Understanding the molecular basis of Zymoseptoria tritici-wheat interactions

Abstract

Zymoseptoria tritici is causal to one of the most devastating diseases of wheat, Septoria tritici blotch, STB, in Europe. An estimated 70% of fungicides deployed in Europe is for managing STB. Fundamental research has been undertaken with a focus on understanding the mechanistic basis of STB disease with partial success. However, lack of molecular tools has withheld functional genomics leading to extensive gaps in knowledge of infection biology of Z.tritici. Current research was conducted to develop molecular tools to facilitate forward and reverse genetic screens in Z.tritici for characterizing putative effector genes involved in STB disease. As a part of this study, a genome editing tool, CRISPR/Cas9, was employed to facilitate high frequency targeted genome manipulations in Z.tritici. Protoplasts were transformed with Cas9 protein and gRNA in form of preassembled ribonucleoprotein (RNP) complex targeting nitrate reductase (NiaD) and Orotidine 5/-phosphate-decarboxylase (PyrG). Subsequent screening revealed no mutations in either gene. In attempt to enhance efficacy, RNP complex was co-transformed with homologous directed repair (HDR) cassette with 1kb homologous DNA flanking targeted cut site in NiaD. Again though, no CRISPR-induced mutations were identified questioning suitability of CRISPR/Cas9 in Z.tritici. In contrast, transformation of RNP into Parastagonospora nodorum targeting Tox3 effector gene resulted in 100% editing of mutants. Efficacy of RNP was further tested when co-transformed with a Tox3-HDR cassette harbouring 1kb of homologous flanks which demonstrated homologous recombination (HR) efficiencies exceeding 70%. Further transformation of Tox3-HDR cassette with 50bp microhomology flanks was also successful with 25% HR efficacy. These data highlight a significant potential of CRISPR/Cas9 in expediting transgene-free gene knockouts in P.nodorum. However, its efficacy is dependent on targeted pathogen. Contrary to expectations set by successful application of CRISPR/Cas9 for targeted gene deletions in Z.tritici, four effector candidates were identified based on previous RNA sequencing data in Solomon lab. Agrobacterium-mediated transformation was carried out for successful mutant generation. Two candidate genes, Zt10 and Zt61 were found to play a potential role in pathogenicity inplanta. It was found that Zt10 gene might be important for necrotic lesion development as deletion of this gene resulted in significantly reduced necrotic lesions over time across different wheat cultivars. Once mutated, Zt61 candidate gene showed reduction in disease symptoms inplanta. Zt61 has a predicted cellulose binding domain and shares high structure similarity to bacterial expansin protein, BsEXLX1. Hence, Zt61 gene was cloned and expressed in E.coli, to purify Zt61 protein, further confirmed via mass spectrometry. Upon wheat infiltration, it caused a general necrotic and chlorotic activity, NCA. Mutation in its three amino acids led to decrease NCA suggesting their partial role. Discovery of candidates like Zt10 and Zt61 provide an insight into disease mechanism of Z.tritici. Complementary to these reverse genetics approaches, a forward genetics approach was also employed to screen for Z.tritici potential avirulence genes that are recognized by Stb19 gene in Lorikeet. For this, UV-mutagenesis was performed to generate a library of Z.tritici mutants and screened for pycnidia development. Four mutants were able to develop pycnidia on Lorikeet indicating a potential mutation in the encoding AvrStb19 gene. Subsequent analysis of these four mutants using Illumina sequencing failed to identify a potential AvrStb19. Besides due to a number of unforeseen challenges, a list of nine putative candidates was obtained to be characterized functionally in future studies. Hence, this project did shed light on the technical limitations of this approach and provided important information on how to improve such experiment in Z. tritici in future.