The wheat sheath miner, Cerodontha australis Malloch (Diptera: Agromyzidae), is a small leaf- and stem-mining fly that is native to New Zealand and eastern Australia where it feeds on grasses and cereals. Although not considered to be a major insect pest of pastures in New Zealand, it can be abundant during spring and summer in Waikato, New Zealand, and infests tillers of the introduced pasture plant, perennial ryegrass, Lolium perenne L. (Poaceae). Cerodontha australis is not deterred from feeding on L. perenne infected with commercially available strains of the fungal endophyte Epichloe festucae var. lolii, which have been shown to provide protection from a range of other insect pests. In this study, three-pot trials and periodic examination over 18 months of field-collected L. perenne tillers showed that two Epichloe endophyte strains, AR47 and AR48, reduced the survival and feeding damage by C. australis larvae, but there was no evidence of an effect on adult female feeding or oviposition. Compared to other strains of E. festucae var. lolii endophytes tested, plants infected with AR47 and AR48 had fewer and smaller larvae and less larval mining. The number of tillers with mining was reduced in AR47 relative to endophyte-free (Nil) by 57.1% (ranging from 25.0 to 92.5% across trials) and in AR48 by 67.1% (26.3-100%). Larvae in AR47 and AR48 were found further from the base of the tiller where they were less likely to damage the meristem and cause tiller death. The number of larvae and pupae in pseudostems was reduced in AR47 and AR48 compared with Nil by 78.4 and 82.0%, respectively, in the field trial. Very few larvae survived to pupation in AR47 and AR48 plants, with a reduction compared to Nil of over 81.5% in AR47 and over 97% in AR48 in each of the four trials presented here.

Abstract Fungi from the genus Epichloë form systemic endobiotic infections of cool season grasses, producing a range of host-protective natural products in return for access to nutrients. These infections are asymptomatic during vegetative host growth, with associations between asexual Epichloë spp. and their hosts considered mutualistic. However, the sexual cycle of Epichloë spp. involves virulent growth, characterized by the envelopment and sterilization of a developing host inflorescence by a dense sheath of mycelia known as a stroma. Microscopic analysis of stromata revealed a dramatic increase in hyphal propagation and host degradation compared with asymptomatic tissues. RNAseq was used to identify differentially expressed genes in asymptomatic vs stromatized tissues from 3 diverse Epichloë–host associations. Comparative analysis identified a core set of 135 differentially expressed genes that exhibited conserved transcriptional changes across all 3 associations. The core differentially expressed genes more strongly expressed during virulent growth encode proteins associated with host suppression, digestion, adaptation to the external environment, a biosynthetic gene cluster, and 5 transcription factors that may regulate Epichloë stroma formation. An additional 5 transcription factor encoding differentially expressed genes were suppressed during virulent growth, suggesting they regulate mutualistic processes. Expression of biosynthetic gene clusters for natural products that suppress herbivory was universally suppressed during virulent growth, and additional biosynthetic gene clusters that may encode production of novel host-protective natural products were identified. A comparative analysis of 26 Epichloë genomes found a general decrease in core differentially expressed gene conservation among asexual species, and a specific decrease in conservation for the biosynthetic gene cluster expressed during virulent growth and an unusual uncharacterized gene.

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network and also grows as an epiphyte. However, little is known about the cell wall-remodeling mechanisms required to avoid host defense and maintain intercalary growth within the host. Here, we use a suite of molecular probes to show that the E. festucae cell wall is remodeled by conversion of chitin to chitosan during infection of L. perenne seedlings, as the hyphae switch from free-living to endophytic growth. When hyphae transition from endophytic to epiphytic growth, the cell wall is remodeled from predominantly chitosan to chitin. This conversion from chitin to chitosan is catalyzed by chitin deacetylase. The genome of E. festucae encodes three putative chitin deacetylases, two of which (cdaA and cdaB) are expressed in planta. Deletion of either of these genes results in disruption of fungal intercalary growth in the intercellular spaces of plants infected with these mutants. These results establish that these two genes are required for maintenance of the mutualistic symbiotic interaction between E. festucae and L. perenne.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonize their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens, but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonization with their hosts. Epichloë festucae forms mutualistic associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome-wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship, and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 abundance is not altered during the mutualistic interaction and it mainly inhibits LpCP2. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1, we propose that additional inhibitors, likely of fungal origin, are involved in the suppression of apoplastic PLCPs during E. festucae infection.

Recent studies have identified key genes in Epichloë festucae that control the symbiotic interaction of this filamentous fungus with its grass host. Here we report on the identification of specific fungal genes that determine its ability to infect and colonize the host. Deletion of setB , which encodes a homolog of the H3K36 histone methyltransferase Set2/KMT3, specifically reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae Δ clrD mutant, which lacks the gene encoding the homolog of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect the host grass, and mutational studies of key residues in the catalytic SET domains from these proteins show that these phenotypes are dependent on the methyltransferase activities of SetB and ClrD. A comparison of the differences in the host transcriptome between seedlings inoculated with wild-type versus mutants suggests that the inability of these mutants to infect the host was not due to an aberrant host defense response. Co-inoculation of either Δ setB or Δ clrD with the wild-type strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild-type. Deletion of crbA , which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae . Author Summary The filamentous fungus Epichloë festucae is an endophyte that forms highly regulated symbiotic interactions with the perennial ryegrass. Proper maintenance of such interactions is known to involve several signalling pathways, but much less is understood about the infection capability of this fungus in the host. In this study, we uncovered two epigenetic marks and their respective histone methyltransferases that are required for E. festucae to infect perennial ryegrass. Null mutants of the histone H3 lysine 9 and lysine 36 methyltransferases are completely defective in colonizing the host intercellular space, and these defects are dependent on the methyltransferase activities of these enzymes. Importantly, we observed no evidence for increased host defense response to these mutants that can account for their non-infection. Rather, these infection defects can be rescued by the wild-type strain in co-inoculation experiments, suggesting that failure of the mutants to infect is due to altered expression of genes encoding infection factors that are under the control of the above epigenetic marks that can be supplied by the wild-type strain. Among genes differentially expressed in the mutants at the early infection stage is a putative small-secreted protein with a carbohydrate binding function, which deletion in E. festucae severely reduced infection efficiency.

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyse the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa , and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne . Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne , were analysed by light-, confocal- and electron microscopy. Changes in PA distribution were analysed in E. festucae using a PA biosensor and the impact of these changes on endocytic recycling and superoxide production investigated. We found that E. festucae PldB and the N. crassa ortholog, PLA-7, are required for polarized growth, cell fusion and ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae . PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne . This study identifies a new component of the cell-cell communication and cell fusion signaling network that controls hyphal morphogenesis and growth in filamentous fungi.

Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signalling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localise the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloë festucae in axenic culture and during interaction with its grass host Lolium perenne . We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homologue of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did uncover the presence of PI[3,4,5]P3 in septa of E. festucae and showed that TepA is required for chemotropism in F. oxysporum . The identification of PI[3,4,5]P3 in septa of ΔtepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 using an alternative biosynthetic pathway and that fungal PTEN homologues are functional lipid phosphatases. The F. oxysporum chemotropism defect demonstrates a conserved role of PTEN homologues in chemotaxis across protists, fungi and mammals. ### Competing Interest Statement The authors have declared no competing interest.

The endophytic fungus Epichloë festucae systemically colonizes the intercellular spaces of cool‐season grasses to establish a mutualistic symbiosis. Hyphal growth of the endophyte within the host plant is tightly regulated and synchronized with the growth of the host plant. A genetic screen to identify symbiotic genes identified mutant FR405 that had an antagonistic interaction with the host plant. Perennial ryegrass infected with the FR405 mutant were stunted and underwent premature senescence and death. The disrupted gene in FR405 encodes a nuclear‐localized protein, designated as NsiA for nuclear protein for symbiotic infection. Like previously isolated symbiotic mutants the nsiA mutant is defective in hyphal cell fusion. NsiA interacts with Ste12, a C2H2 zinc‐finger transcription factor, and a MAP kinase MpkB. Both are known as essential components for cell fusion in other fungal species. In E. festucae, MpkB, but not Ste12, is essential for cell fusion. Expression of several genes required for cell fusion and symbiosis, including proA/adv‐1, pro41/ham‐6, ham7, ham8, and ham9 were downregulated in the nsiA mutant. However, the NsiA ortholog in Neurospora crassa was not essential for hyphal cell fusion. These results demonstrate that the roles of NsiA and Ste12 orthologs in hyphal cell fusion are distinctive between fungal species.

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonise their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonisation with their hosts. Epichloë festucae forms mutualist associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 inhibits LpCP2, indicating that LpCys1 might play a role in the suppression of PLCP activity during the interaction with E. festucae . However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1 we propose that additional inhibitors are involved in the suppression of apoplastic PLCPs during E. festucae infection.

SummaryPeramine 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.

© 2019 John Wiley & Sons Ltd Studies on the regulation of fungal secondary metabolism highlight the importance of histone H3K4 methylation regulators Set1, CclA (Ash2) and KdmB (KDM5), but it remains unclear whether these proteins act by direct modulation of H3K4me3 at the target genes. In filamentous fungi, secondary metabolite genes are frequently located near telomeres, a site where H3K4 methylation is thought to have a repressive role. Here we analyzed the role of CclA, KdmB and H3K4me3 in regulating the subtelomeric EAS and LTM cluster genes in Epichloë festucae. Depletion of H3K4me3 correlated with transcriptional activation of these genes in ΔcclA, similarly enrichment of H3K4me3 correlated with transcriptional repression of the genes in ΔkdmB which was accompanied by significant reduction in the levels of the agriculturally undesirable lolitrems. These transcriptional changes could only be explained by the alterations in H3K4me3 and not in the subtelomerically-important marks H3K9me3/K27me3. However, H3K4me3 changes in both mutants were not confined to these regions but occurred genome-wide, and at other subtelomeric loci there were inconsistent correlations between H3K4me3 enrichment and gene repression. Our study suggests that CclA and KdmB are crucial regulators of secondary metabolite genes, but these proteins likely act via means independent to, or in conjunction with the H3K4me3 mark.

Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3' end of ppzA We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.

Epichloë festucae is an endophyte of the agriculturally important perennial ryegrass. This species systemically colonises the aerial tissues of this host where its growth is tightly regulated thereby maintaining a mutualistic symbiotic interaction. Recent studies have suggested that small secreted proteins, termed effectors, play a vital role in the suppression of host defence responses. To date only a few effectors with important roles in mutualistic interactions have been described. Here we make use of the fully assembled E. festucae genome and EffectorP to generate a suite of 141 effector candidates. These were analysed with respect to their genome location and expression profiles in planta and in several symbiosis-defective mutants. We found an association between effector candidates and a class of transposable elements known as MITEs, but no correlation with other dynamic features of the E. festucae genome, such as transposable element-rich regions. Three effector candidates and a small GPI-anchored protein were chosen for functional analysis based on their high expression in planta compared to in culture and their differential regulation in symbiosis defective E. festucae mutants. All three candidate effector proteins were shown to possess a functional signal peptide and two could be detected in the extracellular medium by western blotting. Localization of the effector candidates in planta suggests that they are not translocated into the plant cell, but rather, are localized in the apoplastic space or are attached to the cell wall. Deletion and overexpression of the effector candidates, as well as the putative GPI-anchored protein, did not affect the plant growth phenotype or restrict growth of E. festucae mutants in planta. These results indicate that these proteins are either not required for the interaction at the observed life stages or that there is redundancy between effectors expressed by E. festucae.

Epichloë festucae forms mutualistic symbiotic interactions with grasses of the Lolium and Festuca genera. Protection from insect and mammalian herbivory are the best-documented host benefits of these associations. The two main classes of anti-mammalian alkaloids synthesized by E. festucae are the ergot alkaloids and indole diterpenes, of which ergovaline and lolitrems are the principal terminal products. Synthesis of both metabolites require multiple gene products encoded by clusters of 11 genes located at the subtelomeric regions of chromosomes I and III respectively. These loci are essentially unexpressed in axenic culture but among the most highly expressed genes in planta. We show here that heterochromatin 1 protein (HepA) is an important component of the regulatory machinery that maintains these loci in a silent state in culture. Deletion of this gene led to derepression of eas and ltm gene expression under non-symbiotic culture conditions. Although there was no obvious culture phenotype, RNAseq analysis revealed that around 1000 genes were differentially expressed in the ΔhepA mutant compared to wild type with just one-third upregulated. Inoculation of the ΔhepA mutants into seedlings of Lolium perenne led to a severe host interaction phenotype characterized by a reduction in tiller length but an increase in tiller number. Hyphae within the leaves of these associations were much more abundant in the intercellular spaces of the leaves and aberrantly colonized the vascular bundles. This physiological change was accompanied by a dramatic change in the transcriptome with around 900 genes differentially expressed, with two thirds of these upregulated. This major physiological change was accompanied by a decrease in ltm gene expression and loss of the ability to synthesize lolitrems. These results show that HepA has an important role in controlling the chromatin state of these sub-telomeric secondary metabolite genes, including their symbiosis-specific regulation.

Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-house Epichloë metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

Epichloe endophytes (Clavicipitaceae) infect pooid grass genera worldwide but predominantly in the Northern Hemisphere, but appear to be rare in native grasses of the Southern Hemisphere. Because of benefits that hosts may receive from the symbiosis, Epichloe endophytes have been extensively studied and are considered important components of sustainable agriculture. There are only a few studies available on the incidence of endophyte infection in grasses of the Southern Hemisphere and most grass species have never been examined. Here we report on a survey of native grasses of New Zealand including 25 endemic or indigenous species. We sampled up to 10 plants per species at different sites from both the North and South Island of New Zealand and examined tissues microscopically for endophyte infection. Overall, only two species were found to be infected, Poa matthewsii (Matthew's bluegrass) and Dichelachne micrantha (short-hair plume grass). Based on analyses of tefA and tubB genes, both endophytes were found to be interspecific hybrids. The endophyte of the new host D. micrantha was previously described as Epichloe australiensis, while the endophyte of P. matthewsii is a new species named here E. novae-zelandiae. The new species is a hybrid derived from E. amarillans, E. bromicola and E. typhina subsp. poae. Alkaloid analyses in planta suggested that E. novae-zelandiae can produce small amounts of peramine, early pathway indole-diterpenes and ergot alkaloids, but no lolines or lolitrems. Target specific primers suggested the presence of genes for ergot alkaloids and peramine, but genes of only early pathway steps for the other alkaloids. Furthermore, genes for both mating type idiomorphs (MTA and MTB) were present, a single copy of MTA and two copies of MTB. Endophytes of native grasses may provide a genetic resource that could be exploited for developing pasture grass cultivars with improved performance.

SummaryEpichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with the grass host Lolium perenne. Endophytic hyphae exit the host by an appressorium‐like structure known as an expressorium. In plant‐pathogenic fungi, the tetraspanin Pls1 and the NADPH oxidase component Nox2 are required for appressorium development. Previously we showed that the homologue of Nox2, NoxB, is required for E. festucae expressorium development and establishment of a mutualistic symbiotic interaction with the grass host. Here we used a reverse genetics approach to functionally characterize the role of the E. festucae homologue of Pls1, PlsA. The morphology and growth of ΔplsA in axenic culture was comparable to wild‐type. The tiller length of plants infected with ΔplsA was significantly reduced. Hyphae of ΔplsA had a proliferative pattern of growth within the leaves of L. perenne with increased colonization of the intercellular spaces and the vascular bundles. The ΔplsA mutant was also defective in expressorium development although the phenotype was not as severe as for ΔnoxB, highlighting potentially distinct roles for PlsA and NoxB in signalling through the NoxB complex. Hyphae of ΔplsA proliferate below the cuticle surface but still occasionally form an expressorium‐like structure that enables the mutant hyphae to exit the leaf to grow on the surface. These expressoria still form a septin ring‐like structure at the point of cuticle exit as found in the wild‐type strain. These results establish that E. festucae PlsA has an important, but distinct, role to NoxB in expressorium development and plant symbiosis.

SummaryEpichloë festucaeis an endophytic fungus that forms a mutualistic symbiotic association withLolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates ofE. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infectedL. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-houseEpichloëmetabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novelEpichloë-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressedin planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.