Requirements of Qa-SNARE LjSYP132s for nodulation and seed development in Lotus japonicus.

SNARE (soluble N-ethyl maleimide sensitive factor attachment protein receptor) mediates membrane fusion of vesicle transport in eukaryotic cells. LjSYP132s is a member of Qa-SNAREs in Lotus japonicus, and two isoforms, LjSYP132a and LjSYP132b, are generated by alternative splicing. Immunoblot analysis showed LjSYP132s antibody was expressed strongly in infected root nodules and seeds by post-transcriptional modification. In LjSYP132a or LjSYP132b silencing roots (RNAi-LjSYP132a, RNAi-LjSYP132b), infection thread (IT) was not elongated, suggesting that both LjSYP132a and LjSYP132b have a role in IT progression. The results were consistent with the data of qRT-PCR that both genes expressed at the early stage of infection. However, during the nodulation only LjSYP132a was induced. LjSYP132s protein was observed in the M. loti-inoculated roots of mutants, nfr1, castor, and pollux, suggesting that LjSYP132s can be induced without Nod factor signaling. Accumulation of LjSYP132s in the PBM suggests the function of not only IT formation but also nutrient transport. In contrast, qRT-PCR showed that LjSYP132b was expressed in the seeds. A stable transgenic plant of LjSYP132b, R132b, was produced by RNAi silencing. In the R132b plants, small pods with a few seeds and abnormal tip growth of the pollen tubes were observed, suggesting LjSYP132b has a role in pollen tube growth as well as nutrient transport in the plasma membrane of seeds.