LjCOCH interplays with LjAPP1 to maintain the nodule development in Lotus japonicus
Legume plants develop nodules during their symbiotic interaction with rhizobia, and much progress has been made towards understanding Nod factor perception and downstream signaling pathways, while our knowledge about the maintenance of nodule organogenesis was limited. We report here the knockdown mutants of LjCOCH, an ortholog of COCHLEATA in Pisum sativum, cause severe defects in nodule organogenesis in Lotus japonicus. The mature nodule number was drastically decreased accompanied with abnormal lenticel and vascular bundle developmental defects, but not produce roots from nodules in both Ljcoch mutants and LjCOCH-RNAi transgenic hairy roots. LjAPP1, a membrane-associated soluble aminopeptidase P1, was identified to interact with LjCOCH through yeast two-hybrid screening. Unlike that of Ljcoch mutants, insertion mutants of LjAPP1 and LjAPP1-RNAi transgenic hairy roots showed increased nodule number, while the lenticel and vascular development were not affected. Gene expression analysis indicated that LjCOCH and LjAPP1 were differentially upregulated by rhizobia inoculation, and LjNF-YA1 was the major downstream target of LjCOCH and LjAPP1. Our findings suggested that LjCOCH acts as a key factor involved in determinate nodule development through direct interaction with LjAPP1 to regulate the expression of LjNF-YA1, opposite effects of LjCOCH and LjAPP1 provide a dynamic regulation of nodule development in L. japonicus.
KeywordsNodule Organogenesis LjCOCH LjAPP1 Lenticel LjNF-YA1 Lotus japonicus
This research was funded by the National Key R & D Program of China (2016YFA0500502), National 973 Project (2010CB126501) and the Ministry of Agriculture of the People’s Republic of China for Transgenic Research (Grant No. 2014ZX0800943B). We thank Prof. Nan Yao in Sun Yat-sen University for providing ER-RB plasmids and Prof. Ertao Wang in Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences for his kind help during our LORE1 mutants’ phenotyping. We also thank Prof. Yanzhang Wang in Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences for providing M. loti strain MAFF303099 DsRed and M. loti strain NZP2235.
Y.C. Liu, D. Luo and J. Yang designed the experiments. Y.C. Liu performed the hairy roots, yeast two-hybrids, in situ hybridization in nodules and Co-IP experiments. Y.W. Lei performed Ljcoch and Ljapp1 LORE1 mutants’ analysis and qPCR experiments. W. Liu performed the experiment on subcellular localization of the proteins. L. Weng and M.J. Lei cloned LjCOCH gene and did the in situ hybridization of COCH and LjCOCH in vegetative and reproductive SAM. X.H. Hu prepared the seeds of Lotus japonicus. Y.C. Liu, Y.W. Lei, Z.C. Dong, D. Luo and J. Yang wrote the paper.
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