Effects of alfalfa nod gene-inducing flavonoids on nodABC transcription in Rhizobium meliloti strains containing different nodD genes

Abstract

Transcription of the nodulation genes nodABC in Rhizobium meliloti requires a plant flavonoid signal and nodD, a family of bacterial regulatory genes (nodDl, nodD2, and nodD3). Results from this study show that all previously identified nod gene inducers released by alfalfa seeds and roots induced nodABC-IacZ transcription in R. meliloti containing extra copies of nodDI, but only 4,4'-dihydroxy-2'-methoxychalcone gave high levels of induction with extra copies of nodD2. While mixtures of the methoxychalcone and luteolin showed a positive synergism with extra NodDl protein, they apparently competed for binding to the NodD2 protein.Rhizobium meliloti infect alfalfa (Medicago sativa L.) plants and induce the formation of root nodules that reduce N2 to ammonia. Several sets of bacterial genes are involved in the early stages of nodule formation. One well-described transcriptional unit, the nodABC genes, initiates cortical cell division (2, 26), root hair deformation (15), and production of a receptorlike protein (12,13). The nodABC genes in R. meliloti are expressed primarily in the presence of particular flavonoids exuded by alfalfa seeds or roots (7, 18, 21,23).Precisely how plant flavonoids induce transcription of nod genes in R. meliloti is not known, but a family of regulatory genes including nodDI, nodD2, and nodD3 is involved (4,5,9,22,25). Studies of the nodDi gene product (NodDl), nod gene-inducing flavonoids, and the promoter region of nod-ABC suggest that NodDl is a DNA-binding protein (3,8,16). Tests with crude seed exudates from different plant species nodulated by R. meliloti indicate that species-specific factors interact with different NodD proteins (5). In those tests, alfalfa seed exudate produced high levels of nod gene induction in the presence of nodDi, nodD2, and nodD3, but it could not be determined whether the same or separate (presumably flavonoid) factors functioned through different nodD genes.A series of flavonoids has now been identified as natural alfalfa nod gene inducers in R. meliloti 1021(pRmM57), which contains a cloned fragment with a nodDIABC-lacZ fusion (21). Luteolin (3',4',5,7-tetrahydroxyflavone; Fig. 1), which was first isolated from alfalfa seeds (23), was not found in root exudates of Moapa 69 alfalfa seedlings (18). However, the root exudates did contain three other nod gene inducers: 4,4'-dihydroxy-2'-methoxychalcone, 4',7-dihydroxyflavone, and 4',7-dihydroxyflavanone ( Fig. 1) (18). Seed rinses from the same alfalfa cultivar contained luteolin and chrysoeriol (3'-methoxyluteolin; Fig. 1) (7). The biological significance of this diversity of nod gene inducers is not clear, but at least three points are noteworthy. First, although luteolin probably is the most prevalent nod gene inducer in the soil around a germinating alfalfa seed (7), the methoxychalcone and chrysoeriol induce half-maximum transcriptional rates of nodABC-lacZ fusion at 10 and 25%, respectively, of the concentration required for luteolin (7, 18). Second, the 4',7 substitution pattern (flavone numberin...

Molecular Basis of Symbiotic Promiscuity

“…nodD products of various Rhizobium species vary in that they respond to different sets of flavonoids (72,249). Moreover, NodD homologues from the same strain may have different flavonoid preferences (112,117,198). In R. meliloti, NodD1 is activated when cells are supplied with a complex plant seed extract or the flavonoid luteolin; NodD2 is activated only with the complex extract, not with purified luteolin; and NodD3 apparently modulates the expression of nod genes even in the absence of any plant factor (180).…”

Trigonelline and Stachydrine Released from Alfalfa Seeds Activate NodD2 Protein in Rhizobium meliloti

“…For example, luteolin, the first nod gene inducer identified from alfalfa (24), is discharged from imbibing seeds (10) but not from roots (19 nod gene inducer that is 10-to 20-fold more active than luteolin (19). A potentially important functional difference between these two natural inducers is that MCh activates both NodDl and NodD2 proteins (11), whereas luteolin activates only NodD1 (11,12). The different nodD regulatory genes (nodD1, nodD2, and nodD3) in R. meliloti affect the rate at which this bacterium nodulates different host plants.…”

“…Alfalfa seed rinse, for example, strongly activated both NodDl and NodD2, but comparable fractions from four other species of legumes failed to activate NodD2 significantly (7). Subsequent studies of seed rinses separated on TLC did not locate a NodD2 activator in alfalfa seed rinse (8), and tests of flavonoids identified in alfalfa seed rinse showed only traces of NodD2 activation (11). This project was initiated to identify the unknown NodD2 activator reported previously (7) in crude alfalfa seed rinses.…”

Importance des composés phénoliques dans les interactions entre plantes et microorganismes: exemple des relationsRhizobium/légumineuses

“…• Chez Medicago sativa, au niveau des graines, cinq composes ont ete mis en evidence (Hartwig et al, 1990) (Phillips et al, 1995). Les principaux inducteurs identifies par Maxwell et al ( 1989) dans les exsudats racinaires sont la4' ,7-dihydroxyflavone, Ia 4', 7 -dihydroxyflavanone (liquiritigenine) et Ia 4,4' -dihydroxy-2-methoxychalcone.…”

“…Les deux premieres molecules ont des activites inductrices inferieures a celle de Ia luteoline et le meilleur inducteur est Ia 4,4' -dihydroxy-2-methoxychalcone qui est au moins dix fois plus puissante que Ia luteoline. Une difference importante entre Ia luteoline et Ia 4,4' -dihydroxy-2-methoxychalcone est Ia capacite de cette derniere a activer les proteines codees par les genes nodDJ et nodD2 tandis que Ia luteoline n'est capable d'activer que le produit du gene nodDJ (Hartwig et al, 1990).…”