Jean-Philippe Combier,1,2 and
1Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université Lyon 1, Université de Lyon, F-69622, Villeurbanne France; 2Laboratoire de Recherche en Sciences Végétales, UMR CNRS 5546, Université Toulouse 3, F-31326 Castanet-Tolosan, France
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Accepted 2 June 2014.
We used Agrobacterium-mediated insertional mutagenesis to identify genes in the ectomycorrhizal fungus Hebeloma cylindrosporum that are essential for efficient mycorrhiza formation. One of the mutants presented a dramatically reduced ability to form ectomycorrhizas when grown in the presence of Pinus pinaster. It failed to form mycorrhizas in the presence of glucose at 0.5 g liter–1, a condition favorable for mycorrhiza formation by the wild-type strain. However, it formed few mycorrhizas when glucose was replaced by fructose or when glucose concentration was increased to 1 g liter–1. Scanning electron microscopy examination of these mycorrhizas revealed that this mutant was unable to differentiate true fungal sheath and Hartig net. Molecular analyses showed that the single-copy disrupting T-DNA was integrated 6,884 bp downstream from the start codon, of an open reading frame potentially encoding a 3,096-amino-acid-long protein. This gene, which we named HcMycE1, has orthologs in numerous fungi as well as different other eukaryotic microorganisms. RNAi inactivation of HcMycE1 in the wild-type strain also led to a mycorrhizal defect, demonstrating that the nonmycorrhizal phenotype of the mutant was due to mutagenic T-DNA integration in HcMycE1. In the wild-type strain colonizing P. pinaster roots, HcMycE1 was transiently upregulated before symbiotic structure differentiation. Together with the inability of the mutant to differentiate these structures, this suggests that HcMycE1 plays a crucial role upstream of the fungal sheath and Hartig net differentiation. This study provides the first characterization of a fungal mutant altered in mycorrhizal ability.
© 2014 The American Phytopathological Society