Genomics and Transcriptomics of the sebacinoid fungi Piriformospora indica and Sebacina vermifera

The root endophyte Piriformospora indica and the orchid mycorrhiza Sebacina vermifera (Sebacinales, Basidiomycota) are able to establish a mutualistic symbiosis with plants. Both fungi colonize the root cortex of a wide range of vascular plants, including the monocot barley (Hordeum vulgare) and the dicot Arabidopsis thaliana. Colonization by the fungi results in growth promotion and induced resistance against abiotic and biotic stresses. Fungal development in roots combines biotrophic growth in living plant cells and cell-death associated colonization of dead cortex cells. These features together with the possibility to cultivate the fungi on synthetic media reveal substantial phenotypic plasticity which is reflected in their genomic traits. In this study, the genomes of Piriformospora indica and Sebacina vermifera were characterized. It could be shown that certain gene and functional protein domain expansions occurred in both species. These included proteins predicted to be involved in intra- and extracellular transport (Transporters), proteolysis (Peptidases), degradation of carbohydrates (Hydrolases) and non-destructive carbohydrate binding (Lectins). Additionally, a novel family of small secreted proteins was identified in P. indica which is characterized by regular distributed histidine and alanine residues and a conserved seven amino acid motif ("RSIDELD") at the C-terminus. On the other side, the number proteins involved in secondary metabolism, in particular polyketide and nonribosomal peptide synthetases, were shown to be strongly reduced in both fungi which is indicative of the non pathogenic character of P. indica and S. vermifera. By using microarrays and RNA-sequencing, a time- and host-specific expression of genes could be shown in P. indica during colonization of barley- or Arabidopsis roots. A first comparative analyses of genes expressed in S. vermifera and P. indica during colonization of Arabidopsis suggests that defined differences exist during the colonization of this host by both analysed fungi

Endophytic Life Strategies Decoded by Genome and Transcriptome Analyses of the Mutualistic Root Symbiont Piriformospora indica

Recent sequencing projects have provided deep insight into fungal lifestyle-associated genomic adaptations. Here we report on the 25 Mb genome of the mutualistic root symbiont Piriformospora indica (Sebacinales, Basidiomycota) and provide a global characterization of fungal transcriptional responses associated with the colonization of living and dead barley roots. Extensive comparative analysis of the P. indica genome with other Basidiomycota and Ascomycota fungi that have diverse lifestyle strategies identified features typically associated with both, biotrophism and saprotrophism. The tightly controlled expression of the lifestyle-associated gene sets during the onset of the symbiosis, revealed by microarray analysis, argues for a biphasic root colonization strategy of P. indica. This is supported by a cytological study that shows an early biotrophic growth followed by a cell death-associated phase. About 10% of the fungal genes induced during the biotrophic colonization encoded putative small secreted proteins (SSP), including several lectin-like proteins and members of a P. indica-specific gene family (DELD) with a conserved novel seven-amino acids motif at the C-terminus. Similar to effectors found in other filamentous organisms, the occurrence of the DELDs correlated with the presence of transposable elements in gene-poor repeat-rich regions of the genome. This is the first in depth genomic study describing a mutualistic symbiont with a biphasic lifestyle. Our findings provide a significant advance in understanding development of biotrophic plant symbionts and suggest a series of incremental shifts along the continuum from saprotrophy towards biotrophy in the evolution of mycorrhizal association from decomposer fungi

Genomics and Transcriptomics of the sebacinoid fungi Piriformospora indica and Sebacina vermifera

The root endophyte Piriformospora indica and the orchid mycorrhiza Sebacina vermifera (Sebacinales, Basidiomycota) are able to establish a mutualistic symbiosis with plants. Both fungi colonize the root cortex of a wide range of vascular plants, including the monocot barley (Hordeum vulgare) and the dicot Arabidopsis thaliana. Colonization by the fungi results in growth promotion and induced resistance against abiotic and biotic stresses. Fungal development in roots combines biotrophic growth in living plant cells and cell-death associated colonization of dead cortex cells. These features together with the possibility to cultivate the fungi on synthetic media reveal substantial phenotypic plasticity which is reflected in their genomic traits. In this study, the genomes of Piriformospora indica and Sebacina vermifera were characterized. It could be shown that certain gene and functional protein domain expansions occurred in both species. These included proteins predicted to be involved in intra- and extracellular transport (Transporters), proteolysis (Peptidases), degradation of carbohydrates (Hydrolases) and non-destructive carbohydrate binding (Lectins). Additionally, a novel family of small secreted proteins was identified in P. indica which is characterized by regular distributed histidine and alanine residues and a conserved seven amino acid motif ("RSIDELD") at the C-terminus. On the other side, the number proteins involved in secondary metabolism, in particular polyketide and nonribosomal peptide synthetases, were shown to be strongly reduced in both fungi which is indicative of the non pathogenic character of P. indica and S. vermifera. By using microarrays and RNA-sequencing, a time- and host-specific expression of genes could be shown in P. indica during colonization of barley- or Arabidopsis roots. A first comparative analyses of genes expressed in S. vermifera and P. indica during colonization of Arabidopsis suggests that defined differences exist during the colonization of this host by both analysed fungi

Broad compatibility in fungal root symbioses

Plants associate with a wide range of beneficial fungi in their roots which facilitate plant mineral nutrient uptake in exchange for carbohydrates and other organic metabolites. These associations play a key role in shaping terrestrial ecosystems and are widely believed to have promoted the evolution of land plants. To establish compatibility with their host, root-associated fungi have evolved diverse colonization strategies with distinct morphological, functional and genomic specializations as well as different degrees of interdependence. They include obligate biotrophic arbuscular mycorrhizal (AM), and facultative biotrophic ectomycorrhizal (ECM) interactions but are not restricted to these well-characterized symbioses. There is growing evidence that root endophytic associations, which due to their inconspicuous nature have been often overlooked, can be of mutualistic nature and represent important players in natural and managed environments. Recent research into the biology and genomics of root associations revealed fascinating insight into the phenotypic and trophic plasticity of these fungi and underlined genomic traits associated with biotrophy and saprotrophy. In this review we will consider the commonalities and differences of AM and ECM associations and contrast them with root endophytes

Semantic Focusing Allows Fully Automated Single-Layer Slide Scanning of Cervical Cytology Slides

<div><p>Liquid-based cytology (LBC) in conjunction with Whole-Slide Imaging (WSI) enables the objective and sensitive and quantitative evaluation of biomarkers in cytology. However, the complex three-dimensional distribution of cells on LBC slides requires manual focusing, long scanning-times, and multi-layer scanning. Here, we present a solution that overcomes these limitations in two steps: first, we make sure that focus points are only set on cells. Secondly, we check the total slide focus quality. From a first analysis we detected that superficial dust can be separated from the cell layer (thin layer of cells on the glass slide) itself. Then we analyzed 2,295 individual focus points from 51 LBC slides stained for p16 and Ki67. Using the number of edges in a focus point image, specific color values and size-inclusion filters, focus points detecting cells could be distinguished from focus points on artifacts (accuracy 98.6%). Sharpness as total focus quality of a virtual LBC slide is computed from 5 sharpness features. We trained a multi-parameter SVM classifier on 1,600 images. On an independent validation set of 3,232 cell images we achieved an accuracy of 94.8% for classifying images as focused. Our results show that single-layer scanning of LBC slides is possible and how it can be achieved. We assembled focus point analysis and sharpness classification into a fully automatic, iterative workflow, free of user intervention, which performs repetitive slide scanning as necessary. On 400 LBC slides we achieved a scanning-time of 13.9±10.1 min with 29.1±15.5 focus points. In summary, the integration of semantic focus information into whole-slide imaging allows automatic high-quality imaging of LBC slides and subsequent biomarker analysis.</p></div

Comparison multi-layer scanning with single-layer scanning.

<p>Two cross sections of a LBC slide are shown. The upper one shows the multilayer scanning principle. The lines represent the particular layers. Green line parts represent in-focus regions and red line parts represent the out-of-focus regions. In multilayer scanning, the most parts of the layers are out-of-focus and thereby an unnecessary amount of data is generated. The lower cross section shows the principle of a single-layer scan. A “master-focus layer” (green line) represents the full 3D focus map of the LBC slide. In the optimal case, one focus layer would be sufficient and multi-layering would not be needed anymore or only as a supplement to cover thick cell clusters (transparent green lines).</p

A simplified schematic of the complete workflow for scanning one slide.

<p>The slide is loaded and the area to be scanned is detected automatically. Focus points are set and after autofocussing, the focus point images are analyzed. If the number of valid focus point is higher than five, the slide is scanned and its sharpness is analyzed. From the results of sharpness analysis, a decision is made whether to re-scan the slide or not. The slide is re-scanned until the quality is sufficient for further analysis.</p

Descriptive statistics of the focus point dataset of the particular slides showing the high variations between the z-values within and between the slides.

<p>Descriptive statistics of the focus point dataset of the particular slides showing the high variations between the z-values within and between the slides.</p

Contingency table and overall performance of the focus point analysis of 2295 focus points from 51 LBC slides.

<p>Contingency table and overall performance of the focus point analysis of 2295 focus points from 51 LBC slides.</p

The detailed steps for whole-slide sharpness quantification;

<p>At first, the slide is divided into 16 sub-regions. Then, cells are detected by their color values. In total 200 cells are used to quantify the sharpness of each region. For every cell, five sharpness features are computed and a support vector machine (SVM) is used to classify each cell into the in-focus (class 1) or out-of-focus(class 0) category. The percentage of in-focus cells (0–100%) is used to calculate a score for each region, and a combination of these scores is used to represent slide sharpness.</p