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Nod Factor-Induced Expression of Leghemoglobin to Study the Mechanism of NH₄NO₃ Inhibition on Root Hair Deformation

¹Department of Molecular Biology, Agricultural University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands; ²Avdeling for Plantefysiologi og Mikrobiologi, Institutt for Biologi og Geologi, Universitetet iTromsø, 9037 Tromsø, Norway; and ³Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, Profesor Albareda 1, 18008 Granada, Spain

Nod factors secreted by Rhizobium leguminosarum bv. viciae induce root hair deformation, the formation of nodule primordia, and the expression of early nodulin genes in Vicia sativa (vetch). Root hair deformation is induced within 3 h in a small, susceptible zone (±2 mm) of the root. NH₄NO₃, known to be a potent blocker of nodule formation, inhibits root hair deformation, initial cortical cell divisions, and infection thread formation. To test whether NH₄NO₃ affects the formation of a component of the Nod factor perception-transduction system, we studied Nod factor-induced gene expression. The differential display technique was used to search for marker genes, which are induced within 1 to 3 h after Nod factor application. Surprisingly, one of the isolated cDNA clones was identified as a leghemoglobin gene (VsLb1), which is induced in vetch roots within 1 h after Nod factor application. By using the drug brefeldin A, it was then shown that VsLb1 activation does not require root hair deformation. The pVsLb1 clone was used as a marker to show that in vetch plants grown in the presence of NH₄NO₃ Nod factor perception and transduction leading to gene expression are unaffected.