Current Biology Vol 21 No 14 R550

Type 1 inflammation

2.

Monocyte

Increased recruitment Inflamed tissue Macrophage M1 M1

3.

M1

4.

Macrophage accumulation

5.

6.

Type 2 inflammation
7.

Monocyte

8.

9.

M2 Local proliferation

M2

M2

10.

Macrophage accumulation Macrophage accumulation

Current Biology

11.

Figure 2. Local macrophage accumulation during inammation. For type 1 inammation, macrophage accumulation involves the enhanced recruitment of monocytes from the bloodstream, whereas increased macrophage numbers in type 2 inammation are a result of enhanced local proliferation [1].

Macdonald, A.S., and Allen, J.E. (2011). Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inammation. Science 332, 12841288. van Furth, R., Cohn, Z.A., Hirsch, J.G., Humphrey, J.H., Spector, W.G., and Langevoort, H.L. (1972). The mononuclear phagocyte system: a new classication of macrophages, monocytes, and their precursor cells. Bull. World Health Organ. 46, 845852. van Furth, R., and Cohn, Z.A. (1968). The origin and kinetics of mononuclear phagocytes. J. Exp. Med. 128, 415435. Gordon, S., and Taylor, P.R. (2005). Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 5, 953964. Geissmann, F., Jung, S., and Littman, D.R. (2003). Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, 7182. Soehnlein, O., and Lindbom, L. (2010). Phagocyte partnership during the onset and resolution of inammation. Nat. Rev. Immunol. 10, 427439. Voehringer, D., Shinkai, K., and Locksley, R.M. (2004). Type 2 immunity reects orchestrated recruitment of cells committed to IL-4 production. Immunity 20, 267277. Martinez, F.O., Helming, L., and Gordon, S. (2009). Alternative activation of macrophages: an immunologic functional perspective. Annu. Rev. Immunol. 27, 451483. Gordon, S., and Martinez, F.O. (2010). Alternative activation of macrophages: mechanism and functions. Immunity 32, 593604. Hoffmann, W.H., Petit, G., Schulz-Key, H., Taylor, D.W., Bain, O., and Le Goff, L. (2000). Litomosoides sigmodontis in mice: reappraisal of an old model for larial research. Parasitol. Today 16, 387389. Milner, J.D., Orekov, T., Ward, J.M., Cheng, L., Torres-Velez, F., Junttila, I., Sun, G., Buller, M., Morris, S.C., Finkelman, F.D., et al. (2010). Sustained IL-4 exposure leads to a novel pathway for hemophagocytosis, inammation, and tissue macrophage accumulation. Blood 116, 24762483.

Importantly, this notion is substantiated by a previous study that reported increased macrophage accumulation in the spleen and liver after continuous application of IL-4 using an osmotic mini-pump in mice [11]. These surprising ndings point towards a completely novel mechanism of local inammation in the context of parasite infections and wound healing responses in which the recruitment of potentially tissue-destructive neutrophils and inammatory macrophages is avoided. Instead, under conditions in which the immune system does not attempt to kill pathogens but instead tries to wall off large parasites and promote healing processes, the cells required for these processes are generated from the local pool of tissue macrophages (Figure 2). In conclusion, depending on the situation, a cost-effective solution based on the use and expansion of local, existing resources can be more useful than recruiting new, highly specialized

Inflamed tissue

Blood

Blood

players that potentially cause damage by unnecessarily aggressive and overshooting actions.
References
1. Jenkins, S.J., Ruckerl, D., Cook, P.C., Jones, L.H., Finkelman, F.D., van Rooijen, N.,

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universitat Munchen, Munich, Germany. E-mail: laura.helming@mikrobio.med.tum.de

DOI: 10.1016/j.cub.2011.06.005

Mycorrhizal Symbioses: How to Be Seen as a Good Fungus

Plants continually encounter many microorganisms. Some are good, but many are bad. Two studies show how benecial fungi tell the plant to let them in and how the fungus avoids setting off the plants defense reaction. Ian R. Sanders Plants form a variety of different associations with microorganisms. They are continually challenged by fungal pathogens but also form mutualistic associations with benecial microorganisms. Of these benecial interactions the mycorrhizal symbiosis, an association between plant roots and soil fungi, is the most abundant and is of major importance for plant ecology,

Dispatch R551

plant growth, agriculture and forestry [1]. There are several types of mycorrhizal symbiosis and at least 80% of plant species worldwide form such associations. By far the commonest is the arbuscular mycorrhizal symbiosis (AM) that occurs between the majority of plants and arbuscular mycorrhizal fungi (AMF). Most plants form the AM symbiosis; it is found in most terrestrial ecosystems and occurs with all globally important food plants. Ectomycorrhizal symbioses (EM) are formed by other groups of fungi and occur on most trees in temperate and boreal forests. Both of these symbioses are of global importance because they help plants to access essential nutrients such as inorganic nitrogen and phosphate, improve plant growth, promote plant diversity and play an important role in global nutrient cycling. The problem for plants is that their roots continually encounter a vast array of microorganisms, especially fungi. Some of these fungi, like the AM and EM fungi, may be benecial, but many of them are potentially harmful. So the problem for both plants and mycorrhizal fungi is how to tell each other to establish a symbiosis and how to do that without setting off some of the plants complex defense reactions. While considerable progress has been made over the last decade on understanding which plant genes are involved in the establishment of the mycorrhizal symbiosis, much less progress has been made on the fungal side [2,3]. It is, therefore, exciting that in this issue of Current Biology two independent studies by Plett et al. [4] and Kloppholz et al. [5] on the EM and AM symbioses shed light on mutualistic fungal effector proteins, showing that they allow symbiosis formation and allow the fungus to manipulate the plants defense response, respectively. Fungal pathogens of plants are known to release effectors, often small proteins that are usually encoded by avirulence genes [6]. Effector proteins play a part in pathogen attack and act either in the plant extra cellular space or after entering host plant cells [7]. It was long suspected that such proteins would also likely occur in mutualistic fungi such as AM and EM symbionts, although owing to difculties of studying organisms that live together in symbiosis such proteins remained elusive. In 2008, genome-wide transcriptome studies

on the symbiosis between the ectomycorrhizal fungus Laccaria bicolor and poplar revealed a large number of small proteins that were suspected to have a role in signalling. One of the genes that was highly up-regulated in L. bicolor in the symbiotic state encodes a protein of 68 amino acids that accumulates in mycorrhizal root tips [8]. The protein, known as Mycorrhizal induced Small Secreted Protein7 (MiSSP7), was shown to occur only when the fungus made the symbiosis and not when the fungus was in a free-living state. Excitingly, Plett et al. [4] found that if the fungus was grown near a root but not allowed contact with the root, MiSSP7 was produced by the fungus. Interestingly, this also occurred in the presence of the plant Arabidopsis thaliana which is not capable of forming any type of mycorrhizal symbiosis, either EM or AM. This indicates that diffusible plant-produced signals must be released by the root but that they are not specic. Combinations of immunouorescent labeling and inhibitors of endocytosis and endosome vesicular tracking were used to demonstrate that MiSSP7 enters plant cells, probably by endocytosis, and accumulates in the plant nucleus. Interestingly, accumulation occurs in the absence of the fungus, as shown by introducing MiSSP7 to plant cell suspensions. These experiments demonstrate that it is the plant that actively takes up MiSSP7. One very exciting nding of this study is that MiSSP7 also seems to be critical for the formation of the so-called Hartig net, a symbiotic structure that creates a high surface area between the fungus and plant cells and allows an efcient bi-directional exchange of nutrients between the plant and fungus. The Hartig net is, thus, a critical structure that has to be formed for a functional mutualistic symbiosis to occur between the plant and the fungus. Homologous gene replacement has not been achieved in L. bicolor and so Plett et al. [4] used RNA silencing to lower the production of MiSSP7, resulting in very low percentages of mycorrhizal root tip formation and only a very poorly developed Hartig net in those instances where some mycorrhizal development had occurred. MiSSP7 appears to control Hartig net formation by inducing transcripts involved in cell

wall remodeling and auxin homeostasis. It seems, therefore, that MiSSP7 really is a fungal effector necessary for ectomycorrhiza formation. Since it has proved difcult to nd mycorrhizal fungal effectors, it is surprising to see a second study appearing at exactly the same time and also demonstrating the existence of such effectors [5]. However, what is so signicant about this second study is that it has been found in AM fungi, fungi that diverged from other fungal lineages hundreds of millions of years before the evolution of EM fungi and most fungal pathogens of plants. Arbuscular mycorrhizal fungi are exceptionally difcult to work with at the molecular level for a number of reasons, including the lack of a stable transformation system and the inability to grow the fungus without plant roots [3]. Because of the lack of a complete AMF genome or published transcriptome data, the authors used a modied version of the yeast secretion sequence trap method to collect proteins secreted by the AM fungus Glomus intraradices. A small protein that they called secreted protein 7 (SP7) was identied that contained a signal domain, a nuclear localization domain and tandem hydrophilic repeats. SP7 was found to accumulate in the fungus when in contact with a plant root. Because there is no known stable transformation system in G. intraradices, or any other AM fungus, SP7 was expressed in the lamentous fungus Aspergillus nidulans in order to show that, similar to MiSSP7, it would locate to the nucleus. Transgenic plants expressing SP7 fused to green ourescent protein also showed that the protein was localized in the plant nucleus. The truly exciting aspect of this study came when the authors found that SP7 interacts with a transcription factor called ERF19 in the plant Medicago truncatula and that ERF19 normally activates the expression of defense proteins in plants. Kloppholz et al. [5] generated transformed plant roots that constitutively express the fungal protein SP7. In those roots, the AM fungus could more efciently colonize the roots. Interestingly, ERF19 is normally expressed in plant roots challenged with the fungal pathogen Colletotrichum trifolii but such expression is halved when the roots express the fungal protein SP7,

Current Biology Vol 21 No 14 R552

indicating that SP7 interacts with ERF19 to suppress its activity. An intriguing part of the study was that when plants were inoculated with a transgenic form of the hemibiotrophic fungal pathogen Magnaporthe oryzae that expressed SP7, the detrimental effects of the pathogen were less than in plants infected with the wild-type fungus. The authors suggest that SP7-mediated suppression of defense pathways in the plant allows the fungus to remain in a biotrophic state. This, in turn, allows the fungus to keep the plant alive for longer. It does not seem very intuitive that a protein that suppresses plant defense could be benecial for the plant. However, it could allow a fungus like M. oryzae to simply remain undetected inside the host for a longer period until the plant has grown larger and more resources could be obtained from the host. Additionally, defense suppression could open up the possibility for other fungi to attack the plant. Such results also raise the question of whether a plant is more or less susceptible to fungal pathogens when colonized by

an AMF that is expressing SP7 and, thus, suppressing part of the plants ability to defend itself. Indeed, reports about whether AMF increase or decrease the ability of plants to protect themselves against fungal pathogens are rather contradictory. Nevertheless, the nding of an AMF effector protein that switches off parts of the plant defense mechanism is indeed a major milestone in understanding the AM symbiosis. Given the global importance of the mycorrhizal symbiosis, coupled with the difculty of working with the fungal partners, these two studies identifying the role of fungal effector proteins that allow mycorrhiza formation and allow the fungus to switch off the plants defense responses truly further our knowledge of these plantfungal associations.
References
1. Smith, S.E., and Read, D.J. (2008). The Mycorrhizal Symbiosis (San Diego: Academic Press). 2. Parniske, M. (2008). Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nat. Rev. Microbiol. 6, 763775.

3. Sanders, I.R., and Croll, D. (2010). Arbuscular mycorrhiza: The challenge to understand the genetics of the fungal partner. Annu. Rev. Genet. 44, 271292. 4. Plett, J.M., Kemppainen, M., Kale, S.D., Kohler, A., Legue, V., Brun, A., Tyler, B.M., Pardo, A.G., and Martin, M. (2011). A secreted effector protein of Laccaria bicolor is required for symbiosis development. Curr. Biol. 21, 11971203. 5. Kloppholz, S., Kuhn, H., and Requena, N. (2011). A secreted fungal effector of Glomus intraradices promotes symbiotic biotrophy. Curr. Biol. 21, 12041209. 6. Stergiopoulos, I., and de Wit, P.J.G.M. (2009). Fungal effector proteins. Annu. Rev. Phytopathol. 47, 233263. 7. Ellis, J.G., Raqi, M., Gan, P., Chakrabarti, A., and Dodds, P.N. (2009). Recent progress in discovery and functional analysis of effector proteins of fungal and oomycete plant pathogens. Curr. Opin. Plant Biol. 12, 399405. 8. Martin, F., Aerts, A., Ahren, D., Brun, A., Danchin, E.G.J., Duchaussoy, F., Gibon, J., Kohler, A., Lindquist, E., Pereda, V., et al. (2008). The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452, 8892.

Department of Ecology and Evolution, Biophore Building, University of Lausanne, 1015 Lausanne, Switzerland. E-mail: ian.sanders@unil.ch

DOI: 10.1016/j.cub.2011.06.022

Cell Biology: Actin Keeps Endocytosis on a Short Leash

High-resolution structural analysis of branched actin networks at the sites of clathrin-mediated endocytosis sheds light on the role of actin in endocytosis and mechanisms controlling actin assembly. Vladimir Sirotkin The dynamic actin cytoskeleton plays many important roles in fundamental cellular processes, including cell motility, cell division, control of cell shape and endocytosis. Studies in organisms ranging from yeast to man have revealed a burst of actin assembly at the endocytic site or patch, peaking at the internalization of an endocytic vesicle [13]. Actin plays an obligatory role in endocytosis in yeast, while the role of actin in endocytosis in animal cells remains a subject of debate. Given that force generated by actin assembly is sufcient to deform cell membranes and to move particles within dense cytoplasm, actin was proposed to participate at multiple stages of endocytosis, including membrane invagination, scission and propulsion of the endocytic vesicle. In this issue of Current Biology, Collins et al. [4] provide remarkable electron microscopy images of the actin network around clathrin-coated structures in animal cells. The organization of the network helps to dene the role of actin in endocytosis and provides important clues about the mechanisms controlling actin assembly at the endocytic sites. The exact mechanism for the role of actin in endocytosis depends on the arrangement of laments around the endocytic sites (Figure 1A). Several models have been proposed [13,5]. Work in budding yeast favors actin lament organization with fast-growing, barbed ends facing the plasma membrane and slow-growing,

pointed ends anchored at the endocytic vesicle coat. In a second model, lament barbed ends are oriented towards the tip of endocytic invagination, so that growing laments force elongation of the neck of the budding vesicle and propel the vesicle after the scission from plasma membrane. The images in the present study [4] support a third model, in which actin laments form a collar-like structure around the neck of the endocytic vesicle. Growing laments are oriented towards the vesicle neck, providing force for neck elongation, vesicle scission and propulsion. Collins et al. [4] used platinum replica electron microscopy and electron tomography to produce striking images of the actin network at clathrincoated structures in cultured mouse cells that were either de-roofed by sonication or extracted with detergent. In the de-roofed preparations, many of the clathrin-coated structures are at the early stages of invagination and are surrounded by a branched actin network. Electron tomography has revealed that the actin network is located at the neck of the endocytic