Effects of ultra-strong static magnetic field on the gut microbiota of humans and mice.

To explore the effect of ultra-strong static magnetic field on gut microbiota, 16 T static magnetic field was used to study the changes in the structure and composition of human and mouse gut microbiota in this environment. In the mouse gut microbiota, at the genus level, the magnetic field significantly decreased the relative abundances of Escherichia-Shigella, Lactobacillus, Enterococcus, Burkholderia-Caballeronia-Paraburkholderia, Parasutterella, and Ralstonia and significantly increased those of Parabacteroides, Alloprevotella, Alistipes, Odoribacter, Bacteroides, Mucispirillum, Sutterella, and Prevotellaceae_UCG-001. Similarly, at the genus level, the relative abundances of Bacteroides, Parabacteroides, Romboutsia, and Streptococcus significantly decreased in the human gut microbiota. Contrary to the changing trend of the abundance in the mouse gut, the abundances of Bacteroides and Parabacteroides in the human gut were significantly reduced under magnetic field. The BugBase phenotypic prediction analysis showed that the relative abundances of five phenotypes, including anaerobism, mobile elements, potential pathogenicity, stress-tolerant, and biofilm formation, changed significantly in the mouse gut microbiota, while the relative abundances of two phenotypes, including Gram-positive and Gram-negative phenotypes, changed significantly in the human gut microbiota. The 16 T magnetic field could differently affect the composition, structure, and phenotypes of gut microbiota in human and mice, suggesting the importance of model selection in studying the biological effects of magnetic field.

HAG Effector Evolution in Pyricularia Species and Plant Cell Death Suppression by HAG4.

Seventy host-adapted gene (HAG) effector family members from Pyricularia species are found in P. oryzae and three closely related species (isolates LS and 18-2 from an unknown Pyricularia sp., P. grisea, and P. pennisetigena) that share at least eight orthologous HAG family members with P. oryzae. The genome sequence of a more distantly related species, P. penniseti, lacks HAG genes, suggesting a time frame for the origin of the gene family in the genus. In P. oryzae, HAG4 is uniquely found in the genetic lineage that contains populations adapted to Setaria and Oryza hosts. We find a nearly identical HAG4 allele in a P. grisea isolate, suggesting transfer of HAG4 from P. grisea to P. oryzae. HAG4 encodes a suppressor of plant cell death. Yeast two-hybrid screens with several HAG genes independently identify common interacting clones from a rice complementary DNA library, suggesting conservation of protein surface motifs between HAG homologs with as little as 40% protein sequence identity. HAG family orthologs have diverged rapidly and HAG15 orthologs display unusually high rates of sequence divergence compared with adjacent genes suggesting gene-specific accelerated divergence. The sequence diversity of the HAG homologs in Pyricularia species provides a resource for examining mechanisms of gene family evolution and the relationship to structural and functional evolution of HAG effector family activity. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Development of versatile and efficient genetic tools for the marine-derived fungus Aspergillus terreus RA2905.

Marine-derived Aspergillus terreus produces a variety of structurally novel secondary metabolites, most of which show unique biological activities. However, the lack of efficient genetic tools limits the discovery of new compounds, the elucidation of involved biosynthesis mechanism, as well as the strain engineering efforts. Therefore, in this study, we first established both an effective PEG-mediated chemical transformation system of protoplasts and an electroporation system of conidia in a marine-derived fungus A. terreus RA2905. To overcome the insensitivity of RA2905 to fungicides, the uracil auxotrophy strain (pyrG gene deletion mutant, ΔpyrG) was constructed using PEG-mediated transformation system, and using ΔpyrG as the genetic background, the methyltransferase gene laeA-overexpression transformants were further constructed through both PEG- and electroporation-mediated transformations, which showed enhanced terrein production. Besides, in this study, an efficient CRISPR/Cas9 genome-editing system was established for the first time in A. terreus, and a higher gene deletion efficiency of 71% for APSES transcription factor gene stuA could be achieved when using short homologous arms compared with conventional long homologous ones. In addition, using a non-integrative Cas9 plasmid, another efficient and marker-free genome-editing system was established, which allowing repeatable and unlimited genetic manipulation in A. terreus. Using the marker-free genome-editing system, we successfully developed the ΔpyrGΔku70 double-deletion mutant in RA2905, which could further improve gene deletion efficiency. In conclusion, efficient genetic manipulation systems along with a variety of functional mutants were developed in this study, which would significantly expedite both theoretical and applied researches in not only A. terreus but also other marine-derived filamentous fungi.

Response of human gut microbiota under simulated microgravity.

The present study was conducted to investigate the influence of microgravity on human gut microbiota using 16S rRNA gene sequencing in vitro. The diamagnetic material magnetic levitation method was used to simulate weightless environment. The human gut microbiota was cultured under two different conditions: normal gravity (1 g), and simulated microgravity (0 g), which showed that both the richness (P = 0.04) and diversity (P = 0.0002) of human gut microbiota were significantly altered. As compared to the normal gravity, the simulated microgravity significantly reduced abundance of bacteria related to anti-inflammatory effects, such as Subdoligranulum, Faecalibacterium, Fusicatenibacter, Butyricicoccus, and Lachnospiraceae-NK4A136-0 group (P < 0.05), while significantly increased that of Alistipes and Eubacterium-Ventriosum-group (P < 0.05). Moreover, the Spearman's correlation analysis showed that there were more significantly correlated species (|r|≥ 0.5, P < 0.05) in normal gravity than that in the simulated microgravity. KEGG pathway analysis revealed that the microgravity significantly (P < 0.05) affected the metabolism of gut microbiota, such as the metabolism of pyrimidine, fatty acids, glyoxylate and dicarboxylate, peptidoglycan biosynthesis, and carbon fixation in photosynthetic organisms. These results suggested that the exposure to a microgravity environment might induce disturbances in human gut microbiota. KEY POINTS: • Using 16sRNA gene sequencing technology, it was found that magnetic levitation-simulated microgravity had varying degrees of influence on the abundance, diversity, species correlation, and KEGG pathways of human intestinal microbes. • Digital PCR can improve the detection rate of microorganisms with low abundance.

Evolution and Regulation of a Large Effector Family of Pyricularia oryzae.

Plant pathogen effectors play important roles in parasitism, including countering plant immunity. However, investigations of the emergence and diversification of fungal effectors across host-adapted populations has been limited. We previously identified a gene encoding a suppressor of plant cell death in Pyricularia oryzae (syn. Magnaporthe oryzae). Here, we report the gene is one of a 21-member gene family and we characterize sequence diversity in different populations. Within the rice pathogen population, nucleotide diversity is low, however; the majority of gene family members display presence-absence polymorphism or other null alleles. Gene family allelic diversity is greater between host-adapted populations and, thus, we named them host-adapted genes (HAGs). Multiple copies of HAGs were found in some genome assemblies and sequence divergence between the alleles in two cases suggested they were the result of repeat-induced point mutagenesis. Transfer of family members between populations and novel HAG haplotypes resulting from apparent recombination were observed. HAG family transcripts were induced in planta and a subset of HAGs are dependent on a key regulator of pathogenesis, PMK1. We also found differential intron splicing for some HAGs that would prevent ex planta protein expression. For some genes, spliced transcript was expressed in antiphase with an overlapping antisense transcript. Characterization of HAG expression patterns and allelic diversity reveal novel mechanisms for HAG regulation and mechanisms generating sequence diversity and novel allele combinations. This evidence of strong in planta-specific expression and selection operating on the HAG family is suggestive of a role in parasitism.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Magnaporthe Chitinase Interacts with a Rice Jacalin-Related Lectin to Promote Host Colonization.

The genome of rice blast fungus (Magnaporthe oryzae) encodes 15 glycoside hydrolase 18 family chitinases. In this study, we characterized the function of an M. oryzae extracellular chitinase, MoChi1, and its interaction with a host protein, OsMBL1, a jacalin-related Mannose-Binding Lectin (MBL) in rice (Oryza sativa). Deletion of MoChi1 resulted in reduced aerial hyphal formation and reduced virulence in rice by activating the expression of defense-responsive genes. We confirmed MoChi1 interaction with rice OsMBL1 in vitro and in vivo. OsMBL1 was induced by pathogen-associated molecular patterns and M. oryzae infection. Overexpression of OsMBL1 led to activation of rice defense-responsive genes and a chitin-induced reactive oxygen species burst, thereby enhancing resistance to M. oryzae Knockdown of OsMBL1 enhances susceptibility of rice plants to M. oryzae Furthermore, MoChi1 suppressed chitin-induced reactive oxygen species in rice cells and competed with OsMBL1 for chitin binding. Taken together, our study reveals a mechanism in which MoChi1 targets a host lectin to suppress rice immunity.

Transposable elements impact the population divergence of rice blast fungus Magnaporthe oryzae.

Dynamic transposition of transposable elements (TEs) in fungal pathogens has significant impact on genome stability, gene expression, and virulence to the host. In Magnaporthe oryzae, genome plasticity resulting from TE insertion is a major driving force leading to the rapid evolution and diversification of this fungus. Despite their importance in M. oryzae population evolution and divergence, our understanding of TEs in this context remains limited. Here, we conducted a genome-wide analysis of TE transposition dynamics in the 11 most abundant TE families in M. oryzae populations. Our results show that these TEs have specifically expanded in recently isolated M. oryzae rice populations, with the presence/absence polymorphism of TE insertions highly concordant with population divergence on Geng/Japonica and Xian/Indica rice cultivars. Notably, the genes targeted by clade-specific TEs showed clade-specific expression patterns and are involved in the pathogenic process, suggesting a transcriptional regulation of TEs on targeted genes. Our study provides a comprehensive analysis of TEs in M. oryzae populations and demonstrates a crucial role of recent TE bursts in adaptive evolution and diversification of the M. oryzae rice-infecting lineage. IMPORTANCE: Magnaporthe oryzae is the causal agent of the destructive blast disease, which caused massive loss of yield annually worldwide. The fungus diverged into distinct clades during adaptation toward the two rice subspecies, Xian/Indica and Geng/Japonica. Although the role of TEs in the adaptive evolution was well established, mechanisms underlying how TEs promote the population divergence of M. oryzae remain largely unknown. In this study, we reported that TEs shape the population divergence of M. oryzae by differentially regulating gene expression between Xian/Indica-infecting and Geng/Japonica-infecting populations. Our results revealed a TE insertion-mediated gene expression adaption that led to the divergence of M. oryzae population infecting different rice subspecies.

A nonclassically secreted effector of Magnaporthe oryzae targets host nuclei and plays important roles in fungal growth and plant infection.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases and poses a growing threat to food security worldwide. Like many other filamentous pathogens, rice blast fungus releases multiple types of effector proteins to facilitate fungal infection and modulate host defence responses. However, most of the characterized effectors contain an N-terminal signal peptide. Here, we report the results of the functional characterization of a nonclassically secreted nuclear targeting effector in M. oryzae (MoNte1). MoNte1 has no signal peptide, but can be secreted and translocated into plant nuclei driven by a nuclear targeting peptide. It could also induce hypersensitive cell death when transiently expressed in Nicotiana benthamiana. Deletion of the MoNTE1 gene caused a significant reduction of fungal growth and conidiogenesis, partially impaired appressorium formation and host colonization, and also dramatically attenuated the pathogenicity. Taken together, these findings reveal a novel effector secretion pathway and deepen our understanding of rice-M. oryzae interactions.

Apoplastic Cell Death-Inducing Proteins of Filamentous Plant Pathogens: Roles in Plant-Pathogen Interactions.

Filamentous pathogens, such as phytopathogenic oomycetes and fungi, secrete a remarkable diversity of apoplastic effector proteins to facilitate infection, many of which are able to induce cell death in plants. Over the past decades, over 177 apoplastic cell death-inducing proteins (CDIPs) have been identified in filamentous oomycetes and fungi. An emerging number of studies have demonstrated the role of many apoplastic CDIPs as essential virulence factors. At the same time, apoplastic CDIPs have been documented to be recognized by plant cells as pathogen-associated molecular patterns (PAMPs). The recent findings of extracellular recognition of apoplastic CDIPs by plant leucine-rich repeat-receptor-like proteins (LRR-RLPs) have greatly advanced our understanding of how plants detect them and mount a defense response. This review summarizes the latest advances in identifying apoplastic CDIPs of plant pathogenic oomycetes and fungi, and our current understanding of the dual roles of apoplastic CDIPs in plant-filamentous pathogen interactions.

Functional Identification of Novel Cell Death-inducing Effector Proteins from Magnaporthe oryzae.

BACKGROUND: Secreted effector proteins play critical roles in plant-fungal interactions. The Magnaporthe oryzae genome encodes a large number of secreted proteins. However, the function of majority of M. oryzae secreted proteins remain to be characterized. We previously identified 851 in planta-expressed M. oryzae genes encoding putative secreted proteins, and characterized five M. oryzae cell death-inducing proteins MoCDIP1 to MoCDIP5. In the present study, we expand our work on identification of novel MoCDIP proteins. RESULTS: We performed transient expression assay of 98 more in planta-expressed M. oryzae putative secreted protein genes, and identified eight novel proteins, MoCDIP6 to MoCDIP13, that induced plant cell death. Yeast secretion assay and truncation expression analysis revealed that the signal peptides that led the secretion of proteins to the extracellular space, were required for cell death inducing activity of the novel MoCDIPs except for MoCDIP8. Exogenous treatment of rice seedlings with recombinant MoCDIP6 or MoCDIP7 resulted in enhanced resistance to blast fungus, indicating that the two MoCDIPs trigger cell death and elicit defense responses in rice. CONCLUSIONS: The newly identified MoCDIP6 to MoCDIP13, together with previously identified MoCDIP1 to MoCDIP5, provide valuable targets for further dissection of the molecular mechanisms underlying the rice-blast fungus interaction.

Green Fluorescent Protein- and Discosoma sp. Red Fluorescent Protein-Tagged Organelle Marker Lines for Protein Subcellular Localization in Rice.

The subcellular localization of proteins is a fundamental aspect of protein functions. Determining the subcellular localization is important for understanding the biological functions of proteins. Here, we developed a set of rice organelle marker lines, in which the expressing fluorescent organelle markers could be used as comparative standards in determining the subcellular localization of the protein of interest. We constructed green fluorescent protein (GFP)- and/or Discosoma sp. red fluorescent protein (DsRed)-tagged organelle markers targeted to the endoplasmic reticulum (ER), mitochondria, Golgi apparatus, peroxisome, actin cytoskeleton, plastid, tonoplast, plasma membrane, and nucleus, respectively. The utility of the rice marker lines for protein subcellular localization studies was demonstrated by detecting a nucleus-localized OsWRKY45 and a mitochondria-associated NbHxk1 in protoplasts of the GFP-OsH2B and the ScCOX4-DsRed lines, respectively. Using a sheath-inoculation method, followed by a live-cell imaging, we detected co-localization of a Magnaporthe oryzae PWL2:mCherry : NLS fusion with the nucleus marker in the GFP-OsH2B rice epidermal cells, confirming the translocation of the M. oryzae effector PWL2 into host cells, and further demonstrating the feasibility of using the organelle marker lines for studying dynamics of proteins in rice cells in the interactions between rice and pathogens. The set of organelle marker lines developed in the present study, provides a valuable resource for protein subcellular localization studies in rice.

Population genomic analysis of the rice blast fungus reveals specific events associated with expansion of three main clades.

We examined the genomes of 100 isolates of Magnaporthe oryzae (Pyricularia oryzae), the causal agent of rice blast disease. We grouped current field populations of M. oryzae into three major globally distributed groups. A genetically diverse group, clade 1, which may represent a group of closely related lineages, contains isolates of both mating types. Two well-separated clades, clades 2 and 3, appear to have arisen as clonal lineages distinct from the genetically diverse clade. Examination of genes involved in mating pathways identified clade-specific diversification of several genes with orthologs involved in mating behavior in other fungi. All isolates within each clonal lineage are of the same mating type. Clade 2 is distinguished by a unique deletion allele of a gene encoding a small cysteine-rich protein that we determined to be a virulence factor. Clade 3 isolates have a small deletion within the MFA2 pheromone precursor gene, and this allele is shared with an unusual group of isolates we placed within clade 1 that contain AVR1-CO39 alleles. These markers could be used for rapid screening of isolates and suggest specific events in evolution that shaped these populations. Our findings are consistent with the view that M. oryzae populations in Asia generate diversity through recombination and may have served as the source of the clades 2 and 3 isolates that comprise a large fraction of the global population.