Rice blast is one of the most serious diseases affecting rice yield which is caused by Magnaporthe oryzae, a model organism for studies on plant pathogenic fungi. Lipids stored in M. oryzae cells have been shown to be crucial for the development of appressorium turgor and the ability of the pathogen to cause infection. Nile red staining is a common method to study lipid dynamics in phytopathogenic fungi. However, the disadvantages of this dye include its wide spectrum, poor water solubility, and susceptibility to quenching. Boron dipyrromethene (BODIPY) is a new type of fluorescent dye that has a different emission wavelength to that of Nile red as well as many desirable spectral and chemical properties. In this study, we used BODIPY to stain the lipids in M. oryzae cells to seek a possible substitute to Nile red in the study of lipid dynamics in plant pathogenic fungi. Our data showed that through simple and routine procedures, BODIPY was able to distinctly label lipids in the cells of mycelia and conidia. The positions of lipids labeled by BODIPY were essentially identical to those labeled by Nile red, but with more clear fluorescence labelling, lower background, and higher specificity. The use of BODIPY to stain germinating M. oryzae conidia allowed the lipid dynamics to be clearly tracked during this process. We also achieved double and multiple fluorescent staining conidia by combining BODIPY with the red fluorescent protein mCherry and other fluorescent dyes, such as Calcofluor white and DAPI, in conidia, mycelia, and sexual structures of M. oryzae. These results indicate that BODIPY is an ideal fluorescent dye for staining fungal lipids and provide a method for the study of the lipid dynamics and lipid metabolism in plant pathogenic fungi.

The DExD/H-box protein family encompasses a large number of RNA helicases that are involved in RNA metabolism and a variety of physiological functions in different species. However, there is limited knowledge of whether DExD/H-box proteins play a role in the pathogenicity of plant fungal pathogens. In the present work, the DExD/H-box protein MoDHX35, which belongs to the DEAH subfamily, was shown to be crucial in appressoria formation and full virulence of the rice blast fungus, Magnaporthe oryzae. The predicted protein sequence of MoDHX35 had typical DEAH-box domains, showed 47% identity to DHX35 in Homo species, but had no orthologs in Saccharomyces cerevisiae. Deletion of the MoDHX35 gene resulted in reduced tolerance of the mutants to doxorubicin, a nucleic acid synthesis disturbing agent, suggesting the involvement of MoDHX35 in RNA metabolism. MoDHX35-deleted mutants exhibited normal vegetative growth, conidia generation and conidial germination, but showed a reduced appressorium formation rate and attenuated virulence. Our work demonstrates the involvement of DEAH-box protein functions in the pathogenicity of plant fungal pathogens.

Magnaporthe oryzae is the causal agent for the devastating disease rice blast. The avirulence (AVR) genes in M. oryzae are required to initiate robust disease resistance mediated by the corresponding resistance (R) genes in rice. Therefore, monitoring pathogen AVR genes is important to predict the stability of R gene-mediated blast resistance. In the present study, we analyzed the DNA sequence dynamics of five AVR genes, namely, AVR-Pita1, AVR-Pik, AVR-Pizt, AVR-Pia, and AVR-Pii, in field isolates of M. oryzae in order to understand the effectiveness of the R genes, Pi-ta, Pi-k, Pi-zt, Pia, and Pii in the Southern U.S. rice growing region. Genomic DNA of 258 blast isolates collected from commercial fields of the Southern UNITED STATES during 1975–2009 were subjected to PCR amplification with AVR gene-specific PCR markers. PCR products were obtained from 232 isolates. The absence of PCR products in the remaining 26 isolates suggests that these isolates do not contain the tested AVR genes. Amplified PCR products were subsequently gel purified and sequenced. Based on the presence or absence of the five AVR genes, 232 field isolates were classified into 10 haplotype groups. The results revealed that 174 isolates of M. oryzae carried AVR-Pita1, 225 isolates carried AVR-Pizt, 44 isolates carried AVR-Pik, 3 isolates carried AVR-Pia, and one isolate carried AVR-Pii. AVR-Pita1 was highly variable, and 40 AVR-Pita1 haplotypes were identified in avirulent isolates. AVR-Pik had four nucleotide sequence site changes resulting in amino acid substitutions, whereas three other AVR genes, AVR-Pizt, AVR-Pia, and AVR-Pii, were relatively stable. Two AVR genes, AVR-Pik and AVR-Pizt, were found to exist in relatively larger proportions of the tested field isolates, which suggested that their corresponding R genes Pi-k and Pi-zt can be deployed in preventing blast disease in the Southern UNITED STATES in addition to Pi-ta. This study demonstrates that continued AVR gene monitoring in the pathogen population is critical for ensuring the effectiveness of deployed blast R genes in commercial rice fields.

Magnaporthe oryzae, a fungal pathogen that causes rice blast, which is the most destructive disease of rice worldwide, has the potential to perform both asexual and sexual reproduction. MAT loci, consisting of MAT genes, were deemed to determine the mating types of M. oryzae strains. However, investigation was rarely performed on the development and molecular mechanisms of the sexual reproduction of the fungus. In the present work, we analyzed the roles of two MAT loci and five individual MAT genes in the sex determination, sexual development and pathogenicity of M. oryzae. Both of the MAT1-1 and MAT1-2 loci are required for sex determination and the development of sexual structures. MAT1-1-1, MAT1-1-3 and MAT1-2-1 genes are crucial for the formation of perithecium. MAT1-1-2 impacts the generation of asci and ascospores, while MAT1-2-2 is dispensable for sexual development. A GFP fusion experiment indicated that the protein of MAT1-1-3 is distributed in the nucleus. However, all of the MAT loci or MAT genes are dispensable for vegetative growth, asexual reproduction, pathogenicity and pathogenicity-related developments of the fungus, suggesting that sexual reproduction is regulated relatively independently in the development of the fungus. The data and methods of this work may be helpful to further understand the life cycle and the variation of the fungus.

The Triticum pathotype of Magnaporthe oryzae (syn. Pyricularia oryzae) causes wheat blast, which has recently spread to Asia. To assess the potential risk of wheat blast in rice–wheat growing regions, we investigated the pathogenicity of 14 isolates of P. oryzae on 32 wheat cultivars, among which Oryzae pathotype of P. oryzae (MoO) isolates were completely avirulent on the wheat cultivars at 22°C but caused various degrees of infection 25°C. These reactions at 25°C were isolate and cultivar dependent, like race-cultivar specificity, which was also recognized at the heading stage and caused typical blast symptoms on spikes. Microscopic analyses indicated that a compatible MoO isolate produced appressoria and infection hyphae on wheat as on rice. When we compared transcriptomes in wheat–MoO interactions, the bulk of pathogen-related genes were upregulated or downregulated in compatible and incompatible patterns, but changes in gene transcription were more significant in a compatible pattern. These results indicate that temperature could influence the infection ratio of wheat with MoO, and some MoO strains could be potential pathogens that increase the risk of wheat blast outbreaks in wheat–rice growing regions with global warming. In addition, certain wheat cultivars exhibited resistance and are assumed to carry resistance-promoting genes to the MoO strains.

Rice blast, caused by Magnaporthe oryzae, significantly threatens global rice production. Disease control is complicated by the pathogen’s high genetic diversity, which is driven by heterothallic recombination between opposite mating types that underlies variation. However, mechanisms governing sexual reproduction in this fungus remain poorly characterized, largely due to the absence of reliable methods for scalable ascospore progeny production. In this study, we established two novel mating methods, namely Conidial Mixing Mating (CMM) and Hyphal Segments Mixed Mating (HMM). Both methods employed optimized suspensions (5 × 10⁴ conidia/mL or equivalent hyphal density) mixed at 1:1 ratios, incubated under standardized conditions: 20 °C with a 12 h/12 h photoperiod. We characterized perithecia, asci, and ascospore morphology using fluorescence microscopy, paraffin sectioning, cryo-scanning electron microscopy, and transmission electron microscopy. Furthermore, both methods enabled phenotypic characterization of sexual reproduction-deficient mutants, including ΔMopmk1 and ΔMoopy2. In conclusion, we established two efficient methods for investigating M. oryzae sexual reproduction, providing foundational tools to advance studies of sexual mechanisms, pathogenicity evolution, and genetic variation.