EzMAP: Easy Microbiome Analysis Platform.

BACKGROUND: The rapid advances in next-generation sequencing technologies have revolutionized the microbiome research by greatly increasing our ability to understand diversity of microbes in a given sample. Over the past decade, several computational pipelines have been developed to efficiently process and annotate these microbiome data. However, most of these pipelines require an implementation of additional tools for downstream analyses as well as advanced programming skills. RESULTS: Here we introduce a user-friendly microbiome analysis platform, EzMAP (Easy Microbiome Analysis Platform), which was developed using Java Swings, Java Script and R programming language. EzMAP is a standalone package providing graphical user interface, enabling easy access to all the functionalities of QIIME2 (Quantitative Insights Into Microbial Ecology) as well as streamlined downstream analyses using QIIME2 output as input. This platform is designed to give users the detailed reports and the intermediate output files that are generated progressively. The users are allowed to download the features/OTU table (.biom;.tsv;.xls), representative sequences (.fasta) and phylogenetic tree (.nwk), taxonomy assignment file (optional). For downstream analyses, users are allowed to perform relative abundances (at all taxonomical levels), community comparison (alpha and beta diversity, core microbiome), differential abundances (DESeq2 and linear discriminant analysis) and functional prediction (PICRust, Tax4Fun and FunGuilds). Our case study using a published rice microbiome dataset demonstrates intuitive user interface and great accessibility of the EzMAP. CONCLUSIONS: This EzMAP allows users to consolidate the microbiome analysis processes from raw sequence processing to downstream analyses specific for individual projects. We believe that this will be an invaluable tool for the beginners in their microbiome data analysis. This platform is freely available at https://github.com/gnanibioinfo/EzMAP and will be continually updated for adoption of changes in methods and approaches.

Draft Genome Sequences of Elsinoë fawcettii and Elsinoë australis Causing Scab Diseases on Citrus.

Elsinoë fawcettii and E. australis (phylum Ascomycota) are phytopathogenic fungi causing scab diseases on citrus plants. We report here the high-quality draft genome sequences and ab initio gene predictions of two E. fawcettii strains and one E. australis strain, which differ in their host range. This genome sequence information will provide valuable resources to underpin genomic attributes for determining host range through comparative genomic analyses of citrus scab fungi.

Application of an S-layer protein as a self-aggregating tag for cost-effective separation of recombinant human and yeast D-amino acid oxidases in the aqueous two-phase system.

OBJECTIVE: To evaluate whether the surface layer (S-layer) protein of Lactobacillus brevis serves as a self-aggregating protein tag for cost-effective separation of human and yeast D-amino acid oxidases (hDAAO and yDAAO) expressed in E. coli. RESULTS: In aqueous two-phase (PEG-phosphate) system, the S-layer:DAAO fusion proteins (shDAAO and syDAAO) were separated at the interface with a recovery of 82 ± 10.6% for shDAAO and 95 ± 1.9% for syDAAO. Some shDAAO proteins were separated as precipitates with a recovery of 41 ± 0.5% in phosphate (9%, w/w) using PEG 3000 and PEG 4000 (16%, w/w), while some syDAAO proteins were also isolated as precipitates with a recovery of 75 ± 17.5% in phosphate (9%, w/w) using PEG 4000 and PEG 8000 (16%, w/w). CONCLUSIONS: The S-layer of L. brevis was applied to a self-assembled protein tag to enable cost-effective separation of human and yeast D-amino acid oxidases expressed in E. coli cells. Because of the self-assembling properties of S-layer proteins, human and yeast D-amino acid oxidases fused with S-layer proteins could be easily separated by aggregates at the interface and/or in a few conditions by precipitates to the bottom of the PEG-phosphate aqueous system.

In Silico Identification of Potential Inhibitor Against a Fungal Histone Deacetylase, RPD3 from Magnaporthe Oryzae.

Histone acetylation and deacetylation play an essential role in the epigenetic regulation of gene expression. Histone deacetylases (HDAC) are a group of zinc-binding metalloenzymes that catalyze the removal of acetyl moieties from lysine residues from histone tails. These enzymes are well known for their wide spread biological effects in eukaryotes. In rice blast fungus, Magnaporthe oryzae, MoRPD3 (an ortholog of Saccharomyces cerevisiae Rpd3) was shown to be required for growth and development. Thus in this study, the class I HDAC, MoRpd3 is considered as a potential drug target, and its 3D structure was modelled and validated. Based on the model, a total of 1880 compounds were virtually screened (molecular docking) against MoRpd3 and the activities of the compounds were assessed by docking scores. The in silico screening suggested that [2-[[4-(2-methoxyethyl) phenoxy] methyl] phenyl] boronic acid (-8.7 kcal/mol) and [4-[[4-(2-methoxyethyl) phenoxy] methyl] phenyl] boronic acid (-8.5 kcal/mol) are effective in comparison to trichostatin A (-7.9 kcal/mol), a well-known general HDAC inhibitor. The in vitro studies for inhibition of appressorium formation by [2-[[4-(2-methoxyethyl) phenoxy] methyl] phenyl] boronic acid has resulted in the maximum inhibition at lower concentrations (1 µM), while the trichostatin A exhibited similar levels of inhibition at 1.5 µM. These findings thus suggest that 3D quantitative structure activity relationship studies on [2-[[4-(2-methoxyethyl) phenoxy] methyl] phenyl] boronic acid compound can further guide the design of more potential and specific HDAC inhibitors.

Role of the Histone Acetyltransferase Rtt109 in Development and Pathogenicity of the Rice Blast Fungus.

Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 plays important roles in maintaining genome integrity and surviving DNA damage. Here, we investigated the implications of Rtt109-mediated response to DNA damage on development and pathogenesis of the rice blast fungus Magnaporthe oryzae (anamorph: Pyricularia oryzae). The ortholog of Rtt109 in M. oryzae (MoRtt109) was found via sequence homology and its functionality was confirmed by phenotypic complementation of the Saccharomyces cerevisiae Rtt109 deletion strain. Targeted deletion of MoRtt109 resulted in a significant reduction in acetylation of H3K56 and rendered the fungus defective in hyphal growth and asexual reproduction. Furthermore, the deletion mutant displayed hypersensitivity to genotoxic agents, confirming the conserved importance of Rtt109 in genome integrity maintenance and genotoxic stress tolerance. Elevated expression of DNA repair genes and the results of the comet assay were consistent with constitutive endogenous DNA damage. Although the conidia produced from the mutant were not impaired in germination and appressorium morphogenesis, the mutant was significantly less pathogenic on rice leaves. Transcriptomic analysis provided insight into the factors underlying phenotypic defects that are associated with deficiency of H3K56 acetylation. Overall, our results indicate that MoRtt109 is a conserved histone acetyltransferase that affects proliferation and asexual fecundity of M. oryzae through maintenance of genome integrity and response to DNA damage.

Identification of Potential Nematicidal Compounds against the Pine Wood Nematode, Bursaphelenchus xylophilus through an In Silico Approach.

Bursaphelenchus xylophilus is a destructive phytophagous nematode that mainly infects pine species and causes pine wilt disease (PWD). PWD is one of the most devastating diseases that has damaged the pine forests of eastern Asia and Portugal for the last four decades. B. xylophilus infects healthy pine trees through Monochamus beetles and its subsequent proliferation results in destruction of the infected pine trees. The poor water solubility and high cost of currently used trunk-injected chemicals such as avermectin and abamectin for the prevention of PWD are major concerns. Thus, for the identification of new compounds targeting the different targets, five proteins including cathepsin L-like cystein proteinase, peroxiredoxins, hsp90, venome allergen protein and tubulin that are known to be important for development and pathogenicity of B. xylophilus were selected. The compounds were virtually screened against five proposed targets through molecular docking into hypothetical binding sites located in a homology-built protein model. Of the fifteen nematicides screened, amocarzine, mebendazole and flubendazole were judged to bind best. For these best docked compounds, structural and electronic properties were calculated through density functional theory studies. The results emphasize that these compounds could be potential lead compounds that can be further developed into nematicidal chemical against B. xylophilus. However, further studies are required to ascertain the nematicidal activity of these compounds against phytophagous nematode.

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.

Global expression profiling of transcription factor genes provides new insights into pathogenicity and stress responses in the rice blast fungus.

Because most efforts to understand the molecular mechanisms underpinning fungal pathogenicity have focused on studying the function and role of individual genes, relatively little is known about how transcriptional machineries globally regulate and coordinate the expression of a large group of genes involved in pathogenesis. Using quantitative real-time PCR, we analyzed the expression patterns of 206 transcription factor (TF) genes in the rice blast fungus Magnaporthe oryzae under 32 conditions, including multiple infection-related developmental stages and various abiotic stresses. The resulting data, which are publicly available via an online platform, provided new insights into how these TFs are regulated and potentially work together to control cellular responses to a diverse array of stimuli. High degrees of differential TF expression were observed under the conditions tested. More than 50% of the 206 TF genes were up-regulated during conidiation and/or in conidia. Mutations in ten conidiation-specific TF genes caused defects in conidiation. Expression patterns in planta were similar to those under oxidative stress conditions. Mutants of in planta inducible genes not only exhibited sensitive to oxidative stress but also failed to infect rice. These experimental validations clearly demonstrated the value of TF expression patterns in predicting the function of individual TF genes. The regulatory network of TF genes revealed by this study provides a solid foundation for elucidating how M. oryzae regulates its pathogenesis, development, and stress responses.

Role of MoAND1-mediated nuclear positioning in morphogenesis and pathogenicity in the rice blast fungus, Magnaporthe oryzae.

To cause disease on host plants, many phytopathogenic fungi undergo morphological transitions including development of reproductive structures as well as specialized infection structures called appressoria. Such morphological transitions display distinct nuclear dynamics. Here we report the developmental requirement of MoAND1-mediated nuclear positioning for pathogenesis of the rice blast fungus, Magnaporthe oryzae. The MoAND1 gene encodes a protein that shows high similarity to Num1 in Saccharomyces cerevisiae and ApsA in Aspergillus nidulans, both of which are cell cortex proteins involved in nuclear migration and positioning. Targeted deletion of MoAND1 did not affect radial growth of the fungus but impaired nuclear distribution along the hyphae, which is reminiscent of ApsA mutant. In contrast to the wild-type, which produces three to five spores in a sympodial manner on the conidiophore, only a single spore was borne on the conidiophore of ΔMoand1, resulting in ∼65% decrease in conidia production, compared to the wild-type. The mutant conidia displayed abnormalities in septation pattern and nuclear distribution, which were correlated with their inability to germinate. Spores of the mutant that did germinate were capable of differentiating appressoria but were defective in the execution of programmed nuclear migration and positioning during development. Furthermore, mutant appressoria were not fully functional, leading to delay in penetration of host plants. However, the ability of ΔMoand1 to grow inside host tissues was comparable to that of the wild-type. All these defects greatly decreased the virulence of the mutant. Taken together, our data suggest that there is a stringent but incomplete developmental requirement for proper migration and positioning of fungal nuclei mediated by MoAND1 during asexual reproduction and pre-penetration phase of fungal pathogenesis.

Changes in Endophyte Communities across the Different Plant Compartments in Response to the Rice Blast Infection.

The rice blast disease, caused by the fungal pathogen, Magnaporthe oryzae (syn. Pyricularia oryzae), poses a significant threat to the global rice production. Understanding how this disease impacts the plant's microbial communities is crucial for gaining insights into hostpathogen interactions. In this study, we investigated the changes in communities of bacterial and fungal endophytes inhabiting different compartments in healthy and diseased plants. We found that both alpha and beta diversities of endophytic communities do not change significantly by the pathogen infection. Rather, the type of plant compartment appeared to be the main driver of endophytic community structures. Although the overall structure seemed to be consistent between healthy and diseased plants, our analysis of differentially abundant taxa revealed the specific bacterial and fungal operational taxonomic units that exhibited enrichment in the root and leaf compartments of infected plants. These findings suggest that endophyte communities are robust to the changes at the early stage of pathogen infection, and that some of endophytes enriched in infected plants might have roles in the defense against the pathogen.

Antifungal Activity-Guided Analysis of Actinostemma lobatum Extracts through Serial Sub-fractions.

Plants are treasure trove of novel compounds that have potential for antifungal chemicals and drugs. In our previous study, we had screened plant extracts obtained from more than eight hundred plant materials collected in Korea, and found that butanol fraction of the Actinostemma lobatum were most potent in suppressing growth of diverse fungal pathogens of plants. Here in this study, we describe further analysis of the butanol fraction, and summarize the results of subsequent antifungal activity test for the sub-fractions against a selected set of plant pathogenic fungi. This line of analyses allowed us to identify the sub-fractions that could account for a significant proportion of observed antifungal activity of initial butanol fraction from A. lobatum. Further analysis of these sub-fractions and determination of structure would provide the shortlist for novel compounds that can be a lead to new agrochemicals.

Edeine B1 produced by Brevibacillus brevis reduces the virulence of a plant pathogenic fungus by inhibiting mitochondrial respiration.

Plant pathogenic fungi cause serious diseases, which result in the loss of crop yields and reduce the quality of crops worldwide. To counteract the escalating risks of chemical fungicides, interest in biological control agents to manage plant diseases has significantly increased. In this study, we comprehensively screened microbial culture filtrates using a yeast screening system to find microbes exhibiting respiratory inhibition activity. Consequently, we found a soil-borne microbe Brevibacillus brevis HK544 strain exhibiting a respiration inhibitory activity and identified edeine B1 (EB1) from the culture filtrate of HK544 as the active compound of the respiration inhibition activity. Furthermore, against a plant pathogenic fungus Fusarium graminearum, our results showed that EB1 has effects on multiple aspects of respiration with the downregulation of most of the mitochondrial-related genes based on transcriptome analysis, differential EB1-sensitivity from targeted mutagenesis, and the synergistic effects of EB1 with electron transport chain complex inhibitors. With the promising plant disease control efficacy of B. brevis HK544 producing EB1, our results suggest that B. brevis HK544 has potential as a biocontrol agent for Fusarium head blight.IMPORTANCEAs a necrotrophic fungus, Fusarium graminearum is a highly destructive pathogen causing severe diseases in cereal crops and mycotoxin contamination in grains. Although chemical control is considered the primary approach to control plant disease caused by F. graminearum, fungicide-resistant strains have been detected in the field after long-term continuous application of fungicides. Moreover, applying chemical fungicides that trigger mycotoxin biosynthesis is a great concern for many researchers. Biocontrol of Fusarium head blight (FHB) by biological control agents (BCAs) represents an alternative approach and could be used as part of the integrated management of FHB and mycotoxin production. The most extensive studies on bacterial BCAs-fungal communications in agroecosystems have focused on antibiosis. Although many BCAs in agricultural ecology have already been used for fungal disease control, the molecular mechanisms of antibiotics produced by BCAs remain to be elucidated. Here, we found a potential BCA (Brevibacillus brevis HK544) with a strong antifungal activity based on the respiration inhibition activity with its active compound edeine B1 (EB1). Furthermore, our results showed that EB1 secreted by HK544 suppresses the expression of the mitochondria-related genes of F. graminearum, subsequently suppressing fungal development and the virulence of F. graminearum. In addition, EB1 exhibited a synergism with complex I inhibitors such as rotenone and fenazaquin. Our work extends our understanding of how B. brevis HK544 exhibits antifungal activity and suggests that the B. brevis HK544 strain could be a valuable source for developing new crop protectants to control F. graminearum.

Two conidiation-related Zn(II)2Cys6 transcription factor genes in the rice blast fungus.

Regulation of gene expression by transcription factors (TFs) helps plant pathogens to interact with the host plants and to sustain a pathogenic lifestyle in the environmental changes. Elucidating novel functions of TFs is, therefore, crucial for understanding pathogenesis mechanisms of plant pathogens. Magnaporthe oryzae, the rice blast pathogen, undergoes a series of developmental morphogenesis to complete its infection cycle. To understand TF genes implicated in pathogenic development of this fungus, two Zn(II)2Cys6 TF genes, MoCOD1 and MoCOD2, whose expression was notably induced during conidiation, were functionally characterized. Targeted deletion of MoCOD1 resulted in defects in conidiation and pathogenicity due to defects in appressorium formation and invasive growth within the host cells. MoCOD2 was also a critical regulator in conidiation and pathogenicity, but not in conidial germination and appressorium formation. When rice plants were inoculated with conidia of the ΔMocod2 mutant, rapid accumulation of dark brown granules was observed around the infection sites in the plant cells and no visible disease symptom was incited. Taken together, both MoCOD1 and MoCOD2 play important roles in conidiation and pathogenicity of the rice blast fungus.

Phyllosphere Microbiome in Plant Health and Disease.

The phyllosphere refers to the aboveground surface of plants colonized by diverse microorganisms. Microbes inhabiting this environment play an important role in enhancing the host's genomic and metabolic capabilities, including defense against pathogens. Compared to the large volume of studies on rhizosphere microbiome for plant health and defense, our understanding of phyllosphere microbiome remains in its infancy. In this review, we aim to explore the mechanisms that govern the phyllosphere assembly and their function in host defence, as well as highlight the knowledge gaps. These efforts will help develop strategies to harness the phyllosphere microbiome toward sustainable crop production.

A Nucleolar Protein, MoRRP8 Is Required for Development and Pathogenicity in the Rice Blast Fungus.

The nucleolus is the largest, membrane-less organelle within the nucleus of eukaryotic cell that plays a critical role in rRNA transcription and assembly of ribosomes. Recently, the nucleolus has been shown to be implicated in an array of processes including the formation of signal recognition particles and response to cellular stress. Such diverse functions of nucleolus are mediated by nucleolar proteins. In this study, we characterized a gene coding a putative protein containing a nucleolar localization sequence (NoLS) in the rice blast fungus, Magnaporthe oryzae. Phylogenetic and domain analysis suggested that the protein is orthologous to Rrp8 in Saccharomyces cerevisiae. MoRRP8-GFP (translational fusion of MoRRP8 with green fluorescence protein) co-localizes with a nucleolar marker protein, MoNOP1 fused to red fluorescence protein (RFP), indicating that MoRRP8 is a nucleolar protein. Deletion of the MoRRP8 gene caused a reduction in vegetative growth and impinged largely on asexual sporulation. Although the asexual spores of ΔMorrp8 were morphologically indistinguishable from those of wild-type, they showed delay in germination and reduction in appressorium formation. Our pathogenicity assay revealed that the MoRRP8 is required for full virulence and growth within host plants. Taken together, these results suggest that nucleolar processes mediated by MoRRP8 is pivotal for fungal development and pathogenesis.

Distinct dynamics of the nucleolus in response to nutrient availability and during development in the rice blast fungus.

IMPORTANCE: The nucleolus is a dynamic subnuclear structure that is involved in many fundamental processes of the nucleus. In higher eukaryotic cells, the size and shape of nucleoli correlate with nucleolar activities. For fungi, knowledge of the nucleolus and its functions is primarily gleaned from budding yeast. Whether such correlation is conserved and how nucleolar functions are regulated in filamentous fungi including important human and crop pathogens are largely unknown. Our observations reveal that the dynamics of nucleolus in a model plant pathogenic fungus, Magnaporthe oryzae, is distinct from those of animal and yeast nucleoli under low nutrient availability and during pathogenic development. Our data not only provide new insight into the nucleoli in filamentous fungi but also highlight the need for investigating how nucleolar dynamics is regulated in comparison to other eukaryotes.

Plant Protective and Growth Promoting Effects of Seed Endophytes in Soybean Plants.

Seed-borne diseases reduce not only the seed germination and seedling growth but also seed quality, resulting in the significant yield loss in crop production. Plant seed harbors diverse microbes termed endophytes other than pathogens inside it. However, their roles and application to agricultures were rarely understood and explored to date. Recently, we had isolated from soybean seeds culturable endophytes exhibiting in-vitro antagonistic activities against common bacterial and fungal seed-borne pathogens. In this study, we evaluated effects of seed treatment with endophytes on plant growth and protection against the common seed-borne pathogens: four fungal pathogens (Cercospora sojina, C. kikuchii, Septoria glycines, Diaporthe eres) and two bacterial pathogens (Xanthomonas axonopodis pv. glycines, Pseudomonas syringae pv. tabaci). Our experiments showed that treatment of soybean seeds with seed endophytes clearly offer protection against seed-borne pathogens. We also found that some of the endophytes promote plant growth in addition to the disease suppression. Taken together, our results demonstrate agricultural potential of seed endophytes in crop protection.

A Brief Guide to Statistical Analysis and Presentation for the Plant Pathology Journal.

Statistical analysis of data is an integral part of research projects in all scientific disciplines including the plant pathology. Appropriate design, application and interpretation of statistical analysis are also, therefore, at the center of publishing and properly evaluating studies in plant pathology. A survey of research works published in the Plant Pathology Journal, however, cast doubt on high standard of statistical analysis required for scientific rigor and reproducibility in the journal. Here I first describe, based on the survey of published works, what mistakes are commonly made and what components are often lacking during statistical analysis and interpretation of its results. Next, I provide possible remedies and suggestions to help guide researchers in preparing manuscript and reviewers in evaluating manuscripts submitted to the Plant Pathology Journal. This is not aiming at delineating technical and practical details of particular statistical methods or approaches.

Nuclear Effectors in Plant Pathogenic Fungi.

The nuclear import of proteins is a fundamental process in the eukaryotes including plant. It has become evident that such basic process is exploited by nuclear effectors that contain nuclear localization signal (NLS) and are secreted into host cells by fungal pathogens of plants. However, only a handful of nuclear effectors have been known and characterized to date. Here, we first summarize the types of NLSs and prediction tools available, and then delineate examples of fungal nuclear effectors and their roles in pathogenesis. Based on the knowledge on NLSs and what has been gleaned from the known nuclear effectors, we point out the gaps in our understanding of fungal nuclear effectors that need to be filled in the future researches.

Large-Scale Screening of the Plant Extracts for Antifungal Activity against the Plant Pathogenic Fungi.

Plants produce chemicals of immense diversity that provide great opportunities for development of new antifungal compounds. In search for environment-friendly alternatives to the fungicide of current use, we screened plant extracts obtained from more than eight hundred plant materials collected in Korea for their antifungal activity against the model plant pathogenic fungus, Magnaporthe oryzae. This initial screening identified antifungal activities from the eleven plant extract samples, among which nine showed reproducibility in the follow-up screening. These nine samples were able to suppress not only M. oryzae but also other fungal pathogens. Interestingly, the plant extracts obtained from Actinostemma lobatum comprised five out of eight samples, and were the most effective in their antifungal activity. We found that butanol fraction of the A. lobatum extract is the most potent. Identification and characterization of antifungal substances in the A. lobatum extracts would provide the promising lead compounds for new fungicide.