Regulation of a Chemical Defense against Herbivory Produced by Symbiotic Fungi in Grass Plants

Neotyphodium uncinatum and Neotyphodium siegelii are fungal symbionts (endophytes) of meadow fescue (MF; Lolium pratense), which they protect from insects by producing loline alkaloids. High levels of lolines are produced following insect damage or mock herbivory (clipping). Although loline alkaloid levels were greatly elevated in regrowth after clipping, loline-alkaloid biosynthesis (LOL) gene expression in regrowth and basal tissues was similar to unclipped controls. The dramatic increase of lolines in regrowth reflected the much higher concentrations in young (center) versus older (outer) leaf blades, so LOL gene expression was compared in these tissues. In MF-N. siegelii, LOL gene expression was similar in younger and older leaf blades, whereas expression of N. uncinatum LOL genes and some associated biosynthesis genes was higher in younger than older leaf blades. Because lolines are derived from amino acids that are mobilized to new growth, we tested the amino acid levels in center and outer leaf blades. Younger leaf blades of aposymbiotic plants (no endophyte present) had significantly higher levels of asparagine and sometimes glutamine compared to older leaf blades. The amino acid levels were much lower in MF-N. siegelii and MF-N. uncinatum compared to aposymbiotic plants and MF with Epichloë festucae (a closely related symbiont), which lacked lolines. We conclude that loline alkaloid production in young tissue depleted these amino acid pools and was apparently regulated by availability of the amino acid substrates. As a result, lolines maximally protect young host tissues in a fashion similar to endogenous plant metabolites that conform to optimal defense theory.Loline alkaloids (LAs; Hofmeister, 1892; Siegel et al., 1990; TePaske et al., 1993; Blankenship et al., 2001) are protective secondary metabolites produced by some Epichloë and Neotyphodium spp. (epichloae), fungi that live as systemic symbionts in many cool season grasses (Poaceae subfamily Pooideae). The lolines are active against a broad spectrum of insects (Schardl et al., 2007) and are derived from l-Pro (Pro) and l-homoserine (Hse; Blankenship et al., 2005). Mock herbivory (clipping plants) is reported to induce higher levels of lolines in several grass-epichloë symbiota (Craven et al., 2001; Bultman et al., 2004; Gonthier et al., 2008), suggesting that the epichloae have evolved to regulate their metabolism in a manner appropriate for defense of their hosts. However, little is known of the regulation of LA synthesis in symbio and whether these symbionts follow prevailing models for how plants deploy chemical defenses against herbivores (McKey, 1979; Rhoades, 1979; Barto and Cipollini, 2005).The loline-alkaloid biosynthesis (LOL) gene cluster contains nine genes likely to direct LA production (Spiering et al., 2005). Neotyphodium uncinatum contains two highly similar LOL clusters (LOL1 and LOL2), and a single LOL cluster has been found in each of the LA-producing species, Neotyphodium coenophialum, Neotyphodium siegelii, and some strains of Epichloë festucae, among others (Spiering et al., 2005; Kutil et al., 2007). Fermentation cultures of N. uncinatum produce lolines, and studies involving application of labeled precursors and intermediates have almost completely elucidated the LA biosynthetic pathway (Blankenship et al., 2005; Spiering et al., 2005; Faulkner et al., 2006; Schardl et al., 2007). Putative roles of the LOL gene products—based on sequence relationships to known enzyme classes—fit well with the pathway. Furthermore, an RNA interference knockdown of lolC reduces LA levels, and a lolP knockout prevents conversion of N-methylloline to N-formylloline (Spiering et al., 2005, 2008). Expression kinetics of the LOL genes are tightly correlated with each other and with the LA production phase in N. uncinatum cultures (Zhang et al., 2009). This finding raises the question whether and how LOL gene expression in symbio relates to changes in LA levels in response to development and stresses in host plants.LA production in symbio may be influenced by physiological differences among plant tissues and developmental stages, as well as differences in nutritional status and environmental stresses (Kennedy and Bush, 1983; Belesky et al., 1987; Justus et al., 1997; Tong et al., 2006). Given the anti-insect activity of lolines, effects of plant damage on LA levels are of particular interest. Mock herbivory (clipping of leaves) leads to apparent increases in LA concentrations in regrowth tissues of tall fescue (TF; Lolium arundinaceum) symbiotic with N. coenophialum (Bultman et al., 2004; Sullivan et al., 2007) and of meadow fescue (MF; Lolium pratense) symbiotic with N. uncinatum or N. siegelii (Craven et al., 2001). Despite the higher LA levels, however, clipping or damage of TF-N. coenophialum by the herbivore Spodoptera frugiperda (fall armyworm) was reported to elicit only minor, marginally significant (P = 0.052) effects on expression of lolC (Sullivan et al., 2007). A study of the Glyceria striata-Epichloë glyceriae symbiotum demonstrated significantly higher expression of lolC and higher LA production when the grass was artificially damaged, whereas the effect of damage by S. frugiperda on LA concentrations and lolC expression was not significant (Gonthier et al., 2008).Prevailing concepts about how plants deploy chemical defenses include the optimal defense theory (ODT; McKey, 1979; Rhoades, 1979) and the growth differentiation balance hypothesis (GDBH; Barto and Cipollini, 2005). The ODT addresses the distribution of chemical defenses in the plant, predicting that such defenses will be concentrated in tissues that have relatively little means to physically inhibit herbivory (e.g. in young tissues) and are important in the fitness of the plant. The GBDH addresses the location of biosynthesis and predicts that mature tissues are more likely to produce secondary metabolites than are actively growing tissues, which instead need to use resources for biomass production. It is intriguing to consider whether the epichloae obey the predictions of ODT and GDBH, considering that many epichloae protect their hosts by synthesizing insecticidal alkaloids, but they are also evolutionarily derived from plant-pathogenic fungi (Moon et al., 2004) and do not always enhance host fitness (Faeth et al., 2004). In order to address these questions, it is necessary to understand how secondary metabolism of the epichloae is regulated in symbio. The production of lolines in MF-N. uncinatum and MF-N. siegelii is an ideal test case because the lolines accumulate to very high levels—up to 1.9% dry weight—in regrowth of clipped plants (Craven et al., 2001). Here, we test the hypotheses that LOL gene expression and substrate availability correlate with LA levels in younger versus older leaf tissues and in response to clipping in MF-N. uncinatum and MF-N. siegelii symbiota.     

Distribution of NRPS gene families within the Neotyphodium/Epichloë complex

Neotyphodium and Epichloë spp are closely related asexual and sexual endophytic fungi, respectively, that form mutualistic associations with cool season grasses of the subfamily Pooideae. The endophytes confer a number of advantages to their hosts, but also can cause animal toxicoses and these effects are, in many cases, due to the production of fungal secondary metabolites. In filamentous fungi, secondary metabolite genes are commonly clustered and, for those pathways involved in non-ribosomal peptide synthesis, a non-ribosomal peptide synthetase (NRPS) gene is always found as a key component of the cluster. Members of this gene family encode large multifunctional enzymes that synthesize a diverse range of bioactive compounds and in numerous cases have been shown to serve as pathogenicity or virulence factors, in addition to suggested roles in niche adaptation. We have used a degenerate PCR approach to identify members of the NRPS gene family from symbiotic fungi of the Neotyphodium/Epichloë complex, and have shown that collectively, at least 12 NRPS genes exist within the genomes examined. This suggests that secondary metabolites are important during the life cycles of these fungi with their hosts. Indeed, both the ergovaline and peramine biosynthetic pathways, which confer competitive abilities to Neotyphodium and Epichloë symbioses, contain NRPS genes at their core. The distribution of these genes among different Neotyphodium/Epichloë lineages suggests that a common ancestor contributed most of the complement of NRPS genes, which have been either retained or lost during the evolution of these fungi.     

Orthologous peramine and pyrrolopyrazine-producing biosynthetic gene clusters in Metarhizium rileyi,Metarhizium majus and Cladonia grayi

Peramine is a non-ribosomal peptide-derived pyrrolopyrazine (PPZ)-containing molecule with anti-insect properties. Peramine is known to be produced by fungi from genus Epichloë, which form mutualistic endophytic associations with cool-season grass hosts. Peramine biosynthesis has been proposed to require only the two-module non-ribosomal peptide synthetase (NRPS) peramine synthetase (PerA), which is encoded by the 8.3 kb gene perA, though this has not been conclusively proven. Until recently, both peramine and perA were thought to be exclusive to fungi of genus Epichloë; however, a putative perA homologue was recently identified in the genome of the insect-pathogenic fungus Metarhizium rileyi. We use a heterologous expression system and a hydrophilic interaction chromatography-based analysis method to confirm that PerA is the only pathway-specific protein required for peramine biosynthesis. The perA homologue from M. rileyi (MR_perA) is shown to encode a functional peramine synthetase, establishing a precedent for distribution of perA orthologs beyond genus Epichloë. Furthermore, perA is part of a larger seven-gene PPZ cluster in M. rileyi, Metarhizium majus and the stalked-cup lichen fungus Cladonia grayi. These PPZ genes encode proteins predicted to derivatize peramine into more complex PPZ metabolites, with the orphaned perA gene of Epichloë spp. representing an example of reductive evolution.     

Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid

A convenient method to remove selectable markers from fungal transformants permits the markers to be used for sequential transformations, and should also reduce public concerns and regulatory impediments to applications involving environmental release of genetically modified fungi. We report a method for marker removal that requires no genetic selection. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum and Epichloë festucae transformants containing a hygromycin B phosphotransferase gene (hph) flanked by loxP sites in direct orientation were transiently transfected with a Cre-recombinase expression plasmid, and then cultured without selection. The marker was eliminated in 0.5–2% of the colonies, leaving a single loxP sequence and no other exogenous DNA in the genome. This approach was also applied to the yA gene of Aspergillus nidulans as a laboratory exercise to demonstrate multiple principles of transformation and genome manipulation. Thus, the Cre-expression plasmid and transient transfection approach was rapid, flexible and useful for diverse filamentous fungi.     

A complex gene cluster for indole-diterpene biosynthesis in the grass endophyte Neotyphodium lolii

Lolitrems are a structurally diverse group of indole-diterpene mycotoxins synthesized by Epichloë/Neotyphodium endophytes in association with Pooid grasses. Using suppression subtractive hybridization combined with chromosome walking, two clusters of genes for lolitrem biosynthesis were isolated from Neotyphodium lolii, a mutualistic endophyte of perennial ryegrass. The first cluster contains five genes, ltmP, ltmQ, ltmF, ltmC, and ltmB, four of which appear to be orthologues of functionally characterized genes from Penicillium paxilli. The second cluster contains two genes, ltmE and ltmJ, that appear to be unique to lolitrem biosynthesis. The two clusters are separated by a 16 kb AT-rich sequence that includes two imperfect direct repeats. A previously isolated ltm cluster composed of ltmG, ltmM, and ltmK, is linked to these two new clusters by 35 kb of AT-rich retrotransposon relic sequence. All 10 genes at this complex LTM locus were highly expressed in planta but expression was very low or undetectable in mycelia. ltmM and ltmC were shown to be functional orthologues of P. paxilli paxM and paxC, respectively. This work provides a genetic foundation for elucidating the metabolic grid responsible for the diversity of indole-diterpenes synthesized by N. lolii.     

Sympatric Epichloë species and chemotypic profiles in natural populations of Lolium perenne

The distribution of different Epichloë species within eight natural populations of Lolium perenne was studied. In total, 40.2% of the asymptomatic plants were infected by Epichloë festucae var. lolii or by Epichloë typhina. Both species occurred in sympatry in seven grass populations, and some plants had dual infections by both taxa. No hybrid taxa such as Epichloë hybrida were detected. Epichloë festucae strains were classified into two morphotypes, M1 and M3, according to culture characters, both morphotypes occurred in sympatry in seven populations. Plants bearing stromata produced by Epichloë typhina were observed, but asymptomatic plants infected by this species also occurred in seven populations. The alkaloid profile of Lolium perenne plants was related to the morphotype of their infecting strains: most plants infected by M3-strains were characterized by lolitrem, and those with M1-strains contained either ergovaline or lolitrem. Plants infected by Epichloë typhina were characterized by high peramine content.     

Identification of extracellular siderophores and a related peptide from the endophytic fungus Epichloë festucae in culture and endophyte-infected Lolium perenne

A number of genes encoding non-ribosomal peptide synthetases (NRPSs) have been identified in fungi of Epichloë/Neotyphodium species, endophytes of Pooid grasses, including sidN, putatively encoding a ferrichrome siderophore-synthesizing NRPS. Targeted gene replacement and complementation of sidN in Epichloë festucae has established that extracellular siderophore epichloënin A is the major product of the SidN enzyme complex (Johnson et al., 2007a). We report here high resolution mass spectrometric fragmentation experiments and NMR analysis of an isolated fraction establishing that epichloënin A is a siderophore of the ferrichrome family, comprising a cyclic sequence of four glycines, a glutamine and three N^δ-trans-anhydromevalonyl–N^δ-hydroxyornithine (AMHO) moieties. Epichloënin A is unusual among ferrichrome siderophores in comprising an octapeptide rather than hexapeptide sequence, and in incorporating a glutamine residue. During this investigation we have established that desferrichrome siderophores with pendant trans-AMHO groups can be distinguished from those with pendant cis-AMHO groups by the characteristic neutral loss of an hydroxyornithine moiety in the MS/MS spectrum. A minor component, epichloënin B, has been characterized as the triglycine variant by mass spectrometry. A peptide characterized by mass spectrometry as the putative deoxygenation product, epichloëamide has been detected together with ferriepichloënin A in guttation fluid from ryegrass (Lolium perenne) plants infected with wild-type E. festucae, but not in plants infected with the ΔsidN mutant strain, and also detected at trace levels in wild-type E. festucae fungal culture.     

<Emphasis Type="Italic">Heteroepichloë</Emphasis>, gen. nov. (Clavicipitaceae; Ascomycotina) on bamboo plants in East Asia

The causal agents of witches' broom of bamboo plants in East Asia, Epichloë bambusae and E. sasae, were morphologically and phylogenetically examined. The phylogenetic studies were conducted using ITS 1, 2, and 5.8 S rDNA regions. Both Epichloë species produce Ephelis-type conidia in artificial medium and are phylogenetically situated in different clades from Epichloë and Parepichloë. Here, we propose a new genus Heteroepichloë for these two bambsicolous Epichloë species.     

The effect of duplications in the T-DNA on the stability of manifestation of heterologous genes in transgenic tobacco plants

The duplication of uidA gene within T-DNA was shown to disturb stability of expression of another marker gene, nptII, in the second generation (T₂) of selfed initial transformants and in F₁ hybrids of the crosses with nontransgenic tobacco. Hybridological analysis of the progeny resulting from various crosses involving T₁ plants demonstrated that the expression of nptII gene was impaired in the hybrids that were hemizygous for the inactivated copy of uidA gene.     

Epichloë endophyte affects the root colonization pattern of belowground symbionts in a wild grass

Plants host multiple symbionts that interact with each other affecting plant performance and regulating their establishment. Here, we analyzed how the association with Epichloë endophytes affects belowground colonization by Dark Septate Endophytes (DSE) and arbuscular mycorrhizal fungi (AMF) in the grass Bromus auleticus. Epichloë-symbiotic (E+) and Epichloë-non symbiotic (E−) plants were sampled from a long-term experimental plot and colonization structures were analyzed in the roots. We also examined the influence of Epichloë exudates on the in vitro growth of DSE Microdochium bolleyi isolated from roots. Epichloë symbiosis increased AMF colonization, although differences were not significant. Despite the lack of differences in total DSE colonization, in concordance with in vitro findings, a higher significant abundance of microsclerotia was observed in E+ plants. A negative correlation between total mycorrhizal and DSE was found. Our findings show a more uniform root colonization pattern in E+ plants, suggesting a root symbiosis modulating role.     

The role of host-specificity in the reproductive isolation of Epichloë endophytes revealed by reciprocal infections

Speciation in sexually reproducing organisms hinges on reproductive barriers that reduce gene flow between species or preclude the formation of hybrids. Here, we studied potential reproductive barriers in four members of the Epichloë typhina (Ascomycota, Clavicipitaceae) complex, i.e. Epichloë typhina infecting Dactylis glomerata, E. typhina subsp. clarkii infecting Holcus lanatus, E. typhina subsp. poae infecting Poa nemoralis and E. typhina infecting P. trivialis. Reciprocal inoculation tests showed that these endophytes are host-specific. This suggests that reproductive isolation among Epichloë strains may be the result of specialization to one host, on which mating between different individuals occurs. Furthermore, significantly lower infection frequencies of F1 progeny from crosses between host-strains compared to parental strains and within host-strain progeny suggest that host-dependent effects upon hybrid fitness exist, which would conform to an extrinsic postzygotic isolation barrier. Our results may explain, why members of the E. typhina complex remain genetically differentiated in natural populations.     

Chemotypic diversity of epichloae,fungal symbionts of grasses

The epichloid fungi – comprising sexual Epichloë species and asexual Neotyphodium species – are symbionts of cool-season grasses (subfamily Poöideae), mostly vertically transmissible (seedborne), and well known for production of anti-herbivore alkaloids. Four classes of alkaloids are known to be produced by epichloae: lolines (saturated aminopyrrolizidines), indole–diterpenes, ergot alkaloids, and peramine. There is a wide range of chemotypic diversity among and even within epichloid species. At the molecular level, this diversity may in part reflect the telomeric association of two of the four alkaloid biosynthesis gene clusters. Ecologically, the chemotypic diversity within species may reflect frequency-dependent selection for the alkaloids, which provide defences against insects and, in some cases, vertebrates, but can be expensive to produce. Interspecific hybridization, common among asexual epichloae, can pyramid the alkaloid biosynthesis genes. Compared to sexual epichloae, many asexual epichloae produce high levels of alkaloids – particularly lolines – suggesting that strict vertical transmission selects for enhanced capability of host protection.     

Botanophila flies on Epichloë host species in Europe and North America: no evidence for co-evolution

Species of Epichloë (Ascomycota: Clavicipitaceae), which infect grasses, maintain an obligate symbiotic relationship with flies of the genus Botanophila (Diptera: Anthomyiidae). Sexual reproduction of the fungus usually requires a visit by Botanophila, which serves to ‘pollinate’ the fungus with spermatia of the opposite mating type; the flies in turn deposit their eggs on fungal tissues upon which the larvae feed. For a molecular phylogenetic study, a total of 108 fly larvae were collected from 10 different Epichloë species on various grasses in Europe and North America. Sequence analysis of the mitochondrial cytochrome oxidase gene (COII) detected six distinct Botanophila taxa that were associated with Epichloë. Three were restricted to samples from Europe, two to samples from North America, and one was present on both continents. In North America the common taxon [identified as Botanophila lobata (Collin)] was found on Epichloë hosts of native grasses and on Epichloë typhina (Pers.: Fr.) Tul. and Epichloë festucae Leuchtm., Schardl and M.R. Siegel of the introduced grasses Dactylis glomerata L. and Festuca rubra L., respectively. In a maximum likelihood phylogram, one of the American taxa was most basal, while the other was nested within the European taxa, suggesting that European and American taxa of Epichloë-associated Botanophila spp. may not have separate origins. Analyses of the fly populations further indicated that up to four different Botanophila taxa can be present at a single location. On taxon level there was no specificity of flies for Epichloë host species (except taxon 6 that was found only on Epichloë glyceriae Schardl & Leuchtmann), although several taxa appeared to prefer some hosts over others. Comparison of molecular phylogenetic relationships of Botanophila species with those of the associated Epichloë hosts did not suggest co-evolution of the fungus and the fly. Thus, associations between Botanophila flies and Epichloë hosts may have arisen independently more than once.     

Functional analysis of an indole-diterpene gene cluster for lolitrem B biosynthesis in the grass endosymbiont Epichloë festucae

Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole-diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub-telomeric locus in the genome of E. festucae. Here we resolve the genetic basis for the remarkable indole-diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.