Michael J. Christensen

Reactive Oxygen Species Play a Role in Regulating a Fungus–Perennial Ryegrass Mutualistic Interaction

Although much is known about the signals and mechanisms that lead to pathogenic interactions between plants and fungi, comparatively little is known about fungus–plant mutualistic symbioses. We describe a novel role for reactive oxygen species (ROS) in regulating the mutualistic interaction between a clavicipitaceous fungal endophyte, Epichloë festucae, and its grass host, Lolium perenne. In wild-type associations, E. festucae grows systemically in intercellular spaces of leaves as infrequently branched hyphae parallel to the leaf axis. A screen to identify symbiotic genes isolated a fungal mutant that altered the interaction from mutualistic to antagonistic. This mutant has a single-copy plasmid insertion in the coding region of a NADPH oxidase gene, noxA. Plants infected with the noxA mutant lose apical dominance, become severely stunted, show precocious senescence, and eventually die. The fungal biomass in these associations is increased dramatically, with hyphae showing increased vacuolation. Deletion of a second NADPH oxidase gene, noxB, had no effect on the E. festucae–perennial ryegrass symbiosis. ROS accumulation was detected cytochemically in the endophyte extracellular matrix and at the interface between the extracellular matrix and host cell walls of meristematic tissue in wild-type but not in noxA mutant associations. These results demonstrate that fungal ROS production is critical in maintaining a mutualistic fungus–plant interaction.

Taxonomy of Acremonium endophytes of tall fescue (Festuca arundinacea), meadow fescue (F. pratensis) and perennial ryegrass (Lolium perenne)

Isozyme analysis, morphological characters, sensitivity to benomyl in vitro, and the production of alkaloids in the fungal-host-grass associations were used to examine the relationships between Acremonium endophytes of Festuca arundinacea, F. pratensis and Lolium perenne. Isozyme analysis identified six taxonomic groupings, with each grouping comprising isolates from just one of the three grass species. Three taxonomic groupings occurred in F. arundinacea, two in L. perenne, and one in F. pratensis. Alkaloid production in the natural host grass-Acremonium associations proved a useful taxonomic criterion, with the profile of alkaloids being consistent in the host plants for all isolates within a single isozyme phenotype and for most isolates within a taxonomic grouping. On the basis of morphological, cultural and secondary product criteria, only one of the three taxonomic groupings identified amongst the F. arundinacea endophytes fitted the definition of A. coenophialum, and only one grouping of L. perenne endophytes could be accommodated within A. lolii. All isolates from F. pratensis could be accommodated within A. uncinatum even though the conidia differed between isolates of the two isozyme phenotypes identified during this study.

The evolutionary origins of Epichloe endophytes from annual ryegrasses

The annual Lolium (ryegrass) species were surveyed for seedborne, asexual Epichloe endo- phytes (i.e., Neotyphodium spp.). Endophytes that were extremely slow growing in culture were identi- fied from all seven annual ryegrass spp. Sequences of ,3-tubulin gene (tub2) introns and the internal tran- scribed spacers of the nuclear ribosomal RNA (rDNA- ITS) were determined, as were profiles based on five microsatellite loci. Two taxonomic groups of annual ryegrass endophytes were distinguishable by mor- phology, culturability and genotypes. One group, for which the name Neotyphodium occultans is proposed, comprised only isolates that could not be subcultured on potato dextrose agar medium. These isolates con- tained multiple tub2 genes and multiple alleles at mi- crosatellite loci, suggestive of an interspecific hybrid origin. The results of phylogenetic analysis implied that the ancestors of N. occultans included Epichloe baconii and E. bromicola, or close relatives. The sec- ond taxonomic group of annual ryegrass endophytes, exemplified by isolate Lc4, was found only in L. can- ariense. This taxon had a single tub2 sequence and microsatellite genotype that was nearly identical to those of extant Epichloi typhina isolates that infect Poa nemoralis. Therefore, it is proposed that Lc4 is an asexual derivative of an E. typhina genotype close- ly related to extant strains from P nemoralis. We pro- pose the name Neotyphodium typhinum var. canariense for this taxon. These two new endophyte taxa from the annual ryegrasses are further examples of asexual Neotyphodium species that have arisen from sexual Ep-

Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass

Lolitrems are potent tremorgenic mycotoxins that are synthesised by clavicipitaceous fungal endophytes of the Epichloë/Neotyphodium group in association with grasses. These indole–diterpenes confer major ecological benefits on the grass–endophyte symbiotum. A molecular signature for diterpene biosynthesis is the presence of two geranylgeranyl diphosphate (GGPP) synthases. Using degenerate primers for conserved domains of fungal GGPP synthases, we cloned two such genes, ltmG and ggsA, from Neotyphodium lolii. Adjacent to ltmG are two genes, ltmM and ltmK, that are predicted to encode an FAD-dependent monooxygenase and a cytochrome P450 monooxygenase, respectively. The cluster of ltm genes is flanked by AT-rich retrotransposon DNA that appears to have undergone extensive repeat induced point (RIP) mutation. Epichloë festucae, the sexual ancestor of N. lolii, contains an identical ltm gene cluster, but lacks the retrotransposon “platform’‘ on the right flank. Associations established between perennial ryegrass and an E. festucae mutant deleted for ltmM lack detectable levels of lolitrems. A wild-type copy of ltmM complemented this phenotype, as did paxM from Penicillium paxilli. Northern hybridization and RT-PCR analysis showed that all three genes are weakly expressed in culture but strongly induced in planta. The relative endophyte biomass in these associations was estimated by real-time PCR to be between 0.3 and 1.9%. Taking this difference into account, the steady-state levels of the ltm transcripts are about 100-fold greater than the levels of the endogenous ryegrass β-tubulin (β -Tub1) and actin (Act1) RNAs. Based on these results we propose that ltmG, ltmM and ltmK are members of a set of genes required for lolitrem biosynthesis in E. festucae and N. lolii.

Growth of Epichloe/Neotyphodium and p-endophytes in leaves of Lolium and Festuca grasses

Epichloe spp. (Clavicipitaceae) and their close asexual relatives, Neotyphodium spp., form systemic endophytic associations with Pooideae grasses. Interactions between Lolium and Festuca host grasses and fungal endophytes were examined in studies focusing on leaves of natural associations and also of plants into which the endophyte was introduced by seedling inoculation. Light microscopy as well as fungal isolation was used to locate the position of hyphae, while transmission electron microscopy was used to examine host/fungus interactions at the cellular level. These studies provide support for synchronised plant and endophyte growth. This characteristic pattern of growth was maintained when both Epichloe and Neotyphodium spp. were introduced by seedling inoculation into new host species. Hyphae, with few exceptions, grew rapidly as leaves grew and ceased when leaf growth ceased. This pattern of growth offers an explanation for the characteristic appearance of hyphae in leaf sheaths of host grasses; seldom branched and for all species other than N. occultans, aligned parallel to the leaf axis. Hyphal growth of a second group of endophytic fungi, referred to as p-endophytes, was not regulated in the same way, with growth continuing as leaves aged. This pattern of growth gives rise to high concentrations of branched ramifying hyphae in old leaf sheaths. Although host genotype did not effect the basic pattern of hyphal growth of Epichloe/Neotyphodium endophytes, it strongly influenced the concentration and distribution of hyphae throughout leaves. Examination by TEM revealed no evidence that penetration of hyphae into dense tissue was aided by the secretion of pectic enzymes that loosen the middle lamella connecting host cells. Instead it appeared that penetration occurred by hyphae physically pushing between cells. The absence of enzymatic loosening of the middle lamella during penetration could explain why hyphae of these endophytes apparently do not elicit host defence reactions in natural associations. However, subtle interactions between the hyphae of Epichloe/Neotyphodium and host cells in natural associations were observed.

In planta regulation of extension of an endophytic fungus and maintenance of high metabolic rates in its mycelium in the absence of apical extension.

ABSTRACT The fungus Neotyphodium lolii is an endophytic symbiont. It grows in the intercellular spaces of the perennial ryegrass Lolium perenne, producing secondary metabolites which enhance the fitness of the association over that of uninfectedL. perenne. We report that the average number of hyphal strands in a given section of a leaf remains constant during the life of a leaf, indicating synchrony of leaf and hyphal extension, including cessation of hyphal extension when leaf extension ceases. We used a constitutively expressed reporter gene as an indicator of the myceliums metabolic activity during and after hyphal extension. Reporter gene activity decreased when the mycelium stopped extending in liquid culture but not in planta. This indicates that in planta endophyte hyphae remain metabolically highly active when extension has ceased and throughout the life of the leaf they are colonizing. The behavior of the fungus in planta indicates the existence of signaling pathways which (i) synchronize the extension of leaf and hypha by regulating hyphal extension, (ii) suppress hyphal branching, and (iii) stop apical extension of fungal hyphae, without reducing the myceliums metabolic activity. These signals may be crucial for the symbiosis, by allowing the endophyte to switch the focus of its metabolic activity from extension to the production of secondary metabolites.

Variation in the ability of Acremonium endophytes of Lolium perenne, Festuca arundinacea and F. pratensis to form compatible associations in the three grasses

The ability of representative isolates of six recognized taxonomic groups (TGs) of Acremonium endophytes from perennial rye-grass, tall fescue and meadow fescue to form compatible associations following inoculation of seedlings of the three grasses was examined. The six TGs were not specific to their natural hosts and most representative isolates formed associations with all three host species. Incompatibility resulting in hyphal death was noted in A. coenophialum -perennial rye-grass associations. Incompatibility affecting host tissue, resulting in stunted tillers with necrosis in the region of the apical meristem, was present in associations of tall fescue with either A. uncinatum or LpTG-2 ( Lolium perenne TG-2). Some associations were unstable with endophyte-free tillers being produced as plants developed. This instability was not confined to endophyte-grass associations showing either of the two types of histological incompatibility reactions. The compatibility of endophyte-grass associations was not related to the percentage of successfully inoculated seedlings obtained. This study provides further evidence that the association between Acremonium endophytes and their host grasses is complex.

Fungal and host genotype effects on compatibility and vascular colonization by Epichloë festucae

Parental and progeny isolates of the endophytic fungus Epichloe festucae were tested for compatibility with five grass species: Lolium perenne, Festuca arundinacea, F. longifolia, F. pratensis , and F. rubra subsp. rubra . One parental and some progeny isolates adversely affected the growth of plants, causing increased mortality of inoculated seedlings, stunted growth and chlorotic leaf symptoms. Growth of hyphae from surface-sterilized leaf sheaths and blades was more vigorous and concentrated from stunted than from symptomless plants. In addition, the growth of isolates was influenced by the host species, whereby estimates of hyphal concentration were consistently highest in meadow fescue associations and lowest in tall fescue associations. Light microscopic and transmission electron microscopic examination of stunted plants indicated no changes in host cells. Intercellular hyphae were observed within vascular bundles of leaf blades and sheaths of many of the endophyte–grass associations. The frequency of infected vascular bundles and the concentration of hyphae within them was highest in plants infected with the stunting parental isolate and lowest in plants infected with the symptomless parental isolate. Hyphae within vascular bundles-were typically in close contact with sieve-tube elements and appeared to be functioning as powerful sinks, diverting assimilates and restricting growth.

Incompatibility of some grass−Acremonium endophyte associations

During a long-term project to create novel Acremonium-grass associations through inoculation of seedlings it was noted that the leaf sheaths of Lolium perenne plants inoculated with some isolates of A. coenophialum contained distorted hyphae lacking in cytoplasmic inclusions. Host-endophyte interactions within leaf sheaths were examined using some of these plants, together with natural L. perenne-A. lolii associations, and some novel L. perenne-Acremonium associations in which the hyphae appeared normal. Vital staining and attempts to isolate fungi from leaf sheaths of varying ages revealed that most hyphae in the second and third leaf sheaths of the L. perenne-A. coenophialum associations were dead. Transmission electron microscopy showed that the hyphae had become distorted and collapsed, with degenerated cytoplasm and cell walls which were electron dense and lacked a fibrous outer layer found in most other Acremonium-grass associations. No hypersensitive reactions were observed within the host cells surrounding the degenerated hyphae. However, the intercellular matrix which maintained contact between host cell walls and hyphae had become electron dense. The changes to the intercellular matrix may be accompanied by a decrease in permeability and nutritional value, or both, of the matrix which could have caused the apparent incompatibility observed in this study.

Variation within isolates of Acremonium endophytes from perennial rye-grasses

Endophytes from perennial rye-grass ( Lolium perenne ) infected with fungi having intercellular mycelium characteristic of Acremonium lolii exhibited wide variation in cultural, morphological and physiological characteristics. Most isolates could be accommodated within the taxon A. lolii , although they were unstable in culture and had greater variation than previously described. Some, however, resembled A. typhinum , the anamorph of Epichloe typhina , but symptoms of choke disease were not observed. The variation observed within isolates indicates the need for reappraisal of the taxonomy of Acremonium sect. Albo-lanosa .