Systematic Analysis of Zn ₂Cys ₆ Transcription Factors Required for Development and Pathogenicity by High-Throughput Gene Knockout in the Rice Blast Fungus

Abstract

Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast- Escherichia- Agrobacterium shuttle vector, pKO1B, in the rice blast fungus Magnaporthe oryzae. Using this method, we deleted 104 fungal-specific Zn ₂Cys ₆ transcription factor (TF) genes in M. oryzae. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn ₂Cys ₆TF genes. A large variation in biological functions of Zn ₂Cys ₆TF genes was observed under the conditions tested. Sixty-one of 104 Zn ₂Cys ₆ TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes GPF1 and CNF2 have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of M. oryzae.

Author Summary

Magnaporthe oryzae is not only the fungus causing the rice blast disease, which leads to 20–30% losses in rice production, but also a primary model pathosystem for understanding host-pathogen interactions. However, there is no high-throughput gene knockout system constructed, and little is known about most of the genes in this fungus. We developed a high-throughput gene knockout system, and using this system, we obtained null mutants of 104 fungal-specific Zn ₂Cys ₆ transcription factor (TF) genes by screening 8741 primary transformants in M. oryzae. We analyzed the functions of these TF genes in development, pathogenesis, and stress responses under 9 conditions. We found that 61 Zn ₂Cys ₆ TF genes play indispensable and diversified roles in fungal development and pathogenicity. CNF1 is the first reported TF gene that strongly and negatively regulates asexual development in the rice blast fungus, and CCA1, CNF1, CNF2, CONx1, GPF1, GTA1, MoCOD1 and PCF1 are required for pathogenicity. We further found via RNA-seq that GPF1 and CNF2 have similar mechanisms in gene expression regulation related to pathogenicity. The resulting data provide new insights into how Zn ₂Cys ₆ TF genes regulate important traits during the infection cycle of this rice blast pathogen.