Proteomic analysis of barley powdery mildew haustorial structures to identify candidates of host susceptibility.

The biotrophic fungal pathogen Blumeria graminis is responsible for powdery mildew infection in cereals. B. graminis haustoria are specialized feeding structures which form exclusively within the plant epidermis and are surrounded by a differentiated plant derived extra-haustorial membrane (EHM). While the EHM is required for successful invasion, little is known of its composition. Previous works showed that the relocation of plant proteins to EHM structures is key to infection. This study aimed to unravel EHM located proteins and validate these likely novel mediators of barley-powdery mildew susceptibility.To reach this goal, first differential proteomics approaches were undertaken to identify barley proteins associated with enriched haustoria, prepared from infected barley leaf or leaf epidermis. Then, a selection of proteins associated with infected epidermis or co-enriched in haustoria were evaluated using a transient gene-silencing approach. Two susceptibility genes were overexpressed in Nicotiana benthamiana and subsequently challenged with the biotrophic oomycete pathogen Peronospora hyoscyami f.sp. tabacina , in order to assess their potential cross-kingdom roles in susceptibility and relocation to haustoria structures.Several pathogenesis related proteins were more abundant in infected- compared to non-infected epidermis, including a Blumeria effector BEC1054 interactor thaumatin-like protein 5 (TLP5). Silencing TLP5 and its isoform TLP8 caused marked reduced powdery mildew infection suggesting that TLP5 and TLP8 modulates barley susceptibility. Two additional putative susceptibility proteins were identified as associated with the haustoria; the membrane Aquaporin PIP2;3 and Early nodulin like protein 9 (ENO9). Silencing of PIP2;3 and ENO9 led to reduced B. graminis infection. Preliminary data suggest that TLP5 overexpression of in N. benthamiana caused a small increase in Peronosopora infection. Moreover, overexpression of barley PIP2;3 yielded a similar yet insignificant trend, while silencing of the endogenous NbPIP2;3 had no impact on Peronospora infection.The data presented here suggest that the epidermis associated TLP5 and TLP8, as well as the haustoria associated PIP2;3 and ENO9, are required for virulence during barley infection with B. graminis. Their susceptibility role should be validated by the generation of stable deletion mutants in barley. Confirming these and other susceptibility protein localisation to the EHM will further demonstrate the pivotal role of haustoria in successful biotrophic interactions. In conclusion, characterisation of the EHM proteome remains a promising strategy to further understand host susceptibility mechanisms.