A Secreted Effector Protein of Laccaria bicolor is Required for Symbiosis Development
Jonathan M. Plett, Minna Kemppainen, Shiv D. Kale, Annegret Kohler, Valérie Legué, Annick Brun, Brett M. Tyler, Alejandro G. Pardo, and Francis Martin
2011 January 03, Current Biology 21 (14): 1197-203; doi:10.1016/j.cub.2011.05.033
Soil-borne mutualistic fungi, such as the ectomycorrhizal fungi, have helped shape forest communities worldwide over the last 180 million years through a mutualistic relationship with tree roots in which the fungal partner provides a large array of nutrients to the plant host in return for photosynthetically derived sugars. This exchange is essential for continued growth and productivity of forest trees, especially in nutrient-poor soils. To date, the signals from the two partners that mediate this symbiosis have remained uncharacterized. Here we demonstrate that MYCORRHIZAL iNDUCED SMALL SECRETED PROTEIN 7 (MiSSP7), the most highly symbiosis-upregulated gene from the ectomycorrhizal fungus Laccaria bicolor, encodes an effector protein indispensible for the establishment of mutualism. MiSSP7 is secreted by the fungus upon receipt of diffusible signals from plant roots, imported into the plant cell via phosphatidylinositol 3-phosphate-mediated endocytosis, and targeted to the plant nucleus where it alters the transcriptome of the plant cell. L. bicolor transformants with reduced expression of MiSSP7 do not enter into symbiosis with poplar roots. MiSSP7 resembles effectors of pathogenic fungi, nematodes, and bacteria that are similarly targeted to the plant nucleus to promote colonization of the plant tissues and thus can be considered a mutualism effector.
The first demonstration that mutualistic fungi use effector-like proteins to control the host cell in a manor that mirrors the role of fungal pathogen effectors is shown. This results suggests that the establishment of symbiosis is closer to the colonization of plant tissues by pathogens than previously thought. MYCORRHIZAL iNDUCED SMALL SECRETED PROTEIN7 (MiSSP7), from the mutualistic ectomycorrhizal fungus L. bicolor, encodes a 68 amino acid-long protein secreted by the fungus upon receipt of diffusible signals from the host plant. Its size and induction conditions suggested that it may act similar to effector-like proteins encoded by pathogens. MiSSP7 was found to be one of the factors necessary for the formation and maintenance of the Hartig net, the network of fungal hyphae within the root apoplastic space across whose interface nutrients are exchanged between the host and the fungus. MiSSP7 actively enters the plant cell via endocytosis through the binding of an RXLR-like domain (RALG) to phosphatidylinositol 3-phosphate. MiSSP7 quickly accumulates in the plant nucleus (Fig.1) where transcriptional alteration of the host cell occurs ï¿½ confirming that MiSSP7 is an effector-like protein. Transcriptomic analysis of the plant cell revealed that 225 genes were significantly regulated by MiSSP7. A large proportion of the modulated genes are involved in the alteration of cell wall and root architecture:
MiSSP7 Expression and Accumulation during Mycorrhizal Development(A) Protein sequence of MiSSP7, with the predicted secretory leader in bold. Sequences used in truncation experiments are labeled A, B, and C above the corresponding amino acids. The identified motif required for plant cell entry (RALG) is indicated in red. For further information on constructs used in mutational and truncation experiments, please see Figure S3.(B) Quantification of MiSSP7 transcripts during mycorrhization (solid line) versus percent mycorrhization (dashed line; n = 6).(C) Indirect immunolocalization of MiSSP7 (green signal) in colonized root. The following abbreviations are used: hn, Hartig net; n, nucleus; e, root epidermal cell layer; fm, fungal mantle. Plant cell walls were stained with propidium iodide (red signal). Scale bar represents 20 Î¼m.(D) Cell entry (black bars) and nuclear localization (white bars) of MiSSP7 (KR), a truncated version of MiSSP7 (AC), and MiSSP7 with a nuclear exclusion signal attached (NES) (defined in Figure S3).(E) Incorporation of MiSSP7 into the plant cell nucleus (solid line) and the rate of loss of MiSSP7 from the plant nucleus upon removal of MiSSP7 from the supernatant (dashed line).All values are shown as mean ï¿½ standard error; n = 3 unless otherwise noted.
Jonathan M. Plett, Minna Kemppainen, Shiv D. Kale, Annegret Kohler, Valï¿½rie Leguï¿½, Annick Brun, Brett M. Tyler, Alejandro G. Pardo, Francis Martin, A Secreted Effector Protein of Laccaria bicolor Is Required for Symbiosis Development, Current Biology, Volume 21, Issue 14, 26 July 2011, Pages 1197-1203, ISSN 0960-9822, 10.1016/j.cub.2011.05.033. (http://www.sciencedirect.com/science/article/pii/S0960982211005896)