Bacterial invasion of plant cells - Molecular Biology

During the Rhizobium-legume symbiosis Rhizobium bacteria are taken up into the cytoplasm of nodule cells, where they are hosted in new organelle-like membrane compartments, called symbiosomes. The intracellular accommodation of bacteria in plant cells is unique to the nodule symbiosis and somewhat resembles the invasion of human and animal cells by pathogenic bacteria, such as Salmonella, Legionella or Brucella, which also survive and replicate in new membrane compartments.

The formation of symbiosomes is a crucial step in the symbiosis because it prevents a strong defence response against the bacteria and facilitates the efficient exchange of metabolites between the two partners to allow the bacteria to fix atmospheric nitrogen. However, hardly anything is known about the molecular basis for symbiosome formation.

To study symbiosome formation we use the model legume Medicago. Medicago nodules contain a meristem at their apex that continuously adds new cells to the tissues of the nodule (Figure 1). The cells just below the meristem are invaded by cell wall bound tubular infection threads, through which the bacteria are transported.  There, so-called unwalled infection droplets extrude from the infection threads and the bacteria are taken up into the cells by an endocytosis-like process (Figure 2). After their "endocytosis" the bacteria divide together with the symbiosome membrane, resulting in thousands of symbiosomes that fill the cell as individual units. These then differentiate into their nitrogen fixing form and fix atmospheric nitrogen for several days. Eventually, as the nodule ages, the symbiosomes are not longer maintained as individual units but start to fuse to form lytic compartments and finally the complete infected cell dies.

Figure 1.  Medicago nodule ontology. A) Longitudinal (5 mm) section through a Medicago nodule. M = meristem, ZII =  infection zone, where symbiosome formation starts, ZIII = fixation zone, in this zone nitrogen fixation takes place, S = senescencing cell. B) Close-up of the infection zone from a. Here the bacteria are released from the infection thread (it) and symbiosomes are formed. The symbiosomes then start to divide and differentiate (elongate) to fill the host cells. C) Close-up of cells in the fixation zone containing mature nitrogen fixing symbiosomes (sb). Some cell remain uninfected (uc).
Figure 1. Medicago nodule ontology. A) Longitudinal (5 mm) section through a Medicago nodule. M = meristem, ZII = infection zone, where symbiosome formation starts, ZIII = fixation zone, in this zone nitrogen fixation takes place, S = senescencing cell. B) Close-up of the infection zone from a. Here the bacteria are released from the infection thread (it) and symbiosomes are formed. The symbiosomes then start to divide and differentiate (elongate) to fill the host cells. C) Close-up of cells in the fixation zone containing mature nitrogen fixing symbiosomes (sb). Some cell remain uninfected (uc).
Figure 2. Electron microscopy picture of an unwalled infection droplet. From here bacteria are “endocytosed” into the cytoplasm and symbiosomes are formed. b = rhizobium bacteria, er = endoplasmic reticulum, m = mitochondria, itm = infection thread matrix.
Figure 2. Electron microscopy picture of an unwalled infection droplet. From here bacteria are “endocytosed” into the cytoplasm and symbiosomes are formed. b = rhizobium bacteria, er = endoplasmic reticulum, m = mitochondria, itm = infection thread matrix.

To understand the molecular basis of symbiosome formation we currently focus on two main questions: 1) How are the bacteria taken up into the cells; and 2) How are the symbiosomes maintained and prevent their fusion with lytic compartments/vacuoles.

Symbiosome formation and membrane trafficking

The formation of symbiosomes requires a massive reorganization of the host endomembrane system as they almost completely fill the host cells. It has been calculated that symbiosomes require >40x more membrane than the plasma membrane in infected cells. The symbiosome membrane originates from the plasma membrane, but needs to acquire a special identity to distinguish it from other endomembrane compartments in the cell. Specific vesicles need to be targeted to the symbiosomes to provide the necessary membrane and associated proteins. Therefore, we especially focus on key regulators of membrane trafficking and fusion, such as small GTPases of the ROP, ARF and RAB families and SNARE proteins. The latter two protein families are also key membrane identity markers that are specifically associated with different endomembrane compartments and control fusion specificity. By studying these proteins the relation of symbiosome formation with the endocytotic pathways and vacuole biogenesis pathways is examined (Figure 3).

Figure 3. Localization of syntaxin132 (SNARE protein) to the symbiosome membrane in infected nodule cells using confocal laser scanning microscopy. A fusion of GFP (green fluorescent protein) to syntaxin132 is used to visualize the protein (green). The rhizobium bacteria express a red fluorescent protein (mRFP) and are seen in red.
Figure 3. Localization of syntaxin132 (SNARE protein) to the symbiosome membrane in infected nodule cells using confocal laser scanning microscopy. A fusion of GFP (green fluorescent protein) to syntaxin132 is used to visualize the protein (green). The rhizobium bacteria express a red fluorescent protein (mRFP) and are seen in red.

Specialized membrane domains (lipid rafts) during infection

Recently a new research line was set up that focuses on the involvement of specialized lipid membrane domains (lipid rafts) in the rhizobial infection process; both infection thread formation and symbiosome formation. Such lipid domains play important roles in the penetration resistance against pathogenic fungi in plants and in animal cells lipid rafts form portals for the invasion by pathogenic bacteria. It will be studied whether lipid rafts play a role in the rhizobial infection process. The possible association of the Nod factor signaling receptors with such domains is studied as well as how such domains are formed. Further, it is studied whether the bacteria enter the nodule cells through lipid rafts.

Practical work

  • Construction of fluorescent fusion protein constructs to localize proteins of interest in vivo using confocal microscopy.
  • Functional analyses using RNA interference, over-expression constructs, construction of mutant proteins (dominant-negative / -positive forms).
  • Expression analyses (promoter-GUS reporter constructs, Real-time RT-PCR (qPCR), in situ hybridization).
  • Light, Confocal Laser Scanning and Electron microscopy.
  • Immunolocalization of proteins.
  • Yeast-two-hybrid (split-ubiquitin) screens to identify interacting partners of proteins of interest and verification using GST-pull down assays or FRET analyses.
  • Agrobacterium rhizogenes mediated plant root transformation.
  • Genetic mapping and cloning of identified plant mutants affected in symbiosome formation.