Developing a silencing tool with short antisense oligonucleotides to investigate effectors and susceptibility genes during barley powdery mildew infection

Barley powdery mildew (Blumeria graminis f.sp. hordei) is a prevalent fungal pathogen of barley in the UK. As an obligate pathogen, methods for gene knockouts or silencing have been limited, with no stable transformation method available. A transient assay using a biolisitic delivery system has been previously applied for Host Induced Gene Silencing (HIGS) of Blumeria effector candidates (BECs). However, HIGS requires cloning of a silencing cassette and since only a small proportion of cells are transformed, it forbids downstream whole-tissue analyses. The aim of the project was to overcome these caveats by developing a new silencing strategy of genes from barley and its powdery mildew in order to investigate their roles during infection. The method required the delivery of phosphorothioate-modified, short, antisense oligonucleotides (PTOs) via the vascular tissue of excised leaves. The method was validated by silencing the known Blumeria effectors BEC1011, BEC1019 and BEC1054, and the barley susceptibility factors Mlo and Blufensin1. While the disease phenotype was monitored microscopically by measuring the infection rate in term of successful secondary hyphae formation, relative mRNA and protein amounts of the cognate silenced gene were measured by qRT-PCR and multiple reaction monitoring mass spectrometry. Targeting the aforementioned effectors or susceptibility genes resulted in 40-60% reduction of secondary hyphae formation and up to 30% reduction in transcript. The technology was then applied to silence putative Blumeria Candidate Secreted Effector Proteins (CSEPs) AVRa1 and AVRa13, resulting in 22% and 31% reduction in secondary hyphae formation, respectively. A barley pathogenesis related protein 5 (PR5) was also targeted. Surprisingly, PR5 silencing led to a drastic decrease in Blumeria infection, suggesting that PR5 is a susceptibility factor. To investigate BEC1011 or PR5 roles in compromising plant immune responses, production of reactive oxygen species such as H2O2 was monitored after Blumeria infection in control, BEC1011, and PR5 silenced plants, showing that BEC1011 and PR5 are preventing a sustained oxidative burst response.