Final Activity Report Summary - NODULE REMORIN (Nodulation as a model system to study the role of remorins, plant specific lipid raft proteins, potentially involved in symbiotic signalling)
We studied the role of a plant-specific remorin protein (MtsymREM) that is specifically induced during symbiotic plant-microbe interactions between the model legume Medicago truncatula and its micro-symbiont Sinorhizobium meliloti.
The function of remorin proteins was so far unknown in plants. To elucidate this family in more detail, we performed in depth phylogenetic and structural analysis of all remorin proteins and genes that have been identified in plants so far (Raffaele et al., 2007). We found that the C-terminal region of the proteins is commonly conserved among all family members while their N-terminal part showed high diversity. Thus, we assume that the different isoforms confer different biological functions.
MtsymREM is a group 2 remorin and specifically transcriptionally induced during nodule development with an almost exclusive expression in nodules. This is in sharp contrast to all other remorin genes which are expressed in all plant tissues at similar levels. Thus, the specific expression of MtsymREM opened the opportunity to overcome lethality problems that were reported upon mutating other family members. We used RNA intereference (RNAi) to post-transcriptionally silence expression of MtsymREM. Transgenic plants with reduced levels of MtsymREM failed to develop functional nodules. Most infections were aborted upon entry of infection threads (ITs) into the epidermal or first cortical cell layers. Several plants showed uncontrolled release of symbiotic bacteria from IT into epidermal cells and IT branching and development of sac-like structures. Thus, we successfully demonstrated that MtsymREM is an essential protein for symbiotic interactions between M. truncatula and S. meliloti.
To characterise expression of the gene we generated different cDNAs from infection time-courses using different bacterial mutants. While MtsymREM is induced during nodule development such induction was not observable using bacterial mutants such as nodA and exoA that fail to infect the plant host or are impaired in relase from ITs, respectively. Using bacterial mutants such as bacA and fixJ that lead to the development of non-fixing nodules did not alter MtsymREM induction. These data indicate roles of MtsymREM during bacterial release from ITs.
We localised the protein to the plasma membrane using expression of fluorophore-tagged versions of the MtsymREM protein in heterologous systems such Nicotiana benthamiana. This localisation was confirmed in M. truncatula by using a specific antibody that we raised against two peptides in the N-terminal region of the protein. Using in situ immuno-localisation we could show that the protein localises around nodular ITs and at sites where symbiotic bacteria are released from the ITs into the plants host cells. Such data were obtained on light and electron microscopy level. Using bi-fluorescence complementation (BiFC) and fluorescence lifetime imaging microscopy (FLIM) we demonstrated that MtsymREM proteins oligomerise at the plasma membrane.
In order to get insights into the function we performed several experiments to identify interacting proteins. In summary, we found interactions of MtsymREM with several receptors that were previously shown to be crucial for these symbiotic interactions. From other experiments we currently hypothesise that MtsymREM is involved externalisation of signalling proteins mainly during bacterial release and symbiosome formation and function. Thus, we revealed the function of a protein with so far unknown function and discovered a novel mechanism for spatial regulation of receptor proteins in plants.
The function of remorin proteins was so far unknown in plants. To elucidate this family in more detail, we performed in depth phylogenetic and structural analysis of all remorin proteins and genes that have been identified in plants so far (Raffaele et al., 2007). We found that the C-terminal region of the proteins is commonly conserved among all family members while their N-terminal part showed high diversity. Thus, we assume that the different isoforms confer different biological functions.
MtsymREM is a group 2 remorin and specifically transcriptionally induced during nodule development with an almost exclusive expression in nodules. This is in sharp contrast to all other remorin genes which are expressed in all plant tissues at similar levels. Thus, the specific expression of MtsymREM opened the opportunity to overcome lethality problems that were reported upon mutating other family members. We used RNA intereference (RNAi) to post-transcriptionally silence expression of MtsymREM. Transgenic plants with reduced levels of MtsymREM failed to develop functional nodules. Most infections were aborted upon entry of infection threads (ITs) into the epidermal or first cortical cell layers. Several plants showed uncontrolled release of symbiotic bacteria from IT into epidermal cells and IT branching and development of sac-like structures. Thus, we successfully demonstrated that MtsymREM is an essential protein for symbiotic interactions between M. truncatula and S. meliloti.
To characterise expression of the gene we generated different cDNAs from infection time-courses using different bacterial mutants. While MtsymREM is induced during nodule development such induction was not observable using bacterial mutants such as nodA and exoA that fail to infect the plant host or are impaired in relase from ITs, respectively. Using bacterial mutants such as bacA and fixJ that lead to the development of non-fixing nodules did not alter MtsymREM induction. These data indicate roles of MtsymREM during bacterial release from ITs.
We localised the protein to the plasma membrane using expression of fluorophore-tagged versions of the MtsymREM protein in heterologous systems such Nicotiana benthamiana. This localisation was confirmed in M. truncatula by using a specific antibody that we raised against two peptides in the N-terminal region of the protein. Using in situ immuno-localisation we could show that the protein localises around nodular ITs and at sites where symbiotic bacteria are released from the ITs into the plants host cells. Such data were obtained on light and electron microscopy level. Using bi-fluorescence complementation (BiFC) and fluorescence lifetime imaging microscopy (FLIM) we demonstrated that MtsymREM proteins oligomerise at the plasma membrane.
In order to get insights into the function we performed several experiments to identify interacting proteins. In summary, we found interactions of MtsymREM with several receptors that were previously shown to be crucial for these symbiotic interactions. From other experiments we currently hypothesise that MtsymREM is involved externalisation of signalling proteins mainly during bacterial release and symbiosome formation and function. Thus, we revealed the function of a protein with so far unknown function and discovered a novel mechanism for spatial regulation of receptor proteins in plants.