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1 Gene Clusters for Insecticidal Loline Alkaloids in the Grass-Endophytic Fungus

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1 Gene Clusters for Insecticidal Loline Alkaloids in the Grass-Endophytic Fungus Genetics: Published Articles Ahead of Print, published on January 16, 2005 as 10.1534/genetics.104.035972 1 1 Gene Clusters for Insecticidal Loline Alkaloids in the Grass-Endophytic Fungus 2 Neotyphodium uncinatum 3 4 Martin J. Spiering*, Christina D. Moon*1, Heather H. Wilkinson† and Christopher L. 5 Schardl* 6 7 *Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546- 8 0312 9 10 †Department of Plant Pathology and Microbiology, Texas A&M University, College 11 Station, Texas 77843-2132 12 13 1Present address: Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, 14 United Kingdom 15 16 Sequence data from this article have been deposited with the GenBank database under 17 accession nos. AY723749, AY723750 and AY724686. 18 2 18 Running title: Loline alkaloid gene clusters 19 20 Key words: alkaloids, biosynthesis genes, pyrrolizidines, RNA-interference, symbiosis 21 22 Corresponding author: Christopher L. Schardl, Department of Plant Pathology, 23 University of Kentucky, 201F Plant Science Building, 1405 Veterans Drive, Lexington, 24 KY 40546-0312, USA. Email [email protected]; Tel 859-257-7445 ext 80730; Fax 859- 25 323-1961 26 3 26 ABSTRACT 27 Loline alkaloids are produced by mutualistic fungi symbiotic with grasses, and protect 28 the host plants from insects. Here we identify in the fungal symbiont, Neotyphodium 29 uncinatum, two homologous gene clusters (LOL-1 and LOL-2) associated with loline- 30 alkaloid production. Nine genes were identified in a 25-kb region of LOL-1, and 31 designated (in order) lolF-1, lolC-1, lolD-1, lolO-1, lolA-1, lolU-1, lolP-1, lolT-1, and 32 lolE-1. LOL-2 contained the homologues lolC-2 through lolE-2 in the same order and 33 orientation. Also identified was lolF-2, but its possible linkage with either cluster was 34 undetermined. Most lol genes were regulated in N. uncinatum and Neotyphodium 35 coenophialum, and all were expressed concomitantly with loline-alkaloid biosynthesis. A 36 lolC-2 RNA-interference (RNAi) construct was introduced into N. uncinatum, and in two 37 independent transformants RNAi significantly decreased lolC expression (p < 0.01) and 38 loline-alkaloid accumulation in culture (p < 0.001) compared to vector-only controls, 39 indicating involvement of lolC in biosynthesis of lolines. The predicted LolU protein had 40 a DNA binding site signature, and the relationships of other lol gene products indicated 41 that the pathway has evolved from various different primary and secondary biosynthesis 42 pathways. 43 44 45 4 45 INTRODUCTION 46 Seed-borne endophytic fungi — specifically the Epichloë species (asexual states = 47 Neotyphodium species) — in symbiosis with cool-season grasses (Poaceae subfam. 48 Poöideae) can impart to those grasses a variety of fitness enhancements including 49 resistance to vertebrate and invertebrate herbivores, resistance to pathogens and parasites, 50 enhanced phosphate uptake and nitrogen utilization, and increased tolerance of drought 51 and heat (BUSH et al. 1997; MALINOWSKI and BELESKY 2000; SCHARDL et al. 2004). 52 Loline alkaloids produced by several Neotyphodium and Epichloë species are potent, 53 broad-spectrum insecticides (DOUGHERTY et al. 1998; RIEDELL et al. 1991; WILKINSON et 54 al. 2000), have little or no anti-mammalian activities (JACKSON et al. 1996), and 55 sometimes accumulate in the plant to levels up to 20 mg g-1 plant dry weight (DW) 56 (CRAVEN et al. 2001). These alkaloids have an unusual structure, comprising a saturated 57 1-aminopyrrolizidine-ring system, with a highly strained ether bridge between C-2 and C- 58 7 (PETROSKI et al. 1989). Lolines are almost exclusively found in these grass-endophyte 59 symbioses (BUSH et al. 1997; HARTMANN and WITTE 1995); outside of the grasses they 60 have only been identified in a few plant species in the families Fabaceae and 61 Convolvulaceae (HARTMANN and WITTE 1995; TOFERN et al. 1999). Genetic tests have 62 confirmed linkage between capability of endophytes to produce lolines in planta and their 63 bioprotective effects against aphids (WILKINSON et al. 2000). Improved survival under 64 drought and/or competition of grass-endophyte symbiota possessing lolines has raised the 65 possibility of additional roles of these alkaloids in host plant fitness and persistence 66 (MALINOWSKI and BELESKY 2000; SCHARDL et al. 2004). 5 67 The biochemical pathway for the lolines is so far unknown. Based on certain 68 structural similarities of lolines with plant pyrrolizidines (HARTMANN and WITTE 1995), 69 BUSH et al. (1993) proposed that lolines are produced by a pathway involving polyamines 70 such as spermidine. However, results of precursor feeding studies have now ruled out this 71 possibility, and suggest that lolines are formed by a novel biosynthetic pathway from the 72 amino acids, L-proline and L-homoserine (BLANKENSHIP et al. 2005). 73 We have previously identified two genes, lolA and lolC, showing strong up- 74 regulation in loline-alkaloid-producing N. uncinatum cultures and strict association with 75 in symbio loline-alkaloid-production phenotypes among isolates of different endophyte 76 species (SPIERING et al. 2002). In the present study we show that two gene clusters in the 77 genome of N. uncinatum contain homologues of lolA and lolC, along with several 78 additional genes whose predicted products showed relationships to enzymes typical of 79 primary or secondary metabolic pathways. A functional test performed with RNA 80 interference (RNAi) in N. uncinatum confirmed involvement of lolC in loline-alkaloid 81 production. These findings provide first insight into the molecular genetics of loline- 82 alkaloid production and constitute an important basis for detailed studies of the 83 biosynthetic pathway and ecological roles of the lolines. 84 85 MATERIALS AND METHODS 86 Fungal strains and growth conditions: Neotyphodium uncinatum CBS 102646 87 (deposited in Centraalbureau voor Schimmelculture) and Neotyphodium coenophialum 88 ATCC 62374 (deposited in the American Type Culture Collection) were grown in shake- 89 cultures as described by BLANKENSHIP et al. (2001). Loline-alkaloid production by N. 6 90 uncinatum was induced in minimal medium (MM) (BLANKENSHIP et al. 2001) with 15 91 mM urea and 20 mM sucrose as nitrogen and carbon sources, respectively. To suppress 92 loline-alkaloid production, the fungus was grown in complex medium (CM) consisting of 93 potato dextrose broth (PDB; Difco, Detroit, MI) diluted 1:1 with MM (SPIERING et al. 94 2002). In all experiments, three replicate culture plates of each treatment were used for 95 loline-alkaloid analysis. For in symbio experiments, Lolium pratense (=Festuca 96 pratensis) and Lolium arundinaceum (=Festuca arundinacea) plants symbiotic with N. 97 uncinatum CBS 102646 and N. coenophialum ATCC 90664, respectively, were grown in 98 the greenhouse. 99 100 Bacterial strains and plasmids: The cosmid pMOCosX (ORBACH 1994) and 101 plasmid pCB1004 (CARROLL et al. 1994) were used in genomic library construction and 102 DNA cloning, respectively, in Escherichia coli strain XL1-Blue (BULLOCK et al. 1987). 103 These plasmids contain the hygromycin B-phosphotransferase (hph) gene under control 104 of constitutive promoters of the cpc-1 gene in Neurospora crassa (pMOCosX) and trpC 105 gene in Aspergillus nidulans (pCB1004) (= hph cassette). Bacterial cultures were grown 106 on LB plates or in LB medium with shaking (200 rpm) at 37° for 16 hr. Plasmid DNA 107 was isolated from bacterial cultures by the method of AHN et al. (2000). 108 109 Fungal genomic DNA isolation and genome walking: Fungal genomic DNA 110 was isolated by the method of AL-SAMARRAI and SCHMID (2000). To amplify long (> 1 111 kb) genomic DNA segments, 5 ng of genomic DNA was amplified in 50-µl reaction 112 volumes, by using the LA-PCR Vers 2.1 Kit as per manufacturer’s instructions (Takara 7 113 Shuzo Co, Otsu, Shiga, Japan). Reactions were performed in a GeneAmp PCR System 114 2400 thermocycler (Perkin-Elmer, Boston, MA); PCR conditions were 95° for 60 sec; 7 115 cycles of 95° for 20 sec, 70° for 8 min; 28 cycles of 95° for 20 sec, 67° for 8 min; and a 116 final 16 min at 67°. To PCR-walk into unknown genomic regions, the Universal Genome 117 Walker Kit (Clontech, Palo Alto, CA) and N. uncinatum or N. coenophialum genomic 118 DNA was used and manufacturer’s instructions were followed for generating genome- 119 walker libraries. PCR primers for genome walking were designed to anneal to sites in 120 known genomic DNA regions and synthesized by Integrated DNA Technologies (IDT; 121 Coralville, IA). For gene-copy-specific genome-walking PCRs, a single-nucleotide 122 polymorphism (SNP) was incorporated into the terminal 3’-nucleotide position of each 123 genome-walking primer. The amplified DNA fragments were purified with the QIAquick 124 PCR Purification Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions, 125 and sequenced as described below. 126 127 Genomic DNA library construction and screening, and cosmid sequencing: A 128 partial N. uncinatum genomic DNA library was constructed in cosmid vector pMOCosX 129 as previously described for construction of a genomic library from Claviceps purpurea 130 (WANG et al. 2004). A total of 2613 primary clones was obtained and arrayed in 384-well 131 microtiter plates. To identify cosmid clones containing lolA and lolC genes, plates were 132 PCR-screened by the method of WANG et al. (2004), with primers lolA-3’ and lolA-5’ 133 (for detection of lolA), and primers lolC-3’a and lolC-5’a (for detection of lolC). The 134 TnsABC transposase/Tn7 transposon-based genome-priming system (GPS-1; New 135 England Biolabs; Beverly, MA) was used following the manufacturer’s instructions, to 8 136 obtain random Tn7 insertions for sequencing. GPS-tagged cosmids were electroporated 137 into E. coli XL1-Blue, and colonies were selected on kanamycin and ampicillin.
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