Cloning of Cowpea (Vigna unguiculata) Genes that Are Regulated During Initiation of Nodulation. Arthur T. Trese. Department of Botany, Porter Hall, Ohio University, Athens 45701-2979. Steven G. Pueppke(2). (2)Department of Plant Pathology, University of Missouri-Columbia, Columbia 65211 U.S.A. MPMI 4:46-51. Accepted 24 August 1990. Copyright 1991 The American Phytopathological Society.

Taproots of cowpea plants inoculated with Rhizobium fredii USDA257 produce numerous nodules that are clustered in a region approximating the position of the root tip at the time of inoculation. Inoculation with the Nod‾ mutant strain, 257B3, does not induce symbiosis specific responses, but it should have the same general effects on roots. We have used these strains to inoculate seedlings and have isolated poly(A)⁺ RNA from root segments 6 days after inoculation. A cDNA library was constructed from poly(A)⁺ RNA that had been extracted from USDA257-inoculated root segments, and screened by differential hybridization with homologous cDNA probes and cDNA probes from 257B3-inoculated roots. Two clones that hybridize preferentially to nodulating root cDNA were isolated, VuA and VuB. Two others, VuC and VuD, hybridized preferentially to cDNA from nonnodulated roots. Northern gel analysis of total RNA confirmed differential regulation of the mRNA transcripts corresponding to these clones. The VuA clone hybridized to two transcripts that are independently regulated, and to several restriction fragments of cowpea genomic DNA, suggesting that it is a member of a small gene family. Hybridization of VuA with the coding region of pGmENOD2 suggests that VuA represents the cowpea homologue of this early nodulin clone. The VuB homologous transcript is differentially expressed within 1.5 days, and the VuC and VuD clones represent genes whose expression is altered within 2.5 days of inoculation. This rapid response suggests that their regulation is triggered by some early signal(s) from nodulation-competent rhizobia.