Evolution of symbiosis genes in rhizobia: chickpea mesorhizobia as case study

The present concerns for the depletion of soil nutrients and the incentive to more sustainable agricultural practices are revitalizing the importance of biological nitrogen fixation, increasing the need for better understanding all aspects of rhizobia-legumes symbioses, particularly those involving important legume crops in terms of food and forage. Chickpea is one of these legumes, and the third pulse crop worldwide with an overall production of 9.8 millions of tonnes in 2009 (FAO data).

Our previous projects allowed the investigation of the diversity and the characterization of rhizobia able to nodulate chickpea in Portuguese soils [1, 2]. A national survey showed that all chickpea rhizobia isolates belong to the genus Mesorhizobium, but the diversity of species able to form effective nodules on this legume is high [PP2, PP5]. In fact, most isolated rhizobia belong to Mesorhizobium species other than M. ciceri and M. mediterraneum, which had been considered the typical chickpea microsymbionts.

Rhizobial host range, i.e., the set of legumes that a given rhizobia strain is able to nodulate is in general related to the nature of the rhizobial Nod factors. These molecules are synthesised by proteins encoded by the rhizobial nodulation genes and are known to be very important in the early plant-bacterium interaction [3]. Several studies indicate that common nod genes have been horizontally transferred between rhizobia species. This may explain why distantly related rhizobia carry similar nod genes.

Chickpea has been considered a restrictive legume host for nodulation, mainly because it is not nodulated by broad host range rhizobia strains [4]. Previous studies on Portuguese chickpea rhizobia showed that symbiosis genes (nodC and nifH) are highly conserved across different Mesorhizobium species [PP4]. This further supports chickpea as a very restrictive host. So how did these features evolved? A high diversity of rhizobial species able to nodulate chickpea plants, but at the same time harbouring highly conserved symbiosis genes seems contradictory. Furthermore, up to date no chickpea rhizobium is known to nodulate alternative legumes [5]. In addition, M. ciceri isolates from Biserrula pelecinus are not able to nodulate chickpea [6].

How rhizobial populations evolve and diversify in soils and what are the driving forces of that evolution is largely unknown. Some particular cases of legume introduction in countries where there was a total absence of rhizobia able to nodulate that legume, generated exceptional conditions for the study of rhizobia evolution. Probably the most remarkable cases are the introduction of soybean in Brasil [7] and of biserrula in Australia. This last example is particularly relevant for the present study, since biserrula is nodulated by M. ciceri, a species that typically nodulates chickpea. Five years were enough for the detection of rhizobia able to nodulate biserrula, different from the original inoculant strain, in Australian soils [8]. This means that naturally occurring rhizobia were able to evolve and acquire, by lateral gene transfer, the set of genes that allowed the infection of the newly introduced legume. This rather fast process is interesting and becomes even more intriguing when it was seen that these new isolates have acquired the symbiosis island from the original inoculant, yet showed very low performance in terms of nitrogen fixation on B. pelecinus [8].

Our main goal is to study the symbiosis genes in order to better understand the evolution of the legume-rhizobium symbiosis taking chickpea, a restrictive host for nodulation, as case study. This will be achieved first by evaluating the host range of our lab collection of chickpea rhizobia native isolates, and afterwards through a preliminary characterisation of symbiosis genes, followed by the complete sequencing of symbiosis islands or plasmids. Symbiosis genes encoded in a symbiosis islands seems to be a Mesorhizobium characteristic, so it is expected that chickpea mesorhizobia also harbour such a chromosomal region. The comparative sequence analysis of symbiosis genes (e.g. phylogeny and synteny studies) together with the functional analysis of those genes (knockout mutation, overexpression and transcriptional analysis) will provide more information on the molecular mechanisms underlying the chickpea-rhizobium specificity. Finally, we propose to genetically transform a chickpea non-nodulating strain into a chickpea microsymbiont.

The present project will be the first extensive study on chickpea rhizobia symbiosis genes, intending to clarify their evolution and to understand how a strict host is nodulated by a high number of Mesorhizobium species. Altogether our results may contribute to a better understanding of the evolution of symbiosis genes in rhizobia.