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Genetic Manipulation of the Ergot Alkaloid Pathway in Epichloë Festucae Var

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Genetic Manipulation of the Ergot Alkaloid Pathway in Epichloë Festucae Var toxins Article Genetic Manipulation of the Ergot Alkaloid Pathway in Epichloë festucae var. lolii and Its Effect on Black Beetle Feeding Deterrence Debbie Hudson 1, Wade Mace 1 , Alison Popay 2 , Joanne Jensen 2, Catherine McKenzie 1 , Catherine Cameron 2 and Richard Johnson 1,* 1 AgResearch Limited, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand; [email protected] (D.H.); [email protected] (W.M.); [email protected] (C.M.) 2 AgResearch Limited, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand; [email protected] (A.P.); [email protected] (J.J.); [email protected] (C.C.) * Correspondence: [email protected] Abstract: Epichloë endophytes are filamentous fungi (family Clavicipitaceae) that live in symbiotic associations with grasses in the sub family Poöideae. In New Zealand, E. festucae var. lolii confers significant resistance to perennial ryegrass (Lolium perenne) against insect and animal herbivory and is an essential component of pastoral agriculture, where ryegrass is a major forage species. The fungus produces in planta a range of bioactive secondary metabolites, including ergovaline, which has demonstrated bioactivity against the important pasture pest black beetle, but can also cause mammalian toxicosis. We genetically modified E. festucae var. lolii strain AR5 to eliminate key enzymatic steps in the ergovaline pathway to determine if intermediate ergot alkaloid compounds Citation: Hudson, D.; Mace, W.; can still provide insecticidal benefits in the absence of the toxic end product ergovaline. Four genes Popay, A.; Jensen, J.; McKenzie, C.; (dmaW, easG, cloA, and lpsB) spanning the pathway were deleted and each deletion mutant was Cameron, C.; Johnson, R. Genetic inoculated into five different plant genotypes of perennial ryegrass, which were later harvested for a Manipulation of the Ergot Alkaloid full chemical analysis of the ergot alkaloid compounds produced. These associations were also used Pathway in Epichloë festucae var. lolii in a black beetle feeding deterrence study. Deterrence was seen with just chanoclavine present, but and Its Effect on Black Beetle Feeding was cumulative as more intermediate compounds in the pathway were made available. Ergovaline Deterrence. Toxins 2021, 13, 76. was not detected in any of the deletion associations, indicating that bioactivity towards black beetle https://doi.org/10.3390/ toxins13020076 can be obtained in the absence of this mammalian toxin. Received: 15 December 2020 Keywords: Epichloë; ergot alkaloid; ergovaline; black beetle; Heteronychus arator; perennial ryegrass; Accepted: 15 January 2021 symbiosis; endophyte; secondary metabolite Published: 20 January 2021 Key Contribution: Genetic modification of the ergot alkaloid pathway in E. festucae var. lolii demon- Publisher’s Note: MDPI stays neutral strates that bioactivity towards the important pasture pest black beetle (Heteronychus arator) can with regard to jurisdictional claims in be obtained in the absence of the mammalian toxin ergovaline, and that feeding deterrence was published maps and institutional affil- cumulative as more intermediate compounds in the pathway accumulated. iations. 1. Introduction Copyright: © 2021 by the authors. Epichloë endophytes are filamentous fungi, belonging to the family Clavicipitaceae, Licensee MDPI, Basel, Switzerland. which live in symbiotic associations with grasses in the sub family Poöideae [1,2]. In This article is an open access article New Zealand, E. festucae var. lolii confers significant resistance to perennial ryegrass distributed under the terms and (Lolium perenne) against insect and animal herbivory and, as such, is an essential component conditions of the Creative Commons of pastoral agriculture [3–5], where ryegrass is a major forage species. African black beetle Attribution (CC BY) license (https:// (Heteronychus arator) is an introduced pest in northern New Zealand and is responsible creativecommons.org/licenses/by/ for devasting losses to the pasture industry, costing farmers up to $242 M annually [6]. A 4.0/). Toxins 2021, 13, 76. https://doi.org/10.3390/toxins13020076 https://www.mdpi.com/journal/toxins Toxins 2021, 13, x FOR PEER REVIEW 2 of 11 Toxins 2021, 13, 76 2 of 10 devasting losses to the pasture industry, costing farmers up to $242 M annually [6]. A number of E. festucae var. lolii strains provide biological control against this serious pest [7,8] through thenumber production of E. festucaeof secondaryvar. lolii metabolitesstrains provide in planta biological [9]. Ergovaline, control against which this is serious pest [7,8] the best characterisedthrough Epichloë- the productionproduced of ergot secondary alkaloid metabolites [9–13], has in demonstrated planta [9]. Ergovaline, insecti- which is the cidal activity againstbest characterised black beetleEpichloë [14,15],-produced but can ergot also alkaloidcause mammalian [9–13], has demonstrated toxicosis insecticidal [10,12,16,17]. activity against black beetle [14,15], but can also cause mammalian toxicosis [10,12,16,17]. The biosyntheticThe pathway biosynthetic responsible pathway for responsibleergot alkaloid for production ergot alkaloid has productionbeen well has been well characterized fromcharacterized a number fromof fungi a number [11,13,18–21], of fungi including [11,13,18 –the21], ergovaline including pathway the ergovaline pathway from Epichloë [9,22].from WhileEpichloë the[9 end,22]. product While the of endthis productpathway of is this ergovaline, pathway intermediate is ergovaline, intermediate ergot alkaloid compoundsergot alkaloid are compoundsalso produced are (F alsoigure produced 1) and the (Figure genes1 )responsible and the genes for responsibleeach for each enzymatic step inenzymatic the ergovaline step in pathway the ergovaline have been pathway identified have been(Figure identified 1) [9,23,24]. (Figure 1)[9,23,24]. Figure 1. SimplifiedFigure 1. Simplified ergovaline ergovaline pathway showing pathway pathway showing intermediates pathway intermediates and the key and genes the proposed key genes for pro- each biosynthetic step. DMAPPposed = dimethylallylfor each biosynthetic pyrophosphate, step. DMAPP DMAT = dime = dimethylallylthylallyl pyrophosphate, tryptophan. Underlined DMAT = dimethylallyl genes were deleted in this study. Ergovalinetryptophan. (boxed) Underlined is a mammalian genes were toxin deleted with in bioactivity this study. towards Ergovaline black (boxed) beetle. is A a comprehensive mammalian review of this pathway istoxin reported with inbioactivity Young et towards al., 2015 black [24]. beetle. A comprehensive review of this pathway is reported in Young et al., 2015 [24]. Functional analysis, through genetic modification, of key genes in this pathway Functional hasanalysis, been through achieved genetic for several modifi fungi,cation, including of key genesEpichloë in this, and pathway has provided has a detailed been achieved forunderstanding several fungi, of theincluding key biosynthetic Epichloë, and steps has required provided for ergota detailed alkaloid under- biosynthesis [20,24]. standing of the keyThe biosynthetic gene dmaW encodessteps required dimethylallyl for ergot tryptophan alkaloid biosynthesis synthase, the [20,24]. first enzymeThe in the ergot gene dmaW encodesalkaloid dimethylallyl pathway [tryptophan25], and as synthase, such, deletion the first of enzymedmaW will in the lead ergot to theal- elimination of kaloid pathway all[25], ergot and alkaloids.as such, deletion The gene of dmaWeasG willencodes lead to an the NADPH-dependent elimination of all ergot reductase involved alkaloids. The genein converting, easG encodes via an a non-enzymaticNADPH-dependent adduct reductase with reduced involved glutathione, in convert- the simple ergot ing, via a non-enzymaticalkaloid chanoclavine adduct with to reduced agroclavine glutathione, [26,27]. Deletion the simple of this ergot gene alkaloid should thus lead to the chanoclavine to accumulationagroclavine [26,27]. of chanoclavine Deletion of in this the gene absence should of other thus ergot lead alkaloidto the accumu- compounds. The gene lation of chanoclavinecloA encodes in the absence a cytochrome of other P450 ergot monooxygenase, alkaloid compounds. which isThe proposed gene cloA to be responsible encodes a cytochromefor the P450 oxidation monooxygenase, of both agroclavine which is (to proposed form elymoclavine) to be responsible and elymoclavinefor the (to form oxidation of bothlysergic agroclavine acid) [(to28 ].form Deletion elymoc oflavine) this gene and iselymoclavine thus predicted (to form to lead lysergic to the accumulation acid) [28]. Deletionof chanoclavine of this gene and is agroclavine thus predicted in the to absence lead to of the more accumulation complex ergot of alkaloids. The chanoclavine andgenes agroclavinelpsA and inlpsB theencode absence the of largemore and complex small ergot subunits alkaloids. of a large The non-ribosomal genes peptide lpsA and lpsB encodesynthetase, the large which and is small responsible subunits for of converting a large non-ribosomal lysergic acid peptide to ergovaline, syn- the last step thetase, which isin responsible the pathway for [ 22converting,29]. Deletion lysergic of either acid to of ergovaline, these genes the leads last tostep the in accumulation the of the pathway [22,29].clavines Deletion group of either and of lysergic these ge acidnes [leads22,29 ].to the accumulation of the clavines
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