The biology of Blumeria effector candidates

Abstract

Blumeria graminis f.sp. hordei is an obligate biotrophic fungus which causes the powdery mildew disease on barley. It is generally accepted that the specialized feeding structure called the haustorium secretes effector proteins that suppress and modulate the host defense responses. I used transmission electron microscopy to successfully obtain electron micrographs of the ultrastructure of the B. graminis f.sp. hordei infection, including the haustoria, in barley leaves. Effector proteins typically show signatures of positive diversifying selection as a result of the coevolutionary struggle between hosts and pathogens. In this thesis, the ratio of nonsynonymous to synonymous substitutions in Blumeria Effector Candidate (BEC) genes among 54 isolates was calculated to detect BECs under positive diversifying selection. Polymorphisms were found in the BEC genes but the small sample size tested was too small to detect statistically significant positive diversifying selection. In parallel to research carried out in this thesis, a panel of fifty Blumeria candidate effector (BEC) proteins was screened using host-induced gene silencing (HIGS). Of these fifty candidates, eight reduced the haustoria development and are considered as virulence effectors: BEC1005, BEC1011, BEC1016, BEC1018, BEC1019, BEC1038, BEC1040 and BEC1054. I measured relative abundance of RNA in nine BECs including the eight BECs confirmed to be virulence effectors by qRT-PCR at six time points of infection. The transcript profiling presented here reveals that some BECs are important during the early stages of the infection when the compatibility of the host-pathogen interaction is being established and other BECs play a role during the later stages of the infection that involve biotrophy maintenance. It has been proposed that the roles of BEC1011 and BEC1054 in infection are related to host RNA binding or another function that evolved from a ribonuclease ancestor. I used the differential scanning fluorimetry method to test whether BEC1054 binds RNA. The findings in this thesis reveal that there is binding between BEC1054 and RNA and RNA has a destabilizing effect on BEC1054. To further investigate their role in virulence, BEC1011 and BEC1054 transgenic wheat lines were created at NIAB. I commenced the genotyping of these transgenic lines and preliminary pathogenicity tests with some homozygous BEC1011- and BEC1054-wobble lines and homozygous null lines. The preliminary results presented here show that there is a visibly increased amount of sporulating conidia on homozygous BEC1011- and BEC1054-wobble lines compared with homozygous null lines.