A Nodule-Specific Lipid Transfer Protein AsE246 Participates in Transport of Plant-Synthesized Lipids to Symbiosome Membrane and Is Essential for Nodule Organogenesis in Chinese Milk Vetch

Lei Lei,Dasong Chen,Xiaofeng Shi,Jianyun Wang,Youguo Li,Fuli Xie,Yixing Li,Ling Chen

doi:10.1104/pp.113.232637

Abstract

Rhizobia in legume root nodules fix nitrogen in symbiosomes, organelle-like structures in which a membrane from the host plant surrounds the symbiotic bacteria. However, the components that transport plant-synthesized lipids to the symbiosome membrane remain unknown. This study identified and functionally characterized the Chinese milk vetch (Astragalus sinicus) lipid transfer protein AsE246, which is specifically expressed in nodules. It was found that AsE246 can bind lipids in vitro. More importantly, AsE246 can bind the plant-synthesized membrane lipid digalactosyldiacylglycerol in vivo. Immunofluorescence and immunoelectron microscopy showed that AsE246 and digalactosyldiacylglycerol localize in the symbiosome membrane and are present in infection threads. Overexpression of AsE246 resulted in increased nodule numbers; knockdown of AsE246 resulted in reduced nodule numbers, decreased lipids contents in nodules, diminished nitrogen fixation activity, and abnormal development of symbiosomes. AsE246 knockdown also resulted in fewer infection threads, nodule primordia, and nodules, while AsE246 overexpression resulted in more infection threads and nodule primordia, suggesting that AsE246 affects nodule organogenesis associated with infection thread formation. Taken together, these results indicate that AsE246 contributes to lipids transport to the symbiosome membrane, and this transport is required for effective legume-rhizobium symbiosis.

Highlights

Rhizobia in legume root nodules fix nitrogen in symbiosomes, organelle-like structures in which a membrane from the host plant surrounds the symbiotic bacteria
To investigate whether Lipid transfer proteins (LTPs) function in symbiosis membrane deposition and nodule organogenesis in Chinese milk vetch, we previously identified two candidate LTP genes, AsE246 and AsIB259, via suppressive subtractive hybridization
According to a classification scheme based on primary structure (Boutrot et al, 2008), comparison of the deduced sequence of mature AsE246 to LTPs from Arabidopsis, M. truncatula, Lotus japonicus, and Glycine max showed that AsE246 belongs to the Type I LTPs

Summary

Introduction

Rhizobia in legume root nodules fix nitrogen in symbiosomes, organelle-like structures in which a membrane from the host plant surrounds the symbiotic bacteria. AsE246 can bind the plant-synthesized membrane lipid digalactosyldiacylglycerol in vivo. AsE246 knockdown resulted in fewer infection threads, nodule primordia, and nodules, while AsE246 overexpression resulted in more infection threads and nodule primordia, suggesting that AsE246 affects nodule organogenesis associated with infection thread formation Taken together, these results indicate that AsE246 contributes to lipids transport to the symbiosome membrane, and this transport is required for effective legume-rhizobium symbiosis. Legume crops can act as hosts for nitrogen-fixing soil Rhizobium spp. bacteria, which induce and occupy a specialized organ, the root nodule (Limpens et al, 2009). The symbiosome membrane contains saturated (16:0, palmitic acid; 18:0, stearic acid) and unsaturated fatty acids (16:1D3trans, palmitoleic acid; 18:1D9cis, oleic acid 18:2D9,12, linoleic acid; 18:3D9,12,15, a-linolenic acid), all of which are typically found in higher plants (Whitehead and Day, 1997; Gaude et al, 2004)

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Conclusion