sign in sign up
<<
Home , Arbuscular mycorrhiza, Burkholderia, Castellaniella, Lateral root, Mycorrhiza, Mycorrhizosphere

Impact of endofungal in - interactions Alabid et al.

Curr. Issues Mol. Biol. (2019) 30: 59-74. caister.com/cimb

Endofungal Bacteria Increase Fitness of their Fungi and Impact their Association with Crop

Ibrahim Alabid1, Stefanie P. Glaeser2 and Karl- their role in supporting sustainable by Heinz Kogel1* promoting plant growth, improving plant resistance, and decreasing yield loss caused by many microbial 1Institute of Phytopathology, IFZ Research Centre . for Biosystems, Land Use and Nutrition, Justus Liebig University, D-35392 Giessen, Germany Endobacteria in plant-colonizing fungi 2Institute of Applied Microbiology, IFZ Research Endofungal bacteria inhabit the of fungal Centre for Biosystems, Land Use and Nutrition, cells (Figure 1). They commonly establish beneficial Justus Liebig University, D-35392 Giessen, relationships (positive symbioses) with their plant- Germany colonizing host fungi thereby forming tripartite interactions that comprise the bacterium, the fungus *[email protected] and the plant (Perotto and Bonfante, 1997; Bonfante and Anca, 2009; Desirò et al., 2014; Moebius et al., DOI: https://dx.doi.org/10.21775/cimb.030.059 2014; Erlacher et al., 2015; Glaeser et al., 2016; Salvioli et al., 2016). From the historical perspective, Abstract: Mosse (1970) was the first to describe intracellular Endofungal bacteria are bacterial symbionts of fungi structures very similar to bacteria, called Bacteria- that exist within fungal hyphae and . There is Like Organisms (BLOs) inside fungal hyphae increasing evidence that these bacteria, alone or in (Figure 2). Since then, BLOs and bacteria were combination with their fungal hosts play a critical detected in glomeromycotan arbuscular role in tripartite symbioses with plants, where they may contribute to plant growth and disease resistance to microbial pathogens. As the frequency of bacteria in fungi is commonly very low, breakthroughs in technology such as molecular and laser scanning microscopy were required to establish the functional contribution of these bacteria in complex symbioses. Yet, the overall biological significance of endofungal bacteria is largely unknown and further progress in understanding is hampered by a very few biological systems where endofungal bacteria have been described mechanistically. We review here the current knowledge on endobacteria (EB) and their role in different types of fungal symbioses with plants. We show that various attempts to cure fungal cells from endobacteria failed, further suggesting that they play a crucial role in the . Moreover, isolation of some of the endobacteria from their fungal hosts allowed confirming their autonomous beneficial activity such Figure 1. Detection of endofungal bacteria in fixed fungal mycelia of as plant growth promotion and resistance-inducing vermifera-MAFF305838 by Fluorescence in situ hybridization (FISH) analysis using a universal Bacteria 16S rRNA targeting probe activity. The review addresses the potential (green) and DAPI counter straining (blue). Detection of endobacteria in agricultural significance of endofungal bacteria and this strain was first described in Sharma et al. (2008).

!59 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

Figure 2. Time scale of the published research on endofungal bacteria.

(AM) symbioses (Scannerini and Bonfante, 1991; pathogenic fungi (Scherlach et al., 2006). In Bianciotto et al., 2003; Naumann et al., 2010), the general, complex interactions including bacteria, related fungus pyriforme (Kluge, 1992; fungi, and plants are poorly understood, and the Schüßler et al., 1994), the ectomycorrhizal (EM) global prevalence of these types of interactions is basidiomycete fungus bicolor (Bertaux et largely unknown. In this review we focus on the al., 2003; 2005), the rice pathogenic fungus three most intensively studied biological models (Partida-Martinez and comprising endobacteria in beneficial and parasitic Hertweck, 2005), phylogenetically diverse foliar tripartite interactions (Figure 3). fungal (Hoffman and Arnold, 2010), and the plant symbiotic Endogone fungi Endofungal bacteria in the AM symbiosis (Desirò et al., 2015). Unlike insect , Two types of endofungal bacteria have been which are localized in specialized tissues (Moran et distinguished in AM fungi on the basis of their al., 2008), endofungal bacteria are found in fungal morphological features. The first type of spores and both extra- and intraradical hyphae, the endobacteria is coccoid-shape and present also in latter colonizing the plant tissues (Lumini et al., the cytoplasm of another basal group of fungi, the 2007). Endofungal bacteria also have been Endogone Mucoromycotina species. These bacteria discovered in the Sebacinalean symbiosis, a represent a monophyletic clade of fungal - mutualistic association of fungi of the derived sequences which is phylogenetically placed order Sebacinales with a broad spectrum of plants in-between the Mollicutes and Firmicutes and (Sharma et al., 2008). Although the role of these termed Mollicutes-related endobacteria (MRE; bacteria is not fully understood, increasing evidence Scannerini and Bonfante, 1991; Naumann et al., gathered by studying the model Sebacinalean 2010; Desirò et al., 2015). In contrast to the -wall fungus indica suggests that they free Mollicutes, the MRE endobacteria detected in contribute to the fitness of their fungal hosts (Guo et the cytoplasm of fungal cells contain a Gram- al., submitted). Some reports illustrate that positive like cell wall. endofungal bacteria support the virulence of

!60 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

Figure 3. Comparative summary of the knowledge about the three most extensively studied tripartite symbioses of plant with their colonizing fungi and endobacteria.

The second type of endofungal bacteria are Gram- genus Burkholderia (Bianciotto et al., 1996). The G. negative rod-shaped bacteria that are restricted to margarita isolate BEG 34 contained 250,000 living the AM-forming family Gigasporaceae (Bianciotto et bacterial cells in a single . The bacteria were al., 2004). Those intracellular bacteria were subsequently classified based on genetic features detected in five Gigaspora spp. through all stages of as a novel bacterial taxon, Candidatus the fungal life cycle: spores, germtubes, extra- and Glomeribacter gigasporarum (CaGg; Banciotto et intraradical hyphae. Based on the 16S rRNA al., 2003) next closest related to the genus sequence phylogeny, the endobacteria in Gigaspora Burkholderia. Isolated bacteria could not be grown margarita were initially identified as a member of the in culture media, but kept alive for up to 4 weeks

!61 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

(Jargeat et al., 2004). Sequencing of the genome of other hand, genome annotation also provided an a homogeneous cell population derived from the insight into the molecular basis for CaGg obligate Gigaspora margarita strain BEG 34 representing biotrophic status: The lack of some crucial metabolic CaGg led to a 1.72 Mb assembly with 1,736 coding pathways can explain the failure to grow CaGg as a DNA sequences (CDS), the smallest genome known free-living organism, as it has a metabolic for a Betaproteobacterium (Ghignone et al., 2012). dependence on the fungal host for both energy and Such small genomes are typically found in nutrition. Thus, the bacterial genome provided clear endocellular bacteria living permanently in their host evidence of the energy/ flows of the tripartite with an obligate symbiotic lifestyle, where gene interaction between the bacterium, the fungus and erosion occured in association with metabolic the plant. CaGg's limited capacity to synthesize dependence on the host (Moran et al., 2008; amino acids, the presence of a large set of amino- Castillo and Pawlowska, 2010). The genome acid transporters in the bacterial genome, and its assembly comprised one chromosome and three location inside the -rich fungal plasmids. Although the genome was rather small, highlight that there is most likely a flow of the G+C content was high (54.8%), which is from the fungus to the bacterium. Consistent with unusual for small genomes. the strong dependency, CaGg is at least in part but non-essential vertically transmitted through fungal A phylogenetic multilocus gene analysis carried out vegetative sporulation, indicating that active on concatenated sequences of 21 protein-coding bacterial proliferation occurs in the multinucleate retrieved from 67 completed genomes mycelium of the fungus (Bianciotto et al., 2004). belonging to a wide range of bacterial lineages Consistent with this, the genome in CaGg showed that CaGg clusters within the family is non-degenerative and exemplifies a departure . Members of this family are highly from the model of degenerative evolution in versatile microbes interacting with animals, humans, heritable endosymbionts such as mutualists of plants and fungi (Bontemps et al., 2010). Analysis of insects (Mondo et al., 2016). a data set restricted to and based on 16S and 23S rRNA gene sequences While the occurrence of CaGg is limited to the placed CaGg as a sister group of a Burkholderia Gigasporaceae, coccoid-shaped MRE endobacteria clade, which includes free-living and fungal- are widely distributed across different lineages of associated species showing that CaGg is an ancient AM fungi. A fungus can harbor both types of member of the taxon sharing a common ancestor endobacteria, with MRE population being more with the present-day Burkholderiaceae (Ghignone et abundant, variable and prone to recombination al., 2012). While phylogenetic analyses placed (Desirò et al., 2014), suggesting that Gigasporaceae CaGg in the Burkholderiaceae, metabolic pathway with their comparatively large spores, which are rich analyses clustered it with endosymbiotic bacteria of in reserves of , fats and , can insects. This positioning among different bacterial support the energetic cost of complex bacterial classes let the authors claim that CaGg has communities. MRE were also identified in the undergone to adapt itself to an Endogone Mucoromycotina fungi, an ancient group intracellular lifestyle. Genome annotation further of fungi capable of symbiotically interacting with revealed an unexpected genetic mosaic where plants. Interestingly, Mucoromycotina fungi along typical genetic determinants of symbiotic, with are considered as the unique pathogenic and free living bacteria are integrated in ancestral symbionts of land plants (Desirò et al., a reduced genome. 2015). MRE possess a homogenous cell wall-like envelope, which upon high pressure and freeze Significantly, the bacterial cells are separated from substitution is rather electron-transparent. 16S rRNA the fungal cytoplasm by a membrane of fungal gene sequences of MRE isolated from AM fungi and origin, suggesting that CaGg communicates with the Endogone Mucoromycotina fungi cluster together fungus via transport and secretion systems that and form a separate clade. In both fungal groups, deliver bacterial molecules to the host. This is coccoid-shaped MRE are embedded in the fungal supported by the large number of genes present in cytoplasm without any evidence of the fungal its genome coding for secretion, including type II membrane which surrounds CaGg (Ghignone et al., and type III secretion systems and synthesizes 2012). vitamin B12, antibiotics- and toxin-resistance molecules, which may contribute to the fungal host's ecological fitness (Ghignone et al., 2012). On the

!62 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

Endofungal bacteria impact the AM symbiosis mycelium, possibly affecting its overall ecological The mechanistic role of AM-associated fitness. endobacteria is still unclear, but their presence may be required for successful interaction of the fungi Endofungal bacteria in EM symbiosis with their plant hosts. In vitro studies suggested that In contrast to AM symbiosis, a relatively small sp. may stimulate AM's hyphal growth number of plants live in symbiosis with (Horii and Ishii 2006; Horii et al. 2008), the formation ectomycorrhizal (EM) fungi, mainly forest in of new spores (Hildebrandt et al., 2002), and the temperate and boreal . EM symbioses suppression of pathogens (Budi et al., 1999; Horii et are formed by a large number of fungal species, al., 2008). Though these bacteria were associated mainly Basidiomycetes, but also Ascomycetes. In with the , a true endofungal the early 1990th the concept of mycorrhization lifestyle has not been proven yet. Similarly, when helper bacteria (MHB) emerged to describe bacteria surface-sterilized spores of intraradices that help to establish a mycorrhizal symbiosis Sy167 were germinated on agar plates, slime- (Garbaye, 1994; Frey-Klett et al., 2007). This forming bacteria, identified as Paenibacillus validus, concept comprised all types of mycorrhiza and free frequently grew up. These bacteria supported the as well as endocellular bacteria that support a fungus completing its life cycle in the absence of symbiosis and thus appears somewhat indistinct. plant (Hildebrandt et al., 2006). We focus here mainly on reports suggesting that EM also is associated with true endofungal bacteria. Clear evidence for endofungal bacteria essentially A rather specific though not endocellular association contributing to beneficial AM symbioses is still was described for the bacterium Streptomyces missing because killing or separating bacteria from strain AcH 505 that improves mycelial growth and their AM host by antibiotic treatments or single EM formation between the fly agaric fungus spore proliferation is extremely difficult. The reason () and spruce (Picea abies). for this intimate association between endofungal Auxofuran was identified as the predominant growth bacteria and their hosts is not exactly known and promoting substance which was most effective at a may vary for single interaction systems. Yet, G. concentration of 15 µM. Co-cultivation of the margarita BEG 34 provides a convincing example bacteria and A. muscaria stimulated auxofuran for a cured fungus: repeated passages through production in the fungus. Another example is the single-spore inocula caused dilution of the initial contribution of fluorescens BBc6R8, CaGg bacterial population eventually leading to isolated from a Laccaria bicolor sporocarp cured AM spores. CaGg was not essential to the consistently promoting L. bicolor-Douglas fir survival or reproduction of the fungus. However, (Pseudotsuga menziesii) EM formation. The spores had a distinct phenotype in terms of the prevalence of strain BBc6R8 in the was cytoplasm organization, morphology, cell significantly enhanced by the presence of L. bicolor wall organization, bodies and pigment granules. S238N in either the presence or absence of The absence of bacteria severely affected Douglas-fir roots, while in contrast its survival was presymbiotic fungal growth such as hyphal not supported by non-mycorrhizal roots, suggesting elongation and branching. However, at least under that the strain P. fluorescens BBc6R8 depends more laboratory conditions, cured G. margarita formed on the fungus than on the plant roots (Frey-Klett et mycorrhizal associations and also sporulated in the al., 2007). colonized roots (Lumini et al., 2007). Recent work supported the view that CaGg increases the Bacterial proliferations also have recurrently been environmental fitness and bioenergetics potential of observed in fermentor cultures of the L. bicolor G. margarita by priming mitochondrial metabolic strain S238N, suggesting cryptic bacteria pathways (Salvioli et al., 2016). The endobacterium associated with this fungus. Endofungal bacteria influenced fungal growth, , and calcium were detected by Fluorescence in situ Hybridization signaling by targeting mitochondrial activity, (FISH) in pure fungal subcultures. They were small upregulation of the genes involved in respiration, in size (0.5 µm in diameter), rare, and ATP production and reactive oxygen species (ROS) heterogeneously distributed in the mycelium. 16S detoxification (Vannini et al., 2016). The authors rRNA gene sequence analysis identified those concluded that - at least for the G. margarita BEG endobacteria as Paenibacillus spp. (Bertaux et al., 34 isolate - the absence of endofungal bacteria 2003). However, Paenibacillus spp. have not been causes delays in the growth of germinating recovered from colonized plant samples, although pure fungal cultures served as inoculum (Bertaux et

!63 Impact of endofungal bacteria in fungus-plant interactions Alabid et al. al., 2005). Instead, samples taken from EM roots many important cereals and such as rather contained endofungal Alphaproteobacteria, the model plant Arabidopsis thaliana (for review which vice versa had not been detected in pure L. Qiang et al., 2012). Unlike AM, Serendipita species bicolor S238N cultures. Many Alphaproteobacteria are facultative biotrophic and thus can easily be also were detected outside the hyphae, in addition cultured with synthetic medium in the absence of a to bacteria belonging to other phyla, such as plant (Deshmukh et al., 2006; Oelmüller et al., Actinomycetes and Cytophaga-Flexibacter. Thus, 2009). Endofungal bacteria identified in various the authors speculated about an environmental Serendipita species belong to two genera of Gram- origin of the endofungal Alphaproteobacteria negative (Rhizobium and Acinetobacter) and two (Bertaux et al., 2005). genera of Gram-positive (Paenibacillus and Rhodococcus) bacteria (Sharma et al., 2008; see Endofungal bacteria in the Sebacinalean figure 1). The most comprehensively studied symbiosis example of a tripartite Sebacinalean symbiosis, The order Sebacinales is the most basal including endofungal bacteria, is the association of Basidiomycota group which contains fungi that P. indica with the Alphaproteobacterium Rhizobium undergo endophytic as well as mycorrhizal radiobacter (Rr; syn. Agrobacterium tumefaciens, interactions with a broad spectrum of syn "Agrobacterium fabrum"; Figure 4). FISH using monocotyledonous and dicotyledonous plants found the Rhizobium-specific probe Rhi-1247 confirmed across all continents (Selosse et al., 2007; Weiss et the endocellular association of Rr with fungal spores al., 2011, Riess et al., 2014). The family and hyphae. Using quantitative PCR analysis, a Serendipitaceae comprises endophytes from the ratio of 0.02 - 0.035 ng of bacterial DNA per 100 ng genus Serendipita such as Piriformospora (syn. of P. indica DNA was determined (Sharma et al., Serendipita) indica that constitutes an excellent 2008). This value is consistent with the low number model (Verma et al., 1998), especially of bacteria (2 - 20 per fungal cell) detected in L. due to available genome information, genetic bicolor (Bertaux et al., 2003; 2005). R. radiobacter tractability, and its broad host range that includes was determined in the original strain of P. indica

Figure 4. Colonization pattern upon inoculation of plant roots with the endofungal bacterium R. radiobacter F4 (RrF4) that was isolated from a fungal Piriformospora indica culture. The endobacteria were labelled with the β-glucuronidase (GUS) reporter enzyme. Bacteria are visualized after X-Gluc treatment of roots at 7 (a,b) and 14 (c,d) days after dip inoculation. Staining of barley primary roots (a,b); protrusions and zones of secondary roots of barley (c) and Arabidopsis (d). Scale bar = 2 mm.

!64 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

DSM 11827 isolated from the Indian jar dessert and reduced, which is consistent with the situation in the from three other P. indica cultures by the use of cultivable endobacterium Burkholderia rhizoxinica Denaturing gradient gel electrophoresis (DGGE) of (Lackner et al., 2011). Thus, the data are consistent 16S rRNA gene fragments amplified from fungal with the hypothesis that RrF4 forms a facultative DNA extracts (Sharma et al., 2008). The strain R. symbiosis with P. indica, where the bacterium is still radiobacter F4 (RrF4) was isolated from P. indica able to live independently outside its host. DSM 11827 and propagated in axenic cultures with Supportive for this hypothesis, curing RrC58 from its Luria-Bertani (LB) medium, showing that the pTi plasmid resulted in a non-pathogenic strain with bacterium is not entirely dependent on its fungal weak plant growth-stimulating activity in maize host. However, attempts to cure P. indica of RrF4 seedlings in non-sterile soil (Walker et al., 2013). have failed. Treatments with antibiotics such as spectinomycin and ciprofloxacin, which inhibit the Endofungal bacteria enhance plant growth growth of RrF4 in pure cultures, merely transiently The benefits or costs that fungal endophytes and reduced the number of bacteria in the fungal endophyte-bacterial complexes extend to their plant mycelium as shown by the increase in the number hosts are greatly associated with plant health and of bacteria after prolonged cultivation in the absence production. Thus, it is important to address the of antibiotics. The fact that bacteria could not be effects endofungal bacteria have on both, disease killed inside the hyphae raised the speculation that resistance and yield. Fungi and bacteria can interact RrF4 enters an inactive state where it becomes synergistically to stimulate plant growth through a insensitive to antibiotics. The protection against range of mechanisms that include improved nutrient antibiotics may also be a survival strategy for acquisition and inhibition of fungal pathogens bacteria in the which is commonly rich (Artursson et al., 2006). In general, the beneficial in antimicrobial compounds producing rhizobacteria. potential of bacteria closely associated with the Nevertheless, a combination of protoplastation and plant root can be inferred from the group of plant antibiotics treatments reduced the abundance of growth-promoting rhizobacteria (PGPR) that are in endobacterial cells below the detection limit. Fungi contact with the root surface, or rhizoplane, and regenerated from those showed both increase plant yield by diverse mechanisms such as reduced vegetative growth and chlamydospore improved mineral uptake, disease suppression, or formation suggesting a requirement of endobacteria phytohormone production (Weller, 1988; Kloepperet for the fungal fitness (Guo et al., submitted). This al., 1991; Lugtenberg et al., 1991; Broek and situation is reminiscent of interactions of CaGg with Vanderleyden, 1995; Défago and Keel, 1995). Even G. margarita and Burkholderia spp. with Rhizopus so they are not "endofungal", PGPR have been microspores, where the absence of the respective reported to benefit associated fungi during bacterium also reduced fungal fitness (Lumini et al., interaction with their plant host. Certain groups of 2007; Lackner et al., 2011). bacteria have been shown to accumulate to a higher extent in the mycorrhizosphere compared with other The genome of RrF4 is organized in a circular (2.8 groups (Artursson et al., 2006 and references Mb) and a linear chromosome (2.06 Mb), a tumor- therein). PGPR also interact with AM fungi and have inducing plasmid pTiF4 (0.21 Mb), and an accessory stimulatory impact on the growth of these fungi plasmid pAtF4 (0.54 Mb) and thus shows a high suggesting that they have at least an additional degree of similarity to the plant pathogenic R. indirect effect on plant growth promotion (Garbaye, radiobacter C58 (RrC58; formerly: Agrobacterium 1994). For example, association of Pseudomonas tumefaciens; syn. "A. fabrum" C58; Goodner et al., putida with AM fungi result in increased growth of 2001). The circular and linear chromosomes of RrF4 clover plants, suggesting that PGPR may have had 100 and 80 singleton open reading frames properties that support both mycorrhizal (ORFs), respectively, that are not present in C58. establishment and function (Meyer and Linderman, Most of these ORFs were of unknown function and 1986). Phylogenetically diverse endofungal bacteria may be candidates for future studies to elucidate a detected in hyphae of diverse foliar potential role for the endofungal growth of RrF4 and/ endophytes of trees produced the plant-growth or fitness of its fungal partner P. indica (Glaeser et promoting hormone indole-3-acetic acid (IAA). Most al., 2016). Differences such as the loss of the T-DNA of these bacteria were members of the in the tumor-inducing (pTi) plasmids can explain the (Hoffman and Arnold, 2010). For loss of RrF4's pathogenicity (Glaeser et al., 2016). instance, in vitro production of IAA by a fungal In contrast to obligate endofungal bacteria such as endophyte determined as Pestalotiopsis spp. the non-cultivable CaGg, the genome of RrF4 is not (Pezizomycotina) that was isolated from foliage of a

!65 Impact of endofungal bacteria in fungus-plant interactions Alabid et al. coniferous host (Platycladus orientalis) was changes in roots during mycorrhizal formation enhanced by the presence of Luteibacter sp. of the (Kaska et al., 1999), including the formation of Xanthomonadales. IAA production by the lateral roots and dichotomous branching of short endophyte-bacterial complex required L-tryptophan. roots (Barker and Tagu, 2000). The isolated and axenically cultured Luteibacter sp. did not produce IAA on a standard growth medium. Promotion of lateral root formation also is a However, culture filtrate from the endophyte- commonly observed characteristic of MHB, bacterium complex enhanced growth of tomato essentially leading to an increase in potential plants relative to filtrate from the endophyte alone. contact points at which the plant and the Given that the hormone produced by diverse plant- ectomycorrhizal fungus can interact (Poole et al., associated fungi can enhance plant growth and also 2001; Schrey et al., 2005). MHB can indirectly suppresses, to a certain extent, plant defense such facilitate EM fungi's root colonization by inducing the as the hypersensitive reaction (HR) and production release of signal molecules such as plant flavonoids of pathogenesis-related (PR) proteins, IAA (Frey-Klett et al., 2007) or suppressors of the plant's production could be an important aspect of foliar defence responses (Lehr et al., 2007). Moreover, endophyte-plant symbioses (Maor et al., 2004; root colonization by EM fungi may be facilitated be Spaepen and Vanderleyden, 2011; Hoffman et al., MHB's production of plant cell wall-digesting 2013). enzymes thereby enhancing penetration and spreading of the fungus within the root tissues Inoculation of various plant species such as barley, (Mosse, 1962). , and Arabidopsis with pure cultures of Rhizobium radiobacter F4 (RrF4) promotes shoot Acyl homoserine lactone production and and root growth, including lateral root branching of endofungal bacteria (Sharma et al., 2008; Glaeser et al., 2016). These Acyl homoserine lactones (AHLs) are well-known activities widely mimicked the effects RrF4's host molecules produced by bacteria for their own fungus P. indica has on plants. Yet, as the fungal communication termed quorum sensing (QS). More host could not be completely cured from the recently, AHLs were also implicated in beneficial endobacterium, it is still an unresolved question of activities bacteria have on plant yield and health whether beneficial plant biomass enhancement in (Schuhegger et al., 2006; Schikora et al., 2011; the Sebacinalean symbiosis merely stems from Schenk et al., 2014; Zarkani et al., 2013). Using a RrF4, the fungus P. indica, or the endophyte- bacterial biosensor screening, AHL-producing bacterium complex (Guo et al., submitted). endofungal bacteria were detected in antagonistic soil fungi (Kai et al., 2012). In situ detection by FISH In a forest nursery, the amount of - analysis and electron microscopy confirmed the solubilizing and -producing fluorescent presence of endobacteria in the mycelium of the pseudomonads was much higher in the Douglas-fir zygomycete fungus Mortierella alpina A-178 that ectomycorrhizal fungus L. bicolor than in the produced the QS molecules. Release of these surrounding root-free soil (Frey-Klett et al., 2005). molecules by the fungus was subsequently proven The enhancement of the solubilization of rock by the detection of AHL from the supernatant of the occurred following formation of mixed liquid culture of the fungus. Amplification of 16S between phosphate-solubilizing rRNA gene sequence fragments from fungal saprotrophic fungi and a elkanii mycelium extracts indicated that a Beta- strain (Jayasinghearachchi and Seneviratne, 2005). proteobacterium with 100% 16S rRNA gene The bacterial secondary metabolite responsible for sequence identity to Castellaniella defragrans (100 plant growth promoting activity of Amantia muscaria %) and a Gram-positive bacterium assigned to the through Streptomyces sp. AcH 505 is 5,6,7- genus Cryobacterium (99.8% 16S rRNA gene trihydro-7-hydroxy-3-prolylbenzofuran-4-1, termed sequence identity) were present in the mycelium of auxofuran, because of its -reminiscent M. alpina A-178. Antibiotic treatment enabled to structure (Frey-Klett et al., 2007; Keller et al., 2006; cure the fungus, and the lack of AHL detection in the Riedlinger et al., 2006). Upon treatment with fungus confirmed that the AHL is produced by the auxofuran, expression of the A. muscaria gene endobacterium. Yet, how QS is involved in the acetoacetyl-CoA synthetase (AmAacs) was up- interaction of fungus and bacterium needs to be regulated, indicating activation of sterol biosynthesis elucidated by further studies. Similarly pure cultures (Riedlinger et al., 2006). and ethylene also of endofungal RrF4, isolated from P. indica, also have been implicated in inducing morphological produce various classes of AHL that are known to

!66 Impact of endofungal bacteria in fungus-plant interactions Alabid et al. have growth promoting and induced resistance signaling where not compromised in RrF4-mediated activity in plants (unpublished data). Significantly, resistance (Glaeser et al., 2016). mutants of RrF4 that were compromised in AHL accumulation, showed reduced growth promoting Toxin-producing endofungal bacteria contribute activity when colonizing wheat or Arabidopsis to fungal virulence plants. These merely preliminary findings suggest a The rice seedling blight fungus Rhizopus more rigorous analysis of the role of AHLs in microspores (Mucoromycotina, Mucorales) severely tripartite interactions. Noteworthy, B. rhizoxinica, affects rice fields in Asia that begins with an lacks genes related to known QS systems (Lackner abnormal swelling of seedling roots and eventually et al., 2011b). results in death of the affected tissue or the complete plant. The fungus forms beneficial Complexes of closely associated bacteria and associations with the Betaproteobacteria beneficial fungi enhance local and systemic Burkholderia endofungorum and Burkholderia resistance to microbial pathogens rhizoxinica (Partida-Martinez et al., 2007b), which Fungus-bacterium complexes also contribute to support the fungal host to parasitize plant tissue. B. plant protection against and root pathogens. rhizoxinica produces the phytotoxic polyketide Various studies have demonstrated in vitro metabolite rhizoxin (Partida-Martinez and Hertweck, antagonistic activity exerted by mycorrhiza- 2005; Lackner et al., 2011) which blocks mitosis in associated bacteria on microbial plant pathogens eukaryotic cells, including plants, by binding to β- (Schelkle and Peterson, 1996; Becker et al., 1999; tubulin and thus serves the fungus as virulence Maier et al., 2004; Frey-Klett et al., 2007). The EM- factor (Sato et al. 1983). Due to an amino acid associated Pseudomonas fluorescens strain change in the tubulin protein, Rhizopus itself is BBc6R8 inhibited the growth of various root- tolerant to rhizoxin (Schmitt et al., 2008). pathogenic fungi belonging to the genera Remarkably, rhizoxin had earlier been related to the Rhizoctonia, Fusarium, Phytophthora and fungal metabolism. However, treatment of the Heterobasidion in the Douglas-fir - L. bicolor fungus with ciprofloxacin (40 µg ml-1), an antibiotic interaction (Frey-Klett et al., 2005). In addition, a active against bacteria but not against fungi, cured strain of Paenibacillus spp. isolated from the the fungus of the bacteria and concomitantly rhizosphere of sorghum proved to be compatible extirpated rhizoxin production (Partida-Martinez and with AM development, but inhibited soil-borne fungal Hertweck, 2005; Partida-Martinez et al., 2005). pathogens (Budi et al., 1999). This bacterial strain Consistent with this, B. rhizoxinica contains the produced small peptides that were harmless to the gene cluster encoding rhizoxin biosynthesis symbiotic fungi, but were responsible for the (Partida-Martinez and Hertweck, 2007). antagonistic effects to pathogens (Selim et al., 2005). Cruz and Ishii (2008) isolated two bacteria In contrast to the AM endofungal bacterium CaGg from surface sterilized spores of Gigaspora that resisted cultivation in cell-free medium and margarita. Both isolates that were closest related to could only be investigated by using molecular Janthinobacterium and Paenibacillus spp. inhibited methods, B. rhizoxinica could be isolated, grown in growth of the soil-borne plant pathogens Fusarium pure culture, and eventually reintroduced into a oxysporum f. sp. lactucae, Rhizoctonia solani, and cured fungal host strain (Partida-Martinez and Pythium ultimum (Cruz and Ishii, 2008). Hertweck, 2005; Scherlach et al., 2006; Partida- Martinez et al., 2007; Moebius et al., 2014). The Pure cultures of the endofungal bacterium RrF4 bacterium is vertically transmitted through asexual induced systemic resistance against various reproduction units (vegetative spores) of its fungal microbial leaf pathogens such as the powdery host, a process that was discovered through re- mildew fungus Blumeria graminis f.sp. hordei and infection of fungal mycelia using laser-mediated the bacterium Xanthomonas translucens pv. microinjection or co-cultivation with green translucens (Xtt), when inoculated to barley or fluorescent protein (GFP)-producing bacterial wheat roots, respectively. Similarly, RrF4 also strains (Partida-Martinez et al., 2007a). Interestingly, induced resistance in Arabidopsis to Pseudomonas in the absence of the endofungal bacteria, R. syringae pv. tomato DC3000 (Pst) (Sharma et al., microsporus is compromised in asexual 2008; Glaeser et al., 2016). The underlying reproduction as evidences by a lack of mature resistance mechanism in Arabidopsis depends on sporangia and spores in mycelium of strains lacking an operable jasmonate pathway, while mutants bacteria. Formation of sporangia was restored upon defective in genes governing SA accumulation and reintroduction of bacteria, showing that reproduction

!67 Impact of endofungal bacteria in fungus-plant interactions Alabid et al. of the fungal host is strictly dependent on the intact The endobacterium RrF4 was isolated in a similar association (Partida-Martinez et al., 2007a). manner from crushed mycelium of P. indica Symbiont-dependent sporulation is a hallmark of (Sharma et al., 2008), and the two endosymbiotic close mutualistic relationships (Moran, 2006) and an Burkholderia species, B. rhizoxinica and B. elegant way to prevent formation of symbiont-free endofungorum, were isolated from the supernatant spores, thus securing the persistence of the of disrupted mycelium of two strains of R. symbiosis. Further experiment showed that the microspores. Whether the free living growth state of bacterial rhizoxin is not required for the bacterial endobacteria occurs in as a part of the infection process nor for fungal sporulation: Both the bacterial live cycle is unclear. Glaeser et al. (2016) B. rhizoxinica wild-type strain and a mutant deficient speculated that the endobacterium of P. indica may in rhizoxin biosynthesis supported fungal sporulation be released by root colonizing fungi to penetrate in culture showing that other bacteria factors are into internal root tissue to enhance the biological necessary for the symbiosis. Supporting this notion, activity. A clear proof by in situ detection is still a Type III secretion system (T3SS) was discovered missing. in B. rhizoxinica that controls a range of interactions between the bacterium and its fungal host, including Bacterial invasion into fungal cells invasion, intracellular transport of bacteria to hyphal Although endofungal bacteria have been known for tips and sporulation (Lackner et al., 2011). The four decades, the mechanism by which they enter entire gene cluster spans about 22,000 bp and fungal cells is unresolved. A recent study showed contains 23 open reading frames (ORFs). In terms that a type II secretion system (T2SS) of the of primary sequence conservation and gene order, endofungal bacteria B. rhizoxinica is required for the the gene cluster is similar to the hypersensitive formation of the endosymbiosis (Moebius et al., response protein (hrp) locus coding for a T3SS that 2014), based on genome mining for potential promotes virulence of the plant Ralstonia symbiosis factors and functional analyses. solanacearum (Cunnac et al., 2004). B. rhizoxinica Comparative proteome analyses show that the T3SS- mutants defective in two T3SS genes, DsctC bacterium releases chitinolytic enzymes (chitinase, and DsctT, did not show growth defects or chitosanase) and chitin-binding proteins. Genes morphological phenotypes compared with the wild encoding chitinase and chitosanase are highly type when grown in culture. However, these mutants expressed during the infection. The authors were strongly compromised in their fitness as only suggested that secreting these enzymes and limited zones of infection were visible. As in cured presumably further effector proteins via a T2SS help fungal cultures, the fungal host infected with these to locally soften the fungal cell wall allowing mutants was affected in intracellular survival and bacterial entry and preventing the disintegration of failed to elicit formation of mature sporangia in an fungal hyphae without permanent damage (Moebius infection assay (Lackner et al., 2011). et al., 2014). Consistent with this, a chitinase loss- of-function mutant lost its ability to enter fungal Isolation of fungal endobacteria hyphae. Only few endofungal bacteria have been isolated from their host fungi. Cruz et al. (2008) described Conclusion the isolation of endofungal bacteria from Gigaspora Elucidating the biology of tripartite associations margarita. After bacteria were determined by PCR between plants, higher fungi, and endofungal from surface sterilized spores, they were isolated by bacteria has shown that endofungal bacteria have osmosis from fungal protoplasts. The protoplasts various beneficial activities that support growth and were generated from spores by using two enzymes, development of their fungal hosts. The association a lysing enzyme and catalase. Two oval cells of Rhizopus microsporus with B. rhizoxinica is an forming bacteria were isolated by this cultivation outstanding model, because the fungus can be approach, a Gram-negative bacterium closest cured from the endofungal bacteria and isolated related to Janthinobacterium lividum and a Gram- bacteria can be grown in pure cultures, two positive bacterium closest related to Paracoccus prerequisites to fully elucidate the role of bacteria in polymyxa. Both isolates showed antagonistic fungi. That is why the role of endofungal bacteria in properties against the pathogenic fungi Rosellinia endo-, ecto-, and Sebacinalean symbioses is still necatrix, Pythium ultimum, Fusarium oxysporum hampered as the fungi either cannot by cured or and Rhizoctonia solani. endobacteria cannot be cultured outside the fungal mycelium. Accordingly, our understanding of these tripartite symbioses is far from being complete. New

!68 Impact of endofungal bacteria in fungus-plant interactions Alabid et al. strategies are needed to remove the bacteria from Bianciotto, V., Bandi, C., Minerdi, D., Sironi, M., the fungal cytoplasm to enable comparisons of Tichy, H.V., and Bonfante, P. (1996). An obligately fungal effects on plants in the presence and endosymbiotic mycorrhizal fungus itself harbors absence of the bacterial symbionts. Despite these obligately intracellular bacteria. Appl. Environ. setbacks, it seems a more common phenomenon Microbiol. 62, 3005-3010. that root endophytic fungi host endobacteria that Bianciotto, V., Genre, A., Jargeat, P., Lumini, E., enhance the fungal fitness. In the cases the Becard, G., and Bonfante, P. (2004). Vertical endofungal bacteria could be isolated and grown in transmission of endobacteria in the arbuscular pure culture they show similar biological activities mycorrhizal fungus Gigaspora margarita through than the fungal hosts itself. This makes it feasible generation of vegetative spores. Appl. Environ. that the fungus gains its full plant-colonizing activity Microbiol. 70, 3600-3608. from its intricate interaction with endobacteria. Bianciotto, V., Lumini, E., Bonfante, P., and Vandamme, P. (2003). Candidatus Glomeribacter Beneficial effect of pure endofungal bacteria also gigasporarum gen. nov., sp. nov.an make them potentially interesting as application as of arbuscular mycorrhizal fungi. Int. J. Syst. Evol. biologicals that could further minimize the use of Microbiol. 53, 121-124. chemical and increase the resistance of Bonfante P., and Anca, I.A. (2009). Plants, crop plants to a wider range of pathogens and pest, mycorrhizal fungi, and bacteria: a network of where the mechanism greatly relays on their direct interactions. Annu. Rev. Microbiol. 63, 363-383. antagonistic activity and their resistance-inducing Bontemps, C., Elliott, G.N., Simon, M.F., Dos Reis, potential. The present knowledge on endobacteria F.B.D., Gross, E., Lawton, R.C. et al. (2010). already shows the requirement towards unraveling a Burkholderia species are ancient symbionts of complex network of interphylum interactions, which . Mol. Ecol. 19, 44-52. is expected to have a previously unrecognized Broek, A.V. and Vanderleyden, J. (1995). The role of ecological impact. bacterial , and attachment in bacteria plant interactions, MPMI. 8, 800-810. References: Budi, S.W., van Tuinen, D., Martinotti, G., and Artursson, V., Finlay, R.D., and Jansson, J.K. Gianinazzi, S. (1999). Isolation from the Sorghum (2006). Interactions between arbuscular bicolor mycorrhizosphere of a bacterium mycorrhizal fungi and bacteria and their potential compatible with for stimulating plant growth. Environ. Microbiol. 8, development and antagonistic towards soilborne 1-10. fungal pathogens. Applied and Environmental Barker, S., and Tagu, D. (2000). The roles of auxins Microbiology 65, 5148-5150. and in mycorrhizal symbioses. Journal Castillo D.M., and Pawlowska, T.E. (2010). of Plant Growth Regulation 19, 144-154. Molecular Evolution in Bacterial Endosymbionts of Becker, D.M, Bagley, S.T., and Podila, G.K. (1999). Fungi. Mol. Biol. Evol. 27, 622-636. Effects of mycorrhiza-associated streptomycetes Cruz, A. F., Horii, S., Ochiai, S., Yasuda, A., and on growth of Laccaria bicolor, Cenococcum Ishii, T. (2008). Isolation and analysis of bacteria geophilum, and species and on gene associated with spores of Gigaspora margarita. J. expression in Laccaria bicolor. Mycologia 91, Appl. Microbiol. 104, 1711-1717. 33-40. Cunnac, S., Occhialini, A., Barberis. P., Boucher, C., Bertaux, J., Schmid, M., Hutzler, P., Hartmann, A., and Genin, S. (2004). Inventory and functional Garbaye, J., and Frey-Klett, P. (2005). analysis of the large Hrp regulon in Ralstonia Occurrence and distribution of endobacteria in the solanacearum: identification of novel effector plant-associated mycelium of the proteins translocated to plant host cells through ectomycorrhizal fungus Laccaria bicolor S238N. the type III secretion system. Molecular Environ. Microbiol. 7, 1786-1795. Microbiology 53, 115-128. Bertaux, J., Schmid, M., Prévost-Bourre, N.C., Défago, G., and Keel, C. (1995). Pseudomonads as Churin, J.L., Hartmann, A., Garbaye, J. and et al. biocontrol agents of diseases caused by soil- (2003). In situ identification of intracellular borne pathogens. pp. 137-148 in Hokkanen HMT, bacteria related to Paenibacillus spp. in the Lynch JM (eds), Biological Control: Benefits and mycelium of the ectomycorrhizal fungus Laccaria Risks. Plant and Microbial Biotechnology bicolor S238N. App. Environ. Microbiol. 69, Research Series 4. Cambridge University Press. 4243-4248. ISNB 9780521544054.

!69 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

Deshmukh, S.D., Hückelhoven, R., Schäfer, P., at the onset of the tripartite Sebacinalean Imani, J., Sharma, M., Weiss, M. and et al. symbiosis with higher plants. Front. Microbiol, (2006). The root endophytic fungus Submitted. Piriformospora indica requires host cell death for Hildebrandt, U., Janetta, K., and Bothe, H. (2002). proliferation during mutualistic symbiosis with Towards growth of arbuscular mycorrhizal fungi barley. Proc. Natl. Acad. Sci. USA. 103, independent of a plant host. Appl. Env. Microbiol. 18450-18457. 68, 1919-1924. Desirò, A., Faccio, A., Kaech, A., Bidartondo, M.I, Hildebrandt, U., Ouziad, F., Marner, F.J, and Bothe, and Bonfante, P. (2015). Endogone, one of the H. (2006). The bacterium Paenibacillus validus oldest plant-associated fungi, host unique stimulates growth of the arbuscular mycorrhizal Mollicutes-related endobacteria. fungus Glomus intraradices up to the formation of 205, 1464-1472. fertile spores. FEMS Microbiol. Lett. 254, 258-67. Desirò, A., Salvioli, A., Ngonkeu, E.L, Mondo, S.J., Hoffman M.T., Gunatilaka M.K., Wijeratne, K., Epis, S., Faccio, A., Kaech, A., Pawlowska, T.E., Gunatilaka, L., and Arnold A.E. (2013). and Bonfante, P. (2014). Detection of a novel Endohyphal bacterium enhances production of intracellular microbiome hosted in arbuscular indole-3-acetic acid by a foliar fungal endophyte. mycorrhizal fungi. ISME. J. 8, 257-270. PLOS ONE 8, e73132. Erlacher, A., Cernava, T., Cardinale, M., Soh, J., Hoffman, M.T., and, Arnold, A.E. (2010). Diverse Sensen, C.W., Grube, M., and Berg, G. (2015). bacteria inhabit living hyphae of phylogenetically Rhizobiales as functional and endosymbiontic diverse fungal endophytes. Appl. Environ. members in the symbiosis of Lobaria Microbiol. 76, 4063-4075. pulmonaria L. Front. Microbiol. 6, 53. Horii, S., and Ishii, T. (2006). Identification and Frey-Klett, P., Chavatte, M., Clausse, M.L., Courrier, function of Gigaspora margarita growth- S., Le Roux, C., Raaijmakers, J., Martinotti, M.G., promoting . Symbiosis 41, Pierrat, J.C., and Garbaye, J. (2005). 135-141. Ectomycorrhizal symbiosis affects functional Horii, S., Matsumura, A., Cruz, A., Ishii, T. (2008). diversity of rhizosphere fluorescent Effect of arbuscular mycorrhizal fungi and their pseudomonads. New Phytol. 165, 317-328. helper microorganisms on root colonization and Frey-Klett, P., Garbaye, J., and Tarkka, M. (2007). growth of trifoliate orange seedlings. Proc. Int. Tansley review: the mycorrhiza helper bacteria Soc. Citriculture 1, 567-571. revisited. New Phytologist 176, 22-36. Jargeat, P., Cosseau, C., Ola'h, B., Jauneau, A., Garbaye, J. (1994). Helper bacteria: a new Bonfante, P., Batut, J., and Becard, G. (2004). dimension to the mycorrhizal symbiosis. New Isolation, free-living capacities, and genome Phytol. 128, 197-209. structure of 'Candidatus Glomeribacter Ghignone, S., Salvioli, A., Anca, I., Lumini, E., Ortu, gigasporarum', the endocellular bacterium of the G., Petiti, L., and et al. (2012). The genome of the mycorrhizal fungus Gigaspora margarita. J. obligate endobacterium of an AM fungus reveals Bacteriol. 186, 6876-6884. an interphylum network of nutritional interactions. Jayasinghearachchi, H.S., and Seneviratne, G. ISME. J. 6, 136-145. (2005). Fungal solubilization of rock phosphate is Glaeser, S.P, Imani, J, Alabid, I., Guo, H.J., Kumar, enhanced by forming fungal-rhizobial biofilms. N., Kämpfer, P., Hardt, M., Blom, J., Rothballer, and Biochemistry 38, 405-408. M., Hartmann, A., and Kogel, K.H. (2016). Non- Kai, K., Furuyabu, K., Tani, A., and Hayashi, H. pathogenic Rhizobium radiobacter F4 deploys (2012). Production of the quorum-sensing plant beneficial activity independent of its host molecules N-acylhomoserine lactones by Piriformospora indica. ISME J. 10, 871-884. endobacteria associated with Mortierella alpina A- Goodner, B., Hinkle, G., Gattung, S., Miller, N., and 178. Chembiochem 13, 1776-1784. Blanchard, M. (2001). Genome sequence of the Kaska, D.D., Myllyla, R. and Cooper, J.B. (1999). plant pathogen and biotechnology agent Auxin transport inhibitors act through ethylene to Agrobacterium tumefaciens C58. Science 294, regulate dichotomous branching of lateral root 2323-2328. in . New Phytologist 142, 49-58. Guo, H.J, Glaeser, S.P, Alabid, I., Imani, J., Keller, S., Schneider, K., and Sussmuth, R.D. Haghighi, H., Kämpfer, P., and Kogel, K.H. (2016). (2006). Structure elucidation of auxofuran, a The incidence of endofungal bacterium Rhizobium metabolite involved in stimulating growth of fly radiobacter (syn. Agrobacterium tumefaciens) agaric, produced by the mycorrhiza helper increases in its fungal host Piriformospora indica

!70 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

bacterium Streptomyces AcH 505. Journal of Moebius, N., Üzüm, Z., Dijksterhuis, J., Lackner, G., Antibiotics (Tokyo) 59, 801-803. and Hertweck, C. (2014). Active invasion of Kloepper, J.W., Rodrguez-kbana, R., Mcinroy, J.A., bacteria into living fungal cells. Elife 3, e03007. and Collins, D.J. (1991). Analysis of populations Mondo, S.J, Salvioli, A., Bonfante, P., Morton, J.B, and physiological characterization of and Pawlowska, T.E. (2016). Nondegenerative microorganisms in of plants with Evolution in Ancient Heritable Bacterial antagonistic properties to phytopathogenic Endosymbionts of Fungi. Mol. Biol Evol. 33, . Plant Soil 136, 95-102. 2216-2231. Kluge, M., Mollenhauer, D., Mollenhauer R., and Moran, N.A., McCutcheon, J.P., and Nakabachi, A. Kape R. (1992). Geosyphon pyriforme, an (2008) .Genomics and evolution of heritable endosymbiotic consortium of a fungus and a bacterial symbionts. Annu. Rev. Genet. 42, cyanobacterium (Nostoc), fixes nitrogen. Bot. 165-190. Acta. 105, 343-344. Moran, N.A. (2006). Symbiosis, Curr. Biol. 16, Kluge, M., Mollenhauer, D., and Mollenhauer, R. R866-R871. (1991). Photosynthetic carbon assimilation in Mosse, B. (1962). The establishment of vesicular- Geosiphon pyriforme (Kützing) F. v. Wettstein, an arbuscular mycorrhiza under aseptic conditions. endosymbiotic association of fungus and Journal of General Microbiology 27, 509-520. cyanobacterium. Planta 185, 311-315. Mosse, B. (1970). Honey-coloured, sessile Lackner, G., Moebius, N., and Hertweck, C. (2011). Endogone spores: II. Changes in fine structure Endofungal bacterium controls its host by an hrp during spore development. Arch. Microbiol. 74, type III secretion system. ISME. J. 5, 252-261. 129-145. Lackner, G., Moebius, N., Partida-Martinez, L.P., Naumann, M., Schüssler, A., and Bonfante, P. Boland, S., and Hertweck, C.(2011b). Evolution of (2010). The obligate endobacteria of arbuscular an endofungal Lifestyle: Deductions from the mycorrhizal fungi are ancient heritable Burkholderia rhizoxinica Genome. BMC components related to the Mollicutes. ISME. J. 4, Genomics 12, 210. 862- 871. Lehr, N.A., Schrey, S.D., Bauer, R., Hampp, R., and Oelmüller, R., Sherameti, I., Tripathi, S., and Varma, Tarkka, M.T. (2007). Suppression of plant defense A. (2009). Piriformospora indica, a cultivable root response by a mycorrhiza helper bacterium. New endophyte with multiple biotechnological Phytol. 174, 892-903. applications. Symbiosis 49, 1-17. Lugtenberg, B.J.J., de Weger, L.A., and Bennett, Partida-Martinez, L.P., and Hertweck, C. (2005). J.W. (1991). Microbial stimulation of plant growth Pathogenic fungus harbours endosymbiotic and protection from disease. Current Opinion in bacteria for toxin production. Nature 437, Biotechnology 2, 457-464. 884-888. Lumini, E., Bianciotto, V., Jargeat, P., Novero, M., Partida-Martinez, L.P., and Hertweck, C. (2007a). A Salvioli, A., Faccio, A., et al. (2007). Presymbiotic Gene Cluster Encoding Rhizoxin Biosynthesis in growth and sporal morphology are affected in the "Burkholderia rhizoxina", the Bacterial arbuscular mycorrhizal fungus Gigaspora Endosymbiont of the Fungus Rhizopus margarita cured of its endobacteria. Cellu. microsporus. ChemBioChem 8, 41-45. Microbiol. 9, 1716-1729. Partida-Martinez, L.P., Groth, I., Schmitt, I., Richter, Maier, A., Riedlinger, J., Fiedler, H.P., and Hampp, W., Roth, M., and Hertweck, C. (2007b). R. (2004). Actinomycetales bacteria from a spruce Burkholderia rhizoxinica and Burkholderia stand: characterization and effects on growth of endofungorum, bacterial endosymbionts of the root symbiotic and plant parasitic soil fungi in dual rice pathogenic fungus Rhizopus microsporus. Int. culture. Mycological Progress 3, 129-136. J. Syst. Evol. Microbiol. 57, 2583-2590. Maor, R., Haskin, S., Levi-Kedmi, H., Sharon, A. Partida-Martinez, L.P., Monajembashi, S., Greulich, (2004). In planta production of indole-3-acetic acid K.O., and Hertweck, C. (2007a). Endosymbiont- by Colletotrichum gloeosporioides f sp dependent host reproduction maintains bacterial- aeschynomene. Appl. Environ. Microbiol. 70, fungal . Curr. Biol. 17, 773-777. 1852-1854. Perotto, S., and Bonfante, P. (1997). Bacterial Meyer, R.J., and Linderman, R.G. (1986). Response associations with mycorrhizal fungi: close and of subterranean clover to dual inoculation with distant friends in the rhizosphere. Trends in vesicular-arbuscular mycorrhizal fungi and plant Microbiology 5, 496-503. growth-promoting rhizobacterium, Pseudomonas Poole, E.J., Bending, G.D., Whipps, J.M., and Read, putida. Soil Biol. Biochem. 18, 185-190. D.J. (2001). Bacteria associated with Pinus

!71 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

sylvestris-Lactarius rufus and Schikora, A., Schenk, S.T, Stein, E., Molitor, A., their effects on mycorrhiza formation in vitro. New Zuccaro, A., and Kogel, K.H. (2011). N-acyl- Phytologist 151, 743-751. homoserine lactone confers resistance towards Qiang, X., Weiss, M., Kogel, K.H., and Schäfer, P. biotrophic and hemibiotrophic pathogens via (2012). Piriformospora indica-a mutualistic altered activation of AtMPK6. Plant Physiol. 157, basidiomycete with an exceptionally large plant 1407-1418. host range. Molecular 13, 508- Schmitt, I., Partida-Martinez, L.P., Winkler, R., Voigt, 518. K., Einax, E., and et al. (2008). Evolution of host Riedlinger, J., Schrey, S.D., Tarkka, M.T., Hampp, resistance in a toxin-producing bacterial-fungal R., Kapur, M., and Fiedler, H.P. (2006). Auxofuran, alliance. ISME. J. 2, 632-641. a novel metabolite that stimulates the growth of fly Schrey, S.D., Schellhammer, M., Ecke, M., Hampp, agaric, is produced by the mycorrhiza helper R., and Tarkka, M.T. (2005). Mycorrhiza helper bacterium Streptomyces strain AcH 505. App. bacterium Streptomyces AcH 505 induces Environ. Microbiol. 72, 3550- 3557. differential gene expression in the ectomycorrhizal Riess, K., Oberwinkler, F., Bauer, R., and Garnica, fungus Amanita muscaria. New Phytologist 168, S. (2014). Communities of endophytic 205-216. sebacinales associated with roots of herbaceous Schuhegger, R., Ihring, A., Gantner, S., Bahnweg, plants in agricultural and grassland ecosystems G., Knappe, C., Vogg, G., Hutzler, P., Schmid, M., are dominated by Serendipita herbamans sp. nov. Van Breusegem, F., Eberl, L., and et al. (2006). PLoS One 9, e94676. Induction of systemic resistance in tomato by N- Salvioli, A., Ghignone, S., Novero, M., Navazio, L., acyl-L-homoserine lactone-producing rhizosphere Venice, F., Bagnaresi, P., and Bonfante. P. (2016). bacteria. Plant Cell Environ. 29, 909-918. Symbiosis with an endobacterium increases the Schüßler, A., Mollenhauer, D., Schnepf, E., and fitness of a mycorrhizal fungus, raising its Kluge, M. (1994). Geosiphon pyriforme, an bioenergetics potential. ISME J. 10, 130-144. endosymbiotic association of fungus and Sato, Z., Noda, T., Matsuda, I., Iwasaki, S., cyanobacteria: the spore structure resembles that Kobayashi, H., and et al. (1983). Studies on of arbuscular mycorrhizal (AM) fungi. Botanica rhizoxin, a phytotoxin produced by Rhizopus Acta. 107, 36-45. chinensis causing rice seedling blight. Annu. Selim, S., Negrel, J., Govaerts, C., Gianinazzi, S., Phytopathol. Soc. Japan 49, 128. and van Tuinen, D. (2005). Isolation and partial Scannerini, S., and Bonfante, P. (1991). Bacteria characterization of antagonistic peptides and bacteria like objects in endomycorrhizal fungi produced by Paenibacillus sp. strain B2 isolated (Glomaceae) in: Margulis, L., Fester, R. (eds) from the Sorghum mycorrhizosphere. Applied and Symbiosis as a source of evolutionary innovation: Environmental Microbiology 71, 6501-6507. speciation and morphogenesis. MIT press, Selosse, M.A., Setaro, S., Glatard, F., Richard, F., Cambridge, Mass, pp273-287. Urcelay, C., and Weiß, M. (2007). Sebacinales are Schelkle, M., and Peterson, R.L. (1996). common mycorrhizal associates of . Suppression of common root pathogens by helper New Phytol. 174, 864-878. bacteria and ectomycorrhizal fungi in vitro. Sharma, M., Schmid, M., Rothballer, M., Hause, G., Mycorrhiza 6, 481-485. Zuccaro, A., Imani, J., Kämpfer, P., Domann, E., Schenk, S.T., Hernández-Reyes, C., Samans, B., Schafer, P., Hartmann, A., and Kogel, K.H. (2008). Stein, E., Neumann, C., Schikora, M., Reichelt, Detection and identification of bacteria intimately M., Mithöfer, A., Becker, A., Kogel, K.H., and associated with fungi of the order Sebacinales. Schikora, A. (2014). N-Acyl-homoserine lactone Cellular Microbiology 10, 2235-2246. primes plants for cell wall reinforcement and Spaepen, S, and Vanderleyden, J. (2011). Auxin induces resistance to bacterial pathogens via the and Plant-Microbe Interactions. Cold Spring Harb salicylic acid/oxylipin pathway. Plant Cell 26, Perspect Biol. 3, a001438. 2708-2723. Vannini, C., Carpentieri, A., Salvioli, A., Novero, M., Scherlach, K., Partida-Martinez, L.P., Dahse, H.M., Marsoni, M., Testa, L., Concetta de Pinto, M., and Hertweck, C. (2006). Antimitotic rhizoxin Amoresano, A., Ortolani, F., Bracale, M., and derivatives from a cultured bacterial Bonfante, P. (2016). An interdomain network: the endosymbiont of the rice pathogenic fungus endobacterium of a mycorrhizal fungus promotes Rhizopus microsporus. J. Am. Chem. Soc. 128, antioxidative responses in both fungal and plant 11529-11536. hosts. New Phytol. 211, 265-275.

!72 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

Verma, S., Varma, A., Rexer, K.H., Hassel, A., Kost, everywhere: previously overlooked ubiquitous G., and Sarabhoy, A. (1998). Piriformospora fungal endophytes. PLoS One 108, 1003-1010. indica, gen. et sp. nov., a new root-colonizing Weller, D.M. (1988). Biological control of soilborne fungus. Mycologia 90, 896-903. plant pathogens in the rhizosphere with bacteria. Walker, V., Bruto, M., Bellvert, F., Bally, R., Muller, Annual Review of Phytopathology 26, 379-407. D., Prigent-Combaret, C., Moënne- Loccoz, Y., Zarkani, A.A., Stein, E., Rohrich, C.R., Schikora, M., and Comte, G. (2013). Unexpected phyto- Evguenieva-Hackenberg, E., Degenkolb, T., and stimulatory behavior for Escherichia coli and et al. (2013). Homoserine lactones influence the Agrobacterium tumefaciens model strains. Mol. reaction of plants to . Int. J. Mol. Sci. 14, Plant Microbe Interact. 26, 495-502. 17122-17146. Weiss, M., Selosse, M.A., Rexer, K.H., Urban, A., and Oberwinkler, F. (2011). Sebacinales

!73 Impact of endofungal bacteria in fungus-plant interactions Alabid et al.

!74