Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III effector HopZ2

Abstract

Background: Identification of protein-protein interactions is a fundamental aspect of understanding protein function. A commonly used method for identifying protein interactions is the yeast two-hybrid system.

Results: Here we describe the application of next-generation sequencing to yeast two-hybrid interaction screens and develop Quantitative Interactor Screen Sequencing (QIS-Seq). QIS-Seq provides a quantitative measurement of enrichment for each interactor relative to its frequency in the library as well as its general stickiness (non-specific binding). The QIS-Seq approach is scalable and can be used with any yeast two-hybrid screen and with any next-generation sequencing platform. The quantitative nature of QIS-Seq data make it amenable to statistical evaluation, and importantly, facilitates the standardization of experimental design, data collection, and data analysis. We applied QIS-Seq to identify the Arabidopsis thaliana MLO2 protein as a target of the Pseudomonas syringae type III secreted effector protein HopZ2. We validate the interaction between HopZ2 and MLO2 in planta and show that the interaction is required for HopZ2-associated virulence.

Conclusions: We demonstrate that QIS-Seq is a high-throughput quantitative interactor screen and validate MLO2 as an interactor and novel virulence target of the P. syringae type III secreted effector HopZ2.

Figure 1

Characterization of cDNA library and HopZ2^C/A putative interactors. A. Percentage of primary cDNA library (left) and HopZ2^C/A interactors (right) encoding proteins belonging to gene ontology (GO) terms for biological processes, molecular functions and cellular components. * indicates categories that are missing for HopZ2^C/A interactors. B. Percentage of genes in library that are upregulated in response to biotic stress (from bacteria, oomycetes or elicitors of innate immunity).

Figure 2

Schematic of QIS-Seq protocol. A. Outline of QIS-Seq screening protocol described in text. B. Example of enrichment calculation showing the effect of varying the recovery of a bait of interest (HopZ), the non-specific control (luciferase), and the frequency in the library. The counts and enrichment score are heat-mapped with high numbers in blue to low numbers in yellow.

Figure 3

Disruption of MLO2 compromises P. syringae virulence. A. The virulent pathogen PtoDC3000 was pressure-infiltrated into the leaves of Arabidopsis Col-0 or T-DNA insertion lines in putative interactors of HopZ2^C/A. An insertion in At1g11310 (mlo2-7) results in increased resistance to PtoDC3000. * indicates significant difference from Col-0 by Fisher's Protected Least Significant Difference (PLSD) test. Error bars indicate one standard deviation of the mean. B. Schematic showing the MLO2 gene and protein structure with the point of insertion for the T-DNA insertion lines and the point mutant. The left side shows the gene with introns represented as lines and exons represented as boxes. MLO2 has two splice forms which differ at the 3' end of the gene. The right side shows the transmembrane structure of the MLO2 protein. mlo2-6 and mlo2-7 are T-DNA insertion lines while mlo2-11 is a point mutant. MLO^Δ1-280 indicates the clone identified in the cDNA library screening. C. The weak pathogen Pci0788-9 carrying the empty vector (Ev) or HopZ2 was pressure-infiltrated into the leaves of Arabidopsis Col-0 or mlo2-7. Statistical significance was determined as stated in part A.

Figure 4

HopZ2^C/A, but not HopZ1c, interacts with MLO2^Δ1-280 in planta by bimolecular fluorescence microscopy. Agrobacterium carrying HopZ2^C/A::nYFP or MLO2^Δ1-280::cYFP (or the reciprocal combination) were mixed at equivalent optical densities and pressure-infiltrated into the leaves of N. benthamiana. Expression of the proteins was induced by 20 μM dexamethasone. Sections of leaf tissue were imaged with a Leica SP5 confocal scanning microscope 72-96 hours post-induction. The close-up images in the second column show the reticulate pattern reminiscent of the endoplasmic reticulum. The scale bar indicates 100 μm.