Transcription factors that regulate gene expression in haustoria for powdery mildew virulence

Barley powdery mildew is an economically important disease caused by the obligate biotrophic fungus Blumeria hordei (Bh). Powdery mildews produce specialised haustorial structures which are crucial to support virulence and biotrophy. Haustoria are involved in the uptake of nutrients and the expression and delivery of effectors and virulence factors into host cells. Transcription factors (TFs) and other mechanisms that orchestrate virulence by upregulation of gene expression of effectors in Blumeria haustoria are mostly unknown and therefore need to be studied. Thus, this work has investigated three : TFs that were upregulated in haustoria, i.e. a bZip containing TF, bZip1 (Pap1), an AP1-like TF homologous to the yeast Yap1p known to play a key role in oxidative stress responses), a Zn-knuckle (ZnK) type TF and TF4072 which belongs to the fungal specific Zn(2)-C6 type TFs which include GAL4 and Xylanolytic transcriptional activator(xlnR) whose Zymoseptoria tritici and Magnaporthe oryzae protein homologues play important role in virulence. In addition, BhPacC, which is homologuous to Candida albicans PacC/ RIM101 , is regulating the alkaline pH response and virulence in several human pathogenic fungi, was also investigated. To establish whether these TFs are involved in B. hordei virulence through the regulation of haustoria gene expression, a transient gene silencing approach using short antisense oligodeoxynucleotides (ASOs) was optimised to allow for in planta silencing of genes from this obligate pathogen. Gene silencing of each four TFs investigated led to substantial decreased infection. Gene silencing of these TFs also affected gene expression of previously characterised haustoria expressed virulence factors such as the effector BEC1011 (CSEP0264) and the metalloprotease like BEC1019 virulence factor, a homologue of the well characterised PRA1/ AspF2 in human pathogenic fungi. Moreover, a genome-wide promoter analysis identified TFs binding elements specific to haustoria expressed genes, some of which were, for instance, associated with yeast homologues of PacC or Pap1. Preliminary experiments confirmed the physical binding of BhPacC to pH regulatory element (PRE) - containing promoter regions of BEC1011 and BEC1019 effectors. These data suggest that all four TFs investigated, i.e., BhPacC, bZip1 (Pap1), Zn-knuckle (ZnK) and TF4072 have a key role at orchestrating Blumeria virulence by controlling gene expression in haustoria during barley powdery mildew infection. The next question to address is whether haustoria gene regulation is conserved across powdery mildews, infecting different plant hosts, such as cucumber, pea or strawberry