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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.
Highlights
Magnaporthe oryzae is the best-studied phytopathogenic fungus, which was voted first in the top 10 list of fungal plant pathogens by an international community of molecular plant pathologists [1]
We found that 61 Zn2Cys6 transcription factor (TF) genes play indispensable and diversified roles in fungal development and pathogenicity
We identified the homologs of the 104 M. oryzae Zn2Cys6 TF genes in N. crassa and F. graminearum by Blastp, and compared the phenotypes of the knockout mutants with the homologs studied in N. crassa [29] or F. graminearum [27] (Table S5 in Text S1)
Summary
Magnaporthe oryzae is the best-studied phytopathogenic fungus, which was voted first in the top 10 list of fungal plant pathogens by an international community of molecular plant pathologists [1]. Recent functional analyses of single or several TF genes revealed their critical biological roles in fungal development, pathogenesis and response to the environment, for instance, in hyphal growth (MNH6, MSTU1, MoCRZ1 and MoSWI6) [7,8,9,10], conidiogenesis (COM1, CON7, COS1, MNH6, and MoHOX2/HTF1) [7,11,12,13,14,15,16], conidial germination (TRA1) [17], appressorium formation (MoLDB1, MoSOM1 and MoCDTF1) [18,19], plant infection (COM1, MNH6, MIG1, MST12/MoHOX8, and MoSfl1) [7,11,16,20,21,22], and response to oxidative stress(MoATF1 and MoAP1) [23,24] or light (MgWC-1) [25]. The biological functions of most TFs have not been revealed, mainly because it is difficult to delete genes on a large scale and Author Summary
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