Molecular and genetic analysis of a symbiosis-specific locus of the Lotus japonicus genome and its potential as a tool for dissecting the symbiotic signal transduction pathway

Type

Student thesis: Doctoral ThesisDoctor of Philosophy

Original languageEnglish
Awarding Institution
Supervisors/Advisors
  • Kathleen Webb
Award date2005
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Abstract

Nitrogen-fixing nodules on legume roots result from a symbiotic interaction with soil bacteria known as rhizobia. This relationship is important in sustainable agriculture and the genetic and molecular basis of root nodule formation and functioning is the subject of intense research.

A promoter-tagging programme using the model legume Lotus japonicus and a -glucuronidase (gus) reporter identified one line, T90, which exhibited GUS expression only after inoculation with the plants’ rhizobial symbiont Mesorhizobium loti, and its arbuscular mycorrhizal fungal symbiont Glomus mosseae. Molecular analysis showed that insertion of the reporter gene construct had occurred approximately 1.1 Kb upstream of an open reading frame encoding a putative calcium-binding protein, LjCbp1.

Calcium is a well-known second messenger and is involved in root nodule symbiosis. This thesis describes the in silico analysis of LjCbp1 and putative regulatory elements upstream of the coding region. Altered protein mobility changes on polyacrylamide gels in the presence of calcium suggest that LjCbp1 indeed has calcium-binding properties. The spatiotemporal expression patterns of LjCbp1 before and after inoculation with M. loti correlated with that of gus in T90. This suggests that reporter gene expression accurately reflects that of the endogenous gene. T90 gus expression was up-regulated by calcium treatment and modulated by pharmaceutical agents (for example the calcium channel blocker nifedipine) that modify calcium influx. Such data suggest that LjCbp1 transcription is influenced by calcium.

Various lines of evidence, including the absence of a nodulation deficient phenotype in lines expressing reduced levels of LjCbp1, indicate that LjCbp1 is not essential for symbiotic interactions but that this gene nevertheless plays a role in the early stages of association with rhizobia and arbuscular mycorrhizal fungi.

The specificity of gus expression was also analysed in detail. Nod factor purified from M. loti, as well as chitosan and various rhizobial strains also elicited GUS activity, thus confirming its specificity to both symbionts.

This specificity was exploited in a chemical mutagenesis programme to create a range of symbiotic phenotypes. Significant changes were observed in expression patterns of the gus reporter gene following inoculation with M. loti in three mutants. This emphasises the value of T90 as a powerful tool in dissecting the signalling events in the early stages of nodulation.