Genetic determinants of host range in Mesorhizobium ciceri WSM1284

Georgina R Robbins

Australian soils lacked native rhizobia capable of nodulating and fixing N2 with the newly domesticated, Biserrula pelecinus, leading to the introduction of Mesorhizobium ciceri WSM1284, WSM1271 and WSM1497, the latter now the current commercial inoculant strain for this host. All three strains were originally isolated from B. pelecinus in the Mediterranean basin yet within the literature these strains exhibit differences in their host ranges. WSM1271 and WSM1497 have narrow host ranges known to nodulate only a few legume species, whereas WSM1284 has a broad host range, nodulating species across multiple legume genera. The genetic cause/s for the differences in host specificity between these three strains is unknown. The host range of these strains was confirmed by assessing the nodulation ability across a diverse subset of legumes in a series of glasshouse studies. All three strains formed nodules on two genotypes of B. pelecinus, Astragalus hamosus, and Glycyrrhiza acanthocarpa. While WSM1497 and WSM1271 failed to nodulate Lotus ornithopodioides, Ornithopus sativus and Leucaena leucocephala. WSM1284 exhibited a broader host range and nodulated these three hosts. Genomically, all three strains are highly similar in structure and architecture and all harbour a tripartite symbiosis Integrative and Conjugative Element (ICE3). Transfer of WSM1284, WSM1271 and WSM1497 ICE3’s to non-symbiotic M. japonicum R7ANS enabled these exconjugants to nodulate the same hosts (B. pelecinus, L. ornithopodioides, O. sativus and L. leucocephala) as the donor strain indicating host range determinants are located on these mobile genetic elements in these strains. Importantly, nodulation (nod) genes, which are located on these ICE3 differ between the three strains indicating the strains may respond to different plant inducers or synthesise different Nod factor molecules. Specifically, WSM1284 encodes 21 nod genes, while WSM1271 has 15 and WSM1497 has 13. All three strains harbour multiple nodD (nodD1 and nodD2) and nodA (nodA1 and nodA2) common nodulation genes which vary in their sequence homology within and between strains. Additionally, WSM1284 harbours an additional copy of nodA (nodA3). Six of the additional WSM1284 genes are accessory nodulation genes that may code for fucose biosynthesis (noeL, nolK, noeJ, noeK), transfer (nodZ) and acetylation (nolL). Whereas, both WSM1271 and WSM1497 harbour nodH, encoding a putative sulfotransferase. These accessory genes indicate that the reducing end of the Nod factor may differ between the strains and therefore were targets for identifying host range determinants. Seventeen WSM1284 site-directed mutants targeting both common and accessory nodulation genes encoding putative Nod factor transcriptional regulators (encoded by nodD) and biosynthesis genes (encoded by nodA, nodZ, nodZnoeLnolK and nolL) were evaluated in planta on B. pelecinus, L. ornithopodioides, O. sativus and L. leucocephala. Three genes (nodD1, nodA1, nodZ) and the putative operon (nodZnoeLnolK) have been identified as host range determinants in WSM1284 that extend the host range of this strain to nodulating O. sativus and/or L. leucocephala. Additionally, two genes, nodA2 and nodD2, which are not host range determinants, are involved in regulating nodule number on L. leucocephala. 9 [OFFICIAL] Deletion of two nod genes, nodA3 and nolL, abolished nodulation on three of the four hosts suggesting these genes were significant contributors to WSM1284 host range. However, further interrogation of the genome and complementation studies revealed deletion of this region impacted expression of downstream nodD1. The role of nodA3 and nolL as host range determinants remains unknown. Of the seventeen WSM1284 nod mutants, only those which removed all functional copies of core genes abolished nodulation on L. ornithopodioides indicating other nod genes or host range determinants are responsible for WSM1284 nodulating this host. These three M. ciceri strains are valuable candidates for studying host range in Mesorhizobium symbiosis due to their differences in host specificity, nod gene complement and putative Nod factor structures. This thesis demonstrates that WSM1284 contains both common and accessory nodulation genes which contribute to its broad host specificity. Ultimately, these data provide a basis for developing genetic markers to predict host range that can be utilised to shorten and save costs within the Australian commercial inoculant selection process.

Genetic determinants of host range in Mesorhizobium ciceri WSM1284

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