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Background Emerging knowledge of the impact of small RNAs as important cellular regulators has prompted an explosion of small transcriptome sequencing projects. Although significant progress has been made towards small RNA discovery and biogenesis in higher eukaryotes and other model organisms, knowledge in simple eukaryotes such as filamentous fungi remains limited. Results Here, we used 454 pyrosequencing to present a detailed analysis of the small RNA transcriptome (~ 15 - 40 nucleotides in length) from mycelia and appressoria tissues of the rice blast fungal pathogen, Magnaporthe oryzae . Small RNAs mapped to numerous nuclear and mitochondrial genomic features including repetitive elements, tRNA loci, rRNAs, protein coding genes, snRNAs and intergenic regions. For most elements, small RNAs mapped primarily to the sense strand with the exception of repetitive elements to which small RNAs mapped in the sense and antisense orientation in near equal proportions. Inspection of the small RNAs revealed a preference for U and suppression of C at position 1, particularly for antisense mapping small RNAs. In the mycelia library, small RNAs of the size 18 - 23 nt were enriched for intergenic regions and repetitive elements. Small RNAs mapping to LTR retrotransposons were classified as LTR retrotransposon-siRNAs (LTR-siRNAs). Conversely, the appressoria library had a greater proportion of 28 - 35 nt small RNAs mapping to tRNA loci, and were classified as tRNA-derived RNA fragments (tRFs). LTR-siRNAs and tRFs were independently validated by 3' RACE PCR and northern blots, respectively. Conclusions Our findings suggest M. oryzae small RNAs differentially accumulate in vegetative and specialized-infection tissues and may play an active role in genome integrity and regulating growth and development.

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.

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.

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.

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.

Protein ubiquitination, which is highly selective, regulates many important biological processes including cellular differentiation and pathogenesis in eukaryotic cells. Here, we integrated pharmacological, molecular and proteomic approaches to explore the role of ubiquitination in Magnaporthe oryzae, the leading fungal disease of rice world-wide. Inhibition of ubiquitin-mediated proteolysis using the 26S proteasome inhibitor, Bortezomib, significantly attenuated conidia germination, appressorium formation and pathogenicity in M. oryzae. Gene expression analysis revealed that many genes associated with protein ubiquitination were developmentally regulated during conidia germination. Only a few, including a polyubiquitin encoding gene, MGG_01282, were more abundantly expressed during appressorium formation and under nitrogen starvation. Targeted gene deletion of MGG_01282, in addition to a significant reduction in protein ubiquitination as determined by immuno blot assays, resulted in pleiotropic effects on M. oryzae including reduced growth and sporulation, abnormal conidia morphology, reduced germination and appressorium formation, and the inability to cause disease. Mutants were also defective in sexual development and were female sterile. Using mass spectrometry, we identified 63 candidate polyubiquitinated proteins under nitrogen starvation, which included overrepresentation of proteins involved in translation, transport and protein modification. Our study suggests that ubiquitination of target proteins plays an important role in nutrient assimilation, development and pathogenicity of M. oryzae.

Significant progress has been made in defining the central signaling networks in many organisms, but collectively we know little about the downstream targets of these networks and the genes they regulate. To reconstruct the regulatory circuit of calcineurin signal transduction via MoCRZ1, a Magnaporthe oryzae C2H2 transcription factor activated by calcineurin dephosphorylation, we used a combined approach of chromatin immunoprecipitation - chip (ChIP-chip), coupled with microarray expression studies. One hundred forty genes were identified as being both a direct target of MoCRZ1 and having expression concurrently differentially regulated in a calcium/calcineurin/MoCRZ1 dependent manner. Highly represented were genes involved in calcium signaling, small molecule transport, ion homeostasis, cell wall synthesis/maintenance, and fungal virulence. Of particular note, genes involved in vesicle mediated secretion necessary for establishing host associations, were also found. MoCRZ1 itself was a target, suggesting a previously unreported autoregulation control point. The data also implicated a previously unreported feedback regulation mechanism of calcineurin activity. We propose that calcium/calcineurin regulated signal transduction circuits controlling development and pathogenicity manifest through multiple layers of regulation. We present results from the ChIP-chip and expression analysis along with a refined model of calcium/calcineurin signaling in this important plant pathogen.

Early blight (EB) is one of the dreadful diseases of tomato caused by several species of Alternaria including Alternaria linariae (which includes A. solani and A. tomatophila), as well as A. alternata. In some instances, annual economic yield losses due to EB have been estimated at 79%. Alternaria are known only to reproduce asexually, but a highly-virulent isolate has the potential to overcome existing resistance genes. Currently, cultural practices and fungicide applications are employed for the management of EB due to the lack of strong resistant cultivars. Resistance sources have been identified in wild species of tomato; some breeding lines and cultivars with moderate resistance have been developed through conventional breeding methods. Polygenic inheritance of EB resistance, insufficient resistance in cultivated species and the association of EB resistance with undesirable horticultural traits have thwarted the effective breeding of EB resistance in tomato. Several quantitative trait loci (QTL) conferring EB resistance have been detected in the populations derived from different wild species including Solanum habrochaites, Solanum arcanum and S. pimpinellifolium, but none of them could be used in EB resistance breeding due to low individual QTL effects. Pyramiding of those QTLs would provide strong resistance. More research is needed to identify additional sources of useful resistance, to incorporate resistant QTLs into breeding lines through marker-assisted selection (MAS) and to develop resistant cultivars with desirable horticultural traits including high yielding potential and early maturity. This paper will review the current understanding of causal agents of EB of tomato, resistance genetics and breeding, problems associated with breeding and future prospects.

Significant progress has been made in defining the central signaling networks in many organisms, but collectively we know little about the downstream targets of these networks and the genes they regulate. To reconstruct the regulatory circuit of calcineurin signal transduction via MoCRZ1, a Magnaporthe oryzae C2H2 transcription factor activated by calcineurin dephosphorylation, we used a combined approach of chromatin immunoprecipitation - chip (ChIP-chip), coupled with microarray expression studies. One hundred forty genes were identified as being both a direct target of MoCRZ1 and having expression concurrently differentially regulated in a calcium/calcineurin/MoCRZ1 dependent manner. Highly represented were genes involved in calcium signaling, small molecule transport, ion homeostasis, cell wall synthesis/maintenance, and fungal virulence. Of particular note, genes involved in vesicle mediated secretion necessary for establishing host associations, were also found. MoCRZ1 itself was a target, suggesting a previously unreported autoregulation control point. The data also implicated a previously unreported feedback regulation mechanism of calcineurin activity. We propose that calcium/calcineurin regulated signal transduction circuits controlling development and pathogenicity manifest through multiple layers of regulation. We present results from the ChIP-chip and expression analysis along with a refined model of calcium/calcineurin signaling in this important plant pathogen. All organisms have the innate ability to perceive their environment and respond to it, largely through controlling gene expression. Tailored specificity of a response is primarily achieved through signal cascades involving unique receptors, downstream transcription factors (proteins that bind to DNA to regulate gene expression), and the genes these transcription factors regulate. For fungal plant pathogens, signal transduction cascades are involved in perception of hosts, transgression of physical barriers, suppression or elicitation of host defenses, in vivo nutrient acquisition, and completion of their life cycle. We know that the Ca2+/calcineurin signaling pathway is a central conduit regulating these aspects of the life cycle for fungal pathogens of plants and animals. In this study, we used advanced ChIP-chip and microarray gene expression technologies to identify the genes that the Ca2+/calcineurin responsive transcription factor MoCRZ1 directly binds to and regulates the expression of. Our findings show conservations and divergence in this pathway within the fungal kingdom. It also identifies points of control in the pathway that were previously unidentified. Most importantly, this study implicates this pathway in the establishment of host associations and virulence for the causal agent of rice blast disease, Magnaporthe oryzae, the most important disease of rice worldwide.

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.