Ané Lab
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Background

Although people often tend to think of microbes as pathogens and “germs,” recent research reveals that thousands of human-associated microbes are extremely beneficial and essential to our health. The same is true of microorganisms associated with plants.  Our goal is to understand and engineer beneficial associations between plants and microbes to improve agriculture productivity, protect the environment over the long term, and enhance human welfare. We are studying the signals and the signaling pathways controlling plant - microbe symbioses. We are improving the practical use of these beneficial associations in the field, and actively trying to engineer efficient nitrogen-fixation in cereals. We are communicating with students, scientists and the general public about the benefits of interdisciplinary research on symbioses and, more generally, in microbiology and plant biology.​

Sweet Talks  between Plants and Microbes

Most of our research is dedicated to beneficial associations between plants and microbes. The vast majority of land plants develop mutualistic associations with arbuscular mycorrhizal (AM) fungi that improve the plants ability to acquire nutrients, especially phosphate and nitrogen, from the soil and their resistance to biotic and abiotic stresses. Legumes also enter into a nitrogen-fixing symbiosis with soil bacteria known as rhizobia that results in the formation of root nodules. A combination of microbial genetics and biochemistry elucidated bacterial and plant factors that mediate the often-high level of host specificity observed in the rhizobia-legume symbiosis (Brelles-Mariño and Ané, 2008; Mukherjee and Ané, 2011; Venkateshwaran and Ané, 2011; Venkateshwaran et al., 2012; Delaux et al., 2013). Legume roots are exquisitely sensitive to bacterial signals named Nod factors which are necessary for nodule development and infection of plant roots. A somehow similar “molecular dialog” was recently identified in arbuscular mycorrhization with symbiotic fungi producing diffusible Myc factors. We like to refer to these signals exchanges as “sweet talks” since Nod and Myc factors are lipo-chito-oligosaccharides (LCOs).​
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Plants responses to the sweet talks of their microbial partners

Power of genetic approaches in model systems
Genetic and molecular analyses in the model legume Medicago truncatula have identified plant mutants affected in nodule development, infection thread formation and early Nod factor responses (Riely et al., 2004; Riely et al., 2006). In addition to their inability to form nodules, dmi (Doesn't Make Infections) mutants are blocked for arbuscular mycorrhization demonstrating that the two symbioses share a Common Symbiotic Pathway (CSP).   We are also developing targeted approaches on candidates such as ROPs and  RopGEFs in collaboration with Dr. Douglas Cook (Riely et al., 2011), sarco/endoplasmic reticulum calcium ATPases (SERCA) in collaboration with Dr. Giles Oldroyd (Capoen et al., 2011).
 
Similarities and differences between model legumes
The DMI1 protein shares strong homologies with bacterial ion channels (Ané et al., 2004). Knowing that ion fluxes play an essential role in responses to Nod factors, we hypothesized that DMI1 could be a novel plant ion channel. In collaboration with Dr. Dale Sanders (University of York, UK), we expressed DMI1 in yeast mutants and used this assay to dissect an RCK domain regulating the DMI1 activity (Peiter et al., 2007). In collaboration with Dr. Douglas Cook, we showed that DMI1 is localized to the nuclear envelope (Riely et al., 2007). In collaboration with Dr. Imaizumi-Anraku (NIAS, Japan) and Dr. Martin Parniske (University of Munich, Germany), we sought to explain genetic differences observed between Medicago and Lotus. For instance, we found that DMI1 can functionally replace CASTOR and POLLUX in Lotus and that CASTOR and POLLUX are required to replace DMI1 in Medicago. We identified a single amino-acid in the selectivity filter region of these ion channels conferring this superior ability to DMI1. This mutation appeared recently in legume evolution, altered the ion selectivity of DMI1 and improved its ability to mediate calcium spiking (Venkateshwaran et al., 2012).
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Protein interactions combined with reverse genetics
Another strategy to dissect these signaling pathways is to analyze protein-protein interactions. In collaboration with Dr. Eva Kondorosi (CNRS, France), we identified a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR1) as interacting specifically with DMI2/NORK and required for root nodule formation (Kevei et al., 2007). We found more recently that HMGR1 is required for arbuscular mycorrhization and early symbiotic signaling including calcium spiking, interacts with Nod factors receptors (NFP and LYK3) and is regulated by NORK phosphorylation (manuscript in preparation).

We also identified IPD3 (Interacting Protein of DMI3) by yeast-two-hybrid (Messinese et al., 2007). We analyzed Tnt1 insertion mutants and fast neutron bombardment mutants of IPD3 in collaboration with Dr. Peter Kaló (University of Szeged, Hungary) and Dr. Giles Oldroyd (John Innes Center, UK). These ipd3 mutants are affected in infection thread formation in both legume nodulation and arbuscular mycorrhization indicating that IPD3 is a new component of the CSP (Horváth et al., 2011).

Evolution of symbiotic associations in land plants and beyond.

We analyze the evolution of plant and microbial mechanisms identified through genetic approaches. For instance, we characterized plant symbiotic genes in soybean (Zhu et al., 2006), in rice (Zhu et al., 2006; Chen et al., 2008; Mukherjee and Ané, 2011) and even in early diverging lineages of land plants such as liverworts and hornworts (Wang et al., 2010; Delaux et al, 2013; Delaux et al., 2015 ). We also analyzed the loss of arbuscylar mycorrhizal associations in various angiosperm lineages including the Brassicales (Delaux et al., 2014).  

Interestingly some of these mechanisms are found in Charophyte green algae suggesting that these Charophyte algae may engage in symbiotic associations that have not yet been identified (Knack et al., 2015).

We also use comparative phylogenomic approaches to understand the critical innovations that gave rise to arbuscular mycorrhizae as well as root nodule symbioses (Delaux et al., 2014).

We also analyze the similarities and differences between arbuscular mycorrhizae and ectomycorrhizae  using poplar and pines as model systems (Garcia et al., 2015).
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Development of genetic and molecular tools for the scientific community

Our laboratory is part of the Wisconsin Plant Symbiosis Group. We are working in collaboration with Dr. Michael Sussman (UW Madison, Biotechnology Center), Dr. Sushmita Roy (Wisconsin Institutes for Discovery) and Dr. Joshua Coon (UW Madison, Chemistry) on innovative techniques to characterize the metabolome, proteome and phosphoproteome of M. truncatula (Grimsrud et al., 2010; Jayaraman et al., 2012; Rose et al., 2012; Rose et al., 2012; Volkening et al., 2012).

In addition, we help in the development of innovative tools for metabolite and peptide imaging in plant cells using MALDI-TOF imaging. This is a collaboration with Dr. Lingjun Li (UW Madison, Chemistry)  (Gemperline et al., 2015; Ye et al., 2013).
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These tools are not only serving our research on plant-microbe symbioses but also benefit many other fields in microbiology and plant sciences.​

Practical applications of research on plant-microbe symbioses

Improving the use of bacterial and fungal inoculants in the field
Soybean growers have been inoculating their seeds with rhizobia for almost one hundred years. However, the decision to inoculate is sometimes difficult especially after events such as the floods which occurred in Wisconsin in 2008. Testing the amount of rhizobia in the soil is classically done using the Most Probable Number (MPN) technique which takes more than a month to yield results. In collaboration with Dr. Shawn Conley (UW Madison, Agronomy), we developed a high-throughput procedure based on real-time PCR to quantify soybean rhizobia in the soil in just a few days (Furseth et al., 2010). Such a tool has already proven to be valuable for both farmers and agronomists in order to determine in which conditions seed inoculation with rhizobia is necessary and cost-effective (Furseth et al., 2011; Furseth et al., 2012). The summer of 2012 was characterized by a long drought throughout the United States. We are currently testing the impact of drought on the survival of soybean rhizobia in the soil in order to help farmers in their decision to inoculate their seeds or not for the next growing season.

Along the same lines, we are currently working on improving the use of mycorrhizal inoculants for organic carrot production in collaboration with Dr. Erin Silva (Plant Pathology, UW Madison) and Dr. Phillip Simon (Horticulture, UW Madison).


Engineering efficient nitrogen fixation in poplar and cereals
We are working on the characterization of maize accessions that can acquire 30-70% of their nitrogen from the air by hosting nitrogen-fixing bacteria in the mucilage produced by their aerial roots. This project was initially started by the Mars Inc. company and collaborators at the University of California-Davis, which led to some legal concerns. However, this N-FARM (Nitrogen-Fixation in Aerial Roots of Maize) project is now publicly funded by the USDA and based on publicly-available accessions from the CIMMYT and the USDA ARS GRIN.  We are also working with Dr. Claudia Calderon, Dr. Natalia deLeon and Dr. Shawn Kaeppler on breeding this trait into accessions that can be used in the US but also in developing countries.

​We also found a similar trait in sorghum accessions. We are characterizing publicly-available accessions from the ICRISAT  and working to introduce this trait into sorghum accession for food and biofuel production. We are also characterizing the microbial communities in this mucilage using approaches of synthetic communities and systems biology. This soNar (sorghum Nitrogen-fixation in aerial roots) project is publicly funded by the DOE. It is an exciting collaboration with Dr. Wilfred Vermerris at the University of Florida, and Dr. Ophelia Venturelli and Dr. Sushmita Roy at the University of Wisconsin-Madison.  

            We are also working, in close collaboration with Dr.  Sushmita Roy, Dr. Matias Kirst, Dr. Douglas Soltis, Dr. Pamela Soltis and Dr. Robert Guralnick on a project aiming at engineering nitrogen-fixing symbioses between rhizobia and non-leguminous crops (poplar and cereals). This Nit_Fix project is funded by the DOE.

​These projects could transform the ways in which the nitrogen nutrition of food and biofuel crops are met and reduce the dependence of our agriculture on nitrogen fertilizers.​
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