Guest blog by by Agnieszka I. Siwoszek (link is external) and Professor Hans Thordal-Christensen (link is external)
Unlike animals, plants have not evolved an adaptive immune system and their defence responses are based on innate immunity. A first layer of defence is achieved through recognition of the pathogen by plasma membrane receptors. Nevertheless, pathogens overcome this by secreting small effector proteins into the host. Those proteins manipulate the plant’s immunity and hijack its metabolism to favour the growth of the pathogen. Usually, the repertoire of effectors is quite large and unique to each clade of pathogens. Plants have evolved a second layer of defence, which is mediated by intracellular plant receptors that recognises effector proteins. In turn, pathogens avoid this recognition by modifying the individual effector protein or by knocking out the gene that encodes it. This arms-race has been going on for millions of years and has resulted in a highly specific and very intimate interaction between plants and their pathogen. It has also caused resistance based on this system to be highly vulnerable, and generally it lasts only a few years.
Barley (Hordeum vulgare) is one of the major cereals grown worldwide. It is used not only as a food and fodder source, but also as a major ingredient in beer and whisky production. Grain quality is an important measure for the barley yield. One of the most devastating diseases of barley is powdery mildew. It appears on leaves as white powdery masses and typically causes yield losses up to 15%. Yield loss occurs due to a poor grain filling that is a result of the parasitic fungus overtaking plants resources.
Barley powdery mildew is caused by a fungus, named Blumeria graminis f.sp. hordei (Bgh). It infects the epidermal cell layer of leaves. This fungus has a biotrophic lifestyle, which requires it to feed and proliferate on living plant tissue. So-called haustoria are established in epidermal cells upon successful penetration and serve both as feeding structure and a main disease hub that secretes effectors.
This disease is controlled by a combination of fungicides and use of resistant varieties. Early disease management is crucial for effective control. Yet, the pathogen commonly develops fungicide resistance due to simple point mutations. Resistant varieties of spring barley are widely used; though their exploitation has been hampered by pleiotropic effects of resistance genes (necrosis, reduced grain yield, and enhanced susceptibility to other diseases, e.g. Ramularia leaf spot). Consequently, in our study, we propose alternative resistance to barley powdery mildew based on Affimer® technology.
The genome of Bgh has been sequenced and over 500 secreted effector proteins were predicted. Those proteins are unique to cereal powdery mildew fungi, but their targets and function are generally unknown. Over 60% of those effectors share a short motif, which is the only common feature among them. This motif occurs at the N-terminal of the mature proteins and consists of an aromatic amino acid at the first position, any amino acid at the second position and cysteine at the third position. The motif has been shown to be under purifying selection in those approximately 300 effectors, indicating that it is essential for effector function. We chose this motif as target for Affimer molecules in order to engineer disease resistance.
Initially, we did a yeast two-hybrid screen to identify Affimer reagents targeting the motif. Interactions were confirmed by bimolecular fluorescence complementation assays in tobacco leaf cells. Point mutations within the motif resulted in loss of interaction, confirming that the Affimer reagents specifically target the motif. This suggests that the identified Affimer reagents should be able to target the majority of Bgh effectors having the motif. Localisation studies showed that both Affimer reagents and effectors are localised to the plant cytosol and nucleus and their coexpression did not affect the localisation of either of proteins. Interestingly, when an Affimer was fused to a nuclear localisation signal, then a coexpressed effector shifted localisation to be present exclusively in the nucleus. This substantiated that the Affimer is forming a complex with the fungal effector and that the binding is strong enough to change the localisation of the effector protein. Importantly, transient overexpression of selected Affimer reagents in barley epidermal cells reduced the susceptibility to barley powdery mildew by 40%. Moreover, this resistance is highly specific to barley powdery mildew. Thus, Affimer reagents described in this study will be expressed in transgenic barley lines, aiming at obtaining resistance that is beneficial relative to existing disease control means. It would not be possible for the pathogen to overcome such a resistance by a simple point mutation due to the high abundance of the motif among effectors. Moreover, Affimer-based resistance will be highly specific against the barley powdery mildew, and it should be able to stop the disease development at early stages and hopefully make fungicide use and problematic disease resistance unnecessary.