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

Elmore, James M.; Velásquez-Zapata, Valeria; Wise, Roger P.

doi:10.1007/978-1-0716-3327-4_19

Cited by 4 publications

(3 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To identify host–pathogen PPI, three independent replications of batch Y2H‐NGIS were performed according to Elmore et al. ( 2023 ), Velásquez‐Zapata et al. ( 2021 ), and Velásquez‐Zapata, Elmore, and Wise ( 2023 ).…”

Section: Resultsmentioning

confidence: 99%

“…The Bh effectors AVR A1 and BEC1016 were used as baits in Y2H‐NGIS to mine for novel PPI (Elmore et al., 2023 ; Velásquez‐Zapata, Elmore, & Wise, 2023 ). Bait sequences (without signal peptides) were fused with the GAL4 transcription factor binding domain (GAL4‐BD) in the p97‐BD, Leu2p vector and transformed into Saccharomyces cerevisiae Y8930 (Dreze et al., 2010 ).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Powdery mildew effectors AVR_A1 and BEC1016 target the ER J‐domain protein HvERdj3B required for immunity in barley

Li,

Velásquez‐Zapata,

Elmore

et al. 2024

Molecular Plant Pathology

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Powdery mildew effectors AVR_A1 and BEC1016 target the ER J‐domain protein HvERdj3B required for immunity in barley

Li,

Velásquez‐Zapata,

Elmore

et al. 2024

Molecular Plant Pathology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Conversely, the array strategy, also known as the matrix approach, entails the direct mating of a pool of baits and a pool of prey with various yeast mating types to identify interactions. Although this method is automated, facilitating the exploration of genome‐wide interactomes, it may overlook certain interactions, termed false negatives [117,118] (Fig. 1A).…”

Section: Yeast Two‐hybridmentioning

confidence: 99%

Uncovering domain motif interactions using high‐throughput protein–protein interaction detection methods

Idrees,

Paudel,

Sadaf

et al. 2024

FEBS Letters

View full text Add to dashboard Cite

Protein–protein interactions (PPIs) are often mediated by short linear motifs (SLiMs) in one protein and domain in another, known as domain–motif interactions (DMIs). During the past decade, SLiMs have been studied to find their role in cellular functions such as post‐translational modifications, regulatory processes, protein scaffolding, cell cycle progression, cell adhesion, cell signalling and substrate selection for proteasomal degradation. This review provides a comprehensive overview of the current PPI detection techniques and resources, focusing on their relevance to capturing interactions mediated by SLiMs. We also address the challenges associated with capturing DMIs. Moreover, a case study analysing the BioGrid database as a source of DMI prediction revealed significant known DMI enrichment in different PPI detection methods. Overall, it can be said that current high‐throughput PPI detection methods can be a reliable source for predicting DMIs.

show abstract

Powdery mildew effectors AVR_A1and BEC1016 target the ER J-domain proteinHvERdj3B required for immunity in barley

Velásquez-Zapata

Elmore

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host nucleotide-binding leucine-rich-repeat (NLR) receptors. The barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley NLR, MLA1, and therefore designated AVRA1. Here we show that AVRA1 and the sequence-unrelated Bgh effector BEC1016 (CSEP0491) suppress immunity in barley cells. We then used yeast 2-hybrid next-generation-interaction screens (Y2H-NGIS), followed by binary Y2H and bimolecular fluorescence complementation, to identify a common barley target of AVRA1 and BEC1016, the endoplasmic reticulum (ER)-localized J-domain protein, HvERdj3B. This is an ER protein quality control (ERQC) protein and silencing it increased the Bgh penetration rate in barley. HvERdj3B is localized to the ER lumen, and AVRA1 and BEC106 translocation into the ER was confirmed using a split GFP system. Together, these results suggest that the barley innate immunity, preventing Bgh entry into epidermal cells, is dependent on ERQC, which in turn is regulated by J-domain protein HvERdj3B and the two effectors. Previous work has shown that AVRA1 is directly recognized in the cytosol by the immune receptor, MLA1. We speculate whether 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.

show abstract

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

Cited by 4 publications

References 33 publications

Powdery mildew effectors AVR_A1 and BEC1016 target the ER J‐domain protein HvERdj3B required for immunity in barley

Powdery mildew effectors AVR_A1 and BEC1016 target the ER J‐domain protein HvERdj3B required for immunity in barley

Uncovering domain motif interactions using high‐throughput protein–protein interaction detection methods

Powdery mildew effectors AVR_A1and BEC1016 target the ER J-domain proteinHvERdj3B required for immunity in barley

Contact Info

Product

Resources

About

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

Cited by 4 publications

References 33 publications

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

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

Uncovering domain motif interactions using high‐throughput protein–protein interaction detection methods

Powdery mildew effectors AVRA1and BEC1016 target the ER J-domain proteinHvERdj3B required for immunity in barley

Contact Info

Product

Resources

About

Powdery mildew effectors AVR_A1 and BEC1016 target the ER J‐domain protein HvERdj3B required for immunity in barley

Powdery mildew effectors AVR_A1 and BEC1016 target the ER J‐domain protein HvERdj3B required for immunity in barley

Powdery mildew effectors AVR_A1and BEC1016 target the ER J-domain proteinHvERdj3B required for immunity in barley