AUXIN TRANSPORT INHIBITION PRECEDES ROOT-NODULE FORMATION IN WHITE CLOVER ROOTS AND IS REGULATED BY FLAVONOIDS AND DERIVATIVES OF CHITIN OLIGOSACCHARIDES

The expression of the auxin responsive reporter construct, GH3:gusA, w as examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Re porter gene expression was monitored at marked locations after the app lication of bacteria or signal molecules using two precise inoculation techniques: spot-inoculation and a novel method for ballistic microta rgeting. Changes in GH3:gusA expression were monitored after the inocu lation of Rhizobium leguminosarum biovar trifolii, non-host rhizobia, lipo-chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthe tic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, t he ENOD40-1 peptide or different flavonoids. The results show that clo ver-nodulating rhizobia induce a rapid, transient and local downregula tion of GH3:gusA expression during nodule initiation followed by an up regulation of reporter gene expression at the site of nodule initiatio n. Microtargeting of auxin caused a local and acropetal upregulation o f GH3:gusA expression, whereas NPA caused local and acropetal downregu lation of expression. Both spot-inoculation and microtargeting of R. I . by. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O-acetylated chitin oligosaccharides cause d similar changes to GH3:gusA expression as R. I. by. trifolii spot-in oculation, but only after delivery by microtargeting. Non-O-acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40-1 peptide failed to induce any detectable changes in GH3:gusA expression. GM:gu sA expression patterns during the later stages of nodule and lateral r oot development were similar. These results support the hypothesis tha t LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that end ogenous flavonoids could mediate this response.