John Innes Centre

The Rhizobium - legume symbiosis

Group leaders

Prof. Allan Downie
Prof. Nick Brewin
Prof. Phil Poole

Nitrogen-fixing nodules on leguminous-plant roots
Nitrogen-fixing nodules on leguminous-plant roots

Legumes such as peas, beans, clover and alfalfa play a very important role in agriculture because they enter into a symbiosis with Rhizobium bacteria which induce nitrogen-fixing nodules on their roots. This symbiosis can relieve the requirements for added nitrogenous fertilizer during the growth of leguminous crops.

Two groups at the John Innes Centre are studying the genes involved in initiating the Rhizobium-legume symbiosis. Professor Allan Downie (in the Department of Molecular Microbiology) and Professor Giles Oldroyd (in the Department of Disease and Stress Biology) share a laboratory and work jointly on the interactions between rhizobia and legumes.

The communications that occur between the plant and the rhizobia during nodule formation and maintenance constitutes a novel opportunity to study signal transduction in a plant system. Knowledge from this interaction can be used in applied objectives as well as yielding a wealth of fundamental knowledge with wide implications. Professor Downie's research group is involved in looking at both bacterial and plant genes involved in the symbiosis.

Rhizosphere molecular microbiology

The expression of "nodulation" genes in the bacteria is activated by signals from plant roots and as a result the bacteria synthesise chemical signals (Nod-factors) that induce a nodule meristem and enable the bacteria to enter this meristem via a plant-made infection thread. However in addition to Nod factors, the bacteria make other chemical signals (acyl homoserine lactones, AHLs) that enable individual bacterial cells to sense how many other bacteria are surround them. In this way they can determine whether there are enough bacteria, i.e. a quorum, to initiate the change towards acting in a multicellular fashion. This is known as 'quorum sensing' (see www.nottingham.ac.uk/quorum/ for further information).

Such gene regulatory systems play an important role in optimising the potential of Rhizobium cells to compete in the rhizosphere and therefore to take the opportunity to infect legume roots to form nodules. Rhizobium genes induced by quorum-sensing regulation affect several characteristics including the transfer of nodulation and nitrogen fixation capacity to other bacteria, legume infection, attachment and biofilm formation on roots, and the ability to survive stresses.

Our current research in this area includes understanding how the network of quorum-sensing regulation is coordinated, understanding root attachment and biofilm formation in relation to various bacterial surface polysaccharides and secreted proteins, and understanding how rhizobia respond to different extracellular stresses regulated by a specialised group of RNA polymerase subunits known as extracytoplasmic sigma factors.

Legume nodulation genes, Nod-factor signalling and calcium

Nod-factors made by Rhizobium bacteria activate a signalling cascade in root cells leading to the activation of gene expression. As a consequence cells in the root start to divide eventually forming a growing meristem that develops into a root nodule. In parallel, the rhizobia initiate infections, inducing the plant to produce specialised infection structures (infection threads) along which the bacteria grow, so that they can reach and infect the dividing plant cells of the nodule meristem.

 We have demonstrated that the Nod factors induce two distinct calcium responses in root hairs, a rapid calcium influx and oscillations in intracellular calcium, particularly around the nuclear region. We are interested in understanding how the plant cells generate these responses and then how the changes in calcium are interpreted leading to induction of gene expression. We have identified seven genes in the legume Lotus japonicus which are required for the calcium oscillations.

We also work on related nodulation signalling genes in pea and in Medicago truncatula. In collaboration with Giles Oldroyd's group have identified a kinase that probably integrates the calcium oscillations and we have identified other downstream genes that are required for induction of expression of early nodulation genes in legumes. We previously analysed the changes in intracellular calcium at the single cell level using a micro-injected calcium-sensing fluorescent dye. We have now produced stably transformed legumes expressing a calcium-sensing (cameleon) protein which changes its fluorescence in response to changing calcium levels and this has given us the ability to analyse calcium changes in various cell types, relatively simply and without microinjection. We are particularly interested in understanding the roles of the two different calcium signals (influx and oscillations) in relation to legume infection and nodule development.