Decoding Host-Pathogen Interactions with Transgenic and Genome Edited Barley.

Powdery mildews, caused by obligate fungal pathogens, are a major threat to cereal grain production worldwide. We focus on the interaction of barley with the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), to address fundamental questions in host resistance and susceptibility. Effector proteins secreted by fungal pathogens suppress or induce host processes to promote nutrient acquisition and colonization. As such, effectors serve as optimal probes to decode host signaling pathways. Secreted Blumeria effector candidate (BEC) 1019 is a putative metalloprotease that enhances Bgh virulence in barley.

Plants also use effector triggered immunity involving R proteins to restrict nutrient loss and minimize cost of defense with hypersensitive response (HR). Receptor-like cytoplasmic kinases in plants such as barley orthologs of PBS1 (AvrPphB Susceptible 1) are often targeted by effectors resulting in recognition by certain intracellular receptors to induce HR.

Here we report overexpression of BEC1019 in transgenic barley plants and a genome editing strategy to knockout barley PBS1 using CRISPR/Cas9. Genetically modified barley plants were regenerated following Agrobacterium-mediated genetic transformation of barley cv. Golden Promise and hygromycin selection. Expression levels of BEC1019 gene variants with or without a signal peptide (SP) and an affinity purification tag (HPB tag), and production of homozygous transgenic seed progenies are reported. Transgenic plants will be analyzed with pull-down assay to determine cellular localization and confirm target interactions. Confirmation of targeted mutagenesis of barley PBS1 with sequencing of PCR amplicons is in progress and genome edited plants will be used to further decode host-pathogen interactions.

Research supported by NSF-PGRP/ERA-CAPS #1339348: Host Targets of Fungal Effectors as Keys to Durable Disease Resistance.