Frontiers in Rhizosphere Research
November 20th, 2018
RIKEN Yokohama Lecture Hall
Supported by the auspice of Research Center for Science Systems (RCSS), JSPS andFrontiers in Rhizosphere Research
Date: November 20th, 2018 (Tuesday) Venue: RIKEN Yokohama, Lecture Hall

Program
10:30-10:35 Opening remarks
Chair:Shuta Asai
10:35-11:10 Stephane Hacquard(Multitrophic Plant-Microbe Interactions, Department of Plant Microbe
Interactions, Max Planck Institute for Plant Breeding Research)“Microbial Interkingdom Interactions in Roots Promote Arabidopsis Survival”
11:10-11:45 Yasunori Ichihashi(Plant-Microbe Symbiosis Research and Development Team, RIKEN BRC)
“Soil solarization-induced organic nitrogen increase agricultural crop yield”
11:45-12:10 Arno Germond(Laboratory for Comprehensive Bioimaging, RIKEN BDR)
“Toward predicting gene expression and metabolism from label free spectralimaging.”
12:10-13:00 Lunch
Chair:Yuji Ishigaki
13:00-13:35 Hirokazu Toju
(Center for Ecological Research, Kyoto University)“Core microbiomes for designing sustainable ecosystems”
13:35-14:10 Ruben Garrido-Oter(Integrative Bioinformatics, Department of Plant Microbe Interactions, Max
Planck Institute for Plant Breeding Research)“Association studies of plant-associated microbial genomes”

14:10-14:45 Shuta Asai
(Plant Immunity Research Group, RIKEN CSRS)“Field-Patho-Genomics −for establishment of soil diagnostics−”
14:45-15:15 Coffee Break
Chair: Yasunori Ichihashi
15:15-15:50 Ryohei Thomas Nakano(Department of Plant Microbe Interactions, Max Planck Institute for Plant
Breeding Research)“Ternary interaction assays revealed modular traits of root microbiota that
dictate host growth and immune status”
15:50-16:25 Masahito Hosokawa(Research Institute for Science and Engineering, Waseda University)“Obtaining high-quality draft genomes from uncultured microbes by single-cellgenome sequencing with droplet microfluidics”
16:25-17:00 Yuji Ishigaki(Plant Immunity Research Group, RIKEN CSRS)“Rhizosphere interactions regulate secondary metabolite production of rootassociated microbes”
17:00-17:05 Closing remarks

Abstracts

Stephane Hacquard(Multitrophic Plant-Microbe Interactions, Department of Plant
Microbe Interactions, Max Planck Institute for Plant Breeding
Research)

Microbial Interkingdom Interactions in Roots Promote Arabidopsis Survival

Roots of healthy plants are inhabited by soil-derived bacteria, fungi, and oomycetes that have
evolved independently in distinct kingdoms of life. How these microorganisms interact and towhat extent those interactions affect plant health are poorly understood. We examined rootassociated microbial communities from three Arabidopsis thaliana populations and detected
mostly negative correlations between bacteria and filamentous microbial eukaryotes. We
established microbial culture collections for reconstitution experiments using germ-free A.

thaliana. In plants inoculated with mono- or multi-kingdom synthetic microbial consortia, weobserved a profound impact of the bacterial root microbiota on fungal and oomycetal
community structure and diversity. We demonstrate that the bacterial microbiota is essential
for plant survival and protection against root-derived filamentous eukaryotes. Deconvolutionof 2,862 binary bacterial-fungal interactions ex situ, combined with community perturbation
experiments in planta, indicate that biocontrol activity of bacterial root commensals is aredundant trait that maintains microbial inter-kingdom balance for plant health.

Yasunori Ichihashi(Plant-Microbe Symbiosis Research and Development Team, RIKEN
BRC)

Soil solarization-induced organic nitrogen increase agricultural crop yield

Organic agriculture is of increasing importance in global food and ecosystem security. Soilsolarization, one of environmentally friendly organic farming method, is the disinfestation ofsoil borne plant pathogens. Even in the absence of major pathogens, soil solarization has an
unexpected beneficial effect to improve plant growth, but the underling mechanisms havebeen unclear. Here we utilized an agricultural filed of Japanese mustard spinach (Brassicarapa var. perviridis) using several farming methods including soil solarization, together withan integrated omics approach. We show that soil solarization assembles and maintains thespecific rhizosphere microbiota despite soil microbiota homogenized during plant cultivation.
Our integrated omics data suggests that the accumulation of several rhizosphere microbes aswell as organic nitrogen correlate with the crop yield. Using a germ-free plant system, weidentified that an organic nitrogen alanine was directly absorbed into plant body to increase
their biomass as much as inorganic nitrogen does. This suggests a possible mechanism forhow soil solarization induces complex material circulation between rhizosphere microbiota
and soil metabolites to increase crop yield. Our findings provide new insight into the theoryof plant nutrition underling the organic agriculture, potentially leading a sustainable
innovation in agriculture.

Arno Germond(Laboratory for Comprehensive Bioimaging, RIKEN BDR)

Toward predicting gene expression and metabolism from label free spectral imaging.

Gene expression and metabolic information are invaluable sources of information forbiological and biomedical investigations. Here we report a quantitative method to integratetranscriptomic data with the label-free spectral data of living cells. Our multivariate analysis
enables the prediction of gene expression from spectral data of unknown cells while
highlighting overrepresented metabolic shifts with a cell-line specificity. Escherichia coli celllines exhibiting antibiotic resistance are used as a case study.

Hirokazu Toju
(Center for Ecological Research, Kyoto University)

Core microbiomes for designing sustainable ecosystems

In an era of ecosystem degradation and climate change, maximizing microbial functions innatural and agricultural ecosystems has become a prerequisite for the future of humanity.
However, managing species-rich communities of plant-associated microbiomes remains amajor challenge. Informatics now allows us to identify members and characteristics of “coremicrobiomes”, which may be deployed to organize otherwise uncontrollable dynamics ofresident microbiomes. After the emergence of high-throughput sequencers, my colleagues
and I have uncovered root microbiome structures of hundreds of plants species across various
types of ecosystems. We then found that diverse taxonomic groups of endophytic fungiubiquitously interacted with plant communities, potentially playing crucial, but often
overlooked, roles at the ecosystem level. Based on the patterns found in microbe-plant andmicrobe-microbe networks, we have begun to design optimal core microbiomes for managing
agroecosystems with high resource-efficiency and stress-resistance. I will discuss how
interdisciplinary research will advance our ability for managing sustainable agroecosystems
and that for restoring degraded ecosystems.

Ruben Garrido-Oter(IntegrativeBioinformatics,DepartmentofPlant

MicrobeInteractions, Max Planck Institute for Plant Breeding Research)

Association studies of plant-associated microbial genomes

Determining functional and co-evolutionary relationships between genes based on sequencealone is a crucial step in integrating large genomic datasets and understanding how bacterial
populations and communities adapt to diverse environments. Here, we propose a phylogenetic
approach for determining clusters of co-evolving genes and their network organization bymodeling gene gain and loss as a continuous process along the branches of the species tree.
Our method accounts for uncertainty in the reconstruction of the ancestral states as well as in
the inference of the species tree and robustly identifies clusters of co-evolving genes thatsignificantly enrich for functional categories
and pathways and which are relevant for adaptation to diverse environments. We demonstrate
its ability to detect biologically meaningful gene family interactions by analyzing a total of
2,737 bacterial genomes, including diverse populations of multiple plant-associated andsymbiotic Rhizobia and a phylogenetically wide collection of bacterial genomes representativeof the Arabidopsis thaliana leaf and root microbiota.

Shuta Asai(Plant Immunity Research Group, RIKEN CSRS)

Field-Patho-Genomics‒ for establishment of soil diagnostics ‒Plant disease results only if all of three essential factors (susceptible host plant, pathogen, andfavorable environment for disease) occur simultaneously. Certain pathogens can infect only
specific plant species, so-called host specificity. Therefore, soilborne disease can be avoided
if the host specificity of pathogens living in the planting soil can be specified. Fusarium

oxysporum species complex consists of ubiquitous soil inhabiting fungi that can infect andcause disease in over 120 different plant species, which has been classified into more than 120
lineages (forma specialis; f. sp.). Since mechanisms for determining host specificity in F.

oxysporum are largely unknown, it is difficult to evaluate which lineage of F. oxysporum
inhabits the soil. Here, we report and discuss the methods to specify F. oxysporum lineages
living in the soil (soil diagnostics) and to predict the occurrence of Fusarium disease based on
the field environmental data (disease prediction model).

Ryohei Thomas Nakano(Department of Plant Microbe Interactions, Max Planck Institute forPlant Breeding Research)

Ternary interaction assays revealed modular traits of root microbiota that dictate host growthand immune status

Plant-associated microorganisms, constituting the plant microbiota, are capable ofmanipulating host physiology, including host growth, immunity, and abiotic stress tolerance.
Despite decades of efforts, however, application of these beneficial capacities to the crops
growing in natural field remains challenging, likely due to the interference by the othermicrobes that are already present in the field and in the plant niche. We recently found a root
growth promotion (RGP) activity that is conserved among root-associated Rhizobiales species
and that stimulation of host immunity disables this RGP capacity. Remarkably, we recorded astrong RGP activity even under immune-activating conditions when plants were coinoculated with two bacterial strains, one with RGP activity and another strain that is able tosuppress host immune response. Both isolates are unable to promote root growth under
immune-stimulative conditions, thereby demonstrating the emergent properties of rootmicrobiota. This further delineates the importance of ternary or higher-order interaction
assays to elucidate the community-level functional governance of host-associated microbialcommunities. We are currently investigating the molecular mechanisms by which Rhizobiales
isolates promote host root growth and root-associated bacteria suppress host immunity,
toward ultimate understanding of their impacts on plant fitness and nutrition, as well as theirevolutionary trajectories.

Masahito Hosokawa(Research Institute for Science and Engineering, Waseda University)

Obtaining high-quality draft genomes from uncultured microbes by single-cell genome
sequencing with droplet microfluidics

Since more than 99% of environmental bacteria are uncultivable, single-cell genomics is thepowerful tool for understanding their diversity and function without cultivation. For genome
sequencing of single microbial cells, whole genome amplification (WGA) is required because
the quantity of DNA contained in a single cell is quite small. However, conventional WGA
usually resulted in uneven genome sequence coverage because of amplification bias,
background amplification of contaminating DNA, and formation of chimeric sequences. Thus,
to maximize the quality and throughput of single-cell sequencing, there is a great demand fornovel techniques, which enable massively parallel reactions with high accuracy. Recently, we
have developed droplet-based microfluidics for massively parallel single-cell genomics(Nishikawa et al, PLoS ONE, 2015, Hosokawa et al, Sci. Rep., 2017), to overcome thelimitations of conventional techniques. In addition, we have also developed the sequencing
analysis pipeline, termed ccSAG (Cleaning and Co-assembly of single amplified genomes)(Kogawa et al, Sci. Rep., 2018), for de novo assembly of single-cell amplified genome toeliminate artefact sequences and contaminants. Our results demonstrated that microfluidicgenerated droplets showed a potential for contamination-free reaction and enhancing
sequencing coverage from single microbial cells isolated from various environments. Theseadvantages will greatly reduce the cost and labor investment required for single-cell genomics
study of environmental microbial cells. The integrated workflow will enable the comparative
genomic analyses of uncultured microbes at single-cell levels, as well as genetic and functional
investigation of microbial dark matters

Yuji Ishigaki(Plant Immunity Research Group, RIKEN CSRS)

Rhizosphere interactions regulate secondary metabolite production of root-associatedmicrobes

Many plant roots-associated actinomycetes are known antagonists toward plant pathogens,often by producing various type of antibiotics. To investigate whether plants influence theroot-associated actinomycetes for antibiotic production, we assayed the antibiotic activities ofthe strains isolated from plant roots. The isolates altered their antibiotic activities in thepresence of the Arabidopsis root extracts. In particular, Streptosporangium sp., produced thered pigment when cultured with the root extracts but not with shoot extracts. Interestingly,
the red pigment production was not triggered by the family Solanaceae roots. Scanningelectron microscopy (SEM) revealed that the hyphae extensively colonized the Arabidopsis
root surface, especially around the root hairs. Genome sequencing and antiSMASH analysis
identified at least one type-II polyketide synthases (PKSs) that is predicted to synthesize thered pigment. Disruption of genes encoding a ketosynthase and a chain length factor in one ofthe type-II PKS clusters abolished the red pigment production, suggesting the only one typeII PKS cluster is responsible for the production of the red pigment. In addition, overexpressionof a transcriptional activator in the cluster resulted in increased the production of the redpigment. The Arabidopsis-Streptosporangium pair and the genomic information of thebacterium provide a novel model system for studying of plant-actinomycete interactions.