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1.  Single Nucleus Genome Sequencing Reveals High Similarity among Nuclei of an Endomycorrhizal Fungus 
PLoS Genetics  2014;10(1):e1004078.
Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya.
Author Summary
Endomycorrhizal fungi are known for their symbiosis with the vast majority of land plants. The fungus penetrates the root and facilitates uptake of nutrients for the plant. For a long time it was hypothesized that endomycorrhizal fungi have a complex genetic makeup, as they are asexual organisms. Their hyphae do not consist of individual cells, but rather form a continuous compartment in which numerous nuclei migrate. Several studies indicated that these nuclei are genetically highly diverse, suggesting that endomycorrhizal fungi evolved a unique genome structure. By sequencing individual nuclei of a single individual of the reference fungus Rhizophagus, we demystify this hypothesis and show that the nuclei are highly similar. Furthermore, we created the first genome sequence of these ancient fungi that will serve as a valuable resource to further understand and exploit this agriculturally and ecologically vital symbiosis.
PMCID: PMC3886924  PMID: 24415955
2.  Interaction of Medicago truncatula Lysin Motif Receptor-Like Kinases, NFP and LYK3, Produced in Nicotiana benthamiana Induces Defence-Like Responses 
PLoS ONE  2013;8(6):e65055.
Receptor(-like) kinases with Lysin Motif (LysM) domains in their extracellular region play crucial roles during plant interactions with microorganisms; e.g. Arabidopsis thaliana CERK1 activates innate immunity upon perception of fungal chitin/chitooligosaccharides, whereas Medicago truncatula NFP and LYK3 mediate signalling upon perception of bacterial lipo-chitooligosaccharides, termed Nod factors, during the establishment of mutualism with nitrogen-fixing rhizobia. However, little is still known about the exact activation and signalling mechanisms of MtNFP and MtLYK3. We aimed at investigating putative molecular interactions of MtNFP and MtLYK3 produced in Nicotiana benthamiana. Surprisingly, heterologous co-production of these proteins resulted in an induction of defence-like responses, which included defence-related gene expression, accumulation of phenolic compounds, and cell death. Similar defence-like responses were observed upon production of AtCERK1 in N. benthamiana leaves. Production of either MtNFP or MtLYK3 alone or their co-production with other unrelated receptor(-like) kinases did not induce cell death in N. benthamiana, indicating that a functional interaction between these LysM receptor-like kinases is required for triggering this response. Importantly, structure-function studies revealed that the MtNFP intracellular region, specific features of the MtLYK3 intracellular region (including several putative phosphorylation sites), and MtLYK3 and AtCERK1 kinase activity were indispensable for cell death induction, thereby mimicking the structural requirements of nodulation or chitin-induced signalling. The observed similarity of N. benthamiana response to MtNFP and MtLYK3 co-production and AtCERK1 production suggests the existence of parallels between Nod factor-induced and chitin-induced signalling mediated by the respective LysM receptor(-like) kinases. Notably, the conserved structural requirements for MtNFP and MtLYK3 biological activity in M. truncatula (nodulation) and in N. benthamiana (cell death induction) indicates the relevance of the latter system for studies on these, and potentially other symbiotic LysM receptor-like kinases.
PMCID: PMC3672211  PMID: 23750228
3.  One-Step Agrobacterium Mediated Transformation of Eight Genes Essential for Rhizobium Symbiotic Signaling Using the Novel Binary Vector System pHUGE 
PLoS ONE  2012;7(10):e47885.
Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.
PMCID: PMC3480454  PMID: 23112864
4.  Evolutionary origin of rhizobium Nod factor signaling 
Plant Signaling & Behavior  2011;6(10):1510-1514.
For over two decades now, it is known that the nodule symbiosis between legume plants and nitrogen fixing rhizobium bacteria is set in motion by the bacterial signal molecule named nodulation (Nod) factor.1 Upon Nod factor perception a signaling cascade is activated that is also essential for endomycorrhizal symbiosis (Fig. 1). This suggests that rhizobium co-opted the evolutionary far more ancient mycorrhizal signaling pathway in order to establish an endosymbiotic interaction with legumes.2 As arbuscular mycorrhizal fungi of the Glomeromycota phylum can establish a symbiosis with the vast majority of land plants, it is most probable that this signaling cascade is wide spread in the plant kingdom.3 However, Nod factor perception generally is considered to be unique to legumes. Two recent breakthroughs on the evolutionary origin of rhizobium Nod factor signaling demonstrate that this is not the case.4,5 The purification of Nod factor-like molecules excreted by the mycorrhizal fungus Glomus intraradices and the role of the LysM-type Nod factor receptor PaNFP in the non-legume Parasponia andersonii provide novel understanding on the evolution of rhizobial Nod factor signaling.
PMCID: PMC3256379  PMID: 21904113
parasponia; legumes; rhizobium; mycorrhizae; Nod factor
5.  Modeling a Cortical Auxin Maximum for Nodulation: Different Signatures of Potential Strategies 
Lateral organ formation from plant roots typically requires the de novo creation of a meristem, initiated at the location of a localized auxin maximum. Legume roots can form both root nodules and lateral roots. From the basic principles of auxin transport and metabolism only a few mechanisms can be inferred for increasing the local auxin concentration: increased influx, decreased efflux, and (increased) local production. Using computer simulations we investigate the different spatio-temporal patterns resulting from each of these mechanisms in the context of a root model of a generalized legume. We apply all mechanisms to the same group of preselected cells, dubbed the controlled area. We find that each mechanism leaves its own characteristic signature. Local production by itself can not create a strong auxin maximum. An increase of influx, as is observed in lateral root formation, can result in an auxin maximum that is spatially more confined than the controlled area. A decrease of efflux on the other hand leads to a broad maximum, which is more similar to what is observed for nodule primordia. With our prime interest in nodulation, we further investigate the dynamics following a decrease of efflux. We find that with a homogeneous change in the whole cortex, the first auxin accumulation is observed in the inner cortex. The steady state lateral location of this efflux reduced auxin maximum can be shifted by slight changes in the ratio of central to peripheral efflux carriers. We discuss the implications of this finding in the context of determinate and indeterminate nodules, which originate from different cortical positions. The patterns we have found are robust under disruption of the (artificial) tissue layout. The same patterns are therefore likely to occur in many other contexts.
PMCID: PMC3361061  PMID: 22654886
root nodules; auxin transport manipulation; modeling
6.  RDML: structured language and reporting guidelines for real-time quantitative PCR data 
Nucleic Acids Research  2009;37(7):2065-2069.
The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium ( to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and between experimenters and journals or public repositories. We here also propose data related guidelines as a subset of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to guarantee inclusion of key data information when reporting experimental results.
PMCID: PMC2673419  PMID: 19223324
7.  Primer3Plus, an enhanced web interface to Primer3 
Nucleic Acids Research  2007;35(Web Server issue):W71-W74.
Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present-day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like designing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software packages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at
PMCID: PMC1933133  PMID: 17485472

Results 1-7 (7)