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.

Tissues and mechanisms associated with Verticillium wilt resistance in tomato using bi-grafted near-isogenic lines.

Host resistance is the primary means to control Verticillium dahliae, a soil-borne pathogen causing major losses on a broad range of plants, including tomato. The tissues and mechanisms responsible for resistance remain obscure. In the field, resistant tomato used as rootstocks does not confer resistance. Here, we created bi-grafted plants with near-isogenic lines (NILs) exhibiting (Ve1) or lacking (ve1) resistance to V. dahliae race 1. Ten days after inoculation, scion and rootstock tissues were subjected to differential gene expression and co-expression network analyses. Symptoms only developed in susceptible scions regardless of the rootstock. Infection caused more dramatic alteration of tomato gene expression in susceptible compared with resistant tissues, including pathogen receptor, signaling pathway, pathogenesis-related protein, and cell wall modification genes. Differences were observed between scions and rootstocks, primarily related to physiological processes in these tissues. Gene expression in scions was influenced by the rootstock genotype. A few genes were associated with the Ve1 genotype, which was independent of infection or tissue type. Several were physically clustered, some near the Ve1 locus on chromosome 9. Transcripts mapped to V. dahliae were dominated by secreted candidate effector proteins. These findings advance knowledge of molecular mechanisms underlying the tomato-V. dahliae interaction.

Host induced gene silencing of Magnaporthe oryzae by targeting pathogenicity and development genes to control rice blast disease.

Rice blast disease caused by the hemi-biotrophic fungus Magnaporthe oryzae is the most destructive disease of rice world-wide. Traditional disease resistance strategies for the control of rice blast disease have not proved durable. HIGS (host induced gene silencing) is being developed as an alternative strategy. Six genes (CRZ1, PMC1, MAGB, LHS1, CYP51A, CYP51B) that play important roles in pathogenicity and development of M. oryzae were chosen for HIGS. HIGS vectors were transformed into rice calli through Agrobacterium-mediated transformation and T0, T1 and T2 generations of transgenic rice plants were generated. Except for PMC1 and LHS1, HIGS transgenic rice plants challenged with M. oryzae showed significantly reduced disease compared with non-silenced control plants. Following infection with M. oryzae of HIGS transgenic plants, expression levels of target genes were reduced as demonstrated by Quantitative RT-PCR. In addition, treating M. oryzae with small RNA derived from the target genes inhibited fungal growth. These findings suggest RNA silencing signals can be transferred from host to an invasive fungus and that HIGS has potential to generate resistant rice against M. oryzae.

Soybean Cyst Nematodes Influence Aboveground Plant Volatile Signals Prior to Symptom Development.

Soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive soybean pests worldwide. Unlike many diseases, SCN doesn't show above ground evidence of disease until several weeks after infestation. Knowledge of Volatile Organic Compounds (VOCs) related to pests and pathogens of foliar tissue is extensive, however, information related to above ground VOCs in response to root damage is lacking. In temporal studies, gas chromatography-mass spectrometry analysis of VOCs from the foliar tissues of SCN infested plants yielded 107 VOCs, referred to as Common Plant Volatiles (CPVs), 33 with confirmed identities. Plants showed no significant stunting until 10 days after infestation. Total CPVs increased over time and were significantly higher from SCN infested plants compared to mock infested plants post 7 days after infestation (DAI). Hierarchical clustering analysis of expression ratios (SCN: Mock) across all time points revealed 5 groups, with the largest group containing VOCs elevated in response to SCN infestation. Linear projection of Principal Component Analysis clearly separated SCN infested from mock infested plants at time points 5, 7, 10 and 14 DAI. Elevated Styrene (CPV11), D-Limonene (CPV32), Tetradecane (CPV65), 2,6-Di-T-butyl-4-methylene-2,5-cyclohexadiene-1-one (CPV74), Butylated Hydroxytoluene (CPV76) and suppressed Ethylhexyl benzoate (CPV87) levels, were associated with SCN infestation prior to stunting. Our findings demonstrate that SCN infestation elevates the release of certain VOCs from foliage and that some are evident prior to symptom development. VOCs associated with SCN infestations prior to symptom development may be valuable for innovative diagnostic approaches.

Comparative Genome Analyses of 18 Verticillium dahliae Tomato Isolates Reveals Phylogenetic and Race Specific Signatures.

Host resistance is one of the few strategies available to combat the soil borne pathogenic fungus Verticillium dahliae. Understanding pathogen diversity in populations is key to successfully deploying host resistance. In this study the genomes of 18 V. dahliae isolates of races 1 (n = 2), 2 (n = 4), and 3 (n = 12) from Japan, California, and North Carolina were sequenced and mapped to the reference genome of JR2 (from tomato). The genomes were analyzed for phylogenetic and pathogen specific signatures to classify specific strains or genes for future research. Four highly clonal lineages/groups were discovered, including a lineage unique to North Carolina isolates, which had the rare MAT1-1 mating type. No evidence for recombination between isolates of different mating types was observed, even in isolates of different mating types discovered in the same field. By mapping these 18 isolates genomes to the JR2 reference genome, 193 unique candidate effectors were found using SignalP and EffectorP. Within these effectors, 144 highly conserved effectors, 42 mutable effectors (truncated or present in some isolates but absent in others), and 7 effectors present in highly variable regions of the chromosomes were discovered. Of the 144 core effectors, 21 were highly conserved in V. alfalfae and V. longisporum, 7 of which have no known function. Within the non-core effectors 30 contained large numbers of non-synonymous mutations, while 15 of them contained indels, frameshift mutations, or were present on highly variable regions of the chromosome. Two of these highly variable region effectors (HVREs) were only present in race 2 isolates, but not in race 3 isolates. The race 1 effector Ave1 was also present in a highly variable region. These data may suggest that these highly variable regions are enriched in race determinant genes, consistent with the two-speed genome hypothesis.

Opportunities and Challenges in Studies of Host-Pathogen Interactions and Management of Verticillium dahliae in Tomatoes.

Tomatoes (Solanum lycopersicum L.) are a valuable horticultural crop that are grown and consumed worldwide. Optimal production is hindered by several factors, among which Verticillium dahliae, the cause of Verticillium wilt, is considered a major biological constraint in temperate production regions. V. dahliae is difficult to mitigate because it is a vascular pathogen, has a broad host range and worldwide distribution, and can persist in soil for years. Understanding pathogen virulence and genetic diversity, host resistance, and plant-pathogen interactions could ultimately inform the development of integrated strategies to manage the disease. In recent years, considerable research has focused on providing new insights into these processes, as well as the development and integration of environment-friendly management approaches. Here, we discuss the current knowledge on the race and population structure of V. dahliae, including pathogenicity factors, host genes, proteins, enzymes involved in defense, and the emergent management strategies and future research directions for managing Verticillium wilt in tomatoes.

Dynamic Changes in the Microbiome of Rice During Shoot and Root Growth Derived From Seeds.

Microbes form close associations with host plants including rice as both surface (epiphytes) and internal (endophytes) inhabitants. Yet despite rice being one of the most important cereal crops agriculturally and economically, knowledge of its microbiome, particularly core inhabitants and any functional properties bestowed is limited. In this study, the microbiome in rice seedlings derived directly from seeds was identified, characterized and compared to the microbiome of the seed. Rice seeds were sourced from two different locations in Arkansas, USA of two different rice genotypes (Katy, M202) from two different harvest years (2013, 2014). Seeds were planted in sterile media and bacterial as well as fungal communities were identified through 16S and ITS sequencing, respectively, for four seedling compartments (root surface, root endosphere, shoot surface, shoot endosphere). Overall, 966 bacterial and 280 fungal ASVs were found in seedlings. Greater abundance and diversity were detected for the microbiome associated with roots compared to shoots and with more epiphytes than endophytes. The seedling compartments were the driving factor for microbial community composition rather than other factors such as rice genotype, location and harvest year. Comparison with datasets from seeds revealed that 91 (out of 296) bacterial and 11 (out of 341) fungal ASVs were shared with seedlings with the majority being retained within root tissues. Core bacterial and fungal microbiome shared across seedling samples were identified. Core bacteria genera identified in this study such as Rhizobium, Pantoea, Sphingomonas, and Paenibacillus have been reported as plant growth promoting bacteria while core fungi such as Pleosporales, Alternaria and Occultifur have potential as biocontrol agents.

Comparative Proteomic Analysis of Wild Type and Mutant Lacking an SCF E3 Ligase F-Box Protein in Magnaporthe oryzae.

Magnaporthe oryzae (M. oryzae) is a pathogenic, filamentous fungus that is a primary cause of rice blast disease. The M. oryzae protein MGG_13065, SCF E3 ubiquitin ligase complex F-box protein, has been identified as playing a crucial role in the infection process, specifically, as part of the ubiquitin mediated proteolysis pathway. Proteins targeted by MGG_13065 E3 ligase are first phosphorylated and then ubiquitinated by E3 ligase. In this study, we used a label-free quantitative global proteomics technique to probe the role of ubiquitination and phosphorylation in the mechanism of how E3 ligase regulates change in virulence of M. oryzae. To do this, we compared the WT M. oryzae 70-15 strain with a gene knock out (E3 ligase KO) strain. After applying a ≥ 5 normalized spectral count cutoff, a total of 4432 unique proteins were identified comprised of 4360 and 4372 in the WT and E3 ligase KO samples, respectively. Eighty proteins drastically increased in abundance, while 65 proteins decreased in abundance in the E3 ligase KO strain. Proteins (59) were identified only in the WT strain; 13 of these proteins had both phosphorylation and ubiquitination post-translational modifications. Proteins (71) were revealed to be only in the E3 ligase KO strain; 23 of the proteins have both phosphorylation and ubiquitination post-translational modifications. Several of these proteins were associated with key biological processes. These data greatly assist in the selection of future genes for functional studies and enable mechanistic insight related to virulence.

Movement of small RNAs in and between plants and fungi.

RNA interference is a biological process whereby small RNAs inhibit gene expression through neutralizing targeted mRNA molecules. This process is conserved in eukaryotes. Here, recent work regarding the mechanisms of how small RNAs move within and between organisms is examined. Small RNAs can move locally and systemically in plants through plasmodesmata and phloem, respectively. In fungi, transportation of small RNAs may also be achieved by septal pores and vesicles. Recent evidence also supports bidirectional cross-kingdom communication of small RNAs between host plants and adapted fungal pathogens to affect the outcome of infection. We discuss several mechanisms for small RNA trafficking and describe evidence for transport through naked form, combined with RNA-binding proteins or enclosed by vesicles.

Investigating host-pathogen meta-metabolic interactions of Magnaporthe oryzae infected barley using infrared matrix-assisted laser desorption electrospray ionization mass spectrometry.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging is a useful tool for identifying important meta-metabolomic features pertinent for enhancing our understanding of biological systems. Magnaporthe oryzae (M. oryzae) is a filamentous fungus that is the primary cause of rice blast disease. True to its name, M. oryzae primarily destroys rice crops and can also destroy other cereal crops as well. In a previous study, the F-box E3 ligase protein in M. oryzae was noted to be crucial for its growth and pathogenicity. In this study, we inoculated three separate sets of barley with wild-type M. oryzae, an F-box E3 ligase protein knock out of M. oryzae, and a control solution. Over the course of the infection (8 days), we imaged each treatment after development of an advanced polarity switching method, which allowed for the detection of low and high molecular weight compounds that ionize in positive or negative polarities. A set of features from initial experiments were chosen for another analysis using tandem mass spectrometry. Serotonin, a barley defense metabolite, was a compound identified in both positive and negative modes. Serotonin was putatively identified using MS1 data including carbon estimation and sulfur counting then confirmed based on tandem mass spectrometry fragmentation patterns. Metabolites in the melanin pathway, important for infection development of M. oryzae, were also identified using MS1 data but were unable to be confirmed with MS/MS due to their low abundances.

Characterizing Small RNAs in Filamentous Fungi Using the Rice Blast Fungus, Magnaporthe oryzae, as an Example.

The goal of this chapter is to provide a framework of sequential steps for small RNA (sRNA) analysis in filamentous fungi. Here, we present protocols for (1) comparative analysis of sRNAs in different conditions, (2) comparisons of sRNA libraries to RNAseq data and (3) identification and analysis of methylguanosine-capped and polyadenylated sRNAs (CPA-sRNAs). This species of small RNA is particularly interesting in Magnaporthe oryzae, as they map to transcription start and end sites of protein-coding genes. While we do not provide specific command lines for scripts, we provide a general framework for steps needed to carry out all three types of analyses, including relevant references, websites and free online tools. Screenshots are provided from our own customized interface using M. oryzae as an example, to assist the reader in visualizing many of the steps.

Sequential Phosphopeptide Enrichment for Phosphoproteome Analysis of Filamentous Fungi: A Test Case Using Magnaporthe oryzae.

A number of challenges have to be overcome to identify a complete complement of phosphorylated proteins, the phosphoproteome, from cells and tissues. Phosphorylated proteins are typically of low abundance and moreover, the proportion of phosphorylated sites on a given protein is generally low. The challenge is further compounded when the tissue from which protein can be recovered is limited. Global phosphoproteomics primarily relies on efficient enrichment methods for phosphopeptides involving affinity binding coupled with analysis by fast high-resolution mass spectrometry (MS) and subsequent identification using various software packages. Here, we describe an effective protocol for phosphopeptide enrichment using an Iron-IMAC resin in combination with titanium dioxide (TiO2) beads from trypsin digested protein samples of the filamentous fungus Magnaporthe oryzae. Representative protocols for LC-MS/MS analysis and phosphopeptide identification are also described.

Comparative proteomic analysis between nitrogen supplemented and starved conditions in Magnaporthe oryzae.

BACKGROUND: Fungi are constantly exposed to nitrogen limiting environments, and thus the efficient regulation of nitrogen metabolism is essential for their survival, growth, development and pathogenicity. To understand how the rice blast pathogen Magnaporthe oryzae copes with limited nitrogen availability, a global proteome analysis under nitrogen supplemented and nitrogen starved conditions was completed. METHODS: M. oryzae strain 70-15 was cultivated in liquid minimal media and transferred to media with nitrate or without a nitrogen source. Proteins were isolated and subjected to unfractionated gel-free based liquid chromatography-tandem mass spectrometry (LC-MS/MS). The subcellular localization and function of the identified proteins were predicted using bioinformatics tools. RESULTS: A total of 5498 M. oryzae proteins were identified. Comparative analysis of protein expression showed 363 proteins and 266 proteins significantly induced or uniquely expressed under nitrogen starved or nitrogen supplemented conditions, respectively. A functional analysis of differentially expressed proteins revealed that during nitrogen starvation nitrogen catabolite repression, melanin biosynthesis, protein degradation and protein translation pathways underwent extensive alterations. In addition, nitrogen starvation induced accumulation of various extracellular proteins including small extracellular proteins consistent with observations of a link between nitrogen starvation and the development of pathogenicity in M. oryzae. CONCLUSION: The results from this study provide a comprehensive understanding of fungal responses to nitrogen availability.

Identification and characterization of suppressors of plant cell death (SPD) effectors from Magnaporthe oryzae.

Phytopathogenic microorganisms, including the fungal pathogen Magnaporthe oryzae, secrete a myriad of effector proteins to facilitate infection. Utilizing the transient expression of candidate effectors in the leaves of the model plant Nicotiana benthamiana, we identified 11 suppressors of plant cell death (SPD) effectors from M. oryzae that were able to block the host cell death reaction induced by Nep1. Ten of these 11 were also able to suppress BAX-mediated plant cell death. Five of the 11 SPD genes have been identified previously as either essential for the pathogenicity of M. oryzae, secreted into the plant during disease development, or as suppressors or homologues of other characterized suppressors. In addition, of the remaining six, we showed that SPD8 (previously identified as BAS162) was localized to the rice cytoplasm in invaded and surrounding uninvaded cells during biotrophic invasion. Sequence analysis of the 11 SPD genes across 43 re-sequenced M. oryzae genomes revealed that SPD2, SPD4 and SPD7 have nucleotide polymorphisms amongst the isolates. SPD4 exhibited the highest level of nucleotide diversity of any currently known effector from M. oryzae in addition to the presence/absence polymorphisms, suggesting that this gene is potentially undergoing selection to avoid recognition by the host. Taken together, we have identified a series of effectors, some of which were previously unknown or whose function was unknown, that probably act at different stages of the infection process and contribute to the virulence of M. oryzae.

Comparative genome analysis and genome evolution of members of the magnaporthaceae family of fungi.

BACKGROUND: Magnaporthaceae, a family of ascomycetes, includes three fungi of great economic importance that cause disease in cereal and turf grasses: Magnaporthe oryzae (rice blast), Gaeumannomyces graminis var. tritici (take-all disease), and Magnaporthe poae (summer patch disease). Recently, the sequenced and assembled genomes for these three fungi were reported. Here, the genomes were compared for orthologous genes in order to identified genes that are unique to the Magnaporthaceae family of fungi. In addition, ortholog clustering was used to identify a core proteome for the Magnaporthaceae, which was examined for diversifying and purifying selection and evidence of two-speed genome evolution. RESULTS: A genome-scale comparative study was conducted across 74 fungal genomes to identify clusters of orthologous genes unique to the three Magnaporthaceae species as well as species specific genes. We found 1149 clusters that were unique to the Magnaporthaceae family of fungi with 295 of those containing genes from all three species. Gene clusters involved in metabolic and enzymatic activities were highly represented in the Magnaporthaceae specific clusters. Also highly represented in the Magnaporthaceae specific clusters as well as in the species specific genes were transcriptional regulators. In addition, we examined the relationship between gene evolution and distance to repetitive elements found in the genome. No correlations between diversifying or purifying selection and distance to repetitive elements or an increased rate of evolution in secreted and small secreted proteins were observed. CONCLUSIONS: Taken together, these data show that at the genome level, there is no evidence to suggest multi-speed genome evolution or that proximity to repetitive elements play a role in diversification of genes.

Genome Sequences of Three Phytopathogenic Species of the Magnaporthaceae Family of Fungi.

Magnaporthaceae is a family of ascomycetes that includes three fungi of great economic importance: Magnaporthe oryzae, Gaeumannomyces graminis var. tritici, and Magnaporthe poae. These three fungi cause widespread disease and loss in cereal and grass crops, including rice blast disease (M. oryzae), take-all disease in wheat and other grasses (G. graminis), and summer patch disease in turf grasses (M. poae). Here, we present the finished genome sequence for M. oryzae and draft sequences for M. poae and G. graminis var. tritici. We used multiple technologies to sequence and annotate the genomes of M. oryzae, M. poae, and G. graminis var. tritici. The M. oryzae genome is now finished to seven chromosomes whereas M. poae and G. graminis var. tritici are sequenced to 40.0× and 25.0× coverage respectively. Gene models were developed by the use of multiple computational techniques and further supported by RNAseq data. In addition, we performed preliminary analysis of genome architecture and repetitive element DNA.