Suberin deficiency and its effect on the transport physiology of young poplar roots.

The precise functions of suberized apoplastic barriers in root water and nutrient transport physiology have not fully been elucidated. While lots of research has been performed with mutants of Arabidopsis, little to no data are available for mutants of agricultural crop or tree species. By employing a combined set of physiological, histochemical, analytical, and transport physiological methods as well as RNA-sequencing, this study investigated the implications of remarkable CRISPR/Cas9-induced suberization defects in young roots of the economically important gray poplar. While barely affecting overall plant development, contrary to literature-based expectations significant root suberin reductions of up to 80-95% in four independent mutants were shown to not evidently affect the root hydraulic conductivity during non-stress conditions. In addition, subliminal iron deficiency symptoms and increased translocation of a photosynthesis inhibitor as well as NaCl highlight the involvement of suberin in nutrient transport physiology. The multifaceted nature of the root hydraulic conductivity does not allow drawing simplified conclusions such as that the suberin amount must always be correlated with the water transport properties of roots. However, the decreased masking of plasma membrane surface area could facilitate the uptake but also leakage of beneficial and harmful solutes.

ENHANCED GRAVITROPISM 2 encodes a STERILE ALPHA MOTIF-containing protein that controls root growth angle in barley and wheat.

The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain-containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.

Transcriptome profiling at the transition to the reproductive stage uncovers stage and tissue-specific genes in wheat.

BACKGROUND: The transition from vegetative to floral phase is the result of complex crosstalk of exogenous and endogenous floral integrators. This critical physiological event is the response to environmental interaction, which causes biochemical cascades of reactions at different internal tissues, organs, and releases signals that make the plant moves from vegetative status to a reproductive phase. This network controlling flowering time is not deciphered largely in bread wheat. In this study, a comparative transcriptome analysis at a transition time in combination with genetic mapping was used to identify responsible genes in a stage and tissue-specific manner. For this reason, two winter cultivars that have been bred in Germany showing contrasting and stable heading time in different environments were selected for the analysis. RESULTS: In total, 670 and 1075 differentially expressed genes in the shoot apical meristem and leaf tissue, respectively, could be identified in 23 QTL intervals for the heading date. In the transition apex, Histone methylation H3-K36 and regulation of circadian rhythm are both controlled by the same homoeolog genes mapped in QTL TaHd112, TaHd124, and TaHd137. TaAGL14 gene that identifies the floral meristem was mapped in TaHd054 in the double ridge. In the same stage, the homoeolog located on chromosome 7D of FLOWERING TIME LOCUS T mapped on chr 7B, which evolved an antagonist function and acts as a flowering repressor was uncovered. The wheat orthologue of transcription factor ASYMMETRIC LEAVES 1 (AS1) was identified in the late reproductive stage and was mapped in TaHd102, which is strongly associated with heading date. Deletion of eight nucleotides in the AS1 promoter could be identified in the binding site of the SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1) gene in the late flowering cultivar. Both proteins AS1 and SOC1 are inducing flowering time in response to gibberellin biosynthesis. CONCLUSION: The global transcriptomic at the transition phase uncovered stage and tissue-specific genes mapped in QTL of heading date in winter wheat. In response to Gibberellin signaling, wheat orthologous transcription factor AS1 is expressed in the late reproductive phase of the floral transition. The locus harboring this gene is the strongest QTL associated with the heading date trait in the German cultivars. Consequently, we conclude that this is another indication of the Gibberellin biosynthesis as the mechanism behind the heading variation in wheat.

MuWU: Mutant-seq library analysis and annotation.

MOTIVATION: Insertional mutagenesis allows for the creation of loss-of-function mutations on a genome-wide scale. In theory, every gene can be 'knocked out' via the insertion of an additional DNA sequence. Resources of sequence-indexed mutants of plant and animal model organisms are instrumental for functional genomics studies. Such repositories significantly speed up the acquisition of interesting genotypes and allow for the validation of hypotheses regarding phenotypic consequences in reverse genetics. To create such resources, comprehensive sequencing of flanking sequence tags using protocols such as Mutant-seq requires various downstream computational tasks, and these need to be performed in an efficient and reproducible manner. RESULTS: Here, we present MuWU, an automated Mutant-seq workflow utility initially created for the identification of Mutator insertion sites of the BonnMu resource, representing a reverse genetics mutant collection for functional genetics in maize (Zea mays). MuWU functions as a fast, one-stop downstream processing pipeline of Mutant-seq reads. It takes care of all complex bioinformatic tasks, such as identifying tagged genes and differentiating between germinal and somatic mutations/insertions. Furthermore, MuWU automatically assigns insertions to the corresponding mutated seed stocks. We discuss the implementation and how parameters can easily be adapted to use MuWU for other species/transposable elements. AVAILABILITY AND IMPLEMENTATION: MuWU is a Snakemake-based workflow and freely available at https://github.com/tgstoecker/MuWU. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley (Hordeum vulgare) and analysis of the cuticular barrier functions.

The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3. Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding ß-ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54. The amounts of intracuticular waxes were strongly decreased in cer-ye.267. The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.

Single-parent expression complementation contributes to phenotypic heterosis in maize hybrids.

The dominance model of heterosis explains the superior performance of F1-hybrids via the complementation of deleterious alleles by beneficial alleles in many genes. Genes active in one parent but inactive in the second lead to single-parent expression (SPE) complementation in maize (Zea mays L.) hybrids. In this study, SPE complementation resulted in approximately 700 additionally active genes in different tissues of genetically diverse maize hybrids on average. We established that the number of SPE genes is significantly associated with mid-parent heterosis (MPH) for all surveyed phenotypic traits. In addition, we highlighted that maternally (SPE_B) and paternally (SPE_X) active SPE genes enriched in gene co-expression modules are highly correlated within each SPE type but separated between these two SPE types. While SPE_B-enriched co-expression modules are positively correlated with phenotypic traits, SPE_X-enriched modules displayed a negative correlation. Gene ontology term enrichment analyses indicated that SPE_B patterns are associated with growth and development, whereas SPE_X patterns are enriched in defense and stress response. In summary, these results link the degree of phenotypic MPH to the prevalence of gene expression complementation observed by SPE, supporting the notion that hybrids benefit from SPE complementation via its role in coordinating maize development in fluctuating environments.

Populus × canescens root suberization in reaction to osmotic and salt stress is limited to the developing younger root tip region.

Populus is a valuable and fast-growing tree species commonly cultivated for economic and scientific purposes. But most of the poplar species are sensitive to drought and salt stress. Thus, we compared the physiological effects of osmotic stress (PEG8000) and salt treatment (NaCl) on poplar roots to identify potential strategies for future breeding or genetic engineering approaches. We investigated root anatomy using epifluorescence microscopy, changes in root suberin composition and amount using gas chromatography, transcriptional reprogramming using RNA sequencing, and modifications of root transport physiology using a pressure chamber. Poplar roots reacted to the imposed stress conditions, especially in the developing younger root tip region, with remarkable differences between both types of stress. Overall, the increase in suberin content was surprisingly small, but the expression of key suberin biosynthesis genes was strongly induced. Significant reductions of the radial water transport in roots were only observed for the osmotic and not the hydrostatic hydraulic conductivity. Our data indicate that the genetic enhancement of root suberization processes in poplar might be a promising target to convey increased tolerance, especially against toxic sodium chloride.

BonnMu: A Sequence-Indexed Resource of Transposon-Induced Maize Mutations for Functional Genomics Studies.

Sequence-indexed insertional libraries in maize (Zea mays) are fundamental resources for functional genetics studies. Here, we constructed a Mutator (Mu) insertional library in the B73 inbred background designated BonnMu A total of 1,152 Mu-tagged F2-families were sequenced using the Mu-seq approach. We detected 225,936 genomic Mu insertion sites and 41,086 high quality germinal Mu insertions covering 16,392 of the annotated maize genes (37% of the B73v4 genome). On average, each F2-family of the BonnMu libraries captured 37 germinal Mu insertions in genes of the Filtered Gene Set (FGS). All BonnMu insertions and phenotypic seedling photographs of Mu-tagged F2-families can be accessed via MaizeGDB.org Downstream examination of 137,410 somatic and germinal insertion sites revealed that 50% of the tagged genes have a single hotspot, targeted by Mu By comparing our BonnMu (B73) data to the UniformMu (W22) library, we identified conserved insertion hotspots between different genetic backgrounds. Finally, the vast majority of BonnMu and UniformMu transposons was inserted near the transcription start site of genes. Remarkably, 75% of all BonnMu insertions were in closer proximity to the transcription start site (distance: 542 bp) than to the start codon (distance: 704 bp), which corresponds to open chromatin, especially in the 5' region of genes. Our European sequence-indexed library of Mu insertions provides an important resource for functional genetics studies of maize.

High light induces species specific changes in the membrane lipid composition of Chlorella.

Algae have evolved several mechanisms to adjust to changing environmental conditions. To separate from their surroundings, algal cell membranes form a hydrophobic barrier that is critical for life. Thus, it is important to maintain or adjust the physical and biochemical properties of cell membranes which are exposed to environmental factors. Especially glycerolipids of thylakoid membranes, the site of photosynthesis and photoprotection within chloroplasts, are affected by different light conditions. Since little is known about membrane lipid remodeling upon different light treatments, we examined light induced alterations in the glycerolipid composition of the two Chlorella species, C. vulgaris and C. sorokiniana, which differ strongly in their ability to cope with different light intensities. Lipidomic analysis and isotopic labeling experiments revealed differences in the composition of their galactolipid species, although both species likely utilize galactolipid precursors originated from the endoplasmic reticulum. However, in silico research of de novo sequenced genomes and ortholog mapping of proteins putatively involved in lipid metabolism showed largely conserved lipid biosynthesis pathways suggesting species specific lipid remodeling mechanisms, which possibly have an impact on the response to different light conditions.

Transcriptome profiling at osmotic and ionic phases of salt stress response in bread wheat uncovers trait-specific candidate genes.

BACKGROUND: Bread wheat is one of the most important crops for the human diet, but the increasing soil salinization is causing yield reductions worldwide. Improving salt stress tolerance in wheat requires the elucidation of the mechanistic basis of plant response to this abiotic stress factor. Although several studies have been performed to analyze wheat adaptation to salt stress, there are still some gaps to fully understand the molecular mechanisms from initial signal perception to the onset of responsive tolerance pathways. The main objective of this study is to exploit the dynamic salt stress transcriptome in underlying QTL regions to uncover candidate genes controlling salt stress tolerance in bread wheat. The massive analysis of 3'-ends sequencing protocol was used to analyze leave samples at osmotic and ionic phases. Afterward, stress-responsive genes overlapping QTL for salt stress-related traits in two mapping populations were identified. RESULTS: Among the over-represented salt-responsive gene categories, the early up-regulation of calcium-binding and cell wall synthesis genes found in the tolerant genotype are presumably strategies to cope with the salt-related osmotic stress. On the other hand, the down-regulation of photosynthesis-related and calcium-binding genes, and the increased oxidative stress response in the susceptible genotype are linked with the greater photosynthesis inhibition at the osmotic phase. The specific up-regulation of some ABC transporters and Na+/Ca2+ exchangers in the tolerant genotype at the ionic stage indicates their involvement in mechanisms of sodium exclusion and homeostasis. Moreover, genes related to protein synthesis and breakdown were identified at both stress phases. Based on the linkage disequilibrium blocks, salt-responsive genes within QTL intervals were identified as potential components operating in pathways leading to salt stress tolerance. Furthermore, this study conferred evidence of novel regions with transcription in bread wheat. CONCLUSION: The dynamic transcriptome analysis allowed the comparison of osmotic and ionic phases of the salt stress response and gave insights into key molecular mechanisms involved in the salt stress adaptation of contrasting bread wheat genotypes. The leveraging of the highly contiguous chromosome-level reference genome sequence assembly facilitated the QTL dissection by targeting novel candidate genes for salt tolerance.

Robust non-syntenic gene expression patterns in diverse maize hybrids during root development.

Distantly related maize (Zea mays L.) inbred lines exhibit an exceptional degree of structural genomic diversity, which is probably unique among plants. This study systematically investigated the developmental and genotype-dependent regulation of the primary root transcriptomes of a genetically diverse panel of maize F1-hybrids and their parental inbred lines. While we observed substantial transcriptomic changes during primary root development, we demonstrated that hybrid-associated gene expression patterns, including differential, non-additive, and allele-specific transcriptome profiles, are particularly robust to these developmental fluctuations. For instance, differentially expressed genes with preferential expression in hybrids were highly conserved during development in comparison to their parental counterparts. Similarly, in hybrids a major proportion of non-additively expressed genes with expression levels between the parental values were particularly conserved during development. Importantly, in these expression patterns non-syntenic genes that evolved after the separation of the maize and sorghum lineages were systemically enriched. Furthermore, non-syntenic genes were substantially linked to the conservation of all surveyed gene expression patterns during primary root development. Among all F1-hybrids, between ~40% of the non-syntenic genes with unexpected allelic expression ratios and ~60% of the non-syntenic differentially and non-additively expressed genes were conserved and therefore robust to developmental changes. Hence, the enrichment of non-syntenic genes during primary root development might be involved in the developmental adaptation of maize roots and thus the superior performance of hybrids.

Stability of Single-Parent Gene Expression Complementation in Maize Hybrids upon Water Deficit Stress.

Heterosis is the superior performance of F1 hybrids compared with their homozygous, genetically distinct parents. In this study, we monitored the transcriptomic divergence of the maize (Zea mays) inbred lines B73 and Mo17 and their reciprocal F1 hybrid progeny in primary roots under control and water deficit conditions simulated by polyethylene glycol treatment. Single-parent expression (SPE) of genes is an extreme instance of gene expression complementation, in which genes are active in only one of two parents but are expressed in both reciprocal hybrids. In this study, 1,997 genes only expressed in B73 and 2,024 genes only expressed in Mo17 displayed SPE complementation under control and water deficit conditions. As a consequence, the number of active genes in hybrids exceeded the number of active genes in the parental inbred lines significantly independent of treatment. SPE patterns were substantially more stable to expression changes by water deficit treatment than other genotype-specific expression profiles. While, on average, 75% of all SPE patterns were not altered in response to polyethylene glycol treatment, only 17% of the remaining genotype-specific expression patterns were not changed by water deficit. Nonsyntenic genes that lack syntenic orthologs in other grass species, and thus evolved late in the grass lineage, were significantly overrepresented among SPE genes. Hence, the significant overrepresentation of nonsyntenic genes among SPE patterns and their stability under water limitation might suggest a function of these genes during the early developmental manifestation of heterosis under fluctuating environmental conditions in hybrid progeny of the inbred lines B73 and Mo17.

The Medicago genome provides insight into the evolution of rhizobial symbioses.

Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.

Analysis of the Arabidopsis shoot meristem transcriptome during floral transition identifies distinct regulatory patterns and a leucine-rich repeat protein that promotes flowering.

Flowering of Arabidopsis thaliana is induced by exposure to long days (LDs). During this process, the shoot apical meristem is converted to an inflorescence meristem that forms flowers, and this transition is maintained even if plants are returned to short days (SDs). We show that exposure to five LDs is sufficient to commit the meristem of SD-grown plants to flower as if they were exposed to continuous LDs. The MADS box proteins SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and FRUITFULL (FUL) play essential roles in this commitment process and in the induction of flowering downstream of the transmissible FLOWERING LOCUS T (FT) signal. We exploited laser microdissection and Solexa sequencing to identify 202 genes whose transcripts increase in the meristem during floral commitment. Expression of six of these transcripts was tested in different mutants, allowing them to be assigned to FT-dependent or FT-independent pathways. Most, but not all, of those dependent on FT and its paralog TWIN SISTER OF FT (TSF) also relied on SOC1 and FUL. However, this dependency on FT and TSF or SOC1 and FUL was often bypassed in the presence of the short vegetative phase mutation. FLOR1, which encodes a leucine-rich repeat protein, was induced in the early inflorescence meristem, and flor1 mutations delayed flowering. Our data contribute to the definition of LD-dependent pathways downstream and in parallel to FT.

PEP1 regulates perennial flowering in Arabis alpina.

Annual plants complete their life cycle in one year and initiate flowering only once, whereas perennials live for many years and flower repeatedly. How perennials undergo repeated cycles of vegetative growth and flowering that are synchronized to the changing seasons has not been extensively studied. Flowering is best understood in annual Arabidopsis thaliana, but many closely related species, such as Arabis alpina, are perennials. We identified the A. alpina mutant perpetual flowering 1 (pep1), and showed that PEP1 contributes to three perennial traits. It limits the duration of flowering, facilitating a return to vegetative development, prevents some branches from undergoing the floral transition allowing polycarpic growth habit, and confers a flowering response to winter temperatures that restricts flowering to spring. Here we show that PEP1 is the orthologue of the A. thaliana gene FLOWERING LOCUS C (FLC). The FLC transcription factor inhibits flowering until A. thaliana is exposed to winter temperatures, which trigger chromatin modifications that stably repress FLC transcription. In contrast, PEP1 is only transiently repressed by low temperatures, causing repeated seasonal cycles of repression and activation of PEP1 transcription that allow it to carry out functions characteristic of the cyclical life history of perennials. The patterns of chromatin modifications at FLC and PEP1 differ correlating with their distinct expression patterns. Thus we describe a critical mechanism by which flowering regulation differs between related perennial and annual species, and propose that differences in chromatin regulation contribute to this variation.

Marker2sequence, mine your QTL regions for candidate genes.

UNLABELLED: Marker2sequence (M2S) aims at mining quantitative trait loci (QTLs) for candidate genes. For each gene, within the QTL region, M2S uses data integration technology to integrate putative gene function with associated gene ontology terms, proteins, pathways and literature. As a typical QTL region easily contains several hundreds of genes, this gene list can then be further filtered using a keyword-based query on the aggregated annotations. M2S will help breeders to identify potential candidate genes for their traits of interest. AVAILABILITY: Marker2sequence is freely accessible at http://www.plantbreeding.wur.nl/BreeDB/marker2seq/. The source code can be obtained at https://github.com/PBR/Marker2Sequence. CONTACT: richard.finkers@wur.nl

A2TEA: Identifying trait-specific evolutionary adaptations.

Background: Plants differ in their ability to cope with external stresses (e.g., drought tolerance). Genome duplications are an important mechanism to enable plant adaptation. This leads to characteristic footprints in the genome, such as protein family expansion. We explore genetic diversity and uncover evolutionary adaptation to stresses by exploiting genome comparisons between stress tolerant and sensitive species and RNA-Seq data sets from stress experiments. Expanded gene families that are stress-responsive based on differential expression analysis could hint at species or clade-specific adaptation, making these gene families exciting candidates for follow-up tolerance studies and crop improvement. Software: Integration of such cross-species omics data is a challenging task, requiring various steps of transformation and filtering. Ultimately, visualization is crucial for quality control and interpretation. To address this, we developed A2TEA: Automated Assessment of Trait-specific Evolutionary Adaptations, a Snakemake workflow for detecting adaptation footprints in silico. It functions as a one-stop processing pipeline, integrating protein family, phylogeny, expression, and protein function analyses. The pipeline is accompanied by an R Shiny web application that allows exploring, highlighting, and exporting the results interactively. This allows the user to formulate hypotheses regarding the genomic adaptations of one or a subset of the investigated species to a given stress. Conclusions: While our research focus is on crops, the pipeline is entirely independent of the underlying species and can be used with any set of species. We demonstrate pipeline efficiency on real-world datasets and discuss the implementation and limits of our analysis workflow as well as planned extensions to its current state. The A2TEA workflow and web application are publicly available at: https://github.com/tgstoecker/A2TEA.Workflow and https://github.com/tgstoecker/A2TEA.WebApp, respectively.

Transcriptome profiling of barley and tomato shoot and root meristems unravels physiological variations underlying photoperiodic sensitivity.

The average sowing date of crops in temperate climate zones has been shifted forwards by several days, resulting in a changed photoperiod regime at the emergence stage. In the present study, we performed a global transcriptome profiling of plant development genes in the seedling stage of root and shoot apical meristems of a photoperiod-sensitive species (barley) and a photoperiod insensitive species (tomato) in short-day conditions (8h). Variant expression indicated differences in physiological development under this short day-length regime between species and tissues. The barley tissue transcriptome revealed reduced differentiation compared to tomato. In addition, decreased photosynthetic activity was observed in barley transcriptome and leaf chlorophyll content under 8h conditions, indicating a slower physiological development of shoot meristems than in tomatoes. The photomorphogenesis controlling cryptochrome gene cry1, with an effect on physiological differentiation, showed an underexpression in barley compared to tomato shoot meristems. This might lead to a cascade of suspended sink-source activities, which ultimately delay organ development and differentiation in barley shoot meristems under short photoperiods.

Transcriptome and Parasitome Analysis of Beet Cyst Nematode Heterodera schachtii.

Beet cyst nematodes depend on a set of secretory proteins (effectors) for the induction and maintenance of their syncytial feeding sites in plant roots. In order to understand the relationship between the beet cyst nematode H. schachtii and its host, identification of H. schachtii effectors is crucial and to this end, we sequenced a whole animal pre-infective J2-stage transcriptome in addition to pre- and post-infective J2 gland cell transcriptome using Next Generation Sequencing (NGS) and identified a subset of sequences representing putative effectors. Comparison between the transcriptome of H. schachtii and previously reported related cyst nematodes and root-knot nematodes revealed a subset of esophageal gland related sequences and putative effectors in common across the tested species. Structural and functional annotation of H. schachtii transcriptome led to the identification of nearly 200 putative effectors. Six putative effector expressions were quantified using qPCR and three of them were functionally analyzed using RNAi. Phenotyping of the RNAi nematodes indicated that all tested genes decrease the level of nematodes pathogenicity and/or the average female size, thereby regulating cyst nematode parasitism. These discoveries contribute to further understanding of the cyst nematode parasitism.

Protein function prediction and annotation in an integrated environment powered by web services (AFAWE).

Many sequenced genes are mainly annotated through automatic transfer of annotation from similar sequences. Manual comparison of results or intermediate results from different tools can help avoid wrong annotations and give hints to the function of a gene even if none of the automated tools could return any result. AFAWE simplifies the task of manual functional annotation by running different tools and workflows for automatic function prediction and displaying the results in a way that facilitates comparison. Because all programs are executed as web services, AFAWE is easily extensible and can directly query primary databases, thereby always using the most up-to-date data sources. Visual filters help to distinguish trustworthy results from non-significant results. Furthermore, an interface to add detailed manual annotation to each gene is provided, which can be displayed to other users.