Materials and methods
Metarhizium brunneum ARSEF 9354 was cultured at 30° C on sucrose yeast extract agar or malt extract broth [5]. To maximize ergot alkaloid yield, 20 µL of a conidial suspension (40,000 conidia per µL) were injected into larvae of the model insect Galleria mellonella [5, 13].
The easP locus of M. brunneum was knocked out via a transient CRISPR/Cas9-mediated approach based on the protocol described by Davis et al. [14]. An sgRNA was synthesized from the template 5′-TTCTAATACGACTCACTATAGTCTGCTCCATGGAGGCTCCTGTTTTAGAGCTAGA-3′ (the 20-nt target sequence is underlined and an additional G was inserted immediately preceding the target sequence) with the EnGen sgRNA synthesis kit (New England Biolabs, Ipswich, MA, USA). The sgRNA was complexed with EnGen Spy Cas9 NLS (New England Biolabs) and co-transformed into protoplasts along with a phosphinothricin-resistance conferring fragment [14]. Transformants were screened for mutations at easP in PCRs primed with oligonucleotides PcrspF (5′-CACACTCTACTCCCTCACAAGG-3′) and PcrspR (5′-CCGCTCCAGGCATCGTCAC-3′). Reaction conditions included an initial denaturation at 98 °C for 30 s, followed by 35 cycles of 98 °C for 15 s, 66 °C for 15 s, and 72 °C for 80 s. PCR products that differed in length from that of wild-type M. brunneum were Sanger sequenced at Eurofins Genomics (Louisville, KY, USA).
The estA gene was knocked out via a similar approach, except the template for the sgRNA was 5′-TTCTAATACGACTCACTATAGTCCAAGACGTAGCCGACTTCGTTTTAGAGCTAGA-3′ (where the target sequence is underlined and preceded by an additional G), and the selectable marker was the hygromycin resistance-conferring plasmid pBC-hygro [15]. Transformants were screened by PCR with oligonucleotides estAF (5′-GAACACAACTTCACCACATCGC-3′) and estAR (5′-GACGTCACCGAGCTCTCTG-3′) in reactions with conditions as described above, except the annealing temperature was 64 °C and the extension time 180 s.
Ergot alkaloids and ergosterol were extracted from infected larvae of G. mellonella by bead beating in methanol [5, 6]. To detect ergot alkaloids, 20 µL of extract was analyzed by high performance liquid chromatography (HPLC) with fluorescence detection (excitation 310 nm/emission 410 nm) [5, 6, 16]. Ergot alkaloid quantities in each sample were normalized to the quantity of the fungus-unique sterol ergosterol in that same extract as described by Steen et al. [6]. Data were compared by Brown-Forsythe tests to assess equality of variances. Data that passed a Brown-Forsythe test (P > 0.05) were analyzed by single-factor ANOVA. For data sets involving multiple comparisons, a Tukey–Kramer Honestly Significant Difference test was employed. In three comparisons (ergonovine and lysergyl-alanine in the easP knockout compared to wild type, and ergine in the easP/estA double knockout), data did not pass a Brown-Forsythe test (P < 0.05) and subsequently were compared nonparametrically, initially with a Wilcoxon rank sums test and then (for the ergine data) a Steel–Dwass nonparametric multiple comparison test. Statistical analyses were conducted in JMP Pro 14 (SAS, Cary, NC, USA).
Results
Introduction of Cas9 complexed with an sgRNA targeting easP, which was co-transformed with a selectable marker for phosphinothricin resistance, resulted in knockout of easP. As a haploid fungus, M. brunneum contains only a single allele of easP. Sequence analysis of the easP locus in two independent transformants indicated Cas9 cut three bp before the PAM site and a portion of the cotransformed phosphinothricin-resistance marker was ligated into the easP locus during repair (Additional file 1: Figure S1). Knockout of easP had a significant effect on accumulation of LAH relative to fungal biomass (estimated by measuring the fungus-unique sterol ergosterol) in M. brunneum-infected larvae of the model insect G. mellonella (Fig. 2). The easP knockout resulted in a significant decrease (approximately 70%) in accumulation of LAH (P < 0.0001). The concentrations of alternate lysergic acid amides, ergonovine and lysergyl-alanine were not affected in the easP knockout. Ergine, the simple amide of lysergic acid, accumulated to a higher concentration in the easP knockout than in the wild type (P = 0.003). Ergine can arise as a spontaneous hydrolysis product of LAH [17, 18] (Fig. 1) and also from hydrolysis of other lysergic acid derivatives [19]. A similar biochemical phenotype was observed qualitatively in the second easP knockout. Whereas the primary purpose of this study was to assess the contribution of easP to LAH biosynthesis, the easP knockout also was investigated for changes in radial growth and sporulation in vitro and colonization of G. mellonella larvae (measured by ergosterol accumulation). No significant differences were detected in these traits (Additional file 2: Table S1).
Because the easP knockout mutation led to reduction as opposed to elimination of LAH, we investigated the possibility that a similar enzyme with redundant function was encoded in the M. brunneum genome. Since the eas cluster contained no additional genes with unidentified function, we queried the haploid genome of M. brunneum ARSEF 3297 for genes with the capacity to encode proteins similar to EasP by tblastn search. The M. brunneum genome encoded only one additional significant match (E value 2e−05; no other match had an E value less than 1). The homolog’s translation product corresponds to the α/β hydrolase fold protein under GenBank accession XP_014542068. We named this gene estA, for esterase A, and knocked it out by a CRISPR/Cas9 approach in the easP knockout strain while selecting for a co-transformed hygromycin resistance marker (Additional file 3: Figure S2). Knockout of estA did not affect ergot alkaloid accumulation relative to the easP knockout (Fig. 3), indicating that EstA plays no role in the ergot alkaloid pathway. In this data set, LAH was reduced by a mean of 54% in the easP and easP/estA knockouts relative to wild type. Concentrations of ergonovine and lysergyl-alanine did not differ significantly among strains. The concentration of ergine was again higher in the easP knockout (and in the easP/estA double knockout) than in the wild type, with P values of 0.035 and 0.014, respectively, for the two easP mutant strains compared to the wild type in a Steel–Dwass multiple comparison test.