Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca2+ spiking in the legume and nonlegume root epidermis.

• The aim of this study was to investigate Ca(2+) responses to endosymbiotic arbuscular mycorrhizal (AM) fungi in the host root epidermis following pre-infection hyphopodium formation in both legumes and nonlegumes, and to determine to what extent these responses could be mimicked by germinated fungal spore exudate. • Root organ cultures of both Medicago truncatula and Daucus carota, expressing the nuclear-localized cameleon reporter NupYC2.1, were used to monitor AM-elicited Ca(2+) responses in host root tissues. • Ca(2+) spiking was observed in cells contacted by AM hyphopodia for both hosts, with highest frequencies correlating with the epidermal nucleus positioned facing the fungal contact site. Treatment with AM spore exudate also elicited Ca(2+) spiking within the AM-responsive zone of the root and, in both cases, spiking was dependent on the M. truncatula common SYM genes DMI1/2, but not on the rhizobial Nod factor perception gene NFP. • These findings support the conclusion that AM fungal root penetration is preceded by a SYM pathway-dependent oscillatory Ca(2+) response, whose evolutionary origin predates the divergence between asterid and rosid clades. Our results further show that fungal symbiotic signals are already generated during spore germination, and that cameleon-expressing root organ cultures represent a novel AM-specific bio-assay for such signals.

[1]  C. Gutjahr,et al.  Cell and developmental biology of arbuscular mycorrhiza symbiosis. , 2013, Annual review of cell and developmental biology.

[2]  Hongyan Zhu,et al.  Antiquity and Function of CASTOR and POLLUX, the Twin Ion Channel-Encoding Genes Key to the Evolution of Root Symbioses in Plants1[W][OA] , 2008, Plant Physiology.

[3]  A. Kereszt,et al.  A receptor kinase gene regulating symbiotic nodule development , 2002, Nature.

[4]  D. Soltis,et al.  Rosid radiation and the rapid rise of angiosperm-dominated forests , 2009, Proceedings of the National Academy of Sciences.

[5]  F. J. Bruijn.  Molecular microbial ecology of the rhizosphere , 2013 .

[6]  N. Requena,et al.  Membrane steroid-binding protein 1 induced by a diffusible fungal signal is critical for mycorrhization in Medicago truncatula. , 2010, The New phytologist.

[7]  R. Akashi,et al.  Super roots in Lotus corniculatus : A unique tissue culture and regeneration system in a legume species , 2003 .

[8]  P. Rougé,et al.  The Medicago truncatula Lysine Motif-Receptor-Like Kinase Gene Family Includes NFP and New Nodule-Expressed Genes1[W] , 2006, Plant Physiology.

[9]  Katharina Markmann,et al.  Functional Adaptation of a Plant Receptor- Kinase Paved the Way for the Evolution of Intracellular Root Symbioses with Bacteria , 2008, PLoS biology.

[10]  S. Shaw,et al.  The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation. , 2003, The Plant journal : for cell and molecular biology.

[11]  A. Edwards,et al.  A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[12]  P. Bonfante,et al.  Prepenetration Apparatus Assembly Precedes and Predicts the Colonization Patterns of Arbuscular Mycorrhizal Fungi within the Root Cortex of Both Medicago truncatula and Daucus carota[W] , 2008, The Plant Cell Online.

[13]  E. Journet,et al.  Four Genes of Medicago truncatula Controlling Components of a Nod Factor Transduction Pathway , 2000, Plant Cell.

[14]  B. Roe,et al.  Medicago truncatula DMI1 Required for Bacterial and Fungal Symbioses in Legumes , 2004, Science.

[15]  Y. Qiu,et al.  Presence of three mycorrhizal genes in the common ancestor of land plants suggests a key role of mycorrhizas in the colonization of land by plants. , 2010, The New phytologist.

[16]  K. Akiyama,et al.  Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi , 2005, Nature.

[17]  A. Timmers,et al.  A Nuclear-Targeted Cameleon Demonstrates Intranuclear Ca2+ Spiking in Medicago truncatula Root Hairs in Response to Rhizobial Nodulation Factors1[W][OA] , 2009, Plant Physiology.

[18]  E. Journet,et al.  Pharmacological Evidence That Multiple Phospholipid Signaling Pathways Link Rhizobium Nodulation Factor Perception in Medicago truncatula Root Hairs to Intracellular Responses, Including Ca2+ Spiking and Specific ENOD Gene Expression1 , 2004, Plant Physiology.

[19]  J. Downie,et al.  Nuclear calcium changes at the core of symbiosis signalling. , 2006, Current opinion in plant biology.

[20]  D. Cook,et al.  Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Richard J Morris,et al.  Differential and chaotic calcium signatures in the symbiosis signaling pathway of legumes , 2008, Proceedings of the National Academy of Sciences.

[22]  Jean-Michel Ané,et al.  The symbiotic ion channel homolog DMI1 is localized in the nuclear membrane of Medicago truncatula roots. , 2007, The Plant journal : for cell and molecular biology.

[23]  R. Tsien,et al.  Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin , 1997, Nature.

[24]  T. Bisseling,et al.  NSP1 of the GRAS Protein Family Is Essential for Rhizobial Nod Factor-Induced Transcription , 2005, Science.

[25]  G. Foster,et al.  Annual Plant Reviews , 2000 .

[26]  A Miyawaki,et al.  Dynamic and quantitative Ca2+ measurements using improved cameleons. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[27]  G. Bécard,et al.  Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. , 2001, Molecular plant-microbe interactions : MPMI.

[28]  C. Jacquet,et al.  Nod factor perception protein carries weight in biotic interactions. , 2013, Trends in plant science.

[29]  Eva Sciacca,et al.  Automated analysis of calcium spiking profiles with CaSA software: two case studies from root-microbe symbioses , 2013, BMC Plant Biology.

[30]  Andrea Genre,et al.  A Diffusible Signal from Arbuscular Mycorrhizal Fungi Elicits a Transient Cytosolic Calcium Elevation in Host Plant Cells1[W] , 2006, Plant Physiology.

[31]  G. Bécard,et al.  Early events of vesicular-arbuscular mycorrhiza formation on Ri T-DNA transformed roots. , 1988, The New phytologist.

[32]  G. Bécard,et al.  The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates. , 2000, Molecular plant-microbe interactions : MPMI.

[33]  Yi Liu,et al.  Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions. , 2013, Fungal biology.

[34]  J. Downie,et al.  Analysis of calcium spiking using a cameleon calcium sensor reveals that nodulation gene expression is regulated by calcium spike number and the developmental status of the cell. , 2006, The Plant journal : for cell and molecular biology.

[35]  G. Bécard,et al.  Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/DMI2 signalling pathway. , 2005, The Plant journal : for cell and molecular biology.

[36]  O. Alizadeh,et al.  Mycorrhizal Symbiosis , 1986, Forest Science.

[37]  M. Parniske,et al.  Evolution of signal transduction in intracellular symbiosis. , 2002, Trends in plant science.

[38]  M. Holsters,et al.  Sesbania rostrata: a case study of natural variation in legume nodulation. , 2010, The New phytologist.

[39]  G. Bécard,et al.  Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi. , 2002, The New phytologist.

[40]  A. Bottcher,et al.  Elicitation of tobacco alkaloid biosynthesis by disrupted spores and filtrate of germinating spores of the arbuscular mycorrhizal fungi Glomus etunicatum , 2013 .

[41]  M. J. Harrison,et al.  Signaling in the arbuscular mycorrhizal symbiosis. , 2005, Annual review of microbiology.

[42]  Martin Parniske,et al.  Arbuscular mycorrhiza: the mother of plant root endosymbioses , 2008, Nature Reviews Microbiology.

[43]  G. Bécard,et al.  Cytoplasmic autofluorescence of an arbuscular mycorrhizal fungus Gigaspora gigantea and nondestructive fungal observations in planta , 1998 .

[44]  U. Paszkowski,et al.  Divergence of evolutionary ways among common sym genes: CASTOR and CCaMK show functional conservation between two symbiosis systems and constitute the root of a common signaling pathway. , 2008, Plant & cell physiology.

[45]  P. Bonfante,et al.  Arbuscular Mycorrhizal Fungi Elicit a Novel Intracellular Apparatus in Medicago truncatula Root Epidermal Cells before Infection[W] , 2005, The Plant Cell Online.

[46]  Jean-Michel Ané,et al.  Tracing Nonlegume Orthologs of Legume Genes Required for Nodulation and Arbuscular Mycorrhizal Symbioses , 2006, Genetics.

[47]  A. Muñoz,et al.  Nodulation independent of rhizobia induced by a calcium-activated kinase lacking autoinhibition , 2006, Nature.

[48]  Jinyuan Liu,et al.  Fungal Symbiosis in Rice Requires an Ortholog of a Legume Common Symbiosis Gene Encoding a Ca2+/Calmodulin-Dependent Protein Kinase1[OA] , 2007, Plant Physiology.

[49]  C. Gutjahr,et al.  Presymbiotic factors released by the arbuscular mycorrhizal fungus Gigaspora margarita induce starch accumulation in Lotus japonicus roots. , 2009, The New phytologist.

[50]  G. Bécard,et al.  A Diffusible Factor from Arbuscular Mycorrhizal Fungi Induces Symbiosis-Specific MtENOD11 Expression in Roots ofMedicago truncatula 1 , 2003, Plant Physiology.

[51]  T. Bisseling,et al.  A Putative Ca2+ and Calmodulin-Dependent Protein Kinase Required for Bacterial and Fungal Symbioses , 2004, Science.