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Abstract Plant pathogens secrete nuclear effectors into the host nuclei to modulate the host immune system. Although several nuclear effectors of fungal pathogens have been recently reported, the molecular mechanism of NLS-associated transport vehicles of nuclear effectors and the roles of NLS in transcriptional reprogramming of host immunity genes remain enigmatic. We previously reported the MoHTR1, a nuclear effector of the rice blast fungus, Magnaporthe oryzae. MoHTR1 is translocated to rice nuclei but not in fungal nuclei. Here, we identify the core NLS (RxKK) responsible for MoHTR1’s nuclear localization. MoHTR1 is translocated in the host nucleus through interaction with rice importin α. MoHTR1 NLS empowers it to translocate the cytoplasmic effectors of M. oryzae into rice nuclei. Furthermore, other nuclear effector candidates of the blast pathogen and rice proteins which have RxKK also exhibit nuclear localization, highlighting the crucial role of RxKK in this process. We also unveil the importance of SUMOylation in the stability of MoHTR1 and translocation of MoHTR1 to host nuclei. Moreover, MoHTR1 NLS is essential for the pathogenicity of M. oryzae by reprogramming immunity-associated genes in the host. Our findings provide insights into the significance of plant-specific NLS on fungal nuclear effectors and its role in plant-pathogen interactions.

Abstract Background Cell wall integrity (CWI) is crucial for fungal growth, pathogenesis, and adaptation to extracellular environments. Calcofluor white (CFW) is a cell wall perturbant that inhibits fungal growth, yet little is known about how phytopathogenic fungi respond to the CFW-induced stress. Results In this study, we unveiled a significant discovery that CFW triggered the translocation of the transcription factor CgCrzA from the cytoplasm to the nucleus in Colletotrichum gloeosporioides. This translocation was regulated by an interacting protein, CgMkk1, a mitogen-activated protein kinase involved in the CWI pathway. Further analysis revealed that CgMkk1 facilitated nuclear translocation by phosphorylating CgCrzA at the Ser280 residue. Using chromatin immunoprecipitation sequencing, we identified two downstream targets of CgCrzA, namely CgCHS5 and CgCHS6, which are critical for growth, cell wall integrity, and pathogenicity as chitin synthase genes. Conclusions These findings provide a novel insight into the regulatory mechanism of CgMkk1-CgCrzA-CgChs5/6, which enables response of the cell wall inhibitor CFW and facilitates infectious growth for C. gloeosporioides.

ABSTRACT Sexual reproduction is crucial for increasing the genetic diversity of populations and providing overwintering structures, such as perithecia and associated tissue, in the destructive plant pathogenic fungus Fusarium graminearum. While mating-type genes serve as master regulators in fungal sexual reproduction, the molecular mechanisms underlying this process remain elusive. Winged-helix DNA-binding proteins are key regulators of embryogenesis and cell differentiation in higher eukaryotes. These proteins are implicated in the morphogenesis and development of several fungal species. However, their involvement in sexual reproduction remains largely unexplored in F. graminearum. Here, we investigated the function of winged-helix DNA-binding proteins in vegetative growth, conidiation, and sexual reproduction, with a specific focus on the FgWING27, which is highly conserved among Fusarium species. Deletion of FgWING27 resulted in an abnormal pattern characterized by a gradual increase in the expression of mating-type genes during sexual development, indicating its crucial role in the stage-specific genetic regulation of MAT genes in the late stages of sexual development. Furthermore, using chromatin immunoprecipitation followed by sequencing analysis, we identified Fg17056 as a downstream gene of Fgwing27, which is essential for sexual reproduction. These findings underscore the significance of winged-helix DNA-binding proteins in fungal development and reproduction in F. graminearum, and highlight the pivotal role of Fgwing27 as a core genetic factor in the intricate genetic regulatory network governing sexual reproduction.IMPORTANCEFusarium graminearum is a devastating plant pathogenic fungus causing significant economic losses due to reduced crop yields. In Fusarium Head Blight epidemics, spores produced through sexual and asexual reproduction serve as inoculum, making it essential to understand the fungal reproduction process. Here, we focus on winged-helix DNA-binding proteins, which have been reported to play crucial roles in cell cycle regulation and differentiation, and address their requirement in the sexual reproduction of F. graminearum. Furthermore, we identified a highly conserved protein in Fusarium as a key factor in self-fertility, along with the discovery of its direct downstream genes. This provides crucial information for constructing the complex genetic regulatory network of sexual reproduction and significantly contribute to further research on sexual reproduction in Fusarium species.

Abstract We examined potato rhizosphere bacterial and fungal communities across three regions: Cheongju, Pyeongchang, and Gangneung. These regions have varying soil and climate conditions, resulting in different yields. We found that precipitation was the main limiting factor in our study while soil physiochemical factors affect bacterial and fungal microbiota in correlation with yield. Both bacterial and fungal microbiota showed distinct patterns according to the regions. ASVs positively correlated with yield were predominantly found in the Pyeongchang region which also produced the highest yields, while ASVs negatively correlated with yield were associated with Gangneung where the lowest yields were observed. The greatest bacterial and fungal diversity was detected in Pyeongchang consisting of Propionibacteriales, Burkholderiales, and Vicinamibacteriales. Gangneung, on the other hand primarily belong to Sordariales, Mortierellales, Cystofilobasidiales, and Tremellales. The putative yield-negative ASVs detected in Gangneung may have been influenced by drought stress. This work has highlighted key bacterial and fungal taxa as well as core taxa that may potentially be associated with high and low yields of potato in relation to metadata which includes soil chemical and physical parameters as well as weather data. Taken together we suggest that this information can be used to assess site suitability for potato production.

Abstract Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)−1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogens Botrytis cinerea and Magnaporthe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, B. cinerea, M. oryzae, Sclerotinia sclerotiorum and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens.

Genetic and environmental cues affecting seed microbial communities have been investigated to assess the ecological characteristics of seed microbial communities. However, little is known concerning seed-to-seed microbial variations and ecological drivers at the single-seed level. We report rare taxa-associated heterogeneity and robustness of seed bacterial and fungal communities in individual seeds using 63 pooled and 70 single-seed samples from a single field-grown rice plant. Ordination analyses showed that seed-to-seed variation patterns could be clustered according to the originating panicle branch. Bacterial–fungal associations and in silico extinction experiments demonstrated that rare taxa contribute to the connectivity and robustness of the associations. Null modeling-based statistical analysis revealed that the distribution of rare taxa is mainly governed by dispersal limitation, whereas the distribution of prevalent taxa is mainly governed by homogeneous selection and ecological drift. Our findings provide an ecological framework for understanding the heterogeneity of seed microbial communities in a single plant; they will facilitate the development and application of seed microbiota or single microbe-based engineering strategies.

Abstract Background The rhizosphere microbiome, which is shaped by host genotypes, root exudates, and plant domestication, is crucial for sustaining agricultural plant growth. Despite its importance, how plant domestication builds up specific rhizosphere microbiomes and metabolic functions, as well as the importance of these affected rhizobiomes and relevant root exudates in maintaining plant growth, is not well understood. Here, we firstly investigated the rhizosphere bacterial and fungal communities of domestication and wild accessions of tetraploid wheat using amplicon sequencing (16S and ITS) after 9 years of domestication process at the main production sites in China. We then explored the ecological roles of root exudation in shaping rhizosphere microbiome functions by integrating metagenomics and metabolic genomics approaches. Furthermore, we established evident linkages between root morphology traits and keystone taxa based on microbial culture and plant inoculation experiments. Results Our results suggested that plant rhizosphere microbiomes were co-shaped by both host genotypes and domestication status. The wheat genomes contributed more variation in the microbial diversity and composition of rhizosphere bacterial communities than fungal communities, whereas plant domestication status exerted much stronger influences on the fungal communities. In terms of microbial interkingdom association networks, domestication destabilized microbial network and depleted the abundance of keystone fungal taxa. Moreover, we found that domestication shifted the rhizosphere microbiome from slow growing and fungi dominated to fast growing and bacteria dominated, thereby resulting in a shift from fungi-dominated membership with enrichment of carbon fixation genes to bacteria-dominated membership with enrichment of carbon degradation genes. Metagenomics analyses further indicated that wild cultivars of wheat possess higher microbial function diversity than domesticated cultivars. Notably, we found that wild cultivar is able to harness rhizosphere microorganism carrying N transformation (i.e., nitrification, denitrification) and P mineralization pathway, whereas rhizobiomes carrying inorganic N fixation, organic N ammonification, and inorganic P solubilization genes are recruited by the releasing of root exudates from domesticated wheat. More importantly, our metabolite-wide association study indicated that the contrasting functional roles of root exudates and the harnessed keystone microbial taxa with different nutrient acquisition strategies jointly determined the aboveground plant phenotypes. Furthermore, we observed that although domesticated and wild wheats recruited distinct microbial taxa and relevant functions, domestication-induced recruitment of keystone taxa led to a consistent growth regulation of root regardless of wheat domestication status. Conclusions Our results indicate that plant domestication profoundly influences rhizosphere microbiome assembly and metabolic functions and provide evidence that host plants are able to harness a differentiated ecological role of root-associated keystone microbiomes through the release of root exudates to sustain belowground multi-nutrient cycles and plant growth. These findings provide valuable insights into the mechanisms underlying plant-microbiome interactions and how to harness the rhizosphere microbiome for crop improvement in sustainable agriculture. Video Abstract

Alternative splicing (AS) contributes to diversifying and regulating cellular responses to environmental conditions and developmental cues by differentially producing multiple mRNA and protein isoforms from a single gene. Previous studies on AS in pathogenic fungi focused on profiling AS isoforms under a limited number of conditions. We analysed AS profiles in the rice blast fungus Magnaporthe oryzae, a global threat to rice production, using high-quality transcriptome data representing its vegetative growth (mycelia) and multiple host infection stages. We identified 4,270 AS isoforms derived from 2,413 genes, including 499 genes presumably regulated by infection-specific AS. AS appears to increase during infection, with 32.7% of the AS isoforms being produced during infection but absent in mycelia. Analysis of the isoforms observed at each infection stage showed that 636 AS isoforms were more abundant than corresponding annotated mRNAs, especially after initial hyphal penetration into host cell. Many such dominant isoforms were predicted to encode regulatory proteins such as transcription factors and phospho-transferases. We also identified the genes encoding distinct proteins via AS and confirmed the translation of some isoforms via a proteomic analysis, suggesting potential AS-mediated neo-functionalization of some genes during infection. Comprehensive profiling of the pattern of genome-wide AS during multiple stages of rice-M. oryzae interaction established a foundational resource that will help investigate the role and regulation of AS during rice infection.

Ecological mechanisms governing inheritance of bacterial and fungal communities from seed to seed in rice are revealed. The inherited microbes dominate each community at the ripening stage, and parental seeds and stem endosphere are major sources.

Abstract Low power IoT communication signals (e.g., Bluetooth and ZigBee) may seriously suffer from the presence of high‐power co‐channel interference like wireless local area network (WLAN) signal. They may effectively avoid WLAN interference by exploiting dynamic characteristics of WLAN traffic. Representing the arrival/departure of WLAN users using an M/M/m/m queueing structure, the characterization of large‐scale dynamics of WLAN channel occupancy is considered. The characterization of small‐scale dynamics of WLAN channel occupancy is also considered by generating WLAN signal using a two‐state semi‐Markovian process. Simulation results show that the proposed model generates WLAN signal having similar statistical characteristics to those of real WLAN signal.

Abstract Lichen-forming fungi are mutualistic symbionts of green algae or cyanobacteria. We report the comparative analysis of six genomes of lichen-forming fungi in classes Eurotiomycetes and Lecanoromycetes to identify genomic information related to their symbiotic lifestyle. The lichen-forming fungi exhibited genome reduction via the loss of dispensable genes encoding plant-cell-wall-degrading enzymes, sugar transporters, and transcription factors. The loss of these genes reflects the symbiotic biology of lichens, such as the absence of pectin in the algal cell wall and obtaining specific sugars from photosynthetic partners. The lichens also gained many lineage- and species-specific genes, including those encoding small secreted proteins. These genes are primarily induced during the early stage of lichen symbiosis, indicating their significant roles in the establishment of lichen symbiosis.Our findings provide comprehensive genomic information for six lichen-forming fungi and novel insights into lichen biology and the evolution of symbiosis.

Synergid cells undergo programmed cell death following pollen tube reception and successful fertilization. Here the authors show that premature synergid cell death is prevented by the mitogen activated protein kinase MPK4 and the R protein SUMM2 which maintain ROS homeostasis in Arabidopsis.

Abstract Background Long non-coding RNAs (lncRNAs) play essential roles in developmental processes and disease development at the transcriptional and post-transcriptional levels across diverse taxa. However, only few studies have profiled fungal lncRNAs in a genome-wide manner during host infection. Results Infection-associated lncRNAs were identified using lncRNA profiling over six stages of host infection (e.g., vegetative growth, pre-penetration, biotrophic, and necrotrophic stages) in the model pathogenic fungus, Magnaporthe oryzae. We identified 2,601 novel lncRNAs, including 1,286 antisense lncRNAs and 980 intergenic lncRNAs. Among the identified lncRNAs, 755 were expressed in a stage-specific manner and 560 were infection-specifically expressed lncRNAs (ISELs). To decipher the potential roles of lncRNAs during infection, we identified 365 protein-coding genes that were associated with 214 ISELs. Analysis of the predicted functions of these associated genes suggested that lncRNAs regulate pathogenesis-related genes, including xylanases and effectors. Conclusions The ISELs and their associated genes provide a comprehensive view of lncRNAs during fungal pathogen-plant interactions. This study expands new insights into the role of lncRNAs in the rice blast fungus, as well as other plant pathogenic fungi.

ABSTRACTRheumatoid arthritis (RA) is closely associated with the oral and gut microbiomes. Fungal cell wall components initiate inflammatory arthritis in mouse models. However, little is known regarding the role of the fungal community in the pathogenesis of RA. To evaluate the association between RA and the gut microbiome, investigations of bacterial and fungal communities in patients with RA are necessary. Therefore, we investigated the compositions and associations of fecal bacterial and fungal communities in 30 healthy controls and 99 patients with RA. The relative abundances of Bifidobacterium and Blautia decreased, whereas the relative abundance of Streptococcus increased, in patients with RA. The relative abundance of Candida in the fecal fungal community was higher in patients with RA than in healthy controls, while the relative abundance of Aspergillus was higher in healthy controls than in patients with RA. Candida species-specific gene amplification showed that C. albicans was the most abundant species of Candida. Ordination analysis and random forest classification models supported the findings of structural changes in bacterial and fungal communities. Aspergillus was the core fecal fungal genus in healthy controls, although Saccharomyces spp. are typically predominant in Western cohorts. In addition, bacterial–fungal association analyses showed that the hub node had shifted from fungi to bacteria in patients with RA. The finding of fungal dysbiosis in patients with RA suggests that fungi play critical roles in the fecal microbial communities and pathogenesis of RA.

RNA interference (RNAi) is divided into canonical, Dicer-dependent and non-canonical, Dicer-independent pathways according to Dicer protein dependency. However, sRNAs processed in a Dicer-independent manner have not been reported in plant pathogenic fungi, including Magnaporthe oryzae. We comparatively profiled the Dicer-dependent and -independent sRNAs of M. oryzae. Dicer-dependent sRNAs were 19–24-nt in length, had low strand-specificity, and showed a preference for uracil at the 5′-end. By contrast, Dicer-independent sRNAs presented irregular patterns in length distribution, high strand-specificity, and a preference for cytosine at the penultimate position. Dicer-dependent sRNA loci were mainly associated with LTR-transposons, while Dicer-independent sRNAs were associated with protein-coding genes and transposons. We identified MoERI-1, a non-canonical RNAi component, and profiled the sRNA and mRNA transcriptomes of ΔMoeri-1 at the mycelia and conidiation stages, as the mutant showed increased conidiation. We found that genes involved in conidiation and cell cycle were upregulated by MoERI-1 deletion. Furthermore, a comparison between sRNA and mRNA transcriptome revealed that MoERI-1-dependent sRNAs mediate the regulation of gene expression. Overall, these results showed that M. oryzae has non-canonical RNAi pathways distinct to the Dicer-dependent manner and exploits MoERI-1-dependent sRNAs to regulate the conidiation process.

Microbial co-occurrence network analysis is being widely used for data exploration in plant microbiome research. Still, challenges lie in how well these microbial networks represent natural microbial communities and how well we can interpret and extract eco-evolutionary insights from the networks. Although many technical solutions have been proposed, in this perspective, we touch on the grave problem of kingdom-level bias in network representation and interpretation. We underscore the eco-evolutionary significance of using cross-kingdom (bacterial-fungal) co-occurrence networks to increase the network’s representability of natural communities. To do so, we demonstrate how ecosystem-level interpretation of plant microbiome evolution changes with and without multi-kingdom analysis. Then, to overcome oversimplified interpretation of the networks stemming from the stereotypical dichotomy between bacteria and fungi, we recommend three avenues for ecological interpretation: (1) understanding dynamics and mechanisms of co-occurrence networks through generalized Lotka-Volterra and consumer-resource models, (2) finding alternative ecological explanations for individual negative and positive fungal-bacterial edges, and (3) connecting cross-kingdom networks to abiotic and biotic (host) environments.

To establish an infection, fungal pathogens must recognize diverse signals from host surfaces. The rice blast fungus, Magnaporthe oryzae, is one of the best models studying host-pathogen interactions. This fungus recognizes physical or chemical signals from the host surfaces and initiates the development of an infection structure called appressorium. Here, we found that protein MoAfo1(appressorium formation, MGG_10422) was involved in sensing signal molecules such as cutin monomers and long chain primary alcohols required for appressorium formation. The knockout mutant (ΔMoafo1) formed a few abnormal appressoria on the onion and rice sheath surfaces. However, it produced normal appressoria on the surface of rice leaves. MoAfo1 localized to the membranes of the cytoplasm and vacuole-like organelles in conidia and appressoria. Additionally, the ΔMoafo1 mutant showed defects in appressorium morphology, appressorium penetration, invasive growth, and pathogenicity. These multiple defects might be partially due to failure to respond properly to oxidative stress. These findings broaden our understanding of the fungal mechanisms at play in the recognition of the host surface during rice blast infection.

Abstract Background Plant pathogenic isolates of Rhizoctonia solani anastomosis group 1-intraspecific group IA (AG1-IA) infect a wide range of crops causing diseases such as rice sheath blight (ShB). ShB has become a serious disease in rice production worldwide. Additional genome sequences of the rice-infecting R. solani isolates from different geographical regions will facilitate the identification of important pathogenicity-related genes in the fungus. Results Rice-infecting R. solani isolates B2 (USA), ADB (India), WGL (India), and YN-7 (China) were selected for whole-genome sequencing. Single-Molecule Real-Time (SMRT) and Illumina sequencing were used for de novo sequencing of the B2 genome. The genomes of the other three isolates were then sequenced with Illumina technology and assembled using the B2 genome as a reference. The four genomes ranged from 38.9 to 45.0 Mbp in size, contained 9715 to 11,505 protein-coding genes, and shared 5812 conserved orthogroups. The proportion of transposable elements (TEs) and average length of TE sequences in the B2 genome was nearly 3 times and 2 times greater, respectively, than those of ADB, WGL and YN-7. Although 818 to 888 putative secreted proteins were identified in the four isolates, only 30% of them were predicted to be small secreted proteins, which is a smaller proportion than what is usually found in the genomes of cereal necrotrophic fungi. Despite a lack of putative secondary metabolite biosynthesis gene clusters, the rice-infecting R. solani genomes were predicted to contain the most carbohydrate-active enzyme (CAZyme) genes among all 27 fungal genomes used in the comparative analysis. Specifically, extensive enrichment of pectin/homogalacturonan modification genes were found in all four rice-infecting R. solani genomes. Conclusion Four R. solani genomes were sequenced, annotated, and compared to other fungal genomes to identify distinctive genomic features that may contribute to the pathogenicity of rice-infecting R. solani. Our analyses provided evidence that genomic conservation of R. solani genomes among neighboring AGs was more diversified than among AG1-IA isolates and the presence of numerous predicted pectin modification genes in the rice-infecting R. solani genomes that may contribute to the wide host range and virulence of this necrotrophic fungal pathogen.

Abstract Background Lyso-phosphatidylethanolamine (LPE) is a natural phospholipid that functions in the early stages of plant senescence. Plant innate immunity and early leaf senescence share molecular components. To reveal conserved mechanisms that link-up both processes, we tried to unravel to what extent LPE coordinates defense response and by what mode of action. Result We found that LPE-treatment induces signaling and biosynthesis gene expression of the defensive hormone salicylic acid (SA). However, jasmonic acid and ethylene triggered gene induction levels are indistinguishable from the control. In accordance with gene induction for SA, oxidative stress, and reactive oxygen species (ROS) production, we detected raised in-situ hydrogen peroxide levels following LPE-application. Yet, ROS-burst assays of LPE-pretreated plants revealed a reduced release of ROS after PAMP-administration suggesting that LPE interferes with an oxidative burst. Our data refer to a priming effect of LPE on SA/ROS-associated genomic loci that encode pivotal factors in early senescence and considerably improve plant basal immunity. Thus, we challenged Arabidopsis thaliana with the hemibiotrophic pathogen Pseudomonas syringae. Consistently, we found an increased resistance in the LPE-pretreated Arabidopsis plants compared to the mock-pretreated control. Conclusions Our results underscore a beneficial effect of LPE on plant innate immunity against hemibiotrophs. Given the resistance-promoting effect of exogenously applied LPE, this bio-agent bears the potential of being applied as a valuable tool for the genetic activation of defense-associated traits.

Plant pathogens secrete various effectors to manipulate host immunity. Here, Kim et al. describe two Magnaporthe oryzae effectors that translocate into the nuclei of infected rice cells and reprogram expression of immunity-associated genes, increasing susceptibility to hemibiotrophic pathogens.

Laccases (EC 1.10.3.2), a group of multi-copper oxidases (MCOs), play multiple biological functions and widely exist in many species. Fungal laccases have been extensively studied for their industrial applications, however, there was no database specially focused on fungal laccases. To provide a comparative genomics platform for fungal laccases, we have developed a comparative genomics platform for laccases and MCOs (http://laccase.riceblast.snu.ac.kr/). Based on protein domain profiles of characterized sequences, 3,571 laccases were predicted from 690 genomes including 253 fungi. The number of putative laccases and their properties exhibited dynamic distribution across the taxonomy. A total of 505 laccases from 68 genomes were selected and subjected to phylogenetic analysis. As a result, four clades comprised of nine subclades were phylogenetically grouped by their putative functions and analyzed at the sequence level. Our work would provide a workbench for putative laccases mainly focused on the fungal kingdom as well as a new perspective in the identification and classification of putative laccases and MCOs.

Phosphatases are key components in cellular signalling networks. Here, the authors present a systematic functional analysis of phosphatases of the fungal pathogen Cryptococcus neoformans, revealing roles in virulence, stress responses, O-mannosylation, retromer function and other processes.

Alternaria alternata JS-1623 is an endophytic fungus isolated from a stem tissue of Korean fir, Abies koreana. Ethyl acetate extracts of culture filtrates exhibited anti-inflammatory activity in LPS induced microglia BV-2 cell without cytotoxicity. Here we report a 33.67 Mb sized genome assembly of JS-1623 comprised of 13 scaffolds with N50 of 4.96 Mb, and 92.41% of BUSCO completeness. GC contents were 50.97%. Of the 11,197 genes annotated, gene families related to the biosynthesis of secondary metabolites or transcription factors were identified.

The genome of the rice blast fungus Magnaporthe oryzae contains several types of transposable elements (TEs), and some TEs cause genetic variation that allows M. oryzae to evade host detection. We studied how five abundant TEs in rice pathogens, Pot3, Pot2, MAGGY, Line-like element (MGL) and Mg-SINE, are expressed under diverse conditions related to growth, development, and stress. Expression of Pot3 and Pot2 was activated in germinated conidia and mycelia treated with tricyclazole. Retrotransposon MAGGY was highly expressed in appressoria and tricyclazole-treated mycelia. MAGGY and Pot2 were also activated during the early and late stages of perithecia development. MGL was up-regulated in conidia and during conidial germination but not during appressorium formation. No noticeable expression of Mg-SINE was observed under most conditions. Our results should help investigate if and how condition-specific expressions of some TEs contribute to the biology and evolution of M. oryzae.

Abstract Background Plant-associated microbiomes, which are shaped by host and environmental factors, support their hosts by providing nutrients and attenuating abiotic and biotic stresses. Although host genetic factors involved in plant growth and immunity are known to shape compositions of microbial communities, the effects of host evolution on microbial communities are not well understood. Results We show evidence that both host speciation and domestication shape seed bacterial and fungal community structures. Genome types of rice contributed to compositional variations of both communities, showing a significant phylosymbiosis with microbial composition. Following the domestication, abundance inequality of bacterial and fungal communities also commonly increased. However, composition of bacterial community was relatively conserved, whereas fungal membership was dramatically changed. These domestication effects were further corroborated when analyzed by a random forest model. With these changes, hub taxa of inter-kingdom networks were also shifted from fungi to bacteria by domestication. Furthermore, maternal inheritance of microbiota was revealed as a major path of microbial transmission across generations. Conclusions Our findings show that evolutionary processes stochastically affect overall composition of microbial communities, whereas dramatic changes in environments during domestication contribute to assembly of microbiotas in deterministic ways in rice seed. This study further provides new insights on host evolution and microbiome, the starting point of the holobiome of plants, microbial communities, and surrounding environments. Video Abtract.

The rice blast (fungal pathogen: Magnaporthe oryzae and host: Oryza sativa) is one of the most important model pathosystems for understanding plant–microbe interactions. Although both genome sequences were published as the first cases of pathogen and host, only a few in planta transcriptome data during infection are available. Due to technical difficulties, previously reported fungal transcriptome data are not highly qualified to comprehensively profile the expression of fungal genes during infection. Here, we report the high-quality transcriptomes of M. oryzae and rice during infection using a sheath infection-based RNA sequencing approach. This comprehensive expression profiling of the fungal pathogen and its host will provide a better platform for understanding the plant–microbe interactions at the genomic level and serve as a valuable resource for the research community.

An endolichenic fungus, Xylaria grammica strain EL000614, showed strong nematicidal effects against plant pathogenic nematode, Meloidogyne incognita by producing grammicin. We report genome assembly of X. grammica EL000614 comprised of 25 scaffolds with a total length of 54.73 Mb, N50 of 4.60 Mb, and 99.8% of BUSCO completeness. GC contents of this genome were 44.02%. Gene families associated with biosynthesis of secondary metabolites or regulatory proteins were identified out of 13,730 gene models predicted.

The myeloblastosis (MYB) transcription factor family is evolutionarily conserved among plants, animals, and fungi, and contributes to their growth and development. We identified and analyzed 10 putative MYB genes in Magnaporthe oryzae (MoMYB) and determined their phylogenetic relationships, revealing high divergence and variability. Although MYB domains are generally defined by three tandem repeats, MoMYBs contain one or two weakly conserved repeats embedded in extensive disordered regions. We characterized the secondary domain organization, disordered segments, and functional contributions of each MoMYB. During infection, MoMYBs are distinctively expressed and can be subdivided into two clades of being either up- or down-regulated. Among these, MoMYB1 and MoMYB8 are up-regulated during infection and vegetative growth, respectively. We found MoMYB1 localized predominantly to the cytosol during the formation of infection structures. ΔMomyb1 exhibited reduced virulence on intact rice leaves corresponding to the diminished ability to form hypha-driven appressorium (HDA). We discovered that MoMYB1 regulates HDA formation on hard, hydrophobic surfaces, whereas host surfaces partially restored HDA formation in ΔMomyb1. Lipid droplet accumulation in hyphal tips and expression of HDA-associated genes were strongly perturbed in ΔMomyb1 indicating genetic interaction of MoMYB1 with downstream components critical to HDA formation. We also found that MoMYB8 is necessary for fungal growth, dark-induced melanization of hyphae, and involved in higher abiotic stress tolerance. Taken together, we revealed a multifaceted picture of the MoMYB family, wherein a low degree of conservation has led to the development of distinct structures and functions, ranging from fungal growth to virulence.

The soil environment determines plants’ health and performance during their life cycle. Therefore, ecological understanding on variations in soil environments, including physical, chemical, and biological properties, is crucial for managing agricultural fields. Here, we present a comprehensive and extensive blueprint of the bacterial, archaeal, and fungal communities in rice paddy soils with differing soil types and chemical properties. We discovered that natural variations of soil nutrients are important factors shaping microbial diversity. The responses of microbial diversity to soil nutrients were related to the distribution of microbial trophic lifestyles (oligotrophy and copiotrophy) in each community. The compositional changes of bacterial and archaeal communities in response to soil nutrients were mainly governed by oligotrophs, whereas copiotrophs were mainly involved in fungal compositional changes. Compositional shift of microbial communities by fertilization is linked to switching of microbial trophic lifestyles. Random forest models demonstrated that depletion of prokaryotic oligotrophs and enrichment of fungal copiotrophs are the dominant responses to fertilization in low-nutrient conditions, whereas enrichment of putative copiotrophs was important in high-nutrient conditions. Network inference also revealed that trophic lifestyle switching appertains to decreases in intra- and inter-kingdom microbial associations, diminished network connectivity, and switching of hub nodes from oligotrophs to copiotrophs. Our work provides ecological insight into how soil nutrient-driven variations in microbial communities affect soil health in modern agricultural systems.

Seeds harbor not only genetic information about plants but also microbial communities affecting plants’ vigor. Knowledge on the movement and formation of seed microbial communities during plant development remains insufficient. Here, we address this knowledge gap by investigating endophytic bacterial and fungal communities of seeds, seedlings, and adult rice plants. We found that seed coats act as microbial niches for seed bacterial and fungal communities. The presence or absence of the seed coat affected taxonomic composition and diversity of bacterial and fungal communities associated with seeds and seedlings. Ordination analysis showed that niche differentiation between above- and belowground compartments leads to compositional differences in endophytic bacterial and fungal communities originating from seeds. Longitudinal tracking of the composition of microbial communities from field-grown rice revealed that bacterial and fungal communities originating from seeds persist in the leaf, stem, and root endospheres throughout the life cycle. Our study provides ecological insights into the assembly of the initial endophytic microbial communities of plants from seeds.

ABSTRACT Colletotrichum scovillei, an ascomycete phytopathogenic fungus, is the main causal agent of serious yield losses of economic crops worldwide. The fungus causes anthracnose disease on several fruits, including peppers. However, little is known regarding the underlying molecular mechanisms involved in the development of anthracnose caused by this fungus. In an initial step toward understanding the development of anthracnose on pepper fruits, we retrieved 624 transcription factors (TFs) from the whole genome of C. scovillei and comparatively analyzed the entire repertoire of TFs among phytopathogenic fungi. Evolution and proliferation of members of the homeobox-like superfamily, including homeobox (HOX) TFs that regulate the development of eukaryotic organisms, were demonstrated in the genus Colletotrichum. C. scovillei was found to contain 10 HOX TF genes (CsHOX1 to CsHOX10), which were functionally characterized using deletion mutants of each CsHOX gene. Notably, CsHOX1 was identified as a pathogenicity factor required for the suppression of host defense mechanisms, which represents a new role for HOX TFs in pathogenic fungi. CsHOX2 and CsHOX7 were found to play essential roles in conidiation and appressorium development, respectively, in a stage-specific manner in C. scovillei. Our study provides a molecular basis for understanding the mechanisms associated with the development of anthracnose on fruits caused by C. scovillei, which will aid in the development of novel approaches for disease management. IMPORTANCE The ascomycete phytopathogenic fungus, Colletotrichum scovillei, causes serious yield loss on peppers. However, little is known about molecular mechanisms involved in the development of anthracnose caused by this fungus. We analyzed whole-genome sequences of C. scovillei and isolated 624 putative TFs, revealing the existence of 10 homeobox (HOX) transcription factor (TF) genes. We found that CsHOX1 is a pathogenicity factor required for the suppression of host defense mechanism, which represents a new role for HOX TFs in pathogenic fungi. We also found that CsHOX2 and CsHOX7 play essential roles in conidiation and appressorium development, respectively, in a stage-specific manner in C. scovillei. Our study contributes to understanding the mechanisms associated with the development of anthracnose on fruits caused by C. scovillei, which will aid for initiating novel approaches for disease management.

The plant microbiome, the consortium of microbes surrounding a plant, has potential for improving crop productivity and sustainability. Despite the necessity of agriculturally applicable microbiomes, plant microbiome studies have been conducted in noncrop plants because of the relative easiness of research. However, in order to make plant microbiomes useful for agriculture, a crop plant-based model is needed. In parallel, overlooked parts of microbiomes other than the bacteria-centered research need to be considered to expand our understandings on microbiomes to the ecosystem level. Beyond the microbial composition, functional properties of microbiomes over time and space will help us to select appropriate microbes that can support crop plants by providing stage-specific functions. Less explored communities such as fungi and protists also can provide novel insights on compositional and functional dynamics of each community, including interkingdom or multitrophic interactions. Finally, identification of host factors on functional microbiomes using genetic information of both the host and the microbiomes will shift host-centered breeding to parallel breeding of host and microbiome. This review will give basic and collective information on the rice microbiome and foster the establishment of a crop plant-based model to meet agricultural needs.

Abstract Deployment of small moving cells (SMCs) has been considered in advanced cellular systems, where wireless backhaul links are required between base stations and SMCs. In this paper, we consider signal transmission by means of multiuser beamforming in the wireless backhaul link. We generate the beam weight in an eigen-direction of weighted combination of short- and long-term channel information of the backhaul link. The beam weight can maximize the average signal-to-leakage-plus-noise ratio (SLNR), while providing the transmission robust to SMC mobility. We analyze the performance of the proposed scheme in terms of the average signal-to-interference-plus-noise ratio (SINR) and optimize the transmit power by iterative water-filling. Finally, we verify the performance of the proposed scheme by computer simulation.

Objectives The validity of instruments measuring socioeconomic position (SEP) has been a major area of concern in research on cardiovascular health disparities. The purpose of this systematic review is to identify the current status of the methods used to measure SEP in research on cardiovascular health disparities in Korea and to provide directions for future research. Methods Relevant articles were obtained through electronic database searches with manual searches of reference lists and no restriction on the date of publication. SEP indicators were categorized into compositional, contextual, composite, and life-course measures. Results Forty-eight studies published from 2003 to 2018 satisfied the review criteria. Studies utilizing compositional measures mainly relied on a limited number of SEP parameters. In addition, these measures hardly addressed the time-varying and subjective features of SEP. Finding valid contextual measures at the organizational, community, and societal levels that are appropriate to Korea’s context remains a challenge, and these are rarely modeled simultaneously. Studies have rarely focused on composite and life-course measures. Conclusions Future studies should develop and utilize valid compositional and contextual measures and appraise social patterns that vary across time, place, and culture using such measures. Studies should also consider multilevel influences, adding a focus on the interactions between different levels of intertwined SEP factors to advance the design of research. More attention should be given to composite and life-course measures.

Molecular oxygen functions as an electron acceptor for aerobic respiration and a substrate for key metabolisms and cellular processes. Most eukaryotes develop direct or indirect oxygen sensors and reprogram transcriptional and translational metabolisms to adapt to altered oxygen availability under varying oxygen concentrations. Human fungal pathogens manipulate transcriptional levels of genes related to virulence as well as oxygen-dependent metabolisms such as ergosterol homeostasis when they are confronted with oxygen limitation (hypoxia) during infection. Oxygen states in plant tissues also vary depending on site, species, and external environment, potentially providing hypoxia to plant pathogens during infection. In this review, knowledge on the regulation of oxygen sensing and adaptive mechanisms in eukaryotes and nascent understanding of hypoxic responses in plant pathogens are summarized and discussed.

The rice blast pathogen Magnaporthe oryzae is a global threat to rice production. Here we characterized RHO2 gene (MGG_02457) that belongs to the Rho GTPase family, using a deletion mutant. This mutant ΔMorho2 exhibited no defects in conidiation and germination but developed only 6% of appressoria in response to a hydrophobic surface when compared to the wild-type progenitor. This result indicates that MoRHO2 plays a role in appressorium development. Furthermore, exogenous cAMP treatment on the mutant led to appressoria that exhibited abnormal morphology on both hydrophobic and hydrophilic surfaces. These outcomes suggested the involvement of MoRHO2 in cAMP-mediated appressorium development. ΔMorho2 mutation also delayed the development of appressorium-like structures (ALS) at hyphal tips on hydrophobic surface, which were also abnormally shaped. These results suggested that MoRHO2 is involved in morphological development of appressoria and ALS from conidia and hyphae, respectively. As expected, ΔMorho2 mutant was defective in plant penetration, but was still able to cause lesions, albeit at a reduced rate on wounded plants. These results implied that MoRHO2 plays a role in M. oryzae virulence as well.

Colletotrichum has a broad host range and causes major yield losses of crops. The fungus Colletotrichum gloeosporioides is associated with anthracnose on Chinese fir. In this study, we present a high-quality draft genome sequence of C. gloeosporioides sensu stricto SMCG1#C, providing a reference genomic data for further research on anthracnose of Chinese fir and other hosts.

The causal agent of root and butt rot of conifer trees, Heterobasidion annosum, is widespread in boreal forests and economically responsible for annual loss of approximately 50 million euros to forest industries in Finland alone and much more at European level. In order to further understand the pathobiology of this fungus at the genome level, a Finnish isolate of H. annosum sensu stricto (isolate 03012) was sequenced and analyzed with the genome sequences of 23 white-rot and 13 brown-rot fungi. The draft genome assembly of H. annosum has a size of 31.01 Mb, containing 11,453 predicted genes. Whole genome alignment showed that 84.38% of H. annosum genome sequences were aligned with those of previously sequenced H. irregulare TC 32-1 counterparts. The result is further supported by the protein sequence clustering analysis which revealed that the two genomes share 6719 out of 8647 clusters. When sequencing reads of H. annosum were aligned against the genome sequences of H. irregulare, six single nucleotide polymorphisms were found in every 1 kb, on average. In addition, 98.68% of SNPs were found to be homo-variants, suggesting that the two species have long evolved from different niches. Gene family analysis revealed that most of the white-rot fungi investigated had more gene families involved in lignin degradation or modification, including laccases and peroxidase. Comparative analysis of the two Heterobasidion spp. as well as white-/brown-rot fungi would provide new insights for understanding the pathobiology of the conifer tree pathogen.

Abstract Background Transposable elements are major evolutionary forces which can cause new genome structure and species diversification. The role of transposable elements in the expansion of nucleotide-binding and leucine-rich-repeat proteins (NLRs), the major disease-resistance gene families, has been unexplored in plants. Results We report two high-quality de novo genomes (Capsicum baccatum and C. chinense) and an improved reference genome (C. annuum) for peppers. Dynamic genome rearrangements involving translocations among chromosomes 3, 5, and 9 were detected in comparison between C. baccatum and the two other peppers. The amplification of athila LTR-retrotransposons, members of the gypsy superfamily, led to genome expansion in C. baccatum. In-depth genome-wide comparison of genes and repeats unveiled that the copy numbers of NLRs were greatly increased by LTR-retrotransposon-mediated retroduplication. Moreover, retroduplicated NLRs are abundant across the angiosperms and, in most cases, are lineage-specific. Conclusions Our study reveals that retroduplication has played key roles for the massive emergence of NLR genes including functional disease-resistance genes in pepper plants.

Abstract Background The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. Results Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications. Conclusion The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

Abstract 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.

Histone methylation plays important roles in regulating chromatin dynamics and transcription in eukaryotes. Implication of histone modifications in fungal pathogenesis is, however, beginning to emerge. Here, we report identification and functional analysis of a putative JmjC-domain-containing histone demethylase in Magnaporthe oryzae. Through bioinformatics analysis, we identified seven genes, which encode putative histone demethylases containing JmjC domain. Deletion of one gene, MoJMJ1, belonging to JARID group, resulted in defects in vegetative growth, asexual reproduction, appressorium formation as well as invasive growth in the fungus. Western blot analysis showed that global H3K4me3 level increased in the deletion mutant, compared to wild-type strain, indicating histone demethylase activity of MoJMJ1. Introduction of MoJMJ1 gene into ΔMojmj1 restored defects in pre-penetration developments including appressorium formation, indicating the importance of histone demethylation through MoJMJ1 during infection-specific morphogenesis. However, defects in penetration and invasive growth were not complemented. We discuss such incomplete complementation in detail here. Our work on MoJMJ1 provides insights into H3K4me3-mediated regulation of infection-specific development in the plant pathogenic fungus.

Magnaporthe oryzae infects rice, wheat, and many grass species in the Poaceae family by secreting protein effectors. Here, we analyzed the distribution, sequence variation, and genomic context of effector candidate (EFC) genes in 31 isolates that represent five pathotypes of M. oryzae, three isolates of M. grisea, a sister species of M. oryzae, and one strain each for eight species in the family Magnaporthaceae to investigate how the host range expansion of M. oryzae has likely affected the evolution of effectors. We used the EFC genes of M. oryzae strain 70-15, whose genome has served as a reference for many comparative genomics analyses, to identify their homologs in these strains. We also analyzed the previously characterized avirulence (AVR) genes and single-copy orthologous (SCO) genes in these strains, which showed that the EFC and AVR genes evolved faster than the SCO genes. The EFC and AVR repertoires among M. oryzae pathotypes varied widely probably because adaptation to individual hosts exerted different types of selection pressure. Repetitive DNA elements appeared to have caused the variation of some EFC genes. Lastly, we analyzed expression patterns of the AVR and EFC genes to test the hypothesis that such genes are preferentially expressed during host infection. This comprehensive dataset serves as a foundation for future studies on the genetic basis of the evolution and host specialization in M. oryzae.

Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis. Here, the authors study the roles played by 129 putative kinases in the growth and virulence of C. neoformans, identifying potential targets for development of anticryptococcal drugs.

Lesion mimic mutants (LMMs) commonly exhibit spontaneous cell death similar to the hypersensitive defense response that occurs in plants in response to pathogen infection. Several lesion mimic mutants have been isolated and characterized, but their molecular mechanisms remain largely unknown. Here, a spotted leaf sheath (sles) mutant derived from japonica cultivar Koshihikari is described. The sles phenotype differed from that of other LMMs in that lesion mimic spots were observed on the leaf sheath rather than on leaves. The sles mutant displayed early senescence, as shown, by color loss in the mesophyll cells, a decrease in chlorophyll content, and upregulation of chlorophyll degradation-related and senescence-associated genes. ROS content was also elevated, corresponding to increased expression of genes encoding ROS-generating enzymes. Pathogenesis-related genes were also activated and showed improved resistance to pathogen infection on the leaf sheath. Genetic analysis revealed that the mutant phenotype was controlled by a single recessive nuclear gene. Genetic mapping and sequence analysis showed that a single nucleotide substitution in the sixth exon of LOC_Os07g25680 was responsible for the sles mutant phenotype and this was confirmed by T-DNA insertion line. Taken together, our results revealed that SLES was associated with the formation of lesion mimic spots on the leaf sheath resulting early senescence and defense responses. Further examination of SLES will facilitate a better understanding of the molecular mechanisms involved in ROS homeostasis and may also provide opportunities to improve pathogen resistance in rice.

Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK) of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase), demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

Genomes contain a large number of unique genes which have not been found in other species. Although the origin of such “orphan” genes remains unclear, they are thought to be involved in species-specific adaptive processes. Here, we analyzed seven orphan genes (MoSPC1 to MoSPC7) prioritized based on in planta expressed sequence tag data in the rice blast fungus, Magnaporthe oryzae. Expression analysis using qRT-PCR confirmed the expression of four genes (MoSPC1, MoSPC2, MoSPC3 and MoSPC7) during plant infection. However, individual deletion mutants of these four genes did not differ from the wild-type strain for all phenotypes examined, including pathogenicity. The length, GC contents, codon adaptation index and expression during mycelial growth of the four genes suggest that these genes formed during the evolutionary history of M. oryzae. Synteny analyses using closely related fungal species corroborated the notion that these genes evolved de novo in the M. oryzae genome. In this report, we discuss our inability to detect phenotypic changes in the four deletion mutants. Based on these results, the four orphan genes may be products of de novo gene birth processes, and their adaptive potential is in the course of being tested for retention or extinction through natural selection.

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.