RnAse-Like Proteins expressed in Haustoria (RALPHs) in Erysiphe necator, the grapevine powdery mildew

Powdery mildews have a huge economic impact as they cause growth reduction of crops and lead to loss of yield. Powdery mildews are caused by many different species of fungi in the order of Erysiphales which can affect all kinds of plants. Some of the powdery mildew fungi can infect a number of plant hosts (having a broad host range), whereas others are species-specific pathogen. For example, Blumeria graminis hordei (Bgh) is only virulent to barley; Erysiphe necator (E. necator) can infect the genera within the Vitaceae, including Vitis, Cissus and Parthenocissus.Therefore, powdery mildews are probably the most common and widespread plant diseases. The symptom of powdery mildew infected plants is very obvious, the white powder-like patches visible on the aerial organs. Powdery mildews are obligate biotrophs, which rely completely on living host cells to complete their lifecycles. Therefore, powdery mildews secret proteins, called candidate secreted effector proteins (CSEPs) into their hosts which manipulate the metabolism and suppress the defence mechanisms of the hosts.
CSEPs are defined as proteins with predicted signal peptides without predicted transmembrane domains and no homology to proteins outside the Erysiphales. There is a class of CSEPs which adapt structures with similarity to fungal ribonucleases, called RnAse-Like Proteins expressed in Haustoria (RALPHs). In barley powdery mildew, Blumeria graminis hordei, 25% of CSEPs are RALPHs, and two of them (CSEP0264 and CSEP0064) have been identified as bona fide effectors. Their virulence effect has been identified by Host-induced gene silencing (HIGS). The existence of large numbers of RALPHs and their identification in many powdery mildews emphasise the importance of understanding their roles in powdery mildew pathogenicity.
E. necator, grapevine powdery mildew is highly virulent to the cultivated grapevines, Vitis vinifera, which leads to heavy use of fungicides to control this disease. Currently, ten RALPHs have also been identified in E. necator (EnRALPHs), however, their virulence and functions are still unknown. By studying these EnRALPHs we may not only understand how they specifically target the grapevine defence mechanism, but also contribute to the understanding of their general roles in conferring powdery mildew virulence and their conserved functions across many powdery mildews.
Therefore, the aim of this project is to understand the roles of EnRALPHs in grapevine defence modulation.
I have listed a number of objectives and the methods to use which will help me to achieve the aim.
The first objective is the characterisation of EnRALPHs pathogenicity.
All ten EnRALPHs have high structural similarity to fungal ribonucleases as predicted by Phyre2. Also, the presence of predicted signal peptides indicates that they are secreted effectors. However, whether they are avirulent effectors (which can be recognised by host R genes) or virulent effectors (involved in plant defence suppression) is unknown. I will use P. tabacina- N. benthamiana pathosystem to characterise the virulence of these EnRALPHs. P. tabacina, commonly known as tobacco blue mould, causes downy mildew in the genus Nicotiana. N. benthamiana is highly susceptible to P. tabacina. P. tabacina is biotroph which relies on living host cells to complete their life cycle, which leads to the production of sporangia at the last stage. Therefore, I will use the number of sporangia on N. benthamiana as a measure of P. tabacina virulence. I will transiently express the gene of EnRALPHs in N. benthamiana by agroinfiltration. In agroinfiltration, the culture of A. tumefaciens carrying constructs encoding the gene of interest will be vacuum infiltrated into the abaxial surface of leaves. Subsequently, A. tumefaciens will transfer these EnRALPHs gene into the hosts and lead to transient expression. Then, I will inoculate P. tabacina onto these agroinfiltrated leaves of N. benthamiana and determine t