Systematic Analysis of Zn2Cys6 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 Zn2Cys6 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 Zn2Cys6TF genes. A large variation in biological functions of Zn2Cys6TF genes was observed under the conditions tested. Sixty-one of 104 Zn2Cys6 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.

Characterization of 47 Cys2‐His2 zinc finger proteins required for the development and pathogenicity of the rice blast fungus Magnaporthe oryzae

“…We evaluated and compared the functions of the DEGs in four virulence‐defective mutants (Δ vrf1 and Δ vrf2 in this study, and Δ gpf1 and Δ cnf2 from a previous report (Lu et al ., )) using GO enrichment analysis (Alexa et al ., ). Tens of GO terms were enriched in Δ gpf1 , Δ cnf2 and Δ vrf2 , but none was enriched in Δ vrf1 (FDR < 0.05).…”

Mitochondrial fission protein MoFis1 mediates conidiation and is required for full virulence of the rice blast fungus Magnaporthe oryzae

“…The rice blast fungus produces spores that form dome-shaped appressoria. Many genes have been reported to affect the production of conidia and appressoria and disruption of these genes leads to failure of the development of rice blast disease (Jeon et al 2007;Kershaw and Talbot 2009;Kim et al 2009;Lu et al 2014;Nishimura et al 2009;Saitoh et al 2012). In this study, we analyzed the genedeletion mutants for their function in infection-related events such as mycelium growth on CM medium, conidiation, conidial germination, appressorium formation, sexual reproduction, pathogenicity on blast susceptible rice and barley, osmotic stress response, glucose and nitrogen deficiencies, and cell wall integrity test.…”

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

“…In M. oryzae, the TFs containing fungus-specific Zn 2 Cys 6 zinc finger domains represent the largest group of TFs. A systematic analysis showed that these TFs have critical biological roles in vegetative growth, conidial germination, conidiation, appressorium formation, and pathogenicity to rice and barley (1005).…”