Multiple domains in MtENOD8 protein including the signal peptide target it to the symbiosome

Plant Physiol. 2012 May;159(1):299-310. doi: 10.1104/pp.111.191403. Epub 2012 Mar 13.

Abstract

Symbiotic nitrogen fixation occurs in nodules, specialized organs on the roots of legumes. Within nodules, host plant cells are infected with rhizobia that are encapsulated by a plant-derived membrane forming a novel organelle, the symbiosome. In Medicago truncatula, the symbiosome consists of the symbiosome membrane, a single rhizobium, and the soluble space between them, called the symbiosome space. The symbiosome space is enriched with plant-derived proteins, including the M. truncatula EARLY NODULIN8 (MtENOD8) protein. Here, we present evidence from green fluorescent protein (GFP) fusion experiments that the MtENOD8 protein contains at least three symbiosome targeting domains, including its N-terminal signal peptide (SP). When ectopically expressed in nonnodulated root tissue, the MtENOD8 SP delivers GFP to the vacuole. During the course of nodulation, there is a nodule-specific redirection of MtENOD8-SP-GFP from the vacuole to punctate intermediates and subsequently to symbiosomes, with redirection of MtENOD8-SP-GFP from the vacuole to punctate intermediates preceding intracellular rhizobial infection. Experiments with M. truncatula mutants having defects in rhizobial infection and symbiosome development demonstrated that the MtNIP/LATD gene is required for redirection of the MtENOD8-SP-GFP from the vacuoles to punctate intermediates in nodules. Our evidence shows that MtENOD8 has evolved redundant targeting sequences for symbiosome targeting and that intracellular localization of ectopically expressed MtENOD8-SP-GFP is useful as a marker for monitoring the extent of development in mutant nodules.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Blotting, Western
  • Cloning, Molecular
  • Green Fluorescent Proteins / chemistry
  • Medicago truncatula / chemistry*
  • Medicago truncatula / genetics
  • Medicago truncatula / microbiology
  • Molecular Sequence Data
  • Nitrogen Fixation
  • Plant Proteins / chemistry*
  • Plant Root Nodulation
  • Plants, Genetically Modified / chemistry
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / microbiology
  • Protein Sorting Signals*
  • Protein Structure, Tertiary
  • Protein Transport
  • RNA, Plant / analysis
  • RNA, Plant / chemistry
  • Recombinant Fusion Proteins / chemistry
  • Root Nodules, Plant / chemistry
  • Root Nodules, Plant / genetics
  • Root Nodules, Plant / microbiology
  • Sequence Alignment
  • Sinorhizobium meliloti / physiology
  • Symbiosis
  • Vacuoles / chemistry*

Substances

  • Plant Proteins
  • Protein Sorting Signals
  • RNA, Plant
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins

Associated data

  • GENBANK/AF064775
  • GENBANK/AF463407