Nodulation signal specificity and perception in legumes

Author

Jonathan Cohn

Date of Award

8-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Gary Stacey

Abstract

The protein products of the rhizobial nodulation genes synthesize lipo-chitooligosaccharides (LCO) signal molecules, or Nod signals, that induce root hair deformation and induce nodule primordia on legume roots. These LCOs are generally tetra- and penta-lipo-oligosaccharides of N-acetyl-D-glucosamine with an acyl substitution on the non-reducing end and a variety of substitutions on the reducing end,depending upon the rhizobial strain from which they were produced. This study demonstrates that synthetic analogs have the same biological activity as natural productLCO. To determine structure-function relationships, a collection of synthetic and natural product LCOs was assayed on Glycine soja roots. All biologically active LCOs induced both root hair deformations and nodule primordia on G. soja roots. Structure-function studies indicate that there was interdependence on backbone length and the presence of substitutions on the LCO reducing end. Pentameric LCOs were active only if they were modified with a 2-0-methyl fucosyl moiety on the reducing end. However, tetrameric LCOs were only active if they lacked this reducing end modification. Thus, G. sojarecognized LCO without reducing end substitutions, despite the reported importance of these modifications for host range.Structure function studies were also carried out on another tropical legume, ricebean (Vigna umbellata). LCOs containing a pentameric chitin backbone and a reducing-end 2-O-methyl fucosyl moiety were active on V. umbellata. In contrast to the studies with G. soja, a tetrameric LCO without any reducing end modification was not active on v. umbellata. A Bradyrhizobium japonicum nodZ mutant, which produces only LCO without 2-O-methylfucose at the reducing end, was able to induce nodule structures on both G. soja and V. umbellata. Surprisingly, the individual, purified, LCO molecules produced by this mutant were incapable of inducing nodule formation on V. umbellata roots. However, when applied in combination, the LCOs produced by the nodZ mutantacted cooperatively to produce nodule-like structures on V. umbellata roots.A recent study reported that a Nod signal binding protein from Dolichos biflorusis a candidate for a Nod signal receptor. This protein,LNP, was found to be an apyrase.To determine if apyrases might play a role in the early events in nodulation, putative orthologs of LNP were analyzed from the model legume Medicago truncatula. Four Putative apyrase genes were identified from M. truncatula. Two of the genes identified from M.truncatula, Mtapyl and Mtapy4 are expressed in roots, and are inducible within three hours after inoculation with Sinorhizobium meliloti. The level of mRNA expression of the other two putative apyrases, Mtapyl2 and Mtapy3, was unaffected by rhizobial inoculation. Screening of a bacterial artificial chromosome (BAC)library ofM.truncatula genomic DNA showed that Mtapyl, MtapyS, and Mtapy4 are present on a single BAC clone,indicating that these apyrases are clustered on the genome. Screening of nodulation deficient mutant lines of M.truncatula revealed that two such mutant lines do not express apyrases to any detectable level. One of these mutant lines, pdl,is unable to produce cortical cell divisions in response to rhizobia. Conversely, apyrase mRNA is expressed at levels comparable to wild-type in another nodulation deficient mutant line,lin, which forms nodule-like structures in response to rhizobia. These data suggest a role for apyrases early in the nodulation response, before the involvement of root cortical cell division leading to nodule development.