Biology subjects

Velasquez-Zapata, V.

Publications and source records attributed to Velasquez-Zapata, V..

8 recordsLinked to original sources

bioRxiv2026

Effector-host networks in the barley powdery mildew pathosystem reveal significant targeting of ubiquitin-associated proteins and transcriptional regulators

- The ascomycete fungal pathogen Blumeria hordei (Bh) is predicted to secrete over 530 effector proteins to infect barley and cause powdery mildew disease, yet their individual and collective functions are largely unexplored. We used yeast two-hybrid next-generation interaction screening (Y2H-NGIS) to probe the host targets of 48 Bh candidate secreted effectors (CSEPs), including multiple AVRA effectors that are recognized by MLA nucleotide-binding leucine-rich repeat (NLR) receptors. - Y2H-NGIS and subsequent binary retesting identified 356 high-confidence protein-protein interactions (PPI) among Bh CSEPs, MLA NLR fragments, and barley proteins. We uncovered unique and shared host targets, including numerous proteins involved in ubiquitin-associated processes, gene expression, immune and kinase signaling, metabolism, and vesicle trafficking. - We identified a host protein (HvCAP2) that interacts with MLA and at least five Bh effectors, as well as additional effector-host PPI that correlate with loss of recognition of AVRA13 and AVRA6 by MLA13 and MLA6, respectively. - Results were integrated with an interolog-based barley interactome (HvInt) to assemble a host-pathogen network of 1248 proteins and 1701 interactions. This PPI resource can serve as a platform for functional investigations into the molecular basis of powdery mildew pathogenesis and host recognition.

plant biology↗

bioRxiv2026

Powdery mildew fungi block plant vacuolar traffic to suppress immunity

Plant immunity can be activated by membrane-localized pattern recognition receptors or by cytosolic nucleotide-binding leucine-rich repeat (NLR) receptors, while it can be counteracted by pathogen secreted effectors. Manifestation of immunity often involves endomembrane traffic. However, limited evidence is available for such an involvement in NLR-mediated immunity, which typically includes a hypersensitive reaction (HR)-programmed cell death response. Here we show that the barley powdery mildew fungus uses several effectors to target and inhibit the vacuolar trafficking pathway, which causes endomembrane markers to stall in the endoplasmic reticulum (ER). We used this ER-stalling phenotype as a proxy to track fungal manipulation of the vacuolar pathway during different stages of the actual infection. Our data indicate that powdery mildew fungi interfere with the vacuolar pathway, but only temporarily, as the stalling is lifted once the fungal haustorial feeding structures are well-developed for nutrient uptake in epidermal cells. Notably, we show evidence that blocking the vacuolar pathway causes a general inhibition of NLR-mediated HR, and that this mechanism is taken advantage of by the pathogen. Finally, we provide an example that the fungus can secrete a different set of effectors at the haustorial stage that result in the re-opening of the vacuolar pathway by inhibiting the initial vacuolar traffic-suppressing effectors.

plant biology↗

bioRxiv2023

Transcriptome-based host epistasis and pathogen co-expression in barley-powdery mildew interactions

Mildew locus a (Mla) from the Triticeae grain crop barley (Hordeum vulgare L.) encodes a multi-allelic series of nucleotide-binding leucine-rich repeat (NLR) immune receptors. These variable NLRs recognize complementary secreted effectors from the powdery mildew fungus, Blumeria hordei (Bh), to block disease progression. We used a dynamic time-course transcriptome of barley infected with Bh to infer gene effects and epistatic relationships governed by the Mla6 NLR, two other host loci critical to the interaction, Blufensin1 (Bln1) and Required for Mla6 resistance3 (Rar3), and genes that interact with them. Bln1 is an R-gene independent regulator of immunity and the resistant bln1 mutant exhibits enhanced basal defense to compatible Bh. Rar3 is required for MLA6-mediated generation of H2O2 and the hypersensitive reaction; the rar3 mutant contains an in-frame Lys-Leu deletion in the SGT1-specific domain that compromises immunity by a subset of Mla alleles. Interactions of Mla6 and Bln1 resulted in symmetric, suppression and masked epistasis on the Bh-induced barley transcriptome. Likewise, dominant or equal effects were caused by Mla6 and Sgt1. Of a total of 468 barley NLRs, 366 were expressed in our dataset and 115 of those were grouped under different gene effect models, which localized to several chromosome hotspots. The corresponding Bh infection transcriptome was classified into nine co-expressed modules, linking differential expression with pathogen development. Expression of most of 517 Bh effectors exhibited dependence on disease phenotype and was associated with appressorial or haustorial structures, suggesting that disease is regulated by a host-pathogen intercommunication network that diversifies the response.

genetics↗

bioRxiv2022

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 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 the Bh penetration. HvERdj3B is ER luminal, and we showed using split GFP that AVRA1 and BEC1016 translocate into the ER - signal peptide-independently. Silencing of HvERdj3B and expression the two effectors hampered trafficking of a vacuolar marker through the ER as a shared cellular phenotype, agreeing with the effectors targeting this ERQC component. Together, these results suggest that the barley innate immunity, preventing Bh entry into epidermal cells, is dependent on ERQC, which in turn requires the J-domain protein, HvERdj3B, 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. SIGNIFICANCEThe complex plant immune system is highly dependent on fundamental cellular machineries, such as the endomembrane system and the ER quality control (ERQC), essential for delivery of immunity-associated membrane-bound and endomembrane soluble proteins to their destinations. We now find that pathogen effectors can interact with an ERQC component and suppress immunity, thereby adding to the molecular insight in plant-pathogen interactions.

plant biology↗

bioRxiv2021

An interolog-based barley interactome as an integration framework for immune signaling

The barley MLA nucleotide-binding, leucine-rich-repeat (NLR) receptor and its orthologs confer recognition specificity to many cereal diseases, including powdery mildew, stem and stripe rust, Victoria blight, and rice blast. We used interolog inference to construct a barley protein interactome (HvInt) comprising 66133 edges and 7181 nodes, as a foundation to explore signaling networks associated with MLA. HvInt was compared to the experimentally validated Arabidopsis interactome of 11253 proteins and 73960 interactions, verifying that the two 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 an infection-time-course transcriptome, we assembled resistant (R) and susceptible (S) subnetworks. The resulting differentially co-expressed (R-S) interactome is essential to barley immunity, facilitates the flow of signaling pathways and is linked to Mla through trans eQTL associations. Lastly, next-generation, yeast-two-hybrid screens identified fifteen novel MLA interactors, which were incorporated into HvInt, to predict receptor localization, and signaling response. These results link genomic, transcriptomic, and physical interactions during MLA-specified immunity. AUTHOR SUMMARYPowdery mildew fungi infect more than 9,500 agronomic and horticultural plant species. In order to prevent economic loss due to diseases caused by pathogens, plant breeders incorporate resistance genes into varieties that are grown for food, feed, fuel and fiber. One of these resistance genes encodes the barley MLA immune receptor, an ancestral cereal protein that confers recognition to powdery mildew, stem and stripe rust, rice blast and Victoria blight. However, in order to function properly, these immune receptors must interact with additional proteins and protein complexes during the different stages of fungal infection and plant defense. We used a combination of computational- and laboratory-based methods to predict over 66,000 possible protein-protein interactions in barley. This network of proteins was then integrated with various defense-specific datasets to assemble the molecular building blocks associated with resistance to the powdery mildew pathogen, in addition to those proteins that interact with the MLA immune receptor. Our application of genome-scale, protein-protein interaction data provides a foundation to decipher the complex molecular components that control immune responses in crops.

systems biology↗

bioRxiv2020

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 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 recognized all validated interactions. NGPINT can be used in any organism with an available reference, thus facilitating the discovery of protein-protein interactions in non-model organisms.

bioinformatics↗

bioRxiv2020

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.

systems biology↗

bioRxiv2020

Inheritance of Rootstock Effects in Avocado ( Persea americana Mill.) cv. Hass

Grafting is typically utilized to merge adapted seedling rootstocks with highly productive clonal scions. This process implies the interaction of multiple genomes to produce a unique tree phenotype. Yet, the interconnection of both genotypes obscures individual contributions to phenotypic variation (i.e. rootstock-mediated heritability), hampering tree breeding. Therefore, our goal was to quantify the inheritance of seedling rootstock effects on scion traits using avocado (Persea americana Mill.) cv. Hass as model fruit tree. We characterized 240 rootstocks from 8 avocado cv. Hass orchards in three regions of the province of Antioquia, in the northwest Andes of Colombia, using 13 microsatellite markers (simple sequence repeats - SSRs). Parallel to this, we recorded 20 phenotypic traits (including morphological, eco-physiological, and fruit yield and quality traits) in the scions for three years (2015-2017). Relatedness among rootstocks was inferred through the genetic markers and inputted in a genetic prediction model in order to calculate narrow-sense heritabilities (h2) on scion traits. We used three different randomization tests to highlight traits with consistently significant heritability estimates. This strategy allowed us to capture five traits with significant heritability values that ranged from 0.33 to 0.45 and model fits (R2) that oscillated between 0.58 and 0.74 across orchards. The results showed significance in the rootstock effects for four complex harvest and quality traits (i.e. total number of fruits, number of fruits with exportation quality, and number of fruits discarded because of low weight or thrips damage), while the only morphological trait that had a significant heritability value was overall trunk height (an emergent property of the rootstock-scion interaction). These findings suggest the inheritance of rootstock effects, beyond root phenotype, on a surprisingly wide spectrum of scion traits in Hass avocado. They also reinforce the utility of SSR markers for relatedness reconstruction and genetic prediction of complex traits. This research is, up to date, the most cohesive evidence of narrow-sense inheritance of rootstock effects in a tropical fruit tree crop. Ultimately, our work reinforces the importance of considering the rootstock-scion interaction to broaden the genetic basis of fruit tree breeding programs, while enhancing our understanding of the consequences of grafting.

plant biology↗