Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546.

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24-36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes' integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum.

Genome sequence of freshwater sediment isolated fungus Westerdykella aurantiaca strain NNIBRFG27121.

We sequenced the genome of Westerdykella aurantiaca NNIBRFG27121 strain isolated from the wetland of Maehwamarum Habitat in Korea. The final assembly consisted of six scaffolds with a size of 31.96 Mb and an N50 of 8,770,400 bp. This genome will help in comparing species within the Westerdykella genus.

Comparative Pathogenicity and Host Ranges of Magnaporthe oryzae and Related Species.

Host shifting and host expansion of fungal plant pathogens increases the rate of emergence of new pathogens and the incidence of disease in various crops, which threaten global food security. Magnaporthe species cause serious disease in rice, namely rice blast disease, as well as in many alternative hosts, including wheat, barley, and millet. A severe outbreak of wheat blast due to Magnaporthe oryzae occurred recently in Bangladesh, after the fungus was introduced from South America, causing great loss of yield. This outbreak of wheat blast is of growing concern, because it might spread to adjacent wheat-producing areas. Therefore, it is important to understand the host range and population structure of M. oryzae and related species for determining the evolutionary relationships among Magnaporthe species and for managing blast disease in the field. Here, we collected isolates of M. oryzae and related species from various Poaceae species, including crops and weeds surrounding rice fields, in Korea and determined their phylogenetic relationships and host species specificity. Internal transcribed spacer-mediated phylogenetic analysis revealed that M. oryzae and related species are classified into four groups primarily including isolates from rice, crabgrass, millet and tall fescue. Based on pathogenicity assays, M. oryzae and related species can infect different Poaceae hosts and move among hosts, suggesting the potential for host shifting and host expansion in nature. These results provide important information on the diversification of M. oryzae and related species with a broad range of Poaceae as hosts in crop fields.

Isolation and Characterization of Four Unrecorded Mucor Species in Korea.

During an investigation of fungi of the order Mucorales from freshwater and sediment samples in Korea, we isolated six strains, NNIBRFG6649, NNIBRFG6255, NNIBRFG1498, CNUFC-YJ13, CNUFC-YR7, and NNIBRFG2739. The morphology and phylogeny of these strains were analyzed. Based on the morphological characteristics and molecular data from internal transcribed spacer (ITS) region, the isolates NNIBRFG6649 and NNIBRFG6255 were identified as Mucor abundans, and M. aligarensis, respectively. The isolates NNIBRFG1498 and CNUFC-YJ13 were identified as M. moelleri, whereas the isolates CNUFC-YR7 and NNIBRFG2739 were identified as M. heterogamus. To the best of our knowledge, M. abundans, M. aligarensis, M. moelleri, and M. heterogamus have not yet been reported in Korea.

Draft Genome Sequence of the Aquatic Fungus Margaritispora aquatica Strain NNIBRFG339.

Margaritispora aquatica is an aquatic fungal species found in leaf litter. Here, we report the 42.5-Mb draft genome sequence of M. aquatica strain NNIBRFG339, which comprises 61 scaffolds and has an overall G+C content of 45.77% and an N 50 value of 1.856 Mb.

Draft Genome Sequence of the Decomposition Fungus Aquanectria penicillioides Strain NNIBRFG19.

Aquanectria penicillioides is a common aquatic fungal species. Here, we report the 53.7-Mb draft genome sequence of A. penicillioides strain NNIBRFG19, which has an overall G+C content of 47.93%, comprising 13 scaffolds with an N 50 value of 4.932 Mb.

Draft Genome Sequence of the Aquatic Fungus Hymenoscyphus tetracladius Strain NNIBRFG329.

Hymenoscyphus tetracladius (anamorph, Articulospora tetracladia) is a common aquatic hyphomycetous fungus. Here, we report the draft genome sequence of H. tetracladius strain NNIBRFG329, which comprises 41.8 Mb in 20 scaffolds with an overall G+C content of 46.95% and an N 50 value of 3.973 Mb.

ER retention receptor, MoERR1 is required for fungal development and pathogenicity in the rice blast fungus, Magnaporthe oryzae.

ER retention receptor is a seven trans-membrane protein that plays pivotal roles in function and integrity of endoplasmic reticulum (ER). Insertional mutagenesis of Magnaporthe oryzae identified MoERR1 as a pathogenicity gene encoding putative ER retention receptor orthologous to ERD2 in Saccharomyces cerevisiae. Search through the genome identified that M. oryzae possesses another ortholog of ERD2, which is designated as MoERR2. When MoERR1 and MoERR2 were tagged with GFP, both were localized to ER. Targeted disruption of MoERR1 showed pleiotropic effects on phenotypes, while deletion of MoERR2 had no effect on phenotypes we examined. The disruption mutant of MoERR1 showed growth retardation and produced significantly reduced number of conidia with aberrant morphology. Appressoria from the mutant were unable to penetrate into plant tissues presumably due to defect in cell wall integrity, thereby rendering the mutant non-pathogenic. The MoERR1 mutant also appeared to display abnormal ER structure and mis-regulation of genes involved in chaperone function and unfolded protein response under ER stress condition. Taken together, these results suggest that MoERR1 is a ER retention receptor required for function and integrity of ER, and that MoERR1-mediated ER functionalities are essential for fungal development and pathogenesis.

The PEX7-mediated peroxisomal import system is required for fungal development and pathogenicity in Magnaporthe oryzae.

In eukaryotes, microbodies called peroxisomes play important roles in cellular activities during the life cycle. Previous studies indicate that peroxisomal functions are important for plant infection in many phytopathogenic fungi, but detailed relationships between fungal pathogenicity and peroxisomal function still remain unclear. Here we report the importance of peroxisomal protein import through PTS2 (Peroxisomal Targeting Signal 2) in fungal development and pathogenicity of Magnaporthe oryzae. Using an Agrobacterium tumefaciens-mediated transformation library, a pathogenicity-defective mutant was isolated from M. oryzae and identified as a T-DNA insert in the PTS2 receptor gene, MoPEX7. Gene disruption of MoPEX7 abolished peroxisomal localization of a thiolase (MoTHL1) containing PTS2, supporting its role in the peroxisomal protein import machinery. ΔMopex7 showed significantly reduced mycelial growth on media containing short-chain fatty acids as a sole carbon source. ΔMopex7 produced fewer conidiophores and conidia, but conidial germination was normal. Conidia of ΔMopex7 were able to develop appressoria, but failed to cause disease in plant cells, except after wound inoculation. Appressoria formed by ΔMopex7 showed a defect in turgor generation due to a delay in lipid degradation and increased cell wall porosity during maturation. Taken together, our results suggest that the MoPEX7-mediated peroxisomal matrix protein import system is required for fungal development and pathogenicity M. oryzae.

The cell cycle gene MoCDC15 regulates hyphal growth, asexual development and plant infection in the rice blast pathogen Magnaporthe oryzae.

Rice blast, caused by the pathogen Magnaporthe oryzae, is a serious hindrance to rice production and has emerged as an important model for the characterization of molecular mechanisms relevant to pathogenic development in plants. Similar to other pathogenic fungi, conidiation plays a central role in initiation of M.oryzae infection and spread over a large area. However, relatively little is known regarding the molecular mechanisms that underlie conidiation in M. oryzae. To better characterize these mechanisms, we identified a conidiation-defective mutant, ATMT0225B6 (MoCDC15(T-DNA)), in which a T-DNA insertion disrupted a gene that encodes a homolog of fission yeast cdc15, and generated a second strain containing a disruption in the same allele (ΔMoCDC15(T-DNA)). The cdc15 gene has been shown to act as a coordinator of the cell cycle in yeast. Functional analysis of the MoCDC15(T-DNA) and ΔMoCDC15(T-DNA) mutants revealed that MoCDC15 is required for conidiation, preinfection development and pathogenicity in M. oryzae. Conidia from these mutants were viable, but failed to adhere to hydrophobic surface, a crucial step required for subsequent pathogenic development. All phenotypic defects observed in mutants were rescued in a strain complemented with wild type MoCDC15. Together, these data indicate that MoCDC15 functions as a coordinator of several biological processes important for pathogenic development in M. oryzae.

Homeobox transcription factors are required for conidiation and appressorium development in the rice blast fungus Magnaporthe oryzae.

The appropriate development of conidia and appressoria is critical in the disease cycle of many fungal pathogens, including Magnaporthe oryzae. A total of eight genes (MoHOX1 to MoHOX8) encoding putative homeobox transcription factors (TFs) were identified from the M. oryzae genome. Knockout mutants for each MoHOX gene were obtained via homology-dependent gene replacement. Two mutants, DeltaMohox3 and DeltaMohox5, exhibited no difference to wild-type in growth, conidiation, conidium size, conidial germination, appressorium formation, and pathogenicity. However, the DeltaMohox1 showed a dramatic reduction in hyphal growth and increase in melanin pigmentation, compared to those in wild-type. DeltaMohox4 and DeltaMohox6 showed significantly reduced conidium size and hyphal growth, respectively. DeltaMohox8 formed normal appressoria, but failed in pathogenicity, probably due to defects in the development of penetration peg and invasive growth. It is most notable that asexual reproduction was completely abolished in DeltaMohox2, in which no conidia formed. DeltaMohox2 was still pathogenic through hypha-driven appressoria in a manner similar to that of the wild-type. However, DeltaMohox7 was unable to form appressoria either on conidial germ tubes, or at hyphal tips, being non-pathogenic. These factors indicate that M. oryzae is able to cause foliar disease via hyphal appressorium-mediated penetration, and MoHOX7 is mutually required to drive appressorium formation from hyphae and germ tubes. Transcriptional analyses suggest that the functioning of M. oryzae homeobox TFs is mediated through the regulation of gene expression and is affected by cAMP and Ca(2+) signaling and/or MAPK pathways. The divergent roles of this gene set may help reveal how the genome and regulatory pathways evolved within the rice blast pathogen and close relatives.

A putative MAP kinase kinase kinase, MCK1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae.

Insertional mutagenesis of Magnaporthe oryzae led to the identification of MCK1, a pathogenicity gene predicted to encode mitogen-activated protein kinase kinase kinase (MAPKKK) homologous to BCK1 in Saccharomyces cerevisiae. Targeted disruption of MCK1 resulted in the fungus undergoing autolysis and showing hypersensitivity to cell-wall-degrading enzyme. The mck1 produced significantly reduced numbers of conidia and developed appressoria in a slightly retarded manner compared with the wild type. Appressorium of the mck1 mutant was unable to penetrate into plant tissues, thereby rendering the mutant nonpathogenic. Cytorrhysis assay and monitoring of lipid mobilization suggested that the appressorial wall was altered, presumably affecting the level of turgor pressure within appressorium. Furthermore, the mck1 mutant failed to grow inside plant tissue. Complementation of the mutated gene restored its ability to cause disease symptoms, demonstrating that MCK1 is required for fungal pathogenicity. Taken together, our results suggest that MCK1 is an MAPKKK involved in maintaining cell wall integrity of M. oryzae, and that remodeling of the cell wall in response to host environments is essential for fungal pathogenesis.

Genome-wide analysis of T-DNA integration into the chromosomes of Magnaporthe oryzae.

Agrobacterium tumefaciens-mediated transformation (ATMT) has become a prevalent tool for functional genomics of fungi, but our understanding of T-DNA integration into the fungal genome remains limited relative to that in plants. Using a model plant-pathogenic fungus, Magnaporthe oryzae, here we report the most comprehensive analysis of T-DNA integration events in fungi and the development of an informatics infrastructure, termed a T-DNA analysis platform (TAP). We identified a total of 1110 T-DNA-tagged locations (TTLs) and processed the resulting data via TAP. Analysis of the TTLs showed that T-DNA integration was biased among chromosomes and preferred the promoter region of genes. In addition, irregular patterns of T-DNA integration, such as chromosomal rearrangement and readthrough of plasmid vectors, were also observed, showing that T-DNA integration patterns into the fungal genome are as diverse as those of their plant counterparts. However, overall the observed junction structures between T-DNA borders and flanking genomic DNA sequences revealed that T-DNA integration into the fungal genome was more canonical than those observed in plants. Our results support the potential of ATMT as a tool for functional genomics of fungi and show that the TAP is an effective informatics platform for handling data from large-scale insertional mutagenesis.

Genome-wide functional analysis of pathogenicity genes in the rice blast fungus.

Rapid translation of genome sequences into meaningful biological information hinges on the integration of multiple experimental and informatics methods into a cohesive platform. Despite the explosion in the number of genome sequences available, such a platform does not exist for filamentous fungi. Here we present the development and application of a functional genomics and informatics platform for a model plant pathogenic fungus, Magnaporthe oryzae. In total, we produced 21,070 mutants through large-scale insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation. We used a high-throughput phenotype screening pipeline to detect disruption of seven phenotypes encompassing the fungal life cycle and identified the mutated gene and the nature of mutation for each mutant. Comparative analysis of phenotypes and genotypes of the mutants uncovered 202 new pathogenicity loci. Our findings demonstrate the effectiveness of our platform and provide new insights on the molecular basis of fungal pathogenesis. Our approach promises comprehensive functional genomics in filamentous fungi and beyond.