The Brassica napus boron deficient inflorescence transcriptome resembles a wounding and infection response.

Oilseed rape and other crops of Brassica napus have a high demand for boron (B). Boron deficiencies result in the inhibition of root growth, and eventually premature flower abortion. Understanding the genetic mechanisms underlying flower abortion in B-limiting conditions could provide the basis to enhance B-efficiency and prevent B-deficiency-related yield losses. In this study, we assessed transcriptomic responses to B-deficiency in diverse inflorescence tissues at multiple time points of soil-grown plants that were phenotypically unaffected by B-deficiency until early flowering. Whilst transcript levels of known B transporters were higher in B-deficient samples, these remained remarkably stable as the duration of B-deficiency increased. Meanwhile, GO-term enrichment analysis indicated a growing response resembling that of a pathogen or pest attack, escalating to a huge transcriptome response in shoot heads at mid-flowering. Grouping differentially expressed genes within this tissue into MapMan functional bins indicated enrichment of genes related to wounding, jasmonic acid and WRKY transcription factors. Individual candidate genes for controlling the "flowering-without-seed-setting" phenotype from within MapMan biotic stress bins include those of the metacaspase family, which have been implicated in orchestrating programmed cell death. Overall temporal expression patterns observed here imply a dynamic response to B-deficiency, first increasing expression of B transporters before recruiting various biotic stress-related pathways to coordinate targeted cell death, likely in response to as yet unidentified B-deficiency induced damage-associated molecular patterns (DAMPs). This response indicates new pathways to target and dissect to control B-deficiency-induced flower abortion and to develop more B-efficient crops.

Transcriptional Changes in Potato Sprouts upon Interaction with Rhizoctonia solani Indicate Pathogen-Induced Interference in the Defence Pathways of Potato.

Rhizoctonia solani is the causer of black scurf disease on potatoes and is responsible for high economical losses in global agriculture. In order to increase the limited knowledge of the plants' molecular response to this pathogen, we inoculated potatoes with R. solani AG3-PT isolate Ben3 and carried out RNA sequencing with total RNA extracted from potato sprouts at three and eight days post inoculation (dpi). In this dual RNA-sequencing experiment, the necrotrophic lifestyle of R. solani AG3-PT during early phases of interaction with its host has already been characterised. Here the potato plants' comprehensive transcriptional response to inoculation with R. solani AG3 was evaluated for the first time based on significantly different expressed plant genes extracted with DESeq analysis. Overall, 1640 genes were differentially expressed, comparing control (-Rs) and with R. solani AG3-PT isolate Ben3 inoculated plants (+Rs). Genes involved in the production of anti-fungal proteins and secondary metabolites with antifungal properties were significantly up regulated upon inoculation with R. solani. Gene ontology (GO) terms involved in the regulation of hormone levels (i.e., ethylene (ET) and jasmonic acid (JA) at 3 dpi and salicylic acid (SA) and JA response pathways at 8 dpi) were significantly enriched. Contrastingly, the GO term "response to abiotic stimulus" was down regulated at both time points analysed. These results may support future breeding efforts toward the development of cultivars with higher resistance level to black scurf disease or the development of new control strategies.

Draft genome sequence of the potato pathogen Rhizoctonia solani AG3-PT isolate Ben3.

The basidiomycetes fungus Rhizoctonia solani AG3 is responsible for black scurf disease on potato and occurs in each potato growing area world-wide. In this study, the draft genome sequence of the black scurf pathogen R. solani AG3-PT isolate Ben3 is presented. The genome sequence of R. solani AG3-PT isolate Ben3 consists of 1385 scaffolds. These scaffolds amount to a size of approx. 51 Mb. Considering coverage analyses of contigs, the size of the diploid genome was estimated to correspond to 116 Mb. Gene prediction by applying AUGUSTUS (3.2.1.) resulted in 12,567 identified genes. Based on automatic annotation using GenDBE, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the R. solani AG3-PT isolate Ben3 genome. Comparative analyses including the R. solani AG3 isolate Rhs1AP, also originating from potato, revealed first insights into core genes shared by both isolates and unique determinants of each isolate.

Whole-Genome Sequence of Aneurinibacillus sp. Ricciae_BoGa-3, Isolated from Riccia fluitans.

Here, we present the Nanopore-only genome sequence of Aneurinibacillus sp. Ricciae_BoGa-3. It was isolated from Riccia fluitans ecotype BoGa-3 and its source was Botanical Garden Osnabrück (Germany). The complete circular genome is 4,981,254 bp with a GC content of 44.8%.

Whole-Genome Sequence of Paenibacillus marchantiae Isolated from the Liverwort Marchantia polymorpha subsp. ruderalis Ecotype BoGa.

The bacterium Paenibacillus marchantiae was isolated from male plants of the liverwort Marchantia polymorpha subsp. ruderalis ecotype BoGa. Here, we report on the complete genome sequence generated from long Nanopore reads. The genome sequence comprises 6,983,959 bp with a GC content of 46.02% and 6,195 predicted protein-coding genes.

Rapid discovery of terpene tailoring enzymes for total biosynthesis.

Twenty oxygenated aristolochene congeners were rapidly synthesised by combining genes from four different fungal pathways in the fungal host organism Aspergillus oryzae. Compounds produced in a single step include the natural product hypoxylan A and an epimer of guignaderemophilane C. A new fungal aromatase was discovered that produces phenols by oxidative demethylation.

Complete Genome Sequence of Pseudomonas sp. Strain MM213, an Isolate from a Brookside in Bielefeld, Germany.

Here, we report the genome sequence of Pseudomonas sp. strain MM213, isolated from brookside soil in Bielefeld, Germany. The genome is complete and consists of 6,746,355 bp, with a GC content of 59.4% and 6,145 predicted protein-coding sequences. Pseudomonas sp. strain MM213 is part of the Pseudomonas mandelii group.

Whole-Genome Sequence of Pseudomonas sp. Strain MM211, Isolated from Soil in Langenfeld, Germany.

Here, we present the genome sequence of Pseudomonas sp. strain MM211, which was isolated from garden soil. The complete circular genome consists of a 5,281,862-bp chromosome, with a GC content of 61.5%.

Genome Analyses of the Less Aggressive Rhizoctonia solani AG1-IB Isolates 1/2/21 and O8/2 Compared to the Reference AG1-IB Isolate 7/3/14.

Rhizoctonia solani AG1-IB of the phylum Basidiomycota is known as phytopathogenic fungus affecting various economically important crops, such as bean, rice, soybean, figs, cabbage and lettuce. The isolates 1/2/21 and O8/2 of the anastomosis group AG1-IB originating from lettuce plants with bottom rot symptoms represent two less aggressive R. solani isolates, as confirmed in a pathogenicity test on lettuce. They were deeply sequenced on the Illumina MiSeq system applying the mate-pair and paired-end mode to establish their genome sequences. Assemblies of obtained sequences resulted in 2092 and 1492 scaffolds, respectively, for isolate 1/2/21 and O8/2, amounting to a size of approximately 43 Mb for each isolate. Gene prediction by applying AUGUSTUS (v. 3.2.1.) yielded 12,827 and 12,973 identified genes, respectively. Based on automatic functional annotation, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the AG1-IB genomes. The annotated genome sequences of the less aggressive AG1-IB isolates were compared with the isolate 7/3/14, which is highly aggressive on lettuce and other vegetable crops such as bean, cabbage and carrot. This analysis revealed the first insights into core genes of AG1-IB isolates and unique determinants of each genome that may explain the different aggressiveness levels of the strains.

Necrotrophic lifestyle of Rhizoctonia solani AG3-PT during interaction with its host plant potato as revealed by transcriptome analysis.

The soil-borne pathogen Rhizoctonia solani infects a broad range of plants worldwide and is responsible for significant crop losses. Rhizoctonia solani AG3-PT attacks germinating potato sprouts underground while molecular responses during interaction are unknown. To gain insights into processes induced in the fungus especially at early stage of interaction, transcriptional activity was compared between growth of mycelium in liquid culture and the growing fungus in interaction with potato sprouts using RNA-sequencing. Genes coding for enzymes with diverse hydrolase activities were strongly differentially expressed, however with remarkably dissimilar time response. While at 3 dpi, expression of genes coding for peptidases was predominantly induced, strongest induction was found for genes encoding hydrolases acting on cell wall components at 8 dpi. Several genes with unknown function were also differentially expressed, thus assuming putative roles as effectors to support host colonization. In summary, the presented analysis characterizes the necrotrophic lifestyle of R. solani AG3-PT during early interaction with its host.

A comprehensive analysis of the Lactuca sativa, L. transcriptome during different stages of the compatible interaction with Rhizoctonia solani.

The leafy green vegetable Lactuca sativa, L. is susceptible to the soil-born fungus Rhizoctonia solani AG1-IB. In a previous study, we reported on the transcriptional response of R. solani AG1-IB (isolate 7/3/14) during the interspecies interaction with L. sativa cv. Tizian by means of RNA sequencing. Here we present the L. sativa transcriptome and metabolome from the same experimental approach. Three distinct interaction zones were sampled and compared to a blank (non-inoculated) sample: symptomless zone 1, zone 2 showing light brown discoloration, and a dark brown zone 3 characterized by necrotic lesions. Throughout the interaction, we observed a massive reprogramming of the L. sativa transcriptome, with 9231 unique genes matching the threshold criteria for differential expression. The lettuce transcriptome of the light brown zone 2 presents the most dissimilar profile compared to the uninoculated zone 4, marking the main stage of interaction. Transcripts putatively encoding several essential proteins that are involved in maintaining jasmonic acid and auxin homeostasis were found to be negatively regulated. These and other indicator transcripts mark a potentially inadequate defence response, leading to a compatible interaction. KEGG pathway mapping and GC-MS metabolome data revealed large changes in amino acid, lignin and hemicellulose related pathways and related metabolites.

Genetic Potential of the Biocontrol Agent Pseudomonas brassicacearum (Formerly P. trivialis) 3Re2-7 Unraveled by Genome Sequencing and Mining, Comparative Genomics and Transcriptomics.

The genus Pseudomonas comprises many known plant-associated microbes with plant growth promotion and disease suppression properties. Genome-based studies allow the prediction of the underlying mechanisms using genome mining tools and the analysis of the genes unique for a strain by implementing comparative genomics. Here, we provide the genome sequence of the strain Pseudomonas brassicacearum 3Re2-7, formerly known as P. trivialis and P. reactans, elucidate its revised taxonomic classification, experimentally verify the gene predictions by transcriptome sequencing, describe its genetic biocontrol potential and contextualize it to other known Pseudomonas biocontrol agents. The P. brassicacearum 3Re2-7 genome comprises a circular chromosome with a size of 6,738,544 bp and a GC-content of 60.83%. 6267 genes were annotated, of which 6113 were shown to be transcribed in rich medium and/or in the presence of Rhizoctonia solani. Genome mining identified genes related to biocontrol traits such as secondary metabolite and siderophore biosynthesis, plant growth promotion, inorganic phosphate solubilization, biosynthesis of lipo- and exopolysaccharides, exoproteases, volatiles and detoxification. Core genome analysis revealed, that the 3Re2-7 genome exhibits a high collinearity with the representative genome for the species, P. brassicacearum subsp. brassicacearum NFM421. Comparative genomics allowed the identification of 105 specific genes and revealed gene clusters that might encode specialized biocontrol mechanisms of strain 3Re2-7. Moreover, we captured the transcriptome of P. brassicacearum 3Re2-7, confirming the transcription of the predicted biocontrol-related genes. The gene clusters coding for 2,4-diacetylphloroglucinol (phlABCDEFGH) and hydrogen cyanide (hcnABC) were shown to be highly transcribed. Further genes predicted to encode putative alginate production enzymes, a pyrroloquinoline quinone precursor peptide PqqA and a matrixin family metalloprotease were also found to be highly transcribed. With this study, we provide a basis to further characterize the mechanisms for biocontrol in Pseudomonas species, towards a sustainable and safe application of P. brassicacearum biocontrol agents.

Assembly of the Lactuca sativa, L. cv. Tizian draft genome sequence reveals differences within major resistance complex 1 as compared to the cv. Salinas reference genome.

Lettuce (Lactuca sativa, L.) is an important annual plant of the family Asteraceae (Compositae). The commercial lettuce cultivar Tizian has been used in various scientific studies investigating the interaction of the plant with phytopathogens or biological control agents. Here, we present the de novo draft genome sequencing and gene prediction for this specific cultivar derived from transcriptome sequence data. The assembled scaffolds amount to a size of 2.22 Gb. Based on RNAseq data, 31,112 transcript isoforms were identified. Functional predictions for these transcripts were determined within the GenDBE annotation platform. Comparison with the cv. Salinas reference genome revealed a high degree of sequence similarity on genome and transcriptome levels, with an average amino acid identity of 99%. Furthermore, it was observed that two large regions are either missing or are highly divergent within the cv. Tizian genome compared to cv. Salinas. One of these regions covers the major resistance complex 1 region of cv. Salinas. The cv. Tizian draft genome sequence provides a valuable resource for future functional and transcriptome analyses focused on this lettuce cultivar.

The Rhizoctonia solani AG1-IB (isolate 7/3/14) transcriptome during interaction with the host plant lettuce (Lactuca sativa L.).

The necrotrophic pathogen Rhizoctonia solani is one of the most economically important soil-borne pathogens of crop plants. Isolates of R. solani AG1-IB are the major pathogens responsible for bottom-rot of lettuce (Lactuca sativa L.) and are also responsible for diseases in other plant species. Currently, there is lack of information regarding the molecular responses in R. solani during the pathogenic interaction between the necrotrophic soil-borne pathogen and its host plant. The genome of R. solani AG1-IB (isolate 7/3/14) was recently established to obtain insights into its putative pathogenicity determinants. In this study, the transcriptional activity of R. solani AG1-IB was followed during the course of its pathogenic interaction with the host plant lettuce under controlled conditions. Based on visual observations, three distinct pathogen-host interaction zones on lettuce leaves were defined which covered different phases of disease progression on tissue inoculated with the AG1-IB (isolate 7/3/14). The zones were defined as: Zone 1-symptomless, Zone 2-light brown discoloration, and Zone 3-dark brown, necrotic lesions. Differences in R. solani hyphae structure in these three zones were investigated by microscopic observation. Transcriptional activity within these three interaction zones was used to represent the course of R. solani disease progression applying high-throughput RNA sequencing (RNA-Seq) analysis of samples collected from each Zone. The resulting three transcriptome data sets were analyzed for their highest expressed genes and for differentially transcribed genes between the respective interaction zones. Among the highest expressed genes was a group of not previously described genes which were transcribed exclusively during early stages of interaction, in Zones 1 and 2. Previously described importance of up-regulation in R. solani agglutinin genes during disease progression could be further confirmed; here, the corresponding genes exhibited extremely high transcription levels. Most differentially higher expressed transcripts were found within Zone 2. In Zone 3, the zone with the strongest degree of interaction, gene transcripts indicative of apoptotic activity were highly abundant. The transcriptome data presented in this work support previous models of the disease and interaction cycle of R. solani and lettuce and may influence effective techniques for control of this pathogen.

Comparative transcriptome analysis of the biocontrol strain Bacillus amyloliquefaciens FZB42 as response to biofilm formation analyzed by RNA sequencing.

The strain Bacillus amyloliquefaciens FZB42 is a plant growth promoting rhizobacterium (PGPR) and biocontrol agent known to keep infections of lettuce (Lactuca sativa) by the phytopathogen Rhizoctonia solani down. Several mechanisms, including the production of secondary metabolites possessing antimicrobial properties and induction of the host plant's systemic resistance (ISR), were proposed to explain the biocontrol effect of the strain. B. amyloliquefaciens FZB42 is able to form plaques (biofilm-like structures) on plant roots and this feature was discussed to be associated with its biocontrol properties. For this reason, formation of B. amyloliquefaciens biofilms was studied at the transcriptional level using high-throughput sequencing of whole transcriptome cDNA libraries from cells grown under biofilm-forming conditions vs. planktonic growth. Comparison of the transcriptional profiles of B. amyloliquefaciens FZB42 under these growth conditions revealed a common set of highly transcribed genes mostly associated with basic cellular functions. The lci gene, encoding an antimicrobial peptide (AMP), was among the most highly transcribed genes of cells under both growth conditions suggesting that AMP production may contribute to biocontrol. In contrast, gene clusters coding for synthesis of secondary metabolites with antimicrobial properties were only moderately transcribed and not induced in biofilm-forming cells. Differential gene expression revealed that 331 genes were significantly up-regulated and 230 genes were down-regulated in the transcriptome of B. amyloliquefaciens FZB42 under biofilm-forming conditions in comparison to planktonic cells. Among the most highly up-regulated genes, the yvqHI operon, coding for products involved in nisin (class I bacteriocin) resistance, was identified. In addition, an operon whose products play a role in fructosamine metabolism was enhanced in its transcription. Moreover, genes involved in the production of the extracellular biofilm matrix including exopolysaccharide genes (eps) and the yqxM-tasA-sipW operon encoding amyloid fiber synthesis were up-regulated in the B. amyloliquefaciens FZB42 biofilm. On the other hand, highly down-regulated genes in biofilms are associated with synthesis, assembly and regulation of the flagellar apparatus, the degradation of aromatic compounds and the export of copper. The obtained transcriptional profile for B. amyloliquefaciens biofilm cells uncovered genes involved in its development and enabled the assessment that synthesis of secondary metabolites among other factors may contribute to the biocontrol properties of the strain.

Development of a Rhizoctonia solani AG1-IB Specific Gene Model Enables Comparative Genome Analyses between Phytopathogenic R. solani AG1-IA, AG1-IB, AG3 and AG8 Isolates.

Rhizoctonia solani, a soil-born plant pathogenic basidiomycetous fungus, affects various economically important agricultural and horticultural crops. The draft genome sequence for the R. solani AG1-IB isolate 7/3/14 as well as a corresponding transcriptome dataset (Expressed Sequence Tags--ESTs) were established previously. Development of a specific R. solani AG1-IB gene model based on GMAP transcript mapping within the eukaryotic gene prediction platform AUGUSTUS allowed detection of new genes and provided insights into the gene structure of this fungus. In total, 12,616 genes were recognized in the genome of the AG1-IB isolate. Analysis of predicted genes by means of different bioinformatics tools revealed new genes whose products potentially are involved in degradation of plant cell wall components, melanin formation and synthesis of secondary metabolites. Comparative genome analyses between members of different R. solani anastomosis groups, namely AG1-IA, AG3 and AG8 and the newly annotated R. solani AG1-IB genome were performed within the comparative genomics platform EDGAR. It appeared that only 21 to 28% of all genes encoded in the draft genomes of the different strains were identified as core genes. Based on Average Nucleotide Identity (ANI) and Average Amino-acid Identity (AAI) analyses, considerable sequence differences between isolates representing different anastomosis groups were identified. However, R. solani isolates form a distinct cluster in relation to other fungi of the phylum Basidiomycota. The isolate representing AG1-IB encodes significant more genes featuring predictable functions in secondary metabolite production compared to other completely sequenced R. solani strains. The newly established R. solani AG1-IB 7/3/14 gene layout now provides a reliable basis for post-genomics studies.

Improved genome sequence of the phytopathogenic fungus Rhizoctonia solani AG1-IB 7/3/14 as established by deep mate-pair sequencing on the MiSeq (Illumina) system.

The phytopathogenic fungus Rhizoctonia solani AG1-IB of the phylum Basidiomycota affects various economically important crops comprising bean, rice, soybean, figs, cabbage and lettuce. The R. solani isolate 7/3/14 of the anastomosis group AG1-IB was deeply resequenced on the Illumina MiSeq system applying the mate-pair mode to improve its genome sequence. Assembly of obtained sequence reads significantly reduced the amount of scaffolds and improved the genome sequence of the isolate compared to the previous sequencing approach. The genome sequence of the AG1-IB isolate 7/3/14 now provides an up-graded basis to analyze genome features predicted to play a role in pathogenesis and for the development of strategies to antagonize the pathogenic impact of this fungus.

Effect of the strain Bacillus amyloliquefaciens FZB42 on the microbial community in the rhizosphere of lettuce under field conditions analyzed by whole metagenome sequencing.

Application of the plant associated bacterium Bacillus amyloliquefaciens FZB42 on lettuce (Lactuca sativa) confirmed its capability to promote plant growth and health by reducing disease severity (DS) caused by the phytopathogenic fungus Rhizoctonia solani. Therefore this strain is commercially applied as an eco-friendly plant protective agent. It is able to produce cyclic lipopeptides (CLP) and polyketides featuring antifungal and antibacterial properties. Production of these secondary metabolites led to the question of a possible impact of strain FZB42 on the composition of microbial rhizosphere communities after its application. Rating of DS and lettuce growth during a field trial confirmed the positive impact of strain FZB42 on the health of the host plant. To verify B. amyloliquefaciens as an environmentally compatible plant protective agent, its effect on the indigenous rhizosphere community was analyzed by metagenome sequencing. Rhizosphere microbial communities of lettuce treated with B. amyloliquefaciens FZB42 and non-treated plants were profiled by high-throughput metagenome sequencing of whole community DNA. Fragment recruitments of metagenome sequence reads on the genome sequence of B. amyloliquefaciens FZB42 proved the presence of the strain in the rhizosphere over 5 weeks of the field trial. Comparison of taxonomic community profiles only revealed marginal changes after application of strain FZB42. The orders Burkholderiales, Actinomycetales and Rhizobiales were most abundant in all samples. Depending on plant age a general shift within the composition of the microbial communities that was independent of the application of strain FZB42 was observed. In addition to the taxonomic profiling, functional analysis of annotated sequences revealed no major differences between samples regarding application of the inoculant strain.