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Abstract Background Rice 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. Results We 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. Conclusions These 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.

Abstract Background Recent 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. Results The 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. Conclusion Many 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.

While the impacts of arsenic (As) and Magnaporthe oryzae on rice have been well-studied, a dearth of knowledge exists on how rice responds to their combined stress. Moreover, increasing exogenous silicon (Si) can alleviate M. oryzae infection and As uptake, but how increasing exogenous Si affects the combined stress of M. oryzae and As is unknown. We grew three cultivars of rice that varied in their susceptibility to As and M. oryzae under low (50 μM, Si L ) and high (1500 μM, Si H ) Si with and without As (4 μM, 80/20 As (III)/As(V)) and with or without M. oryzae infection and examined the impacts of treatments on plant As and Si concentrations, severity of disease by M. oryzae, and stress via targeted gene expression. Si H treatments generally decreased shoot As concentrations by 20-70% compared to Si L treatments depending on cultivar and M. oryzae exposure. There was no effect of Si or As treatments on percent of leaf diseased in the As-tolerant cultivar M206, but in the As-sensitive cultivar IR66, Si H treatment decreased percent of leaf diseased in the absence of As and had no impact when As was present. In the M. oryzae-susceptible Sariceltik, plants receiving Si H had significantly fewer lesions than those receiving Si L and plants with the fewest lesions were in the Si H  + As treatments. Plants that were exposed to As + M. oryzae were the most stressed when grown under Si L , but this stress response was lowered by Si H treatments. A separate pathogenicity assay with Sariceltik showed that in contrast to our hypothesis, As exposure decreased lesion growth, particularly under Si H treatments, and lessened the impact of M. oryzae on rice. These results suggest that rice grown under replete Si will be able to withstand combined stressors of M. oryzae and As, but will be highly stressed under Si deficient scenarios., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Here, we report the complete genome sequence of Bacillus velezensis strain S4, which was isolated from biochar-amended agricultural soil collected in Smyrna, Delaware. The genome is 4.07 Mbp, encodes 3,918 predicted proteins, and has a GC content of 46.4%., (Copyright © 2020 Hempel et al.)

Coffee leaf rust caused by the fungus Hemileia vastatrix is one of the most important leaf diseases of coffee plantations worldwide. Current knowledge of the H. vastatrix genome is limited and only a small fraction of the total fungal secretome has been identified. In order to obtain a more comprehensive understanding of its secretome, we aimed to sequence and assemble the entire H. vastatrix genome using two next-generation sequencing platforms and a hybrid assembly strategy. This resulted in a 547 Mb genome of H. vastatrix race XXXIII (Hv33), with 13,364 predicted genes that encode 13,034 putative proteins with transcriptomic support. Based on this proteome, 615 proteins contain putative secretion peptides, and lack transmembrane domains. From this putative secretome, 111 proteins were identified as candidate effectors (EHv33) unique to H. vastatrix, and a subset consisting of 17 EHv33 genes was selected for a temporal gene expression analysis during infection. Five genes were significantly induced early during an incompatible interaction, indicating their potential role as pre-haustorial effectors possibly recognized by the resistant coffee genotype. Another nine genes were significantly induced after haustorium formation in the compatible interaction. Overall, we suggest that this fungus is able to selectively mount its survival strategy with effectors that depend on the host genotype involved in the infection process., Competing Interests: The authors have declared that no competing interests exist.

The phytopathogen Spiroplasma phoeniceum was isolated from diseased plants of Madagascar periwinkle [ Catharanthus roseus (L.) G. Don]. Here, we report the nucleotide sequence of the 1,791,576-bp circular chromosome and three plasmids of strain P40 T This information serves as a resource for comparative analyses of spiroplasmal adaptations to diverse ecological niches.

Common bean ( Phaseolus vulgaris L.) is an important legume, useful for its high protein and dietary fiber. The fungal pathogen Uromyces appendiculatus (Pers.) Unger can cause major loss in susceptible varieties of the common bean. The Ur-3 locus provides race specific resistance to virulent strains or races of the bean rust pathogen along with Crg , (Complements resistance gene), which is required for Ur-3 -mediated rust resistance. In this study, we inoculated two common bean genotypes (resistant "Sierra" and susceptible crg) with rust race 53 of U. appendiculatus , isolated leaf RNA at specific time points, and sequenced their transcriptomes. First, molecular markers were used to locate and identify a 250 kb deletion on chromosome 10 in mutant crg (which carries a deletion at the Crg locus). Next, we identified differential expression of several disease resistance genes between Mock Inoculated (MI) and Inoculated (I) samples of "Sierra" leaf RNA within the 250 kb delineated region. Both marker assisted molecular profiling and RNA-seq were used to identify possible transcriptomic locations of interest regarding the resistance in the common bean to race 53. Identification of differential expression among samples in disease resistance clusters in the bean genome may elucidate significant genes underlying rust resistance. Along with preserving favorable traits in the crop, the current research may also aid in global sustainability of food stocks necessary for many populations., Competing Interests: The authors declare no conflict of interest.

Spiroplasma citri causes stubborn disease in Citrus spp. and diseases in other plants. Here, we report the nucleotide sequence of the 1,599,709-bp circular chromosome and two plasmids of S. citri strain R8-A2 T This information will facilitate analyses to understand spiroplasmal pathogenicity and evolutionary adaptations to lifestyles in plants and arthropod hosts., (Copyright © 2017 Davis et al.)

The order Magnaporthales comprises about 200 species and includes the economically and scientifically important rice blast fungus and the take-all pathogen of cereals, as well as saprotrophs and endophytes. Recent advances in phylogenetic analyses of these fungi resulted in taxonomic revisions. In this paper we list the 28 currently accepted genera in Magnaporthales with their type species and available gene and genome resources. The polyphyletic Magnaporthe 1972 is proposed for suppression, and Pyricularia 1880 and Nakataea 1939 are recommended for protection as the generic names for the rice blast fungus and the rice stem rot fungus, respectively. The rationale for the recommended names is also provided. These recommendations are made by the Pyricularia/Magnaporthe Working Group established under the auspices of the International Commission on the Taxonomy of Fungi (ICTF).

Rice suffers dramatic yield losses due to blast pathogen Magnaporthe oryzae. Pseudomonas chlororaphis EA105, a bacterium that was isolated from the rice rhizosphere, inhibits M. oryzae. It was shown previously that pre-treatment of rice with EA105 reduced the size of blast lesions through jasmonic acid (JA)- and ethylene (ETH)-mediated ISR. Abscisic acid (ABA) acts antagonistically toward salicylic acid (SA), JA, and ETH signaling, to impede plant defense responses. EA105 may be reducing the virulence of M. oryzae by preventing the pathogen from up-regulating the key ABA biosynthetic gene NCED3 in rice roots, as well as a β-glucosidase likely involved in activating conjugated inactive forms of ABA. However, changes in total ABA concentrations were not apparent, provoking the question of whether ABA concentration is an indicator of ABA signaling and response. In the rice-M. oryzae interaction, ABA plays a dual role in disease severity by increasing plant susceptibility and accelerating pathogenesis in the fungus itself. ABA is biosynthesized by M. oryzae. Further, exogenous ABA increased spore germination and appressoria formation, distinct from other plant growth regulators. EA105, which inhibits appressoria formation, counteracted the virulence-promoting effects of ABA on M. oryzae. The role of endogenous fungal ABA in blast disease was confirmed through the inability of a knockout mutant impaired in ABA biosynthesis to form lesions on rice. Therefore, it appears that EA105 is invoking multiple strategies in its protection of rice from blast including direct mechanisms as well as those mediated through plant signaling. ABA is a molecule that is likely implicated in both tactics.

Spiroplasma turonicum was isolated from a Haematopota sp. fly in France. We report the nucleotide sequence of the circular chromosome of strain Tab4c(T). The genome information will facilitate evolutionary studies of spiroplasmas, including symbionts of insects and ticks and pathogens of plants, insects, crustaceans, and humans., (Copyright © 2015 Davis et al.)

Spiroplasma kunkelii causes corn stunt disease of Zea mays L. in the Americas. Here, we report the nucleotide sequence of the 1,463,926-bp circular chromosome and four plasmids of strain CR2-3x. This information will facilitate studies of Spiroplasma pathogenicity and evolutionary adaptations to transkingdom parasitism in plants and insect vectors., (Copyright © 2015 Davis et al.)

Background: The 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., Results: Naturally occurring root-associated rhizospheric bacteria were isolated from California field grown rice plants (M-104), eleven of which were taxonomically identified by 16S 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., Conclusions: Out 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.

Background: Rice 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., Results: We 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., Conclusion: We 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.

Towards the goal of disrupting all genes in the genome of Magnaporthe oryzae and identifying their function, a collection of >55,000 random insertion lines of M. oryzae strain 70-15 were generated. All strains were screened to identify genes involved in growth rate, conidiation, pigmentation, auxotrophy, and pathogenicity. Here, we provide a description of the high throughput transformation and analysis pipeline used to create our library. Transformed lines were generated either by CaCl(2)/PEG treatment of protoplasts with DNA or by Agrobacterium tumefaciens-mediated transformation (ATMT). We describe the optimization of both approaches and compare their efficiency. ATMT was found to be a more reproducible method, resulting in predominantly single copy insertions, and its efficiency was high with up to 0.3% of conidia being transformed. The phenotypic data is accessible via a public database called MGOS and all strains are publicly available. This represents the most comprehensive insertional mutagenesis analysis of a fungal pathogen.

Efficient regulation of nitrogen metabolism likely plays a role in the ability of fungi to exploit ecological niches. To learn about regulation of nitrogen metabolism in the rice blast pathogen Magnaporthe grisea, we undertook a genome-wide analysis of gene expression under nitrogen-limiting conditions. Five hundred and twenty genes showed increased transcript levels at 12 and 48 h after shifting the fungus to media lacking nitrate as a nitrogen source. Thirty-nine of these genes have putative functions in amino acid metabolism and uptake, and include the global nitrogen regulator in M. grisea, NUT1. Evaluation of seven nitrogen starvation-induced genes revealed that all were expressed during rice infection. Targeted gene replacement on one such gene, the vacuolar serine protease, SPM1, resulted in decreased sporulation and appressorial development as well as a greatly attenuated ability to cause disease. Data are discussed in the context of nitrogen metabolism under starvation conditions, as well as conditions potentially encountered during invasive growth in planta.

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.

To identify pathogen-induced genes distinct from those involved in systemic acquired resistance, we used cDNA-amplified fragment length polymorphism to examine RNA levels in Arabidopsis thaliana wild type, nim1-1, and salicylate hydroxylase-expressing plants after inoculation with an incompatible isolate of the downy mildew pathogen Peronospora parasitica. Fifteen genes are described, which define three response profiles on the basis of whether their induction requires salicylic acid (SA) accumulation and NIM1/NPR1 activity, SA alone, or neither. Sequence analysis shows that the genes include a calcium binding protein related to TCH3, a protein containing ankyrin repeats and potential transmembrane domains, three glutathione S-transferase gene family members, and a number of small, putatively secreted proteins. We further characterized this set of genes by assessing their expression patterns in each of the three plant lines after inoculation with a compatible P. parasitica isolate and after treatment with the SA analog 2,6-dichloroisonicotinic acid. Some of the genes within subclasses showed different requirements for SA accumulation and NIM1/NPR1 activity, depending upon which elicitor was used, indicating that those genes were not coordinately regulated and that the regulatory pathways are more complex than simple linear models would indicate.

To investigate the impact of induced host defenses on the virulence of a compatible Peronospora parasitica strain on Arabidopsis thaliana, we examined growth and development of this pathogen in nim1-1 mutants and transgenic salicylate hydroxylase plants. These plants are unable to respond to or accumulate salicylic acid (SA), respectively, are defective in expression of systemic acquired resistance (SAR), and permit partial growth of some normally avirulent pathogens. We dissected the P. parasitica life cycle into nine stages and compared its progression through these stages in the defense-compromised hosts and in wild-type plants. NahG plants supported the greatest accumulation of pathogen biomass and conidiophore production, followed by nim1-1 and then wild-type plants. Unlike the wild type, NahG and nim1-1 plants showed little induction of the SAR gene PR-1 after colonization with P parasitica, which is similar to our previous observations. We examined the frequency and morphology of callose deposits around parasite haustoria and found significant differences between the three hosts. NahG plants showed a lower fraction of haustoria surrounded by thick callose encasements and a much higher fraction of haustoria with callose limited to thin collars around haustorial necks compared to wild type, whereas nim1-1 plants were intermediate between NahG and wild type. Chemical induction of SAR in plants colonized by P. parasitica converted the extrahaustorial callose phenotype in NahG to resemble closely the wild-type pattern, but had no effect on nim1-1 plants. These results suggest that extrahaustorial callose deposition is influenced by the presence or lack of SA and that this response may be sensitive to the NIM1/NPR1 pathway. Additionally, the enhanced susceptibility displayed by nim1-1 and NahG plants shows that even wild-type susceptible hosts exert defense functions that reduce disease severity and pathogen fitness.

To test the claim that lesions of left anterior and middle temporal cortical structures specifically impair processing of nouns but not verbs, 56 left-hemisphere-language-dominant patients who had undergone anterior temporal lobectomy (ATL) completed tasks assessing confrontation naming of pictured objects and actions, generation of synonyms for nouns and verbs, and semantic lexical judgments about nouns and verbs. Compared with right ATL patients, left ATL patients were impaired across different tasks that assessed naming and comprehension of high-imageability as well as low-imageability nouns. These groups did not differ, however, in verb naming or comprehension on most tasks. Results are consistent with the hypothesized specialization of left temporal lobe structures for processing nouns and suggest that naming problems commonly seen after left ATL extend beyond difficulties with retrieving object names and may be related to subtle disturbances in comprehension of the meanings underlying nominal word forms.

The MMPI and MMPI-2 have been used extensively in the study of personality correlates in various coronary populations. It has been hypothesized that personality variables are associated with various clinical outcomes, such as quality of life, morbidity, and mortality; however, no data are available in a cardiac transplant sample. This study presents descriptive MMPI and MMPI-2 data for male and female patients awaiting cardiac transplant. Analyses of differences in MMPI-2 depression scales between cardiomyopathy groups for 366 men and 99 women were performed. Significant differences were found between ischemic and nonischemic male patients.

The authors examined interhemispheric memory transfer in 32 patients with lateralized temporal lobe complex partial epilepsy (15 right onsets, 17 left onsets). Visually presented verbal, nonverbal, and two types of dually encodable stimuli were displayed during amobarbital anesthesia, and recognition memory was tested with verbal and nonverbal (pointing) response modalities. No relationship was found between the material specificity of stimuli and response modality. The only significant findings were for poorer recognition memory after injection of the hemisphere contralateral to the seizure focus. Visual information presented predominantly to one hemisphere during anesthesia is available to the other hemisphere for recognition memory on clearing.