MtGA2ox10 encoding C20-GA2-oxidase regulates rhizobial infection and nodule development in Medicago truncatula

Goon-Bo Kim,Jeong-Hwan Mun,Hee-Ju Yu,Seong-Uk Son

doi:10.1038/s41598-019-42407-3

Goon-Bo Kim, Jeong-Hwan Mun + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-42407-3

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Abstract

Gibberellin (GA) plays a controversial role in the legume-rhizobium symbiosis. Recent studies have shown that the GA level in legumes must be precisely controlled for successful rhizobial infection and nodule organogenesis. However, regulation of the GA level via catabolism in legume roots has not been reported to date. Here, we investigate a novel GA inactivating C20-GA2-oxidase gene MtGA2ox10 in Medicago truncatula. RNA sequencing analysis and quantitative polymerase chain reaction revealed that MtGA2ox10 was induced as early as 6 h post-inoculation (hpi) of rhizobia and reached peak transcript abundance at 12 hpi. Promoter::β-glucuronidase fusion showed that the promoter activity was localized in the root infection/differentiation zone during the early stage of rhizobial infection and in the vascular bundle of the mature nodule. The CRISPR/Cas9-mediated deletion mutation of MtGA2ox10 suppressed infection thread formation, which resulted in reduced development and retarded growth of nodules on the Agrobacterium rhizogenes-transformed roots. Over-expression of MtGA2ox10 in the stable transgenic plants caused dwarfism, which was rescued by GA3 application, and increased infection thread formation but inhibition of nodule development. We conclude that MtGA2ox10 plays an important role in the rhizobial infection and the development of root nodules through fine catabolic tuning of GA in M. truncatula.

Highlights

Nodulation is the mutual interaction between legume plants and rhizobial bacteria that forms a symbiotic nitrogen-fixing nodule
GA12, the first GA compound produced by the GA biosynthesis pathway, is imported into the cytosol; it is further oxidized by GA oxidases and converted to the bioactive form of GAs10
The MtGA2ox genes were identified based on a BLASTP search of all M. truncatula reference gene models against the A. thaliana GA2ox gene family, including seven GA2ox genes and two GA2-oxidase like (GAOL) genes, defined in the METACyc database[28]

Summary

Introduction

Nodulation is the mutual interaction between legume plants and rhizobial bacteria that forms a symbiotic nitrogen-fixing nodule. GA-deficient mutant plants recovered normal nodule organogenesis via knockout of DELLA5 Based on these results, a dual role of GA in two distinct stages of nodule organogenesis was proposed; the suppression of infection thread formation and promotion of nodule development[6]. The cellular level of bioactive GA can be regulated in several ways, including transport of precursors or active forms of GA into the cells, inactivation of bioactive GA, or transcriptional regulation of genes involved in the biosynthesis and catabolic pathways (reviewed by Olszewski et al.[10]). GA12, the first GA compound produced by the GA biosynthesis pathway, is imported into the cytosol; it is further oxidized by GA oxidases and converted to the bioactive form of GAs10. GA precursor biosynthesis genes were highly expressed upon rhizobium inoculation of the root hair cells of M. truncatula[21]

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