlap with each other. Remarkably, these short uORFs exhibit strong translational repression as do longer uORFs. We also systematically discovered 594 uORFs regulated by alternative splicing, suggesting widespread isoform-specific translational control. Finally, these prevalent uORFs are associated with numerous important pathways. In summary, our improved Arabidopsis translational landscape provides valuable resources to study gene expression regulation., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The plant pathogenic fungus Fusarium graminearum is the causal agent of Fusarium head blight (FHB) disease on small-grain cereals. F. graminearum produces trichothecene mycotoxins such as deoxynivalenol (DON) that are required for full virulence. DON must be exported outside the cell to cause FHB disease, a process that may require the involvement of membrane-bound transporters. In this study, we show that the deletion of membrane-bound transporters results in reduced DON accumulation as well as reduced FHB symptoms on wheat. Deletion of the ATP-binding cassette (ABC) transporter gene Abc1 results in the greatest reduction in DON accumulation and virulence. Deletion of another ABC transporter gene, Abc6 , also reduces FHB symptoms to a lesser degree. Combining deletions fails to reduce DON accumulation or virulence in an additive fashion, even when a ∆ abc1 deletion is included. Heterologous expression of F. graminearum transporters in a DON-sensitive strain of yeast confirms Abc1 as a major DON resistance mechanism; furthermore, it suggests that Abc1 is directly participating in DON transport rather than facilitating DON transport though other means. Yeast expression further indicates that multiple transporters, including Abc1, play an important role in resistance to the wheat phytoalexin 2-benzoxazolinone (BOA) and other xenobiotics. Thus, Abc1 may contribute to virulence on wheat both by facilitating export of DON and by providing resistance to the wheat phytoalexin BOA. This research provides useful information that may aid in designing novel management techniques of FHB or other destructive plant diseases., Competing Interests: The author(s) declare no conflict of interest.

Yeast two-hybrid next-generation interaction screening (Y2H-NGIS) uses the output of next-generation sequencing to mine for novel protein-protein interactions. Here, we outline the analytics underlying Y2H-NGIS datasets. Different systems, libraries, and experimental designs comprise Y2H-NGIS methodologies. We summarize the analysis in several layers that comprise the characterization of baits and preys, quantification, and identification of true interactions for subsequent secondary validation. We present two software designed for this purpose, NGPINT and Y2H-SCORES, which are used as front-end and back-end tools in the analysis. Y2H-SCORES software can be used and adapted to analyze different datasets not only from Y2H-NGIS but from other techniques ruled by similar biological principles., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Yeast two-hybrid is a powerful approach to discover new protein-protein interactions. Traditional methods involve screening a target protein against a cDNA expression library and assaying individual positive colonies to identify interacting partners. Here we describe a simple approach to perform yeast two-hybrid screens of a cDNA expression library in batch liquid culture. Positive yeast cell populations are enriched under selection and then harvested en masse. Prey cDNAs are amplified and used as input for next-generation sequencing libraries for identification, quantification, and ranking., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Brassinosteroids (BRs) and Target of Rapamycin Complex (TORC) are two major actors coordinating plant growth and stress responses. Brassinosteroids function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies have revealed connections between these two pathways, but a system-wide view of their interplay is still missing. We quantified the level of 23 975 transcripts, 11 183 proteins, and 27 887 phosphorylation sites in wild-type Arabidopsis thaliana and in mutants with altered levels of either BRASSINOSTEROID INSENSITIVE 2 (BIN2) or REGULATORY ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Furthermore, we used the multi-omic data to reconstruct an integrated signaling network. We screened 41 candidate genes identified from the reconstructed network and found that loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results establish a predictive network that defines different layers of molecular interactions between BR- or TORC-regulated growth and autophagy., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

The barley MLA nucleotide-binding leucine-rich-repeat (NLR) receptor and its orthologs confer recognition specificity to many fungal diseases, including powdery mildew, stem-, and stripe rust. We used interolog inference to construct a barley protein interactome (Hordeum vulgare predicted interactome, HvInt) comprising 66,133 edges and 7,181 nodes, as a foundation to explore signaling networks associated with MLA. HvInt was compared with the experimentally validated Arabidopsis interactome of 11,253 proteins and 73,960 interactions, verifying that the 2 networks share scale-free properties, including a power-law distribution and small-world network. Then, by successive layering of defense-specific "omics" datasets, HvInt was customized to model cellular response to powdery mildew infection. Integration of HvInt with expression quantitative trait loci (eQTL) enabled us to infer disease modules and responses associated with fungal penetration and haustorial development. Next, using HvInt and infection-time-course RNA sequencing of immune signaling mutants, we assembled resistant and susceptible subnetworks. The resulting differentially coexpressed (resistant - susceptible) interactome is essential to barley immunity, facilitates the flow of signaling pathways and is linked to mildew resistance locus a (Mla) through trans eQTL associations. Lastly, we anchored HvInt with new and previously identified interactors of the MLA coiled coli + nucleotide-binding domains and extended these to additional MLA alleles, orthologs, and NLR outgroups to predict receptor localization and conservation of signaling response. These results link genomic, transcriptomic, and physical interactions during MLA-specified immunity., (Published by Oxford University Press on behalf of Genetics Society of America 2022. This work is written by a US Government employee and is in the public domain in the US.)

The Mla ( Mildew resistance locus a ) of barley ( Hordeum vulgare L.) is an effective model for cereal immunity against fungal pathogens. Like many resistance proteins, variants of the MLA coiled-coil nucleotide-binding leucine-rich repeat (CC-NLR) receptor often require the HRS complex (HSP90, RAR1, and SGT1) to function. However, functional analysis of Sgt1 has been particularly difficult, as deletions are often lethal. Recently, we identified rar3 ( required for Mla6 resistance 3 ), an in-frame Sgt1 ΔKL308-309 mutation in the SGT1-specific domain, that alters resistance conferred by MLA but without lethality. Here, we use autoactive MLA6 and recombinant yeast-two-hybrid strains with stably integrated HvRar1 and HvHsp90 to determine that this mutation weakens but does not entirely disrupt the interaction between SGT1 and MLA. This causes a concomitant reduction in MLA6 protein accumulation below the apparent threshold required for effective resistance. The ΔKL308-309 deletion had a lesser effect on intramolecular interactions than alanine or arginine substitutions, and MLA variants that display diminished interactions with SGT1 appear to be disproportionately affected by the SGT1 ΔKL308-309 mutation. We hypothesize that those dimeric plant CC-NLRs that appear unaffected by Sgt1 silencing are those with the strongest intermolecular interactions with it. Combining our data with recent work in CC-NLRs, we propose a cyclical model of the MLA-HRS resistosome interactions.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.