Manipulation of the plant microbiome: antimicrobial peptide expression in nicotiana attenuata

Endophytic bacteria are believed to be important for plant growth, plant health and plant resistance. But the exploration of these roles in nature is difficult. To be able to manipulate endophytic bacteria of Nicotiana attenuata, I made 11 different genotypes for the constitutive expression of antimicrobial peptides under a 35S promoter. Several plant lines showed gene expression loss and a promoter methylation analysis by bisulfite sequencing revealed epigenetic gene silencing. The augmentation of the silencing occurred independently from a generational change and plants developed epigenetic changes solely during normal plant development with rapid methylation increase in somatic cells. Lines with low promoter methylation and trans-generational stable gene expression where further analyzed in peptide abundance. The peptide accumulation of each genotype was directly analyzed in the apoplast by a new and versatile nanoUPLC-MSE method suitable for the absolute quantification of peptides allowing the selection of lines with high peptide levels. The in planta activities against 19 different bacterial strains were examined by leaf infiltration and confirmed in vivo activity only for a single genotype, the so called ICE line, expressing a knottin from the common ice plant. This line showed high taxa specific activity against most Bacilli but did not affect proteobacteria and most actinobacteria. The different native Bacillus isolates showed distinct susceptibilities against the peptide indicating a high heterogeneity within wild isolates, which allows bacterial communities to compensate for antimicrobial activity by diversity. Plants were further used for comparative growth studies in the native environment, showed no reduction in growth performance and harbored a similar bacterial community, observed by high throughput pyrosequencing

Antimicrobial peptide expression in a wild tobacco plant reveals the limits of host-microbe-manipulations in the field

Plant-microbe associations are thought to be beneficial for plant growth and resistance against biotic or abiotic stresses, but for natural ecosystems, the ecological analysis of microbiome function remains in its infancy. We used transformed wild tobacco plants (Nicotiana attenuata) which constitutively express an antimicrobial peptide (Mc-AMP1) of the common ice plant, to establish an ecological tool for plant-microbe studies in the field. Transgenic plants showed in planta activity against plant-beneficial bacteria and were phenotyped within the plants´ natural habitat regarding growth, fitness and the resistance against herbivores. Multiple field experiments, conducted over 3 years, indicated no differences compared to isogenic controls. Pyrosequencing analysis of the root-associated microbial communities showed no major alterations but marginal effects at the genus level. Experimental infiltrations revealed a high heterogeneity in peptide tolerance among native isolates and suggests that the diversity of natural microbial communities can be a major obstacle for microbiome manipulations in nature

Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants

BACKGROUND: Genetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. Despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. A transgene can be epigenetically silenced by a process called RNA directed DNA methylation (RdDM), which can be seen as a heritable loss of gene expression. The spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear. RESULTS: Transgenic wild tobacco plants (Nicotiana attenuata) expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. We focused on three independently transformed lines (PNA 1.2, PNA 10.1 and ICE 4.4) as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than 200 fold after only one plant generation. Bisulfite sequencing indicated hypermethylation within the 35S and NOS promoters of these lines. To shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35S promoter methylation during vegetative growth (up to 77% absolute increase within 45 days of growth). The levels of de novo methylation were inherited by the offspring without any visible discontinuation. A secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants. CONCLUSIONS: The unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines. De novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. A secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits

Unsupervised protein embeddings outperform hand-crafted sequence and structure features at predicting molecular function

This work was supported by Keygene N.V., a crop innovation company in the Netherlands and by the Spanish MINECO/FEDER Project TEC201680141-P with the associated FPI grant BES-2017-079792.The authors thank Dr. Elvin Isufi and Chirag Raman for their valuable comments and feedback.Motivation: Protein function prediction is a difficult bioinformatics problem. Many recent methods use deep neural networks to learn complex sequence representations and predict function from these. Deep supervised models require a lot of labeled training data which are not available for this task. However, a very large amount of protein sequences without functional labels is available. Results: We applied an existing deep sequence model that had been pretrained in an unsupervised setting on the supervised task of protein molecular function prediction. We found that this complex feature representation is effective for this task, outperforming hand-crafted features such as one-hot encoding of amino acids, k-mer counts, secondary structure and backbone angles. Also, it partly negates the need for complex prediction models, as a two-layer perceptron was enough to achieve competitive performance in the third Critical Assessment of Functional Annotation benchmark. We also show that combining this sequence representation with protein 3D structure information does not lead to performance improvement, hinting that 3D structure is also potentially learned during the unsupervised pretraining.Keygene N.V., a crop innovation company in the NetherlandsSpanish MINECO/FEDER TEC201680141-PFPI grant BES-2017-07979

Bowel Movement: Integrating Host Mobility and Microbial Transmission Across Host Taxa

The gut microbiota of animals displays a high degree of plasticity with respect to environmental or dietary adaptations and is shaped by factors like social interactions, diet diversity or the local environment. But the contribution of these drivers varies across host taxa and our ability to explain microbiome variability within wild populations remains limited. Terrestrial animals have divergent mobility ranges and can either crawl, walk or fly, from a couple of centimeters toward thousands of kilometers. Animal movement has been little regarded in host microbiota frameworks, though it can directly influence major drivers of the host microbiota: (1) Aggregation movement can enhance social transmissions, (2) foraging movement can extend range of diet diversity, and (3) dispersal movement determines the local environment of a host. Here, I would like to outline how movement behaviors of different host taxa matter for microbial acquisition across mammals, birds as well as insects. Host movement can have contrasting effects and either reduce or enlarge spatial scale. Increased dispersal movement could dissolve local effects of sampling location, while aggregation could enhance inter-host transmissions and uniformity among social groups. Host movement can also extend the boundaries of microbial dispersal limitations and connect habitat patches across plant-pollinator networks, while the microbiota of wild populations could converge toward a uniform pattern when mobility is interrupted in captivity or laboratory settings. Hence, the implementation of host movement would be a valuable addition to the metacommunity concept, to comprehend microbial dispersal within and across trophic levels

Bumble bee microbiota shows temporal succession and increase of lactic acid bacteria when exposed to outdoor environments

QuestionThe large earth bumble bee (Bombus terrestris) maintains a social core gut-microbiota, similar as known from the honey bee, which plays an important role for host health and resistance. Experiments under laboratory conditions with commercial hives are limited to vertically transmitted microbes and neglect influences of environmental factors or external acquisition of microbes. Various environmental and landscape-level factors may have an impact on the gut-microbiota of pollinating insects, with consequences for pollinator health and fitness in agroecosystems. Still, it is not fully clear whether access to different flower diversities will have a significant influence on the bumble bee microbiota. Here, we tested in a semi-field experiment if the bumble bee microbiota changes over time when exposed to different flower diversities within outdoor flight cages. We used commercial hives to distinguish between vertically and horizontally transmitted bacteria, respectively from the nest environment or the exposed outside environment.ResultThe sequential sampling of foraging workers over a period of 35 days indicated a temporal progression of the bumble bee microbiota when placed outside. The microbiota increased in diversity and changed in composition and variability over time. We observed a major increase in relative abundance of the families Lactobacillaceae, Bifidobacteriaceae and Weeksellaceae. In contrast, major core-taxa like Snodgrassella and Gilliamella declined in their relative abundance over time. The genus Lactobacillus showed a high diversity and strain specific turnover, so that only specific ASVs showed an increase over time, while others had a more erratic occurrence pattern. Exposure to different flower diversities had no significant influence on the progression of the bumble bee microbiota.ConclusionThe bumble bee microbiota showed a dynamic temporal succession with distinct compositional changes and diversification over time when placed outdoor. The exposure of bumble bees to environmental conditions, or environmental microbes, increases dissimilarity and changes the gut-community composition. This shows the importance of environmental influences on the temporal dynamic and progression of the bumble bee microbiota

Supplemental Table S2

Results of diagnostic PCRs with 44 different transgenic N. attenuata lines

Data from: Efficient screening of transgenic plant lines for ecological research

Plants stably transformed to manipulate the expression of genes mediating ecological performance have profoundly altered research in plant ecology. Agrobacterium-mediated transformation remains the most effective method of creating plants harboring a limited number of transgene integrations of low complexity. For ecological/physiological research, the following requirements must be met: 1) the regenerated plants should have the same ploidy level as the corresponding wild type plant and 2) contain a single transgene copy in a homozygous state; 3) the T-DNA must be completely inserted without vector backbone sequence and all its elements functional; and 4) the integration should not change the phenotype of the plant by interrupting chromosomal genes or by mutations occurring during the regeneration procedure. The screening process to obtain transformed plants that meet the above criteria is costly and time consuming and an optimized screening procedure is presented. We developed a flow chart that optimizes the screening process to efficiently select transformed plants for ecological research. It consists of segregational analyses, which select transgenic T1 and T2 generation plants with single T-DNA copies that are homozygous. Indispensable molecular genetic tests (flow cytometry, diagnostic PCRs and Southern blotting) are performed at the earliest and most effective times in the screening process. qPCR to quantify changes in transcript accumulation to confirm gene silencing or over-expression, are the last steps in the selection process. Since we routinely transform the wild tobacco, Nicotiana attenuata, with constructs that silence or ectopically over-express ecologically relevant genes, the proposed protocol is supported by examples from this system

Isolating fungal pathogens from a dynamic disease outbreak in a native plant population to establish plant-pathogen bioassays for the ecological model plant Nicotiana attenuata

The wild tobacco species Nicotiana attenuata has been intensively used as a model plant to study its interaction with insect herbivores and pollinators in nature, however very little is known about its native pathogen community. We describe a fungal disease outbreak in a native N. attenuata population comprising 873 plants growing in an area of about 1500 m2. The population was divided into 14 subpopulations and disease symptom development in the subpopulations was monitored for 16 days, revealing a waxing and waning of visible disease symptoms with some diseased plants recovering fully. Native fungal N. attenuata pathogens were isolated from diseased plants, characterized genetically, chemotaxonomically and morphologically, revealing several isolates of the ascomycete genera Fusarium and Alternaria, that differed in the type and strength of the disease symptoms they caused in bioassays on either detached leaves or intact soil-grown plants. These isolates and the bioassays will empower the study of N. attenuata-pathogen interactions in a realistic ecological context

Legacy of a Butterfly's Parental Microbiome in Offspring Performance

An insect's phenotype can be influenced by the experiences of the parental generation. However, the effects of the parental symbiotic microbiome and host plant use on the offspring are unclear. We addressed this gap of knowledge by studying Pieris brassicae, a multivoltine butterfly species feeding on different brassicaceous plants across generations. We investigated how disturbance of the parental bacterial community by antibiotic treatment affects F1 larval traits. We tested the effects depending on whether F1 larvae are feeding on the same plant species as their parents or on a different one. The parental treatment alone had no impact on the biomass of F1 larvae feeding on the parental plant species. However, the parental treatment had a detrimental effect on F1 larval biomass when F1 larvae had a different host plant than their parents. This effect was linked to higher larval prophenoloxidase activity and greater downregulation of the major allergen gene (MA), a glucosinolate detoxification gene of P. brassicae Bacterial abundance in untreated adult parents was high, while it was very low in F1 larvae from either parental type, and thus unlikely to directly influence larval traits. Our results suggest that transgenerational effects of the parental microbiome on the offspring's phenotype become evident when the offspring is exposed to a transgenerational host plant shift.IMPORTANCE Resident bacterial communities are almost absent in larvae of butterflies and thus are unlikely to affect their host. In contrast, adult butterflies contain conspicuous amounts of bacteria. While the host plant and immune state of adult parental butterflies are known to affect offspring traits, it has been unclear whether also the parental microbiome imposes direct effects on the offspring. Here, we show that disturbance of the bacterial community in parental butterflies by an antibiotic treatment has a detrimental effect on those offspring larvae feeding on a different host plant than their parents. Hence, the study indicates that disturbance of an insect's parental microbiome by an antibiotic treatment shapes how the offspring individuals can adjust themselves to a novel host plant.</p