Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhizobium trifolii

Abstract

Rhizobium tifolii mutants containing Escherichia coli lac gene fusions to specific nodulation (nod) genes were used to characterise phenolic compounds secreted from the roots of white clover (Trifolium repens) plants. These compounds either had stimulatory or inhibitory effects upon the induction of the nod genes. The stimulatory compounds were hydroxylated flavones and the most active compound was 7,4'-dihydroxyflavone. The inhibitory compounds present in white clover root exudates were umbelliferone (a coumarin) and formononetin (an isoflavone). Transcriptional activation of nod gene promoters in response to short exposures (3 h) of 7,4'-dihydroxyflavone was growth phase dependent; cells in early log phase were highly responsive to flavone additions in vitro and nod gene induction could be detected within 20 min of exposure at 5 x 10-7 M. Cells in other growth phases were generally unresponsive. A 10-fold molar excess of umbelliferone to 7,4'-dihydroxyflavone resulted in complete inhibition of nod gene induction. Some commerciallyobtained flavones were found to have weak stimulatory activity but could also inhibit nod gene induction by more effective stimulatory compounds. Strong stimulatory and inhibitory compounds all possessed a 7-hydroxy moiety and showed other structural similarities. This suggested that there was one binding site for these compounds. Because the response to these compounds was rapid, we propose that these phenolics act at the bacterial membrane or that an active uptake system is involved.

Interactions among Flavonoid nod Gene Inducers Released from Alfalfa Seeds and Roots

“…Studies with compounds not exuded by the host legume have shown that weak nod gene inducers can restrict the activity of strong inducers in Rhizobium leguminosarum biovar viciae (6), R. leguminosarum biovar trifolii (4), and in R. meliloti (18,19). Other commercially available flavonoids without detectable nod gene-inducing activity inhibited activities of strong nod gene inducers in several systems (4,6,18). In one case, formononetin and umbelliferone, compounds with no nod gene-inducing activity, were identified in clover root extracts (20) and shown to inhibit nod gene induction by 4',7-dihydroxyflavone in R. leguminosarum biovar trifolii (4).…”

Induction of Microbial Genes for Pathogenesis and Symbiosis by Chemicals from Root Border Cells

“…One observation that has been consistent among independent laboratories is that the root tip is rich in chemicals that specifically influence reporter gene expression (Firmin et al, 1986;Redmond et al, 1986;Djordjevic et al, 1987;Peters and Long, 1988). Because this region of intense activity corresponds with some precision to the region where border cells are released, it was of interest to test the hypothesis that border cells constitute one cellular source of signals that induce microbial gene expression.…”

“…Even allowing for loss of some root tip exudate by diffusion into the agar or absorption into paper, the amount from border cells alone could easily account for a significant part of the intense reporter gene activity that occurs at root tips of legumes (Firmin et al, 1986;Redmond et al, 1986;Djordjevic et al, 1987;Peters and Long, 1988).…”

“…Conditions of the plate assay used in previous studies (Firmin et al, 1986;Redmond et al, 1986;Djordjevic et al, 1987;Peters and Long, 1988)-incubation of plant roots with bacteria overnight at 28°C-were duplicated, but in the current study the only source of chemicals was border cells. A quantitative assay was used to measure enzyme activity reflecting the level of the reporter gene product present at the end of the cocultivation period.…”

“…(Firmin et al, 1986;Redmond et al, 1986;Djordjevic et al, 1987;Peters and Long, 1988). In those studies a plate assay in which host plants were cocultivated overnight at 28°C with Rkizobium strains was used (Djordjevic et al, 1987;Peters and Long, 1988).…”