Sequential fungal pretreatment of unsterilized Miscanthus: changes in composition, cellulose digestibility and microbial communities.

A sequential fungal pretreatment of Miscanthus × giganteus was conducted by mixing unsterilized Miscanthus with material previously colonized with the white-rot fungus Ceriporiopsis subvermispora. For three generations, each generation started with inoculation by mixing unsterilized fresh Miscanthus with end material from the previous generation and ended after 28 days of incubation at 28 °C. After the first generation, the cellulose digestibility of the material doubled, compared to that of the unsterilized Miscanthus, but the second and third generations showed no enhancements in cellulose digestibility. Furthermore, high degradation of Miscanthus structural carbohydrates occurred during the first generation. A microbial community study showed that, even though the fungal community of the material previously colonized by C. subvermispora was composed mainly of this fungus (> 99%), by the first generation its relative abundance was down to only 9%, and other microbes had prevailed. Additionally, changes in the bacterial community occurred that might be associated with unwanted cellulose degradation in the system. This reiterates the necessity of feedstock microbial load reduction for the stability and reproducibility of fungal pretreatment of lignocellulosic biomass. KEY POINTS: • Sequential fungal pretreatment of unsterilized Miscanthus was unsuccessful. • Feedstock changes with white-rot fungi favored the growth of other microorganisms. • Feedstock microbial reduction is necessary for pretreatment with C. subvermispora.

Genome Resources for Seven Fungal Isolates That Cause Dollar Spot Disease in Turfgrass, Including Clarireedia jacksonii and C. monteithiana.

Fungi in the genus Clarireedia are widespread and destructive pathogens of grasses worldwide, and are best known as the causal agents of dollar spot disease in turfgrass. Here, we report genome assemblies of seven Clarireedia isolates, including ex-types of the two most widespread species, Clarireedia jacksonii and C. monteithiana. These datasets provide a valuable resource for ongoing studies of the dollar spot pathogens that include population diversity, host-pathogen interactions, marker development, and disease control.

FgHtf1 Regulates Global Gene Expression towards Aerial Mycelium and Conidiophore Formation in the Cereal Fungal Pathogen Fusarium graminearum.

The homeobox gene family of transcription factors (HTF) controls many developmental pathways and physiological processes in eukaryotes. We previously showed that a conserved HTF in the plant-pathogenic fungus Fusarium graminearum, Htf1 (FgHtf1), regulates conidium morphology in that organism. This study investigated the mechanism of FgHtf1-mediated regulation and identified putative FgHtf1 target genes by a chromatin immunoprecipitation assay combined with parallel DNA sequencing (ChIP-seq) and RNA sequencing. A total of 186 potential binding peaks, including 142 genes directly regulated by FgHtf1, were identified. Subsequent motif prediction analysis identified two DNA-binding motifs, TAAT and CTTGT. Among the FgHtf1 target genes were FgHTF1 itself and several important conidiation-related genes (e.g., FgCON7), the chitin synthase pathway genes, and the aurofusarin biosynthetic pathway genes. In addition, FgHtf1 may regulate the cAMP-protein kinase A (PKA)-Msn2/4 and Ca2+-calcineurin-Crz1 pathways. Taken together, these results suggest that, in addition to autoregulation, FgHtf1 also controls global gene expression and promotes a shift to aerial growth and conidiation in F. graminearum by activation of FgCON7 or other conidiation-related genes.IMPORTANCE The homeobox gene family of transcription factors is known to be involved in the development and conidiation of filamentous fungi. However, the regulatory mechanisms and downstream targets of homeobox genes remain unclear. FgHtf1 is a homeobox transcription factor that is required for phialide development and conidiogenesis in the plant pathogen F. graminearum In this study, we identified FgHtf1-controlled target genes and binding motifs. We found that, besides autoregulation, FgHtf1 also controls global gene expression and promotes conidiation in F. graminearum by activation of genes necessary for aerial growth, FgCON7, and other conidiation-related genes.

Genotyping-by-Sequencing-Based Genetic Analysis of African Rice Cultivars and Association Mapping of Blast Resistance Genes Against Magnaporthe oryzae Populations in Africa.

Understanding the genetic diversity of rice germplasm is important for the sustainable use of genetic materials in rice breeding and production. Africa is rich in rice genetic resources that can be utilized to boost rice productivity on the continent. A major constraint to rice production in Africa is rice blast, caused by the hemibiotrophic fungal pathogen Magnaporthe oryzae. In this report, we present the results of a genotyping-by-sequencing (GBS)-based diversity analysis of 190 African rice cultivars and an association mapping of blast resistance (R) genes and quantitative trait loci (QTLs). The 190 African cultivars were clustered into three groups based on the 184K single nucleotide polymorphisms generated by GBS. We inoculated the rice cultivars with six African M. oryzae isolates. Association mapping identified 25 genomic regions associated with blast resistance (RABRs) in the rice genome. Moreover, PCR analysis indicated that RABR_23 is associated with the Pi-ta gene on chromosome 12. Our study demonstrates that the combination of GBS-based genetic diversity population analysis and association mapping is effective in identifying rice blast R genes/QTLs that contribute to resistance against African populations of M. oryzae. The identified markers linked to the RABRs and 14 highly resistant cultivars in this study will be useful for rice breeding in Africa.

Early Detection of Airborne Inoculum of Magnaporthe oryzae in Turfgrass Fields Using a Quantitative LAMP Assay.

Gray leaf spot (GLS) is a destructive disease of perennial ryegrass caused by a host specific pathotype of the ascomycete Magnaporthe oryzae. Early diagnosis is crucial for effective disease management and the implementation of Integrated Pest Management practices. However, a rapid protocol for the detection of low levels of airborne inoculum is still missing. We developed a pathogen-specific quantitative loop-mediated isothermal amplification (qLAMP) assay coupled with a spore trap system for rapid detection and quantification of airborne inoculum of the M. oryzae perennial ryegrass pathotype, and tested its suitability for implementation in GLS-infected turfgrass fields. In summer 2015, two perennial ryegrass plots were artificially inoculated with the pathogen, with four continuously running custom impaction spore traps placed in each plot. Sampling units were replaced daily and tested with the developed qLAMP assay, while plots were monitored for symptom development. Results confirmed that the qLAMP assay-trap system was able to detect as few as 10 conidia up to 12 days before symptoms developed in the field. LAMP technology is particularly appropriate for field implementation by nontechnical users, and has the potential to be a powerful decision support tool to guide timing of fungicide applications for GLS management.

ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.

Cyclophilin 20-3 relays a 12-oxo-phytodienoic acid signal during stress responsive regulation of cellular redox homeostasis.

The jasmonate family of phytohormones plays central roles in plant development and stress acclimation. However, the architecture of their signaling circuits remains largely unknown. Here we describe a jasmonate family binding protein, cyclophilin 20-3 (CYP20-3), which regulates stress-responsive cellular redox homeostasis. (+)-12-Oxo-phytodienoic acid (OPDA) binding promotes CYP20-3 to form a complex with serine acetyltransferase 1, which triggers the formation of a hetero-oligomeric cysteine synthase complex with O-acetylserine(thiol)lyase B in chloroplasts. The cysteine synthase complex formation then activates sulfur assimilation that leads to increased levels of thiol metabolites and the buildup of cellular reduction potential. The enhanced redox capacity in turn coordinates the expression of a subset of OPDA-responsive genes. Thus, we conclude that CYP20-3 is a key effector protein that links OPDA signaling to amino acid biosynthesis and cellular redox homeostasis in stress responses.

ChIP-seq reveals a role for CrzA in the Aspergillus fumigatus high-osmolarity glycerol response (HOG) signalling pathway.

Aspergillus fumigatus is an opportunistic pathogen and allergen of mammals. Calcium signalling is essential for A. fumigatus pathogenicity and is regulated by the CrzA transcription factor. We used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) to explore CrzA gene targets in A. fumigatus. In total, 165 potential binding peaks including 102 directly regulated genes were identified, resulting in the prediction of the A[GT][CG]CA[AC][AG] CrzA-binding motif. The 102 CrzA putatively regulated genes exhibited a diverse array of functions. The phkB (Afu3g12530) histidine kinase and the sskB (Afu1g10940) MAP kinase kinase kinase of the HOG (high-osmolarity glycerol response) pathway were regulated by CrzA. Several members of the two-component system (TCS) and the HOG pathway were more sensitive to calcium. CrzA::GFP was translocated to the nucleus upon osmotic stress. CrzA is important for the phosphorylation of the SakA MAPK in response to osmotic shock. The ΔsskB was more sensitive to CaCl2 , NaCl, and paraquat stress, while being avirulent in a murine model of invasive pulmonary aspergillosis. The presence of CaCl2 and osmotic stresses resulted in synergistic inhibition of ΔcrzA and ΔsskB growth. These results suggest there is a genetic interaction between the A. fumigatus calcineurin-CrzA and HOG pathway that is essential for full virulence.

Histoplasma yeast and mycelial transcriptomes reveal pathogenic-phase and lineage-specific gene expression profiles.

BACKGROUND: The dimorphic fungus Histoplasma capsulatum causes respiratory and systemic disease in mammalian hosts by expression of factors that enable survival within phagocytic cells of the immune system. Histoplasma's dimorphism is distinguished by growth either as avirulent mycelia or as pathogenic yeast. Geographically distinct strains of Histoplasma differ in their relative virulence in mammalian hosts and in production of and requirement for specific virulence factors. The close similarity in the genome sequences of these diverse strains suggests that phenotypic variations result from differences in gene expression rather than gene content. To provide insight into how the transcriptional program translates into morphological variation and the pathogenic lifestyle, we compared the transcriptional profile of the pathogenic yeast phase and the non-pathogenic mycelial phase of two clinical isolates of Histoplasma. RESULTS: To overcome inaccuracies in ab initio genome annotation of the Histoplasma genome, we used RNA-seq methodology to generate gene structure models based on experimental evidence. Quantitative analyses of the sequencing reads revealed 6% to 9% of genes are differentially regulated between the two phases. RNA-seq-based mRNA quantitation was strongly correlated with gene expression levels determined by quantitative RT-PCR. Comparison of the yeast-phase transcriptomes between strains showed 7.6% of all genes have lineage-specific expression differences including genes contributing, or potentially related, to pathogenesis. GFP-transcriptional fusions and their introduction into both strain backgrounds revealed that the difference in transcriptional activity of individual genes reflects both variations in the cis- and trans-acting factors between Histoplasma strains. CONCLUSIONS: Comparison of the yeast and mycelial transcriptomes highlights genes encoding virulence factors as well as those involved in protein glycosylation, alternative metabolism, lipid remodeling, and cell wall glycanases that may contribute to Histoplasma pathogenesis. These studies lay an essential foundation for understanding how gene expression variations contribute to the strain- and phase-specific virulence differences of Histoplasma.

Genome comparison of two Magnaporthe oryzae field isolates reveals genome variations and potential virulence effectors.

BACKGROUND: Rice blast caused by the fungus Magnaporthe oryzae is an important disease in virtually every rice growing region of the world, which leads to significant annual decreases of grain quality and yield. To prevent disease, resistance genes in rice have been cloned and introduced into susceptible cultivars. However, introduced resistance can often be broken within few years of release, often due to mutation of cognate avirulence genes in fungal field populations. RESULTS: To better understand the pattern of mutation of M. oryzae field isolates under natural selection forces, we used a next generation sequencing approach to analyze the genomes of two field isolates FJ81278 and HN19311, as well as the transcriptome of FJ81278. By comparing the de novo genome assemblies of the two isolates against the finished reference strain 70-15, we identified extensive polymorphisms including unique genes, SNPs (single nucleotide polymorphism) and indels, structural variations, copy number variations, and loci under strong positive selection. The 1.75 MB of isolate-specific genome content carrying 118 novel genes from FJ81278, and 0.83 MB from HN19311 were also identified. By analyzing secreted proteins carrying polymorphisms, in total 256 candidate virulence effectors were found and 6 were chosen for functional characterization. CONCLUSIONS: We provide results from genome comparison analysis showing extensive genome variation, and generated a list of M. oryzae candidate virulence effectors for functional characterization.

Combining ChIP-chip and expression profiling to model the MoCRZ1 mediated circuit for Ca/calcineurin signaling in the rice blast fungus.

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.

Diverse and tissue-enriched small RNAs in the plant pathogenic fungus, Magnaporthe oryzae.

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.

The genome sequence of the rice blast fungus Magnaporthe grisea.

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.

Identification and analysis of in planta expressed genes of Magnaporthe oryzae.

BACKGROUND: Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction. RESULTS: A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence. CONCLUSION: This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.

Identification of novel virulence factors associated with signal transduction pathways in Alternaria brassicicola.

Alternaria brassicicola is an important, necrotrophic fungal pathogen that causes black spot disease on Brassicas. In order to study pathogenicity mechanisms, gene deletion mutants were generated for 21 putative regulatory genes including kinases and transcription factors subjectively selected from the annotated A. brassicicola genome. Except for Ste12, the deletion of the SNF1 kinase, XlnR, and CreA homologues that control cell wall-degrading enzyme production did not significantly affect virulence in contrast to other pathogenic fungi. Only deletion of XlnR but not CreA, Ste12 or SNF1 impaired the fungus' ability to utilize sole carbon sources suggesting Alternaria regulates expression of cell wall-degrading enzymes in a novel manner. In addition, two novel virulence factors encoding a transcription factor (AbPro1) and a two-component histidine kinase gene (AbNIK1) were discovered. Deletion of AbPro1 resulted in a 70% reduction in virulence and a 25% reduction in vegetative growth rates in vitro. Deletion of AbNIK1 resulted in a near complete loss of virulence, increased sensitivity to osmotic stress, and no changes in vegetative growth rates in vitro. Interestingly, addition of long polypeptides to spores of both Deltaabste12 and Deltaabnik1 during inoculations resulted in a complete restoration of pathogenicity through a yet to be defined mechanism.

Infection and colonization of turf-type bermudagrass by Ophiosphaerella herpotricha expressing green or red fluorescent proteins.

Spring dead spot, caused by Ophiosphaerella herpotricha, is the most important disease of turf-type bermudagrass (Cynodon spp.) in the transition zone of the United States. Despite the importance of the disease, only limited information is available about the host-pathogen interaction at the cellular level. To evaluate the host plant interaction, an isolate of O. herpotricha expressing green fluorescent proteins (GFP) or red fluorescent proteins (tdTomato) was used to study the infection and colonization of roots and stolons of several bermudagrass cultivars. Roots of cultivars Tifway 419 and Midlawn were colonized similarly, resulting in extensive root necrosis, whereas an accession of Cynodon transvaalensis was less necrotic. The stele of C. transvaalensis roots was colonized but not those of Tifway 419 and Midlawn. For intact stolons, colonization was limited to the epidermis and defined macroscopic necrotic lesions were observed on Tifway 419 and Midlawn while C. transvaalensis stolon tissues remained mostly nonnecrotic. Internal colonization of stolons occurred when hyphae grew into wounds, resulting in necrosis in Tifway 419 and Midlawn, but not in C. transvaalensis. These studies suggest that the interaction of O. herpotricha with bermudagrass varies across host genotypes and the host tissues infected. The limited necrosis in C. transvaalensis tissues, though colonized, suggests an inherent tolerance to O. herpotricha.

Gene Ontology annotation of the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: Magnaporthe oryzae, the causal agent of blast disease of rice, is the most destructive disease of rice worldwide. The genome of this fungal pathogen has been sequenced and an automated annotation has recently been updated to Version 6 http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/MultiDownloads.html. However, a comprehensive manual curation remains to be performed. Gene Ontology (GO) annotation is a valuable means of assigning functional information using standardized vocabulary. We report an overview of the GO annotation for Version 5 of M. oryzae genome assembly. METHODS: A similarity-based (i.e., computational) GO annotation with manual review was conducted, which was then integrated with a literature-based GO annotation with computational assistance. For similarity-based GO annotation a stringent reciprocal best hits method was used to identify similarity between predicted proteins of M. oryzae and GO proteins from multiple organisms with published associations to GO terms. Significant alignment pairs were manually reviewed. Functional assignments were further cross-validated with manually reviewed data, conserved domains, or data determined by wet lab experiments. Additionally, biological appropriateness of the functional assignments was manually checked. RESULTS: In total, 6,286 proteins received GO term assignment via the homology-based annotation, including 2,870 hypothetical proteins. Literature-based experimental evidence, such as microarray, MPSS, T-DNA insertion mutation, or gene knockout mutation, resulted in 2,810 proteins being annotated with GO terms. Of these, 1,673 proteins were annotated with new terms developed for Plant-Associated Microbe Gene Ontology (PAMGO). In addition, 67 experiment-determined secreted proteins were annotated with PAMGO terms. Integration of the two data sets resulted in 7,412 proteins (57%) being annotated with 1,957 distinct and specific GO terms. Unannotated proteins were assigned to the 3 root terms. The Version 5 GO annotation is publically queryable via the GO site http://amigo.geneontology.org/cgi-bin/amigo/go.cgi. Additionally, the genome of M. oryzae is constantly being refined and updated as new information is incorporated. For the latest GO annotation of Version 6 genome, please visit our website http://scotland.fgl.ncsu.edu/smeng/GoAnnotationMagnaporthegrisea.html. The preliminary GO annotation of Version 6 genome is placed at a local MySql database that is publically queryable via a user-friendly interface Adhoc Query System. CONCLUSION: Our analysis provides comprehensive and robust GO annotations of the M. oryzae genome assemblies that will be solid foundations for further functional interrogation of M. oryzae.

Anastomosis is required for virulence of the fungal necrotroph Alternaria brassicicola.

A fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogen Alternaria brassicicola and two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of the A. brassicicola isolates tested, as well as A. mimicula and A. japonica, were capable of self-fusion, with two isolates of A. brassicicola being capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) in A. brassicicola resulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenic A. brassicicola mutant defective for a mitogen-activated protein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity in A. brassicicola.

Transformation of Phomopsis viticola with the green fluorescent protein.

Phomopsis viticola is the causal agent of Phomopsis cane and leaf spot on Vitis spp., a persistent and economically important disease in temperate regions. Here we describe the transformation of this fungus with two different constructs (pBHt2_sGFP and pIGPAPA) containing the green fluorescent protein (GFP) and the hygromycin B resistance gene (hph). Protoplast-mediated transformation yielded mitotically stable transformants with no change in virulence on grape internodes and leaves in comparison to the wild type. These transformants will be critical tools for elucidating fungal penetration of host plants, invasive growth and the nature of its host association.

Expressed sequence tags from phytophthora sojae reveal genes specific to development and infection.

Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.