A genetic approach to study rhizobial Nod factor and mycorrhizal fungi activated signaling

Leguminous plants are able to interact with bacteria of the genera Azorhizobium , Bradyrhizobium , Rhizobium and Sinorhizobium . This is a symbiotic interaction that results in the formation of a complete new organ, the root nodule. In these nodules the bacteria are hosted intracellularly and there they find the proper environment to reduce atmospheric nitrogen into ammonia, a source of nitrogen that can be used by the plant.

Root nodule formation involves growth responses in the epidermis as well as cortex of the root. This implies that the bacteria redirect the development of fully differentiated plant cells. The bacterial signals that set this in motion are the so-called nodulation (Nod) factors. Nod factors of the different Rhizobium species have a common basic structure; aβ-1,4-linked N-acyl-D-glucosamine backbone of mostly 4 or 5 units, containing a fatty acid at the non-reducing terminal sugar. Furthermore, several species-specific decorations can be present at both terminal glucosamine residues and also the structure of the fatty acyl chain can vary. These substitutions play an important role in the host-specificity of the symbiosis.

Nod factors are active at low concentrations and their activity depends on their structure. This implicates that Nod factors are perceived by receptor(s). However, it is unclear how Nod factors are perceived, and how the signals are transduced. The aim of this thesis is to unravel Nod factor perception and transduction mechanisms by using a genetic approach. Furthermore, it is studied whether mycorrhizal fungi and rhizobia use similar mechanisms to establish an endosymbiotic relationship. This can provide insight in the phylogenetic origin of the legume mechanism controlling nodulation.

Leguminous plants mutated in a gene encoding a key component of the Nod factor perception or transduction pathway will not respond to Nod factors. In Chapter 2 , we describe a pea mutant, Sparkle-R25, which shows such phenotype. Sparkle-R25 is mutated in the SYM8 gene. We demonstrate that rhizobial Nod factors are unable to trigger the early nodulin genes PsENOD5 and PsENOD12A , whereas in wild type pea they do. This shows that SYM8 is required for the induction of both genes by Nod factors. Besides that Sparkle-R25 does not respond to Nod factors, it is also unable to establish a mycorrhizal symbiosis, showing that SYM8 is also involved in this endosymbiotic interaction. We demonstrate that mycorrhizal fungi are as well able to trigger the expression of both early nodulin genes in wild type peas, but are unable to do so in Sparkle-R25. This indicates that mycorrhizal signals activate a signal transduction cascade sharing SYM8 in common with a Nod factor induced signal transduction cascade.

The studies in chapter 3, 4, 5 and 6 are focused on the SYM2 gene. SYM2 is first identified in the pea ecotype Afghanistan ( SYM2 ^A), where it inhibits nodulation by Rhizobium strains secreting only Nod factors without a specific substitution at the reducing terminal sugar residue. In Chapter 4 we show that these specific substitutions can either be an acetyl or a fucosyl group. SYM2 is specifically involved in the infection process ( Chapter 3 ). In order to study the mode of action of SYM2 a suppressor mutant has been isolated by mutagenizing a SYM2 ^Aharboring pea line ( Chapter 5 ). Bacteria that do not produce properly substituted Nod factors are blocked in their ability to trigger infection thread formation on SYM2 ^Aharboring peas, whereas other Nod factor controlled responses occur normally.

Genetic analysis showed that SYM2 plays a role in controlling infection thread growth and its activity depends on Nod factor structure. This suggests that SYM2 is involved in a Nod factor recognition mechanism. In Chapter 6 the first steps towards the cloning of the SYM2 gene are described. By using differential RNA display several root hair cDNAs are identified which a genetically linked to the SYM2 locus. One of these clones, encoding a putative receptor kinase, shows tight linkage to SYM2 . This clone can serve as marker for further research to clone SYM2 .

In the Discussion of this theses ( Chapter 7 ) the common aspects between the rhizobial and the mycorrhizal symbiosis are described and it is discussed whether mycorrhizal fungi and rhizobia use similar mechanisms to establish an endosymbiotic relationship.