Research programme Paul Schulze-Lefert
I. Structure and functions of the Arabidopsis root microbiota (supported by ERC advanced grant ROOTMICROBIOTA)
Remarkably little is understood about plant-microbe interactions that are, at first glance, ‘symptomless’. These poorly studied plant microbiomes harbor an unknown reservoir of probiotic and plant protective associations. One gram of soil typically contains ~108 to 1010 bacteria. Microbial DNA fingerprints from plant roots or aerial organs uncover microbial communities thriving on the surface of, or within, healthy plant tissue. Rhizosphere microbiomes attached to, and in the first few millimeters away from, the root surface are distinct from bulk soil, suggesting specific colonization events. Organic carbon flux from roots promotes the growth of microbial decomposers that, in turn, recycle plant nutrients for root uptake by transpiration-driven water fluxes. Seedlings exude 30-40%, and adult plants 20%, of photosynthetically fixed carbon into the rhizosphere in the form of poorly characterized rhizodeposits.
We have developed and applied bacterial 16S rRNA gene pyrosequencing to characterize and compare soil and root-inhabiting bacterial communities of Arabidopsis thaliana. We show that roots of Arabidopsis, grown in different natural soils under controlled environmental conditions, are preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum is represented by a dominating class or family. Soil type defines the composition of root-inhabiting bacterial communities and host genotype determines their ribotype profiles to a limited extent. The identification of soil type-specific members within the root-inhabiting assemblies supports our conclusion that these represent soil-derived root endophytes. Surprisingly, plant cell wall features of other tested plant species appear to provide a sufficient cue for the assembly of ~40% of the Arabidopsis bacterial root-inhabiting microbiota, with a bias for Betaproteobacteria. Thus, this root sub-community may not be Arabidopsis-specific but saprophytic bacteria that would naturally be found on any plant root or plant debris in the tested soils. In contrast, colonization of Arabidopsis roots by members of the Actinobacteria depends on additional cues from metabolically active host cells (Bulgarelli et al., in press).
Schlaeppi, K., Dombrowski, N., Garrido Oter, R., ver Loren van Themaat, E. and Schulze-Lefert, P. (2014) Quantitative divergence of the bacterial root microbiota in Arabidopsis thaliana relatives, PNAS, 111(2), 585-592.
Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver Loren van Themaat, E. and Schulze-Lefert, P. Structure and Functions of the Bacterial Microbiota of Plants. Annual Review of Plant Biology Vol. 64 (2013).
Bulgarelli, D., Rott, M., Schlaeppi, K., Ver Loren van Themaat, E., Ahmadinejad, N, Assenza, F., Rauf, P., Huettel, B., Reinhardt R., Schmelzer, E., Peplies, J., Gloeckner, F., Amann, R., Eickhorst, T., and Schulze-Lefert P. (2012), Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature 488, 91-95 (2012).
Lebeis, S.L., Rott, M., Dangl, J.L.* and Schulze-Lefert, P.*: Culturing a plant microbiome at the cross-Rhodes, New Phytologist 196, 341–344 (2012).
Bisseling, T., Dangl, J. and Schulze-Lefert, P. (2009), Next Generation Communication, Science Editorial, Science 324, p.691, 8 May (2009).
Current and future work
We have begun to systematically purify Arabidopsis bacterial endophytes on the basis of the aforementioned culture-independent 16S rRNA gene survey. We also examine presumed probiotic functions of the root microbiota for plant growth using defined bacterial communities under laboratory conditions.
Stijn Spaepen (post-doc), Haruhiko Inoue (post-doc), Yang Bai (post-doc), Stéphane Hacquard (post-doc), Kei Hiruma (post-doc), Ryohei Thomas Nakano (post-doc), Girish Srinivas (bioinformatician), Nina Dombrowski (PhD), Ruben Garrido Oter (PhD), Petra Köchner (technical assistant)
II. Non-self perception and signaling by intracellular immune sensors
One class of immune receptors, designated NB-LRR proteins, detects isolate-specific pathogen effector molecules inside plant cells. The Mla locus in barley encodes coiled-coil (CC) domain, nucleotide-binding (NB) site, and leucine-rich repeat (LRR)-containing receptor proteins and mediate isolate-specific immunity to the grass powdery mildew fungus (Blumeria graminis f. sp. hordei). Genetic studies identified a large number of allelic variants at the Mla locus each recognizing a cognate race-specific B. graminis effector (AVRA). We have shown that barley MLA partitions between the cytoplasm and nucleus. We have also shown that nucleocytoplasmic partitioning of the receptor results in a bifurcation of MLA‐triggered host cell death and disease resistance signaling in a cell compartment‐dependent manner. MLA-mediated host cell death signaling is initiated in the cytoplasm whereas disease resistance signaling takes place in the nucleus. Recognition of B. graminis AVRA effectors by MLA induces nuclear associations between the receptor and WRKY transcription factors. These WRKY proteins act as repressors of MAMP (also called PAMP-)-triggered basal defense. MLA appears to interfere with the WRKY repressor function in the nucleus, thereby de-repressing MAMP-triggered defense. Collectively, our findings imply a mechanism by which the polymorphic MLA immune receptors integrate distinct pathogen signals and initiate a bifurcated signal transduction cascade. Our structural and biochemical work revealed a CC domain-dependent homodimerization of the immune sensor that is necessary for receptor function. Notably, the CC homodimer module defines a minimal functional unit, which is sufficient to activate host cell death signaling.
Jacob,F.; Vernaldi, S. and Maekawa, T: Evolution and conservation of plant NLR functions. Frontiers in Immunology 4:297, doi 10.3389/fimmu.2013.00297 (2013). abstract
Bai, S., Liu, J., Chang Ch., Zhang, L., Maekawa, T., Wang, Q., Xiao, W., Liu, Y., Chai, J., Takken, FLW., Schulze-Lefert, P., Shen, QH. (2012) Structure-Function Analysis of Barley NLR immune Receptor MLA10 Reveals Its Cell Compartment Specific Activity in Cell Death and Disease Resistance. PLoS Pathogens 8(6), e1002752
Maekawa, T., Kracher, B., Vernaldi, S., Ver Loren van Themaat, E. and Schulze-Lefert, P. (2012), Conservation of NLR-triggered immunity across plant lineages. PNAS 49, 20119-20123 (2012).
Maekawa, T., Cheng, W., Spiridon, LN., Töller, A. Lukasik, E., Saijo, Y., Liu, P.; Shen, Q. Micluta, MA., Somssich, IE., Takken, FL., Petrescu, A.-J., Chai, J. and Schulze-Lefert, P. (2011) Coiled-coil domain-dependent homodimerization of intracellular MLA immune receptors defines a minimal functional module for triggering cell death. Cell Host & Microbe 9, 187-199.
Shen, Q.-H., Saijo Y., Mauch S., Biskup C., Bieri S, Keller B, Seki H., lker B., Somssich I.E., and Schulze-Lefert P. (2007): Nuclear Activity of MLA Immune Receptors Links Isolate-Specific and Basal Disease-Resistance Responses. Science 315: 1098-1103.
(abstract) (full text)
This work was featured in:
- Cell 128: 823-824, 2007. Jen Sheen and Ping He: Nuclear Actions in Innate Immune Signaling
- Science 315: 1088-1089, 2007. Jeff L. Dangl: Nibbling at the Plant Cell Nucleus
Bieri S, Mauch S, Shen Q-H, Peart J, Devoto A, Casais C, Ceron F, Schulze S, Steinbiss H.-H., Shirasu K, and Schulze-Lefert P (2004) RAR1 positively controls steady state levels of barley MLA resistance proteins and enables sufficient MLA6 accumulation for effective resistance. The Plant Cell 16, 3480-3495.
Shen Q.-H., Zhou F., Bieri S., Haizel T., Shirasu K., and Schulze-Lefert P. (2003) Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus. Plant Cell 15: 732-744.
Current and future work
We aim at a deep understanding of MLA immune receptor function and evolution. Our recent experiments revealed that MLA is fully functional in Arabidopsis which diverged from barley ~150 My ago, thereby enabling comparative studies on MLA function in the model plant Arabidopsis and the crop barley. We aim to resolve the structure of individual MLA domains and of the full-length receptor in collaboration with the structural biology group of Jijie Chai, Beijing. We explore cytoplasmic and nuclear functions of MLA and wish to understand how this immune sensor reconfigures host transcription upon recognition of powdery mildew effectors in barley and Arabidopsis (in collaboration with Imre Somssich). We will also clarify whether MLA receptors detect AVRA effectors directly or indirectly.
Takaki Maekawa (Senior scientist), Xunli Lu (post doc), Saskia Vernaldi (PhD), Florence Jacob (PhD), Petra Köchner (technical assistant), Sabine Haigis (Technical assistant), Makoto Yoshikawa-Maekawa (post-doc)
III. Secretory pathways in immune responses
Surprisingly little is known about the cellular mechanisms that mediate the execution of immune responses. Our genetic and biochemical work on Arabidopsis PEN proteins revealed the existence of a secretory machinery that becomes engaged in the execution of extracellular immune responses. At least two vesicle-associated and SNARE protein-dependent exocytosis pathways appear to drive focal and/or non-directional secretion of antimicrobial cocktails comprising proteins, small molecules, and cell wall building blocks into the apoplastic space at pathogen contact sites. Both pathways have additional functions in plant development and might have been co-opted for immune responses. Independently from this, a plant plasma membrane ATP-binding cassette-type (ABC) transporter and an enzymatic machinery for the biosynthesis and hydrolysis of indole glucosinolates act in a parallel defense pathway for the targeted delivery of antimicrobials and/or agents promoting chemical cross-linking of plant cell wall polymers. Our work on PEN proteins has established a critical role of extracellular (pre-invasive) PEN-dependent defense in non-host resistance to fungal pathogens.
Kim, H., O‘Connell, R., Maekawa-Yoshikawa, M., Uemura, T., Neumann, U., Schulze-Lefert, P. (2014) The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes, The Plant Journal, doi: 10.1111/tpj.12591.
Uemura, T., Kim, H., Saito, C, Ebine, K., Ueda, T., Schulze-Lefert, P., and Nakano, A. (2012) Qa-SNAREs localised to the trans-Golgi network regulate multiple transport pathways and extracellular disease resistance in plants. PNAS 109 (5), 1784-1789.
Bednarek, P., Piślewska-Bednarek, M., Ver Loren van Themaat, E., Maddula, R.K., Svatoš, A. and Schulze-Lefert, P. (2011) Conservation and clade-specific diversification of the pathogen-inducible tryptophan and indole glucosinolate metabolism in Arabidopsis thaliana relatives, New Phytologist 192, 713-726
This work was commented in:
- New Phytologist (2011) 192: 566-569 by Nicole K. Clay: Chemical diversity on display in the plant innate immune systems of closely-related species.
Bednarek, P, Pislewska-Bednarek, M., Svatos, A., Schneider, B., Doubsky, J., Mansourova, M., Humphry, M., Consonni, C., Panstruga, R., Sanchez-Vallet, A., Molina, A., and Schulze-Lefert, P. (2009): A glucosinolate metabolism pathway imediates broadspectrum antifungal defense in plants. Science 323, 101-106.
This work was featured in:
- ACS Chem. Biol. 4 (2), 80-84. DOI: 10.1021/cb9000266, pp 82 - 83
- Kwon, C., P. Bednarek, and P. Schulze-Lefert (2008): Secretory Pathways in Plant Immune Responses, Plant Physiology 147, 1575-158
- Kwon, C., R. Panstruga and P. Schulze-Lefert (2008): Les liaisons dangereuses: Immunological synapse formation in animals and plants. Trends in Immunology 29, 159-166
Kwon, C., C. Neu, S. Pajonk, H. S. Yun, U. Lipka, M. E. Humphry, S. Bau, M. Straus, H. Rampelt, F. El Kasmi, G. Jürgens, J. Parker, R. Panstruga, V. Lipka and P. Schulze-Lefert (2008): Co-option of a default secretory pathway for plant immune responses. Nature 451, 835-840.
Lipka, V., Dittgen J., Bednarek P., Bhat R., Wiermer M., Stein M., Lantag J., Brandt W., Rosahl S., Scheel D., Llorente F., Molina A., Parker J., Somervillle S., Schulze-Lefert P. (2005) Pre- and postinvasion defenses both contribute to nonhost resistance in Arabidopsis. Science 310, 1180-1183.
(abstract) (full text)
This work was featured in:
The Plant Cell 18: 523-528, 2006, Ellis J.: Insights into nonhost disease resistance: Can they assist disease control in agriculture?
Collins N.C., H. Thordal-Christensen, V. Lipka, S. Bau, E. Kombrink, J.-L. Qiu, R. H ckelhoven, M. Stein, A. Freialdenhoven, S.C. Somerville and P. Schulze-Lefert (2003): SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425, 973-977.
Current and future work
We aim to characterize cargos transported by VAMP721/722 vesicles as well as regulatory proteins controlling biogenesis, turnover, and directed intracellular transport of these endomembrane compartments to the plant cell periphery. We also study in collaboration with Pawel Bednarek (Poznan, Poland) the evolutionary and functional diversification of glucosinolate biosynthesis and hydrolysis pathways in Arabidopsis relatives and in different plant organs including roots
Hyeran Kim (post-doc), Makoto Maekawa (post-doc), Xunli Lu (post-doc), Sabine Haigis (technical assistant).