Hort 510 Seminar Abstract April 30, 2015 Evaluation of MLO

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Hort 510 Seminar Abstract

April 30, 2015

Evaluation of MLO-mediated powdery mildew resistance in grape and strawberry using gene editing

Kathie L Nicholson, PhD proposal seminar

Powdery mildew affects approximately 10,000 plant species world-wide, including many economically important agricultural and horticultural crops. Grape

(Vitis vinifera)

and strawberry

(Fragaria

x

ananassa),

valuable crops in the State of Washington, are both highly susceptible to powdery mildew. Powdery mildew can severely reduce fruit quality, decrease yield, and in extreme cases kill the host plant. Fungicides are used to combat powdery mildew, however, application is costly, time consuming, and potentially harmful to the environment.

The mildew resistance locus O (MLO) family of genes encode transmembrane proteins which allow for powdery mildew infection by facilitating penetration of the epidermal cells by the fungus. This particular function of the MLO gene was described following the discovery of a mutation in a barley landrace collected in Ethiopia in the 1930s which conferred durable resistance to powdery mildew. Since that time, work has been done in several plant systems to define the MLO genes and the role they play in powdery mildew response, in addition to determining other functions of these genes. The MLO gene family is highly conserved among plant species, with each species having between 8 and 39 MLO genes. Phylogenetic analysis of the MLO proteins has identified six clades, and research to date has shown that the proteins related to powdery mildew response in dicots all fall into Clade V. Other MLO gene functions that have been identified, such as biotic and abiotic stress response, fall into other clades. These phylogenetic analyses provide support for selecting which MLO gene or genes to target for powdery mildew resistance.

The recently developed CRISPR/Cas9 gene editing tool is based on a naturally occurring bacterial adaptive immune response to viral infection. Following repeated attacks, bacterial

RNAs recognize the foreign nucleic acid, bind to it, and guide a nuclease to the invasion site which then cleaves the invading genetic material. Mutations via the CRISPR/Cas9 gene editing tool have been characterized in several species, including

Arabidopsis

, tobacco, rice and tomato.

It has been shown that these mutations are heritable, independent of the CRISPR/Cas9 insert, as backcrosses have been produced that no longer have the insert but carry the mutation. Therefore, using CRISPR/Cas9 it is possible to create a heritable mutation in a targeted MLO gene resulting in a non-GMO plant with powdery mildew resistance.

The hypothesis of this study is that loss of function mutations in selected MLO genes will provide resistance to powdery mildew in grape and strawberry. This hypothesis will be tested using CRISPR/Cas9 gene editing to induce a loss of function mutation in selected grape and strawberry MLO genes, followed by analysis of the transformed plants for resistance to powdery mildew. This study is building on previous research performed in our lab that has established the connection between strawberry MLO genes and powdery mildew response. The MLO genes in grape and strawberry have been selected, and target sites within those genes have been identified.

The CRISPR/Cas9 cassette will be inserted into a plant transformation vector, and delivered into the plant cells via gene bombardment. Detection of successful mutation will be analyzed by targeted sequencing of the selected MLO genes. Disease bioassays will be performed on plants determined to be carrying the desirable mutations to assess resistance to powdery mildew.

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