NGPINT: a next-generation protein-protein interaction software.

Mapping protein-protein interactions at a proteome scale is critical to understanding how cellular signaling networks respond to stimuli. Since eukaryotic genomes encode thousands of proteins, testing their interactions one-by-one is a challenging prospect. High-throughput yeast-two hybrid (Y2H) assays that employ next-generation sequencing to interrogate complementary DNA (cDNA) libraries represent an alternative approach that optimizes scale, cost and effort. We present NGPINT, a robust and scalable software to identify all putative interactors of a protein using Y2H in batch culture. NGPINT combines diverse tools to align sequence reads to target genomes, reconstruct prey fragments and compute gene enrichment under reporter selection. Central to this pipeline is the identification of fusion reads containing sequences derived from both the Y2H expression plasmid and the cDNA of interest. To reduce false positives, these fusion reads are evaluated as to whether the cDNA fragment forms an in-frame translational fusion with the Y2H transcription factor. NGPINT successfully recognized 95% of interactions in simulated test runs. As proof of concept, NGPINT was tested using published data sets and it recognized all validated interactions. NGPINT can process interaction data from any biosystem with an available genome or transcriptome reference, thus facilitating the discovery of protein-protein interactions in model and non-model organisms.

SGT1-Specific Domain Mutations Impair Interactions with the Barley MLA6 Immune Receptor in Association with Loss of NLR Protein.

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 Sgt1ΔKL308-309 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 SGT1ΔKL308-309 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.

Next-generation yeast-two-hybrid analysis with Y2H-SCORES identifies novel interactors of the MLA immune receptor.

Protein-protein interaction networks are one of the most effective representations of cellular behavior. In order to build these models, high-throughput techniques are required. Next-generation interaction screening (NGIS) protocols that combine yeast two-hybrid (Y2H) with deep sequencing are promising approaches to generate interactome networks in any organism. However, challenges remain to mining reliable information from these screens and thus, limit its broader implementation. Here, we present a computational framework, designated Y2H-SCORES, for analyzing high-throughput Y2H screens. Y2H-SCORES considers key aspects of NGIS experimental design and important characteristics of the resulting data that distinguish it from RNA-seq expression datasets. 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 enabled us to identify 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, with an equivalent or superior performance than existing methods. Proof-of-concept was demonstrated by discovery and validation of novel interactions between the barley nucleotide-binding leucine-rich repeat (NLR) immune receptor MLA6, and fourteen proteins, including those that function in signaling, transcriptional regulation, and intracellular trafficking.

FINDER: an automated software package to annotate eukaryotic genes from RNA-Seq data and associated protein sequences.

BACKGROUND: Gene annotation in eukaryotes is a non-trivial task that requires meticulous analysis of accumulated transcript data. Challenges include transcriptionally active regions of the genome that contain overlapping genes, genes that produce numerous transcripts, transposable elements and numerous diverse sequence repeats. Currently available gene annotation software applications depend on pre-constructed full-length gene sequence assemblies which are not guaranteed to be error-free. The origins of these sequences are often uncertain, making it difficult to identify and rectify errors in them. This hinders the creation of an accurate and holistic representation of the transcriptomic landscape across multiple tissue types and experimental conditions. Therefore, to gauge the extent of diversity in gene structures, a comprehensive analysis of genome-wide expression data is imperative. RESULTS: We present FINDER, a fully automated computational tool that optimizes the entire process of annotating genes and transcript structures. Unlike current state-of-the-art pipelines, FINDER automates the RNA-Seq pre-processing step by working directly with raw sequence reads and optimizes gene prediction from BRAKER2 by supplementing these reads with associated proteins. The FINDER pipeline (1) reports transcripts and recognizes genes that are expressed under specific conditions, (2) generates all possible alternatively spliced transcripts from expressed RNA-Seq data, (3) analyzes read coverage patterns to modify existing transcript models and create new ones, and (4) scores genes as high- or low-confidence based on the available evidence across multiple datasets. We demonstrate the ability of FINDER to automatically annotate a diverse pool of genomes from eight species. CONCLUSIONS: FINDER takes a completely automated approach to annotate genes directly from raw expression data. It is capable of processing eukaryotic genomes of all sizes and requires no manual supervision-ideal for bench researchers with limited experience in handling computational tools.

Convergent Evolution of Effector Protease Recognition by Arabidopsis and Barley.

The Pseudomonas syringae cysteine protease AvrPphB activates the Arabidopsis resistance protein RPS5 by cleaving a second host protein, PBS1. AvrPphB induces defense responses in other plant species, but the genes and mechanisms mediating AvrPphB recognition in those species have not been defined. Here, we show that AvrPphB induces defense responses in diverse barley cultivars. We also show that barley contains two PBS1 orthologs, that their products are cleaved by AvrPphB, and that the barley AvrPphB response maps to a single locus containing a nucleotide-binding leucine-rich repeat (NLR) gene, which we termed AvrPphB Response 1 (Pbr1). Transient coexpression of PBR1 with wild-type AvrPphB but not with a protease inactive mutant triggered defense responses, indicating that PBR1 detects AvrPphB protease activity. Additionally, PBR1 coimmunoprecipitated with barley and Nicotiana benthamiana PBS1 proteins, suggesting mechanistic similarity to detection by RPS5. Lastly, we determined that wheat cultivars also recognize AvrPphB protease activity and contain two putative Pbr1 orthologs. Phylogenetic analyses showed, however, that Pbr1 is not orthologous to RPS5. Our results indicate that the ability to recognize AvrPphB evolved convergently and imply that selection to guard PBS1-like proteins occurs across species. Also, these results suggest that PBS1-based decoys may be used to engineer protease effector recognition-based resistance in barley and wheat.

Small RNA discovery in the interaction between barley and the powdery mildew pathogen.

BACKGROUND: Plants encounter pathogenic and non-pathogenic microorganisms on a nearly constant basis. Small RNAs such as siRNAs and miRNAs/milRNAs influence pathogen virulence and host defense responses. We exploited the biotrophic interaction between the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), and its diploid host plant, barley (Hordeum vulgare) to explore fungal and plant sRNAs expressed during Bgh infection of barley leaf epidermal cells. RESULTS: RNA was isolated from four fast-neutron immune-signaling mutants and their progenitor over a time course representing key stages of Bgh infection, including appressorium formation, penetration of epidermal cells, and development of haustorial feeding structures. The Cereal Introduction (CI) 16151 progenitor carries the resistance allele Mla6, while Bgh isolate 5874 harbors the AVRa6 avirulence effector, resulting in an incompatible interaction. Parallel Analysis of RNA Ends (PARE) was used to verify sRNAs with likely transcript targets in both barley and Bgh. Bgh sRNAs are predicted to regulate effectors, metabolic genes, and translation-related genes. Barley sRNAs are predicted to influence the accumulation of transcripts that encode auxin response factors, NAC transcription factors, homeodomain transcription factors, and several splicing factors. We also identified phasing small interfering RNAs (phasiRNAs) in barley that overlap transcripts that encode receptor-like kinases (RLKs) and nucleotide-binding, leucine-rich domain proteins (NLRs). CONCLUSIONS: These data suggest that Bgh sRNAs regulate gene expression in metabolism, translation-related, and pathogen effectors. PARE-validated targets of predicted Bgh milRNAs include both EKA (effectors homologous to AVRk1 and AVRa10) and CSEP (candidate secreted effector protein) families. We also identified barley phasiRNAs and miRNAs in response to Bgh infection. These include phasiRNA loci that overlap with a significant proportion of receptor-like kinases, suggesting an additional sRNA control mechanism may be active in barley leaves as opposed to predominant R-gene phasiRNA overlap in many eudicots. In addition, we identified conserved miRNAs, novel miRNA candidates, and barley genome mapped sRNAs that have PARE validated transcript targets in barley. The miRNA target transcripts are enriched in transcription factors, signaling-related proteins, and photosynthesis-related proteins. Together these results suggest both barley and Bgh control metabolism and infection-related responses via the specific accumulation and targeting of genes via sRNAs.

Correction to: small RNA discovery in the interaction between barley and the powdery mildew pathogen.

Following the publication of the original article [1], the authors noted several typesetting errors which are noted in this Correction article.

A Confounding Effect of Bacterial Titer in a Type III Delivery-Based Assay of Eukaryotic Effector Function.

This letter describes a newly discovered confounding effect of bacterial titer in a previously published type III delivery-based assay of the fungal effector BEC1019. The original publication (Whigham et al. 2015) has been retracted as a consequence of this discovery. Here, we tabulate the affected and unaffected figures and conclusions in the original publication and briefly reflect on potential pitfalls to bear in mind when designing experiments that use bacterial type III secretion to characterize eukaryotic effectors.

A physical, genetic and functional sequence assembly of the barley genome.

Barley (Hordeum vulgare L.) is among the world's earliest domesticated and most important crop plants. It is diploid with a large haploid genome of 5.1 gigabases (Gb). Here we present an integrated and ordered physical, genetic and functional sequence resource that describes the barley gene-space in a structured whole-genome context. We developed a physical map of 4.98 Gb, with more than 3.90 Gb anchored to a high-resolution genetic map. Projecting a deep whole-genome shotgun assembly, complementary DNA and deep RNA sequence data onto this framework supports 79,379 transcript clusters, including 26,159 'high-confidence' genes with homology support from other plant genomes. Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation. Our data provide a platform for both genome-assisted research and enabling contemporary crop improvement.

Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome.

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.

Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene.

Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.

Broadly Conserved Fungal Effector BEC1019 Suppresses Host Cell Death and Enhances Pathogen Virulence in Powdery Mildew of Barley (Hordeum vulgare L.).

The interaction of barley, Hordeum vulgare L., with the powdery mildew fungus Blumeria graminis f. sp. hordei is a well-developed model to investigate resistance and susceptibility to obligate biotrophic pathogens. The 130-Mb Blumeria genome encodes approximately 540 predicted effectors that are hypothesized to suppress or induce host processes to promote colonization. Blumeria effector candidate (BEC)1019, a single-copy gene encoding a putative, secreted metalloprotease, is expressed in haustorial feeding structures, and host-induced gene silencing of BEC1019 restricts haustorial development in compatible interactions. Here, we show that Barley stripe mosaic virus-induced gene silencing of BEC1019 significantly reduces fungal colonization of barley epidermal cells, demonstrating that BEC1019 plays a central role in virulence. In addition, delivery of BEC1019 to the host cytoplasm via Xanthomonas type III secretion suppresses cultivar nonspecific hypersensitive reaction (HR) induced by Xanthomonas oryzae pv. oryzicola, as well as cultivar-specific HR induced by AvrPphB from Pseudomonas syringae pv. phaseolicola. BEC1019 homologs are present in 96 of 241 sequenced fungal genomes, including plant pathogens, human pathogens, and free-living nonpathogens. Comparative analysis revealed variation at several amino acid positions that correlate with fungal lifestyle and several highly conserved, noncorrelated motifs. Site-directed mutagenesis of one of these, ETVIC, compromises the HR-suppressing activity of BEC1019. We postulate that BEC1019 represents an ancient, broadly important fungal protein family, members of which have evolved to function as effectors in plant and animal hosts.

Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ).

Next-generation whole-genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost-efficient establishment of powerful genomic information for many species.

Mla- and Rom1-mediated control of microRNA398 and chloroplast copper/zinc superoxide dismutase regulates cell death in response to the barley powdery mildew fungus.

• Barley (Hordeum vulgare L.) Mildew resistance locus a (Mla) confers allele-specific interactions with natural variants of the ascomycete fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew disease. Significant reprogramming of Mla-mediated gene expression occurs upon infection by this obligate biotrophic pathogen. • We utilized a proteomics-based approach, combined with barley mla, required for Mla12 resistance1 (rar1), and restoration of Mla resistance1 (rom1) mutants, to identify components of Mla-directed signaling. • Loss-of-function mutations in Mla and Rar1 both resulted in the reduced accumulation of chloroplast copper/zinc superoxide dismutase 1 (HvSOD1), whereas loss of function in Rom1 re-established HvSOD1 levels. In addition, both Mla and Rom1 negatively regulated hvu-microRNA398 (hvu-miR398), and up-regulation of miR398 was coupled to reduced HvSOD1 expression. Barley stripe mosaic virus (BSMV)-mediated over-expression of both barley and Arabidopsis miR398 repressed accumulation of HvSOD1, and BSMV-induced gene silencing of HvSod1 impeded Mla-triggered H2O2 and hypersensitive reaction (HR) at barley-Bgh interaction sites. • These data indicate that Mla- and Rom1-regulated hvu-miR398 represses HvSOD1 accumulation, influencing effector-induced HR in response to the powdery mildew fungus.

PLEXdb: gene expression resources for plants and plant pathogens.

PLEXdb (http://www.plexdb.org), in partnership with community databases, supports comparisons of gene expression across multiple plant and pathogen species, promoting individuals and/or consortia to upload genome-scale data sets to contrast them to previously archived data. These analyses facilitate the interpretation of structure, function and regulation of genes in economically important plants. A list of Gene Atlas experiments highlights data sets that give responses across different developmental stages, conditions and tissues. Tools at PLEXdb allow users to perform complex analyses quickly and easily. The Model Genome Interrogator (MGI) tool supports mapping gene lists onto corresponding genes from model plant organisms, including rice and Arabidopsis. MGI predicts homologies, displays gene structures and supporting information for annotated genes and full-length cDNAs. The gene list-processing wizard guides users through PLEXdb functions for creating, analyzing, annotating and managing gene lists. Users can upload their own lists or create them from the output of PLEXdb tools, and then apply diverse higher level analyses, such as ANOVA and clustering. PLEXdb also provides methods for users to track how gene expression changes across many different experiments using the Gene OscilloScope. This tool can identify interesting expression patterns, such as up-regulation under diverse conditions or checking any gene's suitability as a steady-state control.

Quantitative and qualitative stem rust resistance factors in barley are associated with transcriptional suppression of defense regulons.

Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host-pathogen interaction with enhancement of R-gene mediated resistance.

Powdery mildew effectors AVRA1 and BEC1016 target the ER J-domain protein HvERdj3B required for immunity in barley.

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 AVRA1. Here, we show that AVRA1 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 AVRA1 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 AVRA1 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 AVRA1 and BEC1016. Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host NLR receptors. Previous work has shown that AVRA1 is directly recognized in the cytosol by the immune receptor MLA1. We speculate that the AVRA1 J-domain target being inside the ER, where it is inapproachable by NLRs, has forced the plant to evolve this challenging direct recognition.

Host-induced gene silencing in barley powdery mildew reveals a class of ribonuclease-like effectors.

Obligate biotrophic pathogens of plants must circumvent or counteract defenses to guarantee accommodation inside the host. To do so, they secrete a variety of effectors that regulate host immunity and facilitate the establishment of pathogen feeding structures called haustoria. The barley powdery mildew fungus Blumeria graminis f. sp. hordei produces a large number of proteins predicted to be secreted from haustoria. Fifty of these Blumeria effector candidates (BEC) were screened by host-induced gene silencing (HIGS), and eight were identified that contribute to infection. One shows similarity to ß-1,3 glucosyltransferases, one to metallo-proteases, and two to microbial secreted ribonucleases; the remainder have no similarity to proteins of known function. Transcript abundance of all eight BEC increases dramatically in the early stages of infection and establishment of haustoria, consistent with a role in that process. Complementation analysis using silencing-insensitive synthetic cDNAs demonstrated that the ribonuclease-like BEC 1011 and 1054 are bona fide effectors that function within the plant cell. BEC1011 specifically interferes with pathogen-induced host cell death. Both are part of a gene superfamily unique to the powdery mildew fungi. Structural modeling was consistent, with BEC1054 adopting a ribonuclease-like fold, a scaffold not previously associated with effector function.

Next-Generation Yeast Two-Hybrid Screening to Discover Protein-Protein Interactions.

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.

Bioinformatic Analysis of Yeast Two-Hybrid Next-Generation Interaction Screen Data.

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.