Nicole M. Donofrio

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.

Genome Evolution Following Host Jumps in the Irish Potato Famine Pathogen Lineage

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. Many plant pathogens, including those in the lineage of the Irish potato famine organism Phytophthora infestans, evolve by host jumps followed by specialization. However, how host jumps affect genome evolution remains largely unknown. To determine the patterns of sequence variation in the P. infestans lineage, we resequenced six genomes of four sister species. This revealed uneven evolutionary rates across genomes with genes in repeat-rich regions showing higher rates of structural polymorphisms and positive selection. These loci are enriched in genes induced in planta, implicating host adaptation in genome evolution. Unexpectedly, genes involved in epigenetic processes formed another class of rapidly evolving residents of the gene-sparse regions. These results demonstrate that dynamic repeat-rich genome compartments underpin accelerated gene evolution following host jumps in this pathogen lineage.

Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae

BackgroundRice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe oryzae and results in significant annual rice yield losses worldwide. Infection by this and many other fungal plant pathogens requires the development of a specialized infection cell called an appressorium. The molecular processes regulating appressorium formation are incompletely understood.ResultsWe analyzed genome-wide gene expression changes during spore germination and appressorium formation on a hydrophobic surface compared to induction by cAMP. During spore germination, 2,154 (approximately 21%) genes showed differential expression, with the majority being up-regulated. During appressorium formation, 357 genes were differentially expressed in response to both stimuli. These genes, which we refer to as appressorium consensus genes, were functionally grouped into Gene Ontology categories. Overall, we found a significant decrease in expression of genes involved in protein synthesis. Conversely, expression of genes associated with protein and amino acid degradation, lipid metabolism, secondary metabolism and cellular transportation exhibited a dramatic increase. We functionally characterized several differentially regulated genes, including a subtilisin protease (SPM1) and a NAD specific glutamate dehydrogenase (Mgd1), by targeted gene disruption. These studies revealed hitherto unknown findings that protein degradation and amino acid metabolism are essential for appressorium formation and subsequent infection.ConclusionWe present the first comprehensive genome-wide transcript profile study and functional analysis of infection structure formation by a fungal plant pathogen. Our data provide novel insight into the underlying molecular mechanisms that will directly benefit efforts to identify fungal pathogenicity factors and aid the development of new disease management strategies.

The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana.

Majority of plant growth promoting rhizobacteria (PGPR) confer plant immunity against a wide range of foliar diseases by activating plant defences that reduce a plant’s susceptibility to pathogen attack. Here we show that Arabidopsis thaliana (Col-0) plants exposed to Bacillus subtilis strain FB17 (hereafter FB17), results in reduced disease severity against Pseudomonas syringae pv. tomato DC3000 (hereafter DC3000) compared to plants without FB17 treatment. Exogenous application of the B. subtilis derived elicitor, acetoin (3-hydroxy-2-butanone), was found to trigger induced systemic resistance (ISR) and protect plants against DC3000 pathogenesis. Moreover, B. subtilis acetoin biosynthetic mutants that emitted reduced levels of acetoin conferred reduced protection to A. thaliana against pathogen infection. Further analysis using FB17 and defense-compromised mutants of A. thaliana indicated that resistance to DC3000 occurs via NPR1 and requires salicylic acid (SA)/ethylene (ET) whereas jasmonic acid (JA) is not essential. This study provides new insight into the role of rhizo-bacterial volatile components as elicitors of defense responses in plants.

HYR1-Mediated Detoxification of Reactive Oxygen Species Is Required for Full Virulence in the Rice Blast Fungus

During plant-pathogen interactions, the plant may mount several types of defense responses to either block the pathogen completely or ameliorate the amount of disease. Such responses include release of reactive oxygen species (ROS) to attack the pathogen, as well as formation of cell wall appositions (CWAs) to physically block pathogen penetration. A successful pathogen will likely have its own ROS detoxification mechanisms to cope with this inhospitable environment. Here, we report one such candidate mechanism in the rice blast fungus, Magnaporthe oryzae, governed by a gene we refer to as MoHYR1. This gene (MGG_07460) encodes a glutathione peroxidase (GSHPx) domain, and its homologue in yeast was reported to specifically detoxify phospholipid peroxides. To characterize this gene in M. oryzae, we generated a deletion mutantΔhyr1 which showed growth inhibition with increased amounts of hydrogen peroxide (H2O2). Moreover, we observed that the fungal mutants had a decreased ability to tolerate ROS generated by a susceptible plant, including ROS found associated with CWAs. Ultimately, this resulted in significantly smaller lesion sizes on both barley and rice. In order to determine how this gene interacts with other (ROS) scavenging-related genes in M. oryzae, we compared expression levels of ten genes in mutant versus wild type with and without H2O2. Our results indicated that the HYR1 gene was important for allowing the fungus to tolerate H2O2 in vitro and in planta and that this ability was directly related to fungal virulence.

Natural rice rhizospheric microbes suppress rice blast infections

BackgroundThe natural interactions between plant roots and their rhizospheric microbiome are vital to plant fitness, modulating both growth promotion and disease suppression. In rice (Oryza sativa), a globally important food crop, as much as 30% of yields are lost due to blast disease caused by fungal pathogen Magnaporthe oryzae. Capitalizing on the abilities of naturally occurring rice soil bacteria to reduce M. oryzae infections could provide a sustainable solution to reduce the amount of crops lost to blast disease.ResultsNaturally occurring root-associated rhizospheric bacteria were isolated from California field grown rice plants (M-104), eleven of which were taxonomically identified by16S rRNA gene sequencing and fatty acid methyl ester (FAME) analysis. Bacterial isolates were tested for biocontrol activity against the devastating foliar rice fungal pathogen, M. oryzae pathovar 70–15. In vitro, a Pseudomonas isolate, EA105, displayed antibiosis through reducing appressoria formation by nearly 90% as well as directly inhibiting fungal growth by 76%. Although hydrogen cyanide (HCN) is a volatile commonly produced by biocontrol pseudomonads, the activity of EA105 seems to be independent of its HCN production. During in planta experiments, EA105 reduced the number of blast lesions formed by 33% and Pantoea agglomerans isolate, EA106 by 46%. Our data also show both EA105 and EA106 trigger jasmonic acid (JA) and ethylene (ET) dependent induced systemic resistance (ISR) response in rice.ConclusionsOut of 11 bacteria isolated from rice soil, pseudomonad EA105 most effectively inhibited the growth and appressoria formation of M. oryzae through a mechanism that is independent of cyanide production. In addition to direct antagonism, EA105 also appears to trigger ISR in rice plants through a mechanism that is dependent on JA and ET signaling, ultimately resulting in fewer blast lesions. The application of native bacteria as biocontrol agents in combination with current disease protection strategies could aid in global food security.

Transcriptome profiling of the rice blast fungus during invasive plant infection and in vitro stresses

BackgroundRice blast is the most threatening disease to cultivated rice. Magnaporthe oryzae, its causal agent, is likely to encounter environmental challenges during invasive growth in its host plants that require shifts in gene expression to establish a compatible interaction. Here, we tested the hypothesis that gene expression patterns during in planta invasive growth are similar to in vitro stress conditions, such as nutrient limitation, temperature up shift and oxidative stress, and determined which condition most closely mimicked that of in planta invasive growth. Gene expression data were collected from these in vitro experiments and compared to fungal gene expression during the invasive growth phase at 72 hours post-inoculation in compatible interactions on two grass hosts, rice and barley.ResultsWe identified 4,973 genes that were differentially expressed in at least one of the in planta and in vitro stress conditions when compared to fungal mycelia grown in complete medium, which was used as reference. From those genes, 1,909 showed similar expression patterns between at least one of the in vitro stresses and rice and/or barley. Hierarchical clustering of these 1,909 genes showed three major clusters in which in planta conditions closely grouped with the nutrient starvation conditions. Out of these 1,909 genes, 55 genes and 129 genes were induced and repressed in all treatments, respectively. Functional categorization of the 55 induced genes revealed that most were either related to carbon metabolism, membrane proteins, or were involved in oxidoreduction reactions. The 129 repressed genes showed putative roles in vesicle trafficking, signal transduction, nitrogen metabolism, or molecular transport.ConclusionsThese findings suggest that M. oryzae is likely primarily coping with nutrient-limited environments at the invasive growth stage 72 hours post-inoculation, and not with oxidative or temperature stresses.

Physiological stressors and invasive plant infections alter the small RNA transcriptome of the rice blast fungus, Magnaporthe oryzae

BackgroundThe rice blast fungus, Magnaporthe oryzae is a destructive pathogen of rice and other related crops, causing significant yield losses worldwide. Endogenous small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs) are critical components of gene regulation in many eukaryotic organisms. Recently several new species of sRNAs have been identified in fungi. This fact along with the availability of genome sequence makes M. oryzae a compelling target for sRNA profiling. We have examined sRNA species and their biosynthetic genes in M. oryzae, and the degree to which these elements regulate fungal stress responses. To this end, we have characterized sRNAs under different physiological stress conditions, which had not yet been examined in this fungus.ResultsThe resulting libraries are composed of more than 37 million total genome matched reads mapping to intergenic regions, coding sequences, retrotransposons, inverted, tandem, and other repeated regions of the genome with more than half of the small RNAs arising from intergenic regions. The 24 nucleotide (nt) size class of sRNAs was predominant. A comparison to transcriptional data of M. oryzae undergoing the same physiological stresses indicates that sRNAs play a role in transcriptional regulation for a small subset of genes. Support for this idea comes from generation and characterization of mutants putatively involved in sRNAs biogenesis; our results indicate that the deletion of Dicer-like genes and an RNA-Dependent RNA Polymerase gene increases the transcriptional regulation of this subset of genes, including one involved in virulence.ConclusionsVarious physiological stressors and in planta conditions alter the small RNA profile of the rice blast fungus. Characterization of sRNA biosynthetic mutants helps to clarify the role of sRNAs in transcriptional control.

Plant biomass degradation by fungi

Plant biomass degradation by fungi has implications for several fields of science. The enzyme systems employed by fungi for this are broadly used in various industrial sectors such as food & feed, pulp & paper, detergents, textile, wine, and more recently biofuels and biochemicals. In addition, the topic is highly relevant in the field of plant pathogenic fungi as they degrade plant biomass to either gain access to the plant or as carbon source, resulting in significant crop losses. Finally, fungi are the main degraders of plant biomass in nature and as such have an essential role in the global carbon cycle and ecology in general. In this review we provide a global view on the development of this research topic in saprobic ascomycetes and basidiomycetes and in plant pathogenic fungi and link this to the other papers of this special issue on plant biomass degradation by fungi.

'PACLIMS': A component LIM system for high-throughput functional genomic analysis

BackgroundRecent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the ~11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required.ResultsThe platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured.ConclusionMany sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors.