Insertional Mutagenesis to Identify Novel Determinants of Pathogenicity in Magnaporthe oryzae

Abstract

Rice blast disease is caused by the filamentous fungus Magnaporthe oryzae and is the most destructive disease of cultivated rice. It was the first plant pathogenic fungus to have its genome sequence published which opened up the opportunities to discern the principal genetic components that confer pathogenicity on the fungus. The availability of the genome sequence has also presented fresh challenges in terms of converting sequence data into meaningful biological information. Functional genomics studies involve the generation of genome-wide mutant collections and comprehensive screens with potential to identify novel pathogenicity determinants. In this study I utilized Agrobacterium tumefaciens mediated random insertional mutagenesis to study the infection mechanism of M. oryzae. A collection 10,200 M. oryzae T-DNA insertion mutants were generated as part of this study and pathogenicity was assayed by high-throughput disease screening. From the primary qualitative screening I obtained 200 mutants that were reduced or lacking in pathogenicity. Quantitative re-screening allowed selection of 71 T-DNA mutants, including 9 non-pathogenic and 63 reduced virulence mutants exhibiting at least a 50% reduction in disease symptoms. Finally, we selected 8 non-pathogenic mutants for detailed phenotypic and gene functional analysis. A novel approach was used to retrieve T-DNA tagged genes from mutants of interest. Next generation DNA sequencing (NGS) was used to retrieve T-DNA flanking sequences in a high-throughput manner. The efficiency of NGS to facilitate the high-throughput large scale insertional mutagenesis was therefore demonstrated. Out of 8 selected mutants, I identified three novel genes that putatively encode a transcription factor, a PH domain containing signalling protein and a MAP kinase. I also provided evidence that, MGG_05343 is a functional C6 zinc finger transcription factor involved in conidiogenesis. The PH domain containing protein MGG_12956 is involved in vegetative growth, condiogenesis and virulence. The novel kinase MGG_15325 is a S. cerevisiae IME2 homolog that belongs to the Ime2 class of non-classical MAP kinase subfamily. Intriguingly, M. oryzae IME2 seems to have an essential role in growth in planta because the mutant was able to penetrate and colonize plant tissue but failed to cause necrotic rice blast lesions. Identification of these novel genes will allow us greater insight into the processes required for condiogenesis, vegetative and invasive growth and a more integrated understanding of the post-penetration phases of plant tissue colonization. Interestingly, I identified two mutants tagged with T-DNA insertion in the autophagy genes ATG2 and ATG3, reaffirming the importance of infection-associated autophagy in plant infection by M. oryzae and we characterized the ATG3 gene. In addition, I generated a resource of 63 unidentified T-DNA mutants which can potentially lead to identification of more novel determinants of pathogenicity in rice blast disease.