Diversity of MSDIN family members in amanitin-producing mushrooms and the phylogeny of the MSDIN and prolyl oligopeptidase genes.

BACKGROUND: Amanitin-producing mushrooms, mainly distributed in the genera Amanita, Galerina and Lepiota, possess MSDIN gene family for the biosynthesis of many cyclopeptides catalysed by prolyl oligopeptidase (POP). Recently, transcriptome sequencing has proven to be an efficient way to mine MSDIN and POP genes in these lethal mushrooms. Thus far, only A. palloides and A. bisporigera from North America and A. exitialis and A. rimosa from Asia have been studied based on transcriptome analysis. However, the MSDIN and POP genes of many amanitin-producing mushrooms in China remain unstudied; hence, the transcriptomes of these speices deserve to be analysed.
RESULTS: In this study, the MSDIN and POP genes from ten Amanita species, two Galerina species and Lepiota venenata were studied and the phylogenetic relationships of their MSDIN and POP genes were analysed. Through transcriptome sequencing and PCR cloning, 19 POP genes and 151 MSDIN genes predicted to encode 98 non-duplicated cyclopeptides, including α-amanitin, β-amanitin, phallacidin, phalloidin and 94 unknown peptides, were found in these species. Phylogenetic analysis showed that (1) MSDIN genes generally clustered depending on the taxonomy of the genus, while Amanita MSDIN genes clustered depending on the chemical substance; and (2) the POPA genes of Amanita, Galerina and Lepiota clustered and were separated into three different groups, but the POPB genes of the three distinct genera were clustered in a highly supported monophyletic group.
CONCLUSIONS: These results indicate that lethal Amanita species have the genetic capacity to produce numerous cyclopeptides, most of which are unknown, while lethal Galerina and Lepiota species seem to only have the genetic capacity to produce α-amanitin. Additionally, the POPB phylogeny of Amanita, Galerina and Lepiota conflicts with the taxonomic status of the three genera, suggesting that underlying horizontal gene transfer has occurred among these three genera.

Background

Amatoxins, which are lethal substances found in mushrooms, have mainly been reported to be present in species from three distinct genera classified into three different families: Amanita (Amanitaceae), Galerina (Hymenogastraceae) and Lepiota (Agaricaceae) [1-4]. Among these amanitin-producing mushrooms, lethal Amanita species are the best-known and most typical mushrooms that produce three primary groups of cyclopeptide toxins: amatoxins, phallotoxins and virotoxins, which are bicyclic octapeptides, bicyclic heptapeptides and monocyclic heptapeptides, respectively [1-4].

The precursor peptide genes of α-amanitin (α-AMA) and phallacidin (PHD) along with multiple related sequences encoding unknown cyclic peptides were first identified and predicted in Amanita bisporigera by genome shotgun sequencing, indicating that amatoxins and phallotoxins are encoded by the same gene family and are biosynthesized on ribosomes [5]. This gene family is referred to as MSDIN in reference to the first five conserved encoded amino acids, and the precursor peptides of its members contain 33–37 amino acids, consisting of two conserved regions, 10 upstream amino acids and 17 downstream amino acids, a highly variable core region, and a 6–10 amino acid sequence that ultimately forms the corresponding cyclopeptide [6]. GmAMA, which is responsible for producing α-AMA, is also found in the genome of Galerina marginata [7]. Galerina marginata is a specific amanitin-containing species in the genus Galerina. Unlike lethal amanitas, G. marginata does not harbour MSDIN-like family genes other than two copies of α-AMA genes. Additionally, α-AMA of Lepiota brunneoincarnata, which is an amanitin-containing mushroom of the genus Lepiota, has been successfully cloned [8]. The genome sequencing of L. venenata, another newly reported amanitin-containing species, has been completed, and it has been shown to harbour α-AMA genes [9]. Precursor peptide sequence alignment of α-AMA sequences from Amanita, Galerina and Lepiota shows high divergence except in the toxin region.

It has been strongly indicated that a prolyl oligopeptidase (POP) plays an important role in the initial processing of MSDIN precursor peptides. Since the core toxin regions are flanked by two highly conserved proline (Pro) residues, this enzyme can cleave the C-terminus of Pro residues and release the peptide chain of the toxin to form a cyclopeptide [10]. It has been reported that there are two types of POP in amatoxin-producing mushrooms: POPA, which behaves like a conventional housekeeping protein that is present in all species, and POPB, which is the enzyme that actually catalyses the cutting and cyclization of precursor peptides [7, 11, 12].

Increasing numbers of MSDIN family members have been published since the first 15 MSDIN genes were found in the A. bisporigera genome, and four were amplified by using degenerate primers in A. phalloides and A. ocreata [5]. Twenty-four MSDIN members were obtained from 6 Amanita species using degenerate primers [13]. Recently, the draft genome sequences of A. palloides and A. bisporigera showed that each species possessed approximately 30 MSDIN members, but only three of these genes were common to the two fungi [6]. Eighteen and twenty-two MSDIN genes were mined from the A. subjunquillea and A. pallidorosea genomes through PacBio and Illumina sequencing, respectively [8]. However, the MSDIN genes of many amanitin-containing Amanita, Galerina and Lepiota mushrooms have not been investigated in depth to date. Lethal Amanita species are classified in section Phalloideae of the genus Amanita [14, 15]. Approximately 50 lethal Amanita species have been reported worldwide, and the species diversity of lethal amanitas is strongly underestimated under the current taxonomy [15, 16]. Many new lethal Amanita and Lepiota species, including A. rimosa, A. subfuliginea, A. subpallidorosea, and L. venenata, have been discovered over the past decade [17-20]. In addition to the 22 known cyclopeptide toxins, some new cyclopeptide substances, such as cycloamanide E and cycloamanide F in A. phalloides and amanexitide in A. exitialis, have been extracted and identified [6, 21, 22]. It has been reported that A. bisporigera and A. phalloides present high potential for the biosynthesis of a variety of cyclopeptides, most of which are unknown according to predictions. Hence, considering the species diversity of amanitin-containing mushrooms and the broad genetic capacity of lethal amanitas to produce unknown cyclopeptides, there are still many new cyclopeptide genes and corresponding cyclopeptides to be discovered.

Alpha-amanitin and toxin-biosyntheic prolyl oligopeptidase B (POPB) genes have been proven to exist in some lethal Amanita [6, 23, 24], Galerina [7] and Lepiota [9] species. The reason that the biosynthetic pathway for α-amanitin is present in these three phylogenetically disjunct genera classified in different families has been studied in recent years. Recent studies reported that horizontal gene transfer (HGT) is the underlying cause of the distribution of MSDIN and POPB genes in Amanita, Galerina and Lepiota on the basis of phylogenetic analysis [8, 9]. The possibility of convergent evolution was negated because the MSDIN and POPB genes in these three genera show similarity and associations, such as a shared conserved gene structure and the encoding of precursor peptides by MSDIN genes [8].

According to previous research, whole-genome sequencing has proven to be the most comprehensive, in-depth method for identifying MSDIN genes or genes related to the cyclopeptide biosynthetic pathway in amanitin-producing mushrooms [6, 8]. Nevertheless, compared with genome sequencing, transcriptome sequencing provides an alternative efficient and low-cost method to obtain functional gene data. To the best of our knowledge, only A. palloides and A. bisporigera from North America and A. exitialis and A. rimosa from Asia have been studied using transcriptome sequencing [6, 25, 26].

In this study, the transcriptomes of seven amanitin-producing mushrooms (A. exitialis, A. fuliginea, A. molliuscula, A. pallidorosea, A. rimosa, A. subpallidorosea and L. venenata) and an Amanita species producing no amanitin (A. oberwinklerana) were sequenced. MSDIN and POP genes were searched and predicted from the transcriptome data. The genomic and coding sequences of the MSDIN and POP genes were cloned and verified. Similarly, MSDIN and POP sequences were cloned from two Galerina strains (G. marginata and G. sulciceps). In addition to the Amanita species mentioned above, MSDIN genes from A. subfuliginea, A. subjunquillea and A. virosa were cloned using specific and degenerate primers. Furthermore, phylogenetic analysis was performed on the obtained toxin and POP genes. Our study was aimed at (a) identifying MSDIN genes from amanitin-producing mushrooms to guide the isolation and identification of new unknown related cyclopeptides and (b) determining the evolutionary relationships of toxin MSDIN and POP genes in amanitin-producing mushrooms.

Results

Data filtering and assembly of transcriptomes

Transcriptome sequencing of seven amanitin-producing mushrooms was performed on the BGISEQ-500 platform using the combinational probe-anchor synthesis sequencing method. After the removal of ambiguous, adaptor-containing and low-quality sequences, clean data were obtained and de novo assembled using Trinity software. The main transcriptomic features and NCBI accession numbers of the transcriptome data obtained in our study are presented in Table 1.

Table 1

Features and accession numbers of transcriptomes

Species	Total clean bases (Gb)	Q30(%)	Total number of unigene	Total length of unigene (nt)	Mean length of unigene (nt)	N50	GC (%)	accession number
A. exitialis	6.67	86.25	24,578	48,383,999	1968	2891	49.75	SRR9929233
A. fuliginea	6.55	88.20	21,624	36,817,429	1702	2599	49.54	SRR9937194
A. molliuscula	6.32	90.65	46,471	79,566,952	1712	3007	49.71	SRR9937646
A. oberwinklerana	6.59	86.87	24,326	61,864,918	2543	3993	48.83	SRR9937816
A. pallidorosea	6.25	89.78	36,846	79,216,743	2149	3375	49.52	SRR9937866
A. rimosa	6.57	87.93	22,532	36,712,344	1629	2648	49.05	SRR9943992
A. subpallidorosea	10.24	91.21	42,803	110,323,057	2577	3630	49.00	SRR9943549
L. venenata	8.47	93.83	13,859	21,738,818	1569	2994	48.88	SRR9943552

MSDIN and POP genes

Through transcriptome sequencing, 110 MSDIN genes (Table 2) were manually identified in 7 lethal Amanita and Lepiota species using known MSDIN members from A. bisporigera as TBLASTN queries. Additionally, 70 MSDIN genes (Table 3) were obtained from 12 lethal Amanita, Galerina and Lepiota species by PCR cloning using degenerate and specific primers. In general, a total of 151 nonrepetitive MSDIN genes were identified at the genomic and transcriptomic levels by using these methods. All the obtained MSDIN genes were predicted to encode 98 cyclopeptides, including α-amanitin (IWGIGCNP), β-amanitin (IWGIGCDP), phallacidin (AWLVDCP), phalloidin (AWLATCP) and 94 unknown peptides. These predicted cyclopeptides were composed of 6–11 amino acids and included 5 hexapeptides, 30 heptapeptides, 73 octapeptides, 22 nanopeptides, 19 decapeptides, and 2 undecapeptides.

Table 2

MSDIN family members searched from the transcriptomes of seven amanitin-producing mushrooms

Name	No.	Leader peptide	Corepeptide	Recognitionsequence	Product
A. exitialis	Ae1	MTDINDTRLP	FIWLLWIWLP	SVGDDNNILNRGEDLC*
	Ae2	MSDINATRLP	LFFPPDFRPP	CVGDADNFTLTRGENLC*
	Ae3	MSDVNATRLP	FNFFRFPYP	CIGDDSGSALRLGESLC*
	Ae4	MSDINTARLP	IPVPPFFIP	FVGDDIDVVLRRGENLC*
	Ae5	MSDINVTRLP	VFIFFFIPP	CVGDGTADIVRKGENLC*
	Ae6	MSDINTARLP	VFSLPVFFP	FVSDDIQAVLTRGESLC*
	Ae7	MSDINTTRLP	FVFVASPP	CVGDDIAMVLTRGENLC*
	Ae8	MSDINPTRLP	IFWFIYFP	CVSDVDSTLTLCISLS*
	Ae9	MSDINTARLP	IIWIIGNP	CVSDDVERILTRGESLC*
	Ae10	MSDINATRLP	IIWAPVVP	CISDDNDSTLTRGQSLC*
	Ae11	MSDINATRLP	IGRPQLLP	CVGGDVNYILISGENLC*
	Ae12	MSDINATRLP	IWGIGCDP	CVGDDVTALLTRGEALC*	β-amanitin
	Ae13	MSDINATRLP	IWGIGCNP	CVGDDVTSVLTRGEALC*	α-amanitin
	Ae14	MSDINVIRLP	SMLTILPP	CVSDDASNTLTRGENLC*
	Ae15	MSDINATRLP	AWLTDCP	CVGDDVNRLLTRGESLC*	“phallotoxin”
	Ae16	MSDINATRLP	AWLVDCP	CVGDDVNRLLTRGESLC*	phallacidin
	Ae17	MSDINLTRLP	GIIAIIP	CVGDDVNSTLTRGQSLC*
	Ae18	MSDINATRLP	VWIGYSP	CVGDDCIALLTRGEGLC*
	Ae19	MSDINATRLP	GFLFWA	YVGDDVDYILTRGESLA*
A. fuliginea	Af1	MSDINATRLP	IIIVLGLIIP	LCVSDIEMILTRGESLC*
	Af2	MSDLNASRLP	ILSVLGLPVP	HVGEETNSTLARGESLC*
	Af3	MSDINSARLP	LFFPPIFIPP	CVSDDVQVVLTRGENLC*
	Af4	MSDINAARLP	FFPFVFIPP	CIGDDATSIVRQAENLC*
	Af5	MSDINTIRIP	FPWTGPFVP	CVSDDVGSVLMRGESLS*
	Af6	MSDTNATRLP	IWFIQLQIP	CAGDDVNSSLTRGESLC*
	Af7	MSDINVTRLP	VLVFIFFPP	YISDDAVNILKQGENLC*
	Af8	MFDINGSRLP	AFRLIPPP	CVGDDVDSTLTSGESLC*
	Af9	MSDINATRLP	GILIVFPP	CVGDDVNSTLTRGESLC*
	Af10	MSDINATRLP	HLFTWIPP	CISDDSTLTRGESFC*
	Af11	MFDINSSRLP	HLYPNSRP	CVCDDACSTLTSAESLC*
	Af12	MSDINATRLP	IFWFIYFP	CVGDDVDNTLTRGESLS*
	Af13	MSDINATRLP	IWGIGCDP	CVGDDVAALITRGEALC*	β-amanitin
	Af14	MSCINATRLP	LPSRPVFP	FVSDAIEVVLGRGEDLC*
	Af15	MSDINSLRLP	VVNSRFNP	CVGDDVSPTLTRGEGLC*
	Af16	MSDINASRLP	AWLATCP	CIGDDVNPTITRGESLC*	phalloidin
	Af17	MSDINATRLP	AWLVDCP	CVGDDVNRLLARGENLC*	phallacidin
	Af18	MSDINATRLP	AWLVTCP	CVGDDINRLLTRGENLC*	“phallotoxin”
A. molliuscula	Am1	MSDINTARLP	YFLPPIFSPP	CVSDDIEMVLTRGENLC*
	Am2	MTDINATRLP	ILFGFFLLP	CVDGVDNTLHSGENLC*
	Am3	MSNINASRLP	IWAAFFRFP	CVGDEVDGILRSGESLC*
	Am4	MSDINATRLA	IWGIGCDP	CVGDDVTALLTRGEALC*	β-amanitin
	Am5	MSDINASRLP	RLLVPRYP	CIDEDAEGATYLC*
	Am6	MSNINAIRLP	GFFAVVP	YLATSITFSLLGRGESLC*
	Am7	MTDINATRLP	WIFFFPP	CVDDVDNTLHSGENLC*
	Am8	MSNINALRLP	GFGFIP	YASGDVDYTLTRGESLS*
	Am9	MSDINATRFP	GKVNPP	YVGDDVDDIIIRGEKLC*
A. pallidorosea	Ap1	MADINAARLP	FHGLFPFLPPP	FVDDDATSTLTRGESLC*
	Ap2	MADINASRLP	LNILPFHLPP	CVSDDATSTLTRGESLC*
	Ap3	MSDINATRLP	NWHAGPTRPP	CVADDVSLTLTRGESLC*
	Ap4	MSDINTARLP	VFFMPPFIPP	CVSDDIQMVLTRGENLC*
	Ap5	MSDINTARLP	EFIVFGIFP	CVGDDIQTVLTRGEDLC*
	Ap6	MSDINASRLP	FFPEVGFFP	CVGDDTNPILTRGGSLS*
	Ap7	MSDLNATRLP	FNLFRFPYP	CIGDDSGSVLTLGEGLC*
	Ap8	MSDINTIRVP	FPWTGPFVP	CVGDDVGSVLTHGESLS*
	Ap9	MSDINATRLP	HPFPLGLQP	CAGDVDNLTLFRGEGLC*
	Ap10	MSDINATRLP	DPRRLLIP	GSSDDVDSALTRGESLC*
	Ap11	MSDINTTRLP	HFFNLMPP	CVGDDIETVLTRGESLC*
	Ap12	MSDINATRLP	HQHHPFVP	GGSDDVGSTLTRGESLC*
	Ap13	MSDMNVVRLP	ISDPTAYP	CVGDDIQAVLGRGESLC*
	Ap14	MSDINATRLP	IWGIGCDP	CVGDDVTAVLTRGEALC*	β-amanitin
	Ap15	MSDINATRLP	IWGIGCNP	CVGDEVAALLTRGEALC*	α-amanitin
	Ap16	MSDINATRLP	IWGIGCNP	CVGDEVTALITRGEALC*	α-amanitin
	Ap17	MSDINATRLP	LGRPESLP	CVGDDVNYILVSGGNLS*
	Ap18	MSDINAARLP	LVYMILFP	SVGDDIDVVLGRGENLC*
	Ap19	MSDVNATRLP	MAFPEFLA	CVGDDVNHTLTRGERLC*
	Ap20	MSDINTARLP	MHILAPPP	CVSDDIEMVLTRGESLC*
	Ap21	MSDINAARLP	NLFVWIPP	CISDDINSTLTRGESLC*
	Ap22	MSDINTTRLP	YMWDHHLP	CASDDIQMVFTRGENLC*
	Ap23	MSDINASRLP	AWLATCP	CAGDDVNPTLTRGESLC*	phalloidin
	Ap24	MSDINATRLP	AWLMTCP	CVGDDVNPTLTRGESLC*	“phallotoxin”
	Ap25	MSDVNATRLP	AWLVDCP	CVGDDINRLLTRGENLC*	phallacidin
A. rimosa	Ar1	MSDINTSRLP	FIPLGIITILP	CVSDDVNTTITRGESLC*
	Ar2	MTDINDTRLP	FVWILWLWLA	CVGDDTSILNRGEDLC*
	Ar3	MSDINATRLP	IIIVLGLIIP	LCVSDIEMILTRGESLC*
	Ar4	MSDVNTTRLP	FNFFRFPYP	CICDDSEKVLELGENLC*
	Ar5	MSDINATRLP	HPFPLGLQP	CAGDVDNFTVSCHSLC*
	Ar6	MLDINATRFP	LGRPTHLP	CVGDDVNYILIGNGENLC*
	Ar7	MSDINASCLP	LILVANGMA	YVSDDVSPTLTRGENLC*
	Ar8	MPDINVTRLP	LLIIVLLTP	CISDDNNILNRGKDLC*
	Ar9	MSDIHAARLP	FPTRPVFP	SAGDDMIEVVLGRGEDLC*
	Ar10	MSDNNAARLP	FYFYLGIP	SDDAHPILTRGESLC*
	Ar11	MSDINIARLP	IFWFIYFP	CVGDDVDNTLSRGESLS*
	Ar12	MSDINASRLP	ILKKPWAP	SVCDDVNSTLTRGEGLC*
	Ar13	MSDINVARLP	ISDPTAYP	CVGDDIQAVVKRGESLC*
	Ar14	MSDINATRLP	IWGIGCDP	CVGDDVAALTTRGEALC*	β-amanitin
	Ar15	MSDINSTRLP	IWGIGCNP	SVGDEVTALLTRGEALC*	α-amanitin
	Ar16	MSDINATRLP	AWDSKHP	CVGDDVSRLLTRGESLC*
	Ar17	MSDINATRVP	AWLAECP	CVGDDISHLLTRGENLC*	“phallotoxin”
	Ar18	MSDINATRVP	AWLVDCP	CVGDDISRLLTRGENLC*	phallacidin
A. subpallidorosea	Asp1	MTDVNDTRLP	FIWLIWLWLP	SVGDDINILNGGEDLC*
	Asp2	MTDINYARLP	ITLFLFFFIP	CLSDDDNILNRGKDLC*
	Asp3	MSDINTARLP	YFLPPIFSPP	CVSDDIEMVLTRGENLC*
	Asp4	MSDINATRLP	HPFPLGLQP	CAGDVDNFTLTKGEDLC*
	Asp5	MSDINATRLP	GILIVWPP	CVGDDVNFTLTRGESLC*
	Asp6	MSDINTTRLP	IAFPEFIA	RVGDDIHRTLTRGESLC*
	Asp7	MSDINVTRLP	IFWFIYFP	CVGDDVDNTLTRGESLS*
	Asp8	MSDINAIRLP	IGRPENKP	CVGGDVNYILISGEKLC*
	Asp9	MSDINATRLP	IVFLEFYS	CVGDDVNSTLTRGESLC*
	Asp10	MSDINATRLP	IWGIGCDP	CVGDDVAAFLTRGEALC*	β-amanitin
	Asp11	MSDINATRLP	IWGIGCNP	SVGDEVTALLTRGEALC*	α-amanitin
	Asp12	MSDINASRLP	VIGLFGLP	YVSDDVQPILTRGDSLC*
	Asp13	MSDINASRLP	VIPFLLPP	CVSDDVNFTLTRGESLC*
	Asp14	MSDINATRLP	YFRPAPPP	CVSDDINPILTCGESLC*
	Asp15	MSDINAARLP	AWITDCP	CVGDDINRILTRGENIC*	“phallotoxin”
	Asp16	MSDINASRFP	AWLATCP	CVGDDVNPTIARGESLC*	phalloidin
	Asp17	MSDINATRLP	AWLVTCP	CVGDDVNFTLTRGESLC*	“phallotoxin”
	Asp18	MSDINATRLP	AWLVTCP	CVGDDVNPTITRGESLC*	“phallotoxin”
	Asp19	MSDINTIRIP	GPFGFA	YVGDEVENLLKRGESLS*
L. venenata	Lv1	MDANATRLP	IWGIGCNP	WTPESVNDTLTKDLS*	α-amanitin
	Lv2	MDANSTRLP	IWGIGCNP	WAPESVNDTLTRGKDLC*	α-amanitin

The MSDIN members with underlined numbers were verified at the genomic level. “Phallotoxin” means a novel heptapeptide similar to the phallotoxin cyclopeptide and capable of containing tryptathione (Trp-Cys)

Table 3

MSDIN family members cloned from genomic DNA of twelve amanitin-producing mushrooms

Name	No.	Leader peptide	Core peptide	Recognitionsequence	Product	GenBank accession no.
A. exitialis	Ae1a	MSDINATRLP	FIWVFGIP	GDIGTVLTRGENLC*		MN318165
	Ae2a	MSDINATRLP	IIWIIGNP	CVSDDVERILTRGESLC*		MN318166
	Ae3ab	MSDINATRLP	IWGIGCDP	CVGDDVTALLTRGEALC*	β-amanitin	MN264225
	Ae4ab	MSDINATRLP	IWGIGCNP	CVGDDVTSVLTRGEALC*	α-amanitin	MN264220
	Ae5b	MSDINATRLP	AWLTDCP	CVGDDVNRLLTRGESLC*	“phallotoxin”	MN264235
	Ae6b	MSDINATRLP	AWLVDCP	CVGDDVNRLLTRGESLC*	phallacidin	MN264231
	Ae7a	MSDINATRLP	VWIGYSP	CVGDDCIALLTRGEGLC*		MN318167
	Ae8a	MSDINATRLP	GFLFWA	YVGDDVDYILTRGESLA*		MN318168
	Ae9a	MSDINATRLP	GFLLWA	YVGDDVDYILTRGESLA*		MN318169
A. fuliginea	Af1a	MSDINATRLP	FPHFPPYNPP	CVSDDIHMVLTRGENLC*		MN318170
	Af2a	MSDINATRLP	YYLLLILPP	CVSDDLQTVLTRGENLC*		MN318171
	Af3a	MSDINATRLP	IFWFIYFP	CVGDDVDNTLARGESLS*		MN318172
	Af4b	MSDINATRLP	IWGIGCDP	CVGDDVAALITRGEALC*	β-amanitin	MN264226
	Af5a	MSDINATRLP	IWGIGCDP	CVGEDVAALITRGEALC*	β-amanitin	MN318173
	Af6a	MSDINATRLP	IWGIGCNP	SVGDEVTALLTSGEALC*	α-amanitin	MN318174
	Af7a	MSDINATRLP	LPSRPVFP	FVSDAIEVVLGRGEDLC*		MN318175
	Af8b	MSDINASRLP	AWLATCP	CIGDDVNPTITRGESLC*	phalloidin	MN264249
	Af9ab	MSDINATRLP	AWLVDCP	CVGDDVNRLLARGENLC*	phallacidin	MN264232
A. molliuscula	Am1b	MSDINATRLA	IWGIGCDP	CVGDDVTALLTRGEALC*	β-amanitin	MN264227
A. pallidorosea	Ap1a	MSDINATRLP	LIFIPPFIPP	CVSDDIQMVLTRGENLC*		MN318176
	Ap2a	MSDINAPRLP	LIFIPPFIPP	CVSDDIQMVLTRGEGLC*		MN318177
	Ap3a	MSDINATRLP	IPFHIPAP	SVGDDIEVVLGRGENLC*		MN318178
	Ap4a	MSDINATRLP	IWGIGCDP	CVGDDVTAVLTCGEALC*	β-amanitin	MN318179
	Ap5b	MSDINATRLP	IWGIGCDP	CVGDDVTAVLTRGEALC*	β-amanitin	MN264228
	Ap6ab	MSDINATRLP	IWGIGCNP	CVGDEVAALLTRGEALC*	α-amanitin	MN264222
	Ap7b	MSDINATRLP	IWGIGCNP	CVGDEVTALITRGEALC*	α-amanitin	MN264221
	Ap8a	MSDINATRLP	AWLATCP	CAGDDVNPTLTRGESLC*	phalloidin	MN318180
	Ap9a	MSDINATRLP	AWLMTCP	CVGDDVNPILTRGESVC*	“phallotoxin”	MN318181
	Ap10b	MSDINATRLP	AWLMTCP	CVGDDVNPTLTRGESLC*	“phallotoxin”	MN264236
	Ap11ab	MSDVNATRLP	AWLVDCP	CVGDDINRLLTRGENLC*	phallacidin	MN264233
A. rimosa	Ar1a	MSDINATRLP	IWGIGCDP	CVGDDVAALATRGEALC*	β-amanitin	MN318182
	Ar2ab	MSDINATRLP	IWGIGCDP	CVGDDVAALTTRGEALC*	β-amanitin	MN264229
	Ar3a	MSDINATRLP	IWGIGCNP	SVGDEVTALLASGEALC*	α-amanitin	MN318183
	Ar4ab	MSDINSTRLP	IWGIGCNP	SVGDEVTALLTRGEALC*	α-amanitin	MN264223
	Ar5b	MSDINATRVP	AWLAECP	CVGDDISHLLTRGENLC*	“phallotoxin”	MN264237
A. subfuliginea	Asf1a	MSDINATRLP	HPFPLGLQP	CAGDVDNFTLTKGEGLC*		MN318184
	Asf2a	MSDINATRLP	AIFLAWPP	CVGDNVNSTLTRGESLC*		MN318185
	Asf3a	MSDINATRLP	IWGIGCDP	CVSDDVAALLTRGEALC*	β-amanitin	MN318186
	Asf4a	MSDINATRLP	IWGIGCNP	CVGDEVAALLTRGEALC*	α-amanitin	MN318187
	Asf5a	MSDINATRLP	AWLVDCP	CVGDDVNRLITRGENLC*	phallacidin	MN318188
A. subjunquillea	Asj1a	MSDINATRLP	AYLPLFFIPP	CVSDDIEMVLTRGESLC*		MN318189
	Asj2a	MSDINATRLP	AYLPLFFIPP	CVSDDIEVVLTRGESLC*		MN318190
	Asj3a	MSDINATRLP	IWGIGCDP	CIGDDVTALLTRGEALC*	β-amanitin	MH142177
	Asj4a	MSDINATRLP	IWGIGCDP	CVGDEVTALLTRGEALC*	β-amanitin	MH142176
	Asj5a	MSDINATRLP	IWGIGCNP	CVGDEVAALLTRGEALC*	α-amanitin	MH142175
	Asj6ab	MSDINATRLP	AWLATCP	CAGDDVNPTLTRGESLC*	phalloidin	MN264250
	Asj7a	MSDINATRLP	AWLATCP	CVGDDVNPTLSRGESLC*	phalloidin	MN318191
	Asj8ab	MSDINATRLP	AWLVDCP	CVGDDINRLLTRGENLC*	phallacidin	MN264234
A. subpallidorosea	Asp1ab	MSDINATRLP	IWGIGCDP	CVGDDVAAFLTRGEALC*	β-amanitin	MN264230
	Asp2ab	MSDINATRLP	IWGIGCNP	SVGDEVTALLTRGEALC*	α-amanitin	MN264224
	Asp3ab	MSDINAARLP	AWITDCP	CVGDDINRILTRGENIC*	“phallotoxin”	MN272408
	Asp4ab	MSDINASRFP	AWLATCP	CVGDDVNPTIARGESLC*	phalloidin	MN272407
	Asp5a	MSDINATRLP	AWLITCP	CVGDDANPTITRGESLC*	“phallotoxin”	MN318192
	Asp6ab	MSDINATRLP	AWLVTCP	CVGDDVNPTITRGESLC*	“phallotoxin”	MN272409
	Asp7a	MSDINATRLP	AWLVTCP	CVGDDVNSTITRGESLC*	“phallotoxin”	MN318193
A. virosa	Av1a	MSDINATRLP	FLLFIIPP	CVSDDVNSTLTRGESLC*		MN318194
	Av2a	MSDINATRLP	FYFQPGFP	WSVGDDVNPTLTRGESLC*		MN318195
	Av3b	MSDINATRLP	IWGIGCNP	SVGDEATALLTRGEALC*	α-amanitin	MN272412
	Av4a	MSDINATRLP	SILIVWPP	CVGDDVNSTLTRGESLC*		MN318196
	Av5a	MSDINATRLP	SILVVWPP	CVSDDVNSTLTRGESLC*		MN318197
	Av6a	MSDINATRLP	AWLATCP	CVGDDVNPTLARGESLC*	phalloidin	MN318198
	Av7a	MSDINATRLP	AWLVDCP	CVGDDINRLLTRGENLC*	phallacidin	MN318199
	Av8a	MSDINATRLP	AWLVTCP	CVGDDVNPTLTRGESLC*	“phallotoxin”	MN318200
	Av9a	MSDINATRLP	GPFLFFP	FVSDDIEVILRRGEDLC*		MN318201
G. marginata	Gm1b	MFDTNATRLP	IWGIGCNP	WTAEHVDQTLASGNDIC*	α-amanitin	MN272413
	Gm2b	MFDTNSTRLP	IWGIGCNP	WTAEHVDQTLVSGNDIC*	α-amanitin	MN272414
G. sulciceps	Gs1b	MFDTNATRLP	IWGIGCNP	WTAEHVDQTLASGNDIC*	α-amanitin	MN272417
	Gs2b	MFDTNSTRLP	I*GIGCNP	WTAEHIDQTLVSGNDTC*		MN272418
L. venenata	Lv1b	MDANATRLP	IWGIGCNP	WTPESVNDTLTKDLS	α-amanitin	MN272421
	Lv2b	MDANSTRLP	IWGIGCNP	WAPESVNDTLTRGKDLC	α-amanitin	MN272422

Superscripts a and b are for products cloned with degenerate and specific primers, respectively. “Phallotoxin” means a novel heptapeptide similar to phallotoxin cyclopeptide and capable of containing Tryptathione (Trp-Cys)

Among the MSDIN members found in the 9 lethal species of Amanita sect. Phalloideae included in our study, in addition to the common α-amanitin, β-amanitin, phallacidin and phalloidin (PHA) peptides, several unnamed predicted peptides overlapped among different Amanita species, including “FNFFRFPYP” in A. exitialis and A. rimosa; “FPWTGPFVP” in A. fuliginea and A. pallidorosea; “IIIVLGLIIP” in A. fuliginea and A. rimosa; “YFLPPIFSPP” in A. molliuscula and A. subpallidorosea; “ISDPTAYP” in A. pallidorosea and A. rimosa; “IFWFIYFP” in A. exitialis, A. fuliginea, A. rimosa and A. subpallidorosea; and “ISDPTAYP” in A. pallidorosea, A. rimosa, A. subfuliginea and A. subpallidorosea. The remaining 87 core regions were unique to their corresponding species. The MSDIN genes encoding “AWLTDCP” in A. exitialis; “AWLMTCP” in A. pallidorosea; “AWLECP” in A. rimosa; “AWLVTCP” in A. fuliginea, A. subpallidorosea and A. virosa; and “AWITDCP” and “AWLITCP” in A. subpallidorosea probably produce new unknown phallotoxins because their core regions are similar to those of phallacidin (AWLVDCP) and phalloidin (AWLATCP). As expected, no MSDIN genes were found in A. oberwinklerana, a species belonging to Amanita sect. Lepidella [16] or sect. Roanokenses that dose not contain cyclopeptide toxins [15].

In G. marginata, G. sulciceps and L. venenata, only MSDIN genes encoding α-amanitin were found, and such genes were the only genes common to the amanitin-producing genera Amanita, Galerina and Lepiota. Unlike the situation in lethal Amanita species, no MSDIN genes other than the α-amanitin gene were discovered. Interestingly, an MSDIN gene with the full amino acid sequence MFDTNSTRLPWTAEHIDQTLVSGNDTC* (with the core region shown in bold and underlined) was found in G. sulciceps. Due to its similarity to the α-amanitin gene Gs_α-AMA1 (MFDTNATRLPWTAEHVDQTLASGNDIC*) in G. sulciceps, it was designated Gs_α-AMA2.

Similarly, 19 POP genes were identified from the transcriptomes of nine Amanita, two Galerina and one Lepiota species using known POPA and POPB genes of A. bisporigera and G. marginata, respectively, as the TBLASTN queries and these sequences were further verified by PCR amplification. Eleven lethal Amanita, Galerina and Lepiota species contained both POPA and POPB genes, but A. oberwinklerana, an Amanita species producing no cyclopeptide toxins, only exhibited the POPA gene. All of the obtained POP sequences and their accession numbers are listed in Table 4.

Table 4

Gene sequences used in the molecular phylogenetic analyses and their GenBank accession numbers

Taxon	Gene	Source	GenBank accession no.
Agaricus bisporus var. bisporus	POP	NCBI	XM006459721
Agaricus bisporus var. burnettii	POP	NCBI	JH971409
Amanita bisporigera	α-AMA1	Pulman et al., 2016	–
	α-AMA2	Pulman et al., 2016	–
	PHA1	Pulman et al., 2016	–
	PHA2	Pulman et al., 2016	–
	POPA	Pulman et al., 2016	–
	POPB	Pulman et al., 2016	–
Amanita exitialis	α-AMA	Our study	MN264220
	β-AMA	Our study	MN264225
	PHA	Our study	MN264231
	“AWLTDCP”	Our study	MN264235
	POPA	Our study	MN264238
	POPB	Our study	MN264244
Amanita fuliginea	β-AMA	Our study	MN264226
	PHA	Our study	MN264232
	PHD	Our study	MN264249
	POPA	Our study	MN264239
	POPB	Our study	MN264245
Amanita molliuscula	β-AMA	Our study	MN264227
	POPA	Our study	MN264240
	POPB	Our study	MN264246
Amanita muscaria	POPA	NCBI	KN818232
Amanita oberwinklerana	POPA	Our study	MN264241
Amanita pallidorosea	α-AMA1	Our study	MN264221
	α-AMA2	Our study	MN264222
	β-AMA	Our study	MN264228
	PHA	Our study	MN264233
	“AWLMTCP”	Our study	MN264236
	POPA	Our study	MN264242
	POPB	Our study	MN264247
Amanita phalloides	α-AMA	Pulman. et al., 2016	–
	β-AMA1	Pulman. et al., 2016	–
	β-AMA2	Pulman et al., 2016	–
	PHA	Pulman et al., 2016	–
	PHD1	Pulman et al., 2016	–
	PHD2	Pulman et al., 2016	–
	PHD3	Pulman et al., 2016	–
	POPA	Pulman et al., 2016	–
	POPB	Pulman et al., 2016	–
Amanita rimosa	α-AMA	Our study	MN264223
	β-AMA	Our study	MN264229
	“AWLAECP”	Our study	MN264237
	POPA	Our study	MN264243
	POPB	Our study	MN264248
Amanita subjunquillea	α-AMA	Luo et al., 2018	–
	β-AMA1	Luo et al., 2018	–
	β-AMA2	Luo et al., 2018	–
	PHA	Our study	MN264234
	PHD	Our study	MN264250
	POPA	Luo et al., 2018	–
	POPB	Luo et al., 2018	–
Amanita subpallidorosea	α-AMA	Our study	MN264224
	β-AMA	Our study	MN264230
	PHD	Our study	MN272407
	“AWITDCP”	Our study	MN272408
	“AWLVTCP”	Our study	MN272409
	POPA	Our study	MN272410
	POPB	Our study	MN272411
Amanita thiersii	POPA	NCBI	KZ302001
Amanita virosa	α-AMA	Our study	MN272412
Anomoporia bombycina	POP	JGI	–
Auriculariopsis ampla	POP	NCBI	VDMD01000002
Bolbitius vitellinus	POP	JGI	–
Conocybe apala	POP	NCBI	FJ906819
Coprinellus micaceus	POP	NCBI	QPFP01000027
Coprinopsis cinerea	POP	NCBI	XM001841192
Coprinopsis marcescibilis	POP	NCBI	ML210154
Cortinarius glaucopus	POP	JGI	–
Crucibulum laeve	POP	NCBI	ML213591
Cyathus striatus	POP	JGI	–
Fistulina hepatica	POP	NCBI	KN881639
Galerina marginata	α-AMA1	Our study	MN272413
	α-AMA2	Our study	MN272414
	POPA	Our study	MN272415
	POPB	Our study	MN272416
Galerina sulciceps	α-AMA1	Our study	MN272417
	“α-AMA2”	Our study	MN272418
	POPA	Our study	MN272419
	POPB	Our study	MN272420
Gymnopilus chrysopellus	POP	JGI	–
Gymnopilus dilepis	POP	NCBI	NHYE01005597
Hebeloma cylindrosporum	POP	NCBI	KN831777
Hypholoma sublateritium	POP	NCBI	KN817688
Hypsizygus marmoreus	POP	NCBI	LUEZ02000233
Laccaria amethystina	POP	NCBI	KN838546
Laccaria bicolor	POP	NCBI	DS547115
Lepiota brunneoincarnata	POPA	NCBI	MN912699
Lepiota subincarnata	α-AMA1	Luo et al., 2018	–
	α-AMA2	Luo et al., 2018	–
	POPB	Luo et al., 2018	–
Lepiota venenata	α-AMA1	Our study	MN272421
	α-AMA2	Our study	MN272422
	POPA	Our study	MN272423
	POPB	Our study	MN272424
Lepista nuda	POP	JGI	–
Leucoagaricus sp.	POP	NCBI	KQ962668
Macrolepiota fuliginosa	POP	JGI	–
Panaeolus cyanescens	POP	NCBI	NHTK01005903
Pleurotus ostreatus	POP	NCBI	KL198007
Plicaturopsis crispa	POP	JGI	–
Pterula gracilis	POP	NCBI	ML178816
Schizophyllum commune	POP	NCBI	GL377318
Termitomyces sp.	POP	NCBI	KQ412502

Comparison of MSDIN precursor peptide sequences

WebLogo alignment was carried out for 145 MSDIN sequences obtained from 9 Amanita species (Fig. 1a). The composition and structure of these sequences and the relative degree of conservation of the amino acids at each point were analysed. As shown in Fig. 1a, the MSDIN precursor peptides of the Amanita species were 31–38 amino acids in length and could be divided into three regions: a highly conserved upstream leader peptide, a relatively conserved downstream recognition sequence and a highly variable core peptide. The core peptide was located between P10 and P21 and included the latter proline, and its ends were the leader peptide and recognition sequence of MSDIN. The leader peptide contained 10 amino acids, and the M1S2D3I4N5R8L9P10 residues were highly conserved, with conservation rates of 100% (145/145), 91.7% (133/145), 97.2% (141/145), 93.1% (135/145), 99.3% (144/145), 99.3% (144/145), 94.5% (137/145) and 99.3% (144/145), respectively. In the leader peptide, P10 was the first cleavage site for prolyl oligopeptidase (POPB) [12]. The recognition sequence usually contained 17 amino acids, beginning with C22V23G24D25D26, with conservation rates of 76.5% (111/145), 79.3% (115/145), 66.2% (96/145), 93.1% (135/145) and 83.4% (121/145), and ending with L31T32R33G34E35L37C38, with conservation rates of 86.9% (126/145), 73.8% (107/145), 82.1% (119/145), 96.6% (140/145), 93.1% (135/145), 97.9% (142/145) and 91.7% (133/145), respectively. In the recognition sequence, L31 and L37 were conducive to the formation of an alpha helix and substrate recognition by the POPB enzyme [28]; additionally, the C-terminal Cys38 (sometimes replaced with Ser) was indispensable for performing the function of POPB [12]. The core peptides were predicted to form cyclopeptides in which the last amino acid, P21 (the second cleavage site for POPB), was highly conserved, with a conservation rate of 94.5% [12].

Fig. 1

Alignment of MSDIN precursor peptide sequences. a WebLogo [27] alignment of 145 MSDIN members from 9 Amanita species. The letter height of each amino acid represents its conservation degree, and the higher the letter, the more conserved the site. b Alignment of the precursor peptide sequences of α-amanitin from 15 amanitin-producing mushrooms. c Alignment of the precursor peptide sequences of α-, β-amanitin, phallacidin and phalloidin from 12 Amanita species. Letters with white background are variations compared with the consensus sequence. The sequences of A. bisporigera, A. phalloides were from Pulman et al. (2016), the sequences of A. fuligineoides were from Li et al. (2014) and the sequences of L. brunneoincarnata were from Lüli et al. (2019)

The α-AMA precursor peptide sequences of the genera Amanita, Galerina and Lepiota were compared, as shown in Fig. 1b. The α-AMA sequences showed few differences within the same genus but presented more differences between the different genera. The α-AMA leader peptides of the three genera showed few differences and were more conserved than the other sequences. The leader peptides of Amanita and Galerina contained 10 amino acids, while that of the genus Lepiota contained 9, and the sequences of the three genera started with “MSDIN”, “MFDTN”, and “MDAN”, respectively. In the recognition sequences of the three genera, with the exception of several highly conserved amino acids (specifically V, L, G, and LC or LS at the end), many differences were observed. Overall, there were large differences among the α-AMA sequences of Amanita, Galerina and Lepiota, but the Galerina and Lepiota α-AMA sequences were closer to each other than to those of Amanita.

The Amanita MSDIN genes encoding amatoxins (α-, β-amanitin) and phallotoxins (phallacidin and phalloidin), which are the major cyclopeptides in these mushrooms, were aligned, and the highlighted variations were compared to representative consensus sequences (Fig. 1c). In general, the precursor peptide sequences encoding the same toxin shared high identity. There were obviously more variations in the recognition sequences than in the leader peptides. The phallotoxin sequences presented more variations than the amatoxin sequences.

Structures of MSDIN and POP genes

The genomic sequences and coding sequences of the toxin MSDIN and POP genes obtained in this study (Table 2)were subjected to pairwise alignment, and the gene composition of the exons and introns was analysed. As shown in Fig. 2, the POPA genes comprised 19 exons and 18 introns, while the POPB genes comprised 18 exons and 17 introns, which was very similar to other known POP genes. The α-AMA genes of Amanita and Galerina contained three introns, while the α-AMA genes of Lepiota contained two or three introns. In addition to α-AMA, other MSDIN toxin genes in Amanita species, such as β-AMA, PHA and PHD, were also composed of three introns.

Fig. 2

Structures of α-AMA and POP exemplified by four agaric species

Phylogenetic analysis of MSDIN and POP genes

From the phylogenetic analysis, two maximum likelihood (ML) trees based on 46 MSDIN toxin genes from 14 amanitin-producing mushrooms and 58 POP genes from 46 agaric species were constructed. In the MSDIN toxin gene tree (Fig. 3), all MSDIN toxin gene sequences were distributed in four clades. Clade I contained 10 α-amanitin gene sequences and 10 β-amanitin gene sequences from 10 lethal Amanita species forming a cluster with 95% bootstrap support and a Bayesian posterior probability of 1.0. Clade II contained MSDIN genes encoding AWLVDCP (phallacidin, PHA) and the unknown related variants AWLAECP, AWITDCP and AWLTDCP forming a cluster with 95% bootstrap support and a Bayesian posterior probability of 1.0. Clade III contained MSDIN genes encoding AWLATCP (phalloidin, PHD) and the unknown related variants AWLMTCP and AWLVTCP forming a cluster with a 100% bootstrap and a 1.0 Bayesian posterior probabilities. Clade IV contained α-amanitin genes from Galerina and Lepiota species, including G. marginata, G. sulciceps, L. subincarnata and L. venenata, forming a cluster with a 100% bootstrap and a 1.0 Bayesian posterior probabilities. In the POP gene tree (Fig. 4), POPA sequences from Amanita, Galerina and Lepiota were separated from each other in different groups. Amanita POPA sequences (12) were clustered together as a single group, while Galerina POPA sequences (2) were clustered in a group containing Gymnopilus dilepis and Gymnopilus chrysopellus, and Lepiota POPA sequences (2) were clustered in a group containing Agaricus bisporus var. bisporus, Agaricus bisporus var. burnettii, Leucoagaricus sp. and Macrolepiota fuliginosa. However, POPB sequences (13) belonging to three disjunct genera (Amanita, Galerina and Lepiota) were clustered together forming a monophyletic group.

Fig. 3

Phylogenetic trees generated from maximum likelihood analysis based on toxin MSDIN genes. Bootstrap percentages (> 50%) based on 1000 replications and Bayesian posterior probabilities (> 0.90) are shown at nodes. Bar, a substitution per 10 nucleotides

Fig. 4

Phylogenetic trees generated from maximum likelihood analysis based on POP genes. Bootstrap percentages (> 50%) based on 1000 replications and Bayesian posterior probabilities (> 0.90) are shown at nodes. Bar, a substitution per 10 nucleotides

Discussion

It has been proven that lethal Amanita species are classified in section Phalloideae of Amanita and that these species contain members of the MSDIN gene family, allowing them to produce many small cyclopeptides, such as α-amanitin, on ribosomes [6, 16]. In the present study, nine lethal Amanita species from China, including A. exitialis, A. fuliginea, A. mulliuscula, A. pallidorosea, A. rimosa, A. subfuliginea, A. subjunquillea, A. subpallidorosea and A. virosa, were proven to contain MSDIN genes, as found in other lethal Amanita species described previously, such as A. bisporigera. These results further suggest that species of the Amanita section Phalloideae are genetically similar and are able to biosynthesize amatoxins, phallotoxins and some other unknown peptides.

Based on the MSDIN gene data from nine lethal Amanita species from China obtained in our study and some other European and North American species, such as A. bisporigera, A. phalloides and A. ocreata [5, 6], most MSDIN genes have not been found to be common and may even be unique among these lethal Amanita species. In species of Amanita section Phalloideae, the MSDIN gene encoding α-amanitin is present in all species, whereas the MSDIN genes encoding β-amanitin, phallacidin and phalloidin are widely distributed but are not common to all species. This findings suggested that each lethal Amanita species exhibits its own independent MSDIN family and that few overlapping MSDIN genes occur among lethal Amanita species.

In addition to species of Amanita section Phalloideae, some Galerina and Lepiota species, such as G. marginata and L. brunneoincarnata, produce amatoxin [2, 29]. In our study, the MSDIN gene-mining results showed that G. marginata and L. venenata only presented two copies of the α-amanitin gene, and no additional MSDIN genes were found, consistent with the results of Luo et al. (2012) and Lüli et al. (2019) [7, 9]. Lethal Galerina and Lepiota species are considered to have two copies of the α-amanitin gene. However, the analysis of the MSDIN genes of another Galerina species, G. sulciceps, showed that G. sulciceps only presented a single copy of the α-amanitin gene, although it also exhibited an MSDIN gene that was extremely similar to the α-amanitin gene with an I*GIGCNP core region. This MSDIN gene seemed to represent an α-amanitin gene mutation, and we speculated that its tryptophan (W) codon, TGG, in the core region has been mutated to a termination codon, TGA, via a single-base substitution, thus inhibiting the proper expression of the gene. In general, only the α-amanitin gene is found in Amanita, Galerina and Lepiota, which indicates that the α-amanitin genes of the three genera might share a common origin or originate from a single genus. Additionally, MSDIN genes including β-AMA, PHA, PHD, etc., were only found in Amanita, which indicated that these MSDIN genes (except for α-AMA) were likely derived from lethal Amanita species. The higher genetic diversity of MSDIN genes in Amanita than in Galernia and Lepiota causes the lethal Amanita species to produce greater amounts of toxic compounds than Galernia and Lepiota species. For this reason, lethal Amanita species present a greater defence ability to prevent their consumption than Galernia and Lepiota species.

Lethal Amanita species contain three primary kinds of peptide toxins: amatoxins, phallotoxins and virotoxins [21]. MSDIN genes encoding amatoxin and phallotoxin were discovered in 2007 [5], but there has been no related evidence of MSDIN genes encoding virotoxins published to date. It has been reported that A. subpallidorosea and A. virosa contain virotoxins [3, 30]. In this study, toxin genes of the two lethal Amanita species were also identified, and no virotoxin genes were found. Nevertheless, the two species both contain MSDIN genes encoding AWLATCP (PHD) and AWLVTCP, which only show a single amino acid difference in the composition of the virotoxins (AWLATSP or AWLVTSP). Therefore, we speculated that virotoxins might be encoded by the PHD gene or the phallotoxin-like gene AWLVTCP and that cysteine (C) is transformed to serine (S) during posttranslational modification.

Phylogenetic analysis showed that the Galerina α-AMA genes and Lepiota α-AMA genes were homologous but were distant from the Amanita α-AMA gene. In the genus Amanita, α-AMA and β-AMA are mixed and clustered in a clade, which indicates that β-AMA might be derived from α-AMA. PHA genes (AWLVDCP) were clustered with MSDIN genes encoding AWLAECP, AWITDCP and AWLTDCP, and PHD genes (AWLATCP) were clustered with MSDIN genes encoding AWLMTCP and AWLVTCP, which indicated that the encoded products of these MSDIN genes were very likely to correspond to new unknown phallotoxins, considering the similarity of their amino acid composition with those of PHA and PHD and their capacity to contain tryptathione (Trp-Cys). These phallotoxin-like genes might be variants derived from PHA and PHD. For example, we found that the PHA gene (AWLVDCP) sequence in A. exitialis was almost the same as the sequence of the MSDIN gene encoding AWLTDCP, with only a two-nucleotide difference in the core region, and the valine (V) codon GTA is likely mutated into the threonine (T) codon ACA. According to this finding, it can be inferred that the MSDIN genes in Amanita evolved faster than those in Galerina and Lepiota, which led to the generation of a variety of new peptide genes and might also be the reason why the Galerina and Lepiota α-AMA genes differed from the Amanita α-AMA genes.

Horizontal gene transfer (HGT), also known as lateral gene transfer, refers to the transmission of genetic material between distinct organisms, specifically across species boundaries [31, 32]. It has been reported that HGT is very common in prokaryotes and may be an important source of their biological evolution, and HGT also occurs in eukaryotes at a lower frequency than in prokaryotes [33-36]. The most recent reports suggest that HGT may be responsible for the α-amanitin biosynthetic pathway found in the three distantly related genera Amanita, Galerina and Lepiota [8, 9]. It has been reported that in amanitin-producing mushrooms, the POPB gene product catalyses the cleavage and cyclization of the toxin precursor peptide, while the POPA gene is a housekeeping gene that is unrelated to toxin biosynthesis [7, 12]. In our study, phylogenetic analysis based on the POP gene showed that the POPA genes of Amanita, Galerina and Lepiota were distributed in three separate groups, but the POPB genes of the three genera were highly homologous forming a highly monophyletic group, which apparently conflicted with the species taxonomic status and could not be explained by conserved gene inheritance. Additionally, the MSDIN and POP genes were proven to exhibit the same exon and intron structures. These results can be considered to represent evidence of HGT events among Amanita, Galerina and Lepiota. For the complete validation of HGT among amanitin-producing mushrooms in the future, the inclusion more related species and their genomic data will be required to perform a phylogenetic analysis with appropriate taxon sampling and tree-building methodologies.

Conclusions

In conclusion, the MSDIN gene family is abundant and diverse. In addition to the peptide toxins α-amanitin, β-amanitin, phallacidin, phalloidin, etc., the MSDIN family encodes a variety of unknown small cyclopeptides. The amanitin-producing species Amanita, Galerina and Lepiota exhibit a common toxin biosynthetic pathway, and their α-amanitin genes and POPB genes may have a common origin that involving HGT among the three distant genera.

Methods

Sample collection and preparation

Samples of seven Amanita species and Lepiota venenata were collected from the wild for RNA extraction and sequencing, and their fresh basidiocarps were cleaned and placed on dry ice then transported back to the lab and stored at − 80 °C. The mushroom samples intended for DNA extraction were dried with silica gel and then stored at 4 °C. The mycelia of two Galerina strains were cultivated to grow material for DNA and RNA extraction. Detailed information for the mushroom materials used in this study is given in Table 5.

Table 5

Information of the mushroom materials used in this study

Species name	Locality	Collection time	Specimen no.	GenBank accession no.	Nucleic acid extract
A. exitialis	Guangdong, China	2017-03-27	MHHNU 30937	KR996717	DNA, RNA
A. fuliginea	Hunan, China	2017-06-06	MHHNU 9047	MN061271	DNA, RNA
A. molliuscula	Jilin, China	2017-08-07	MHHNU 9142	MN061272	DNA, RNA
A. oberwinklerana	Hunan, China	2017-06-09	MHHNU 9051	MN061273	DNA, RNA
A. pallidorosea	Shandong, China	2018-08-13	MHHNU 31203	MN061274	DNA, RNA
A. rimosa	Hunan, China	2017-06-09	MHHNU 9050	MN061275	DNA, RNA
A. subfuliginea	Chongqing, China	2015-07-01	MHHNU 30946	MN061276	DNA
A. subjunquillea	Hunan, China	2012-09-10	MHHNU 7751	KR996715	DNA
A. subpallidorosea	Hunan, China	2017-09-14	MHHNU 8617	KU601411	DNA, RNA
A. virosa	Hunan, China	2016-09-09	MHHNU 8621	KY472227	DNA
G. marginata	–	–	MHHNU 8380	MN061277	DNA, RNA
G. sulciceps	–	–	MHHNU 7669	KX214585	DNA, RNA
L. venenata	Hubei, China	2017-9-10	MHHNU 31031	MK095189	DNA, RNA

G. marginata and G. sulciceps samples were cultured mycelia, and the other mushroom samples were wild fruiting bodies

Nucleic acid extraction and cDNA preparation

Total genomic DNA was extracted using the Fungal DNA Mini Kit (Omega Bio-tek, Norcross, USA). Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, USA) following the TRIzol User Guide. cDNA was synthesized using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMIX (Transgen Biotech, Beijing, China). The DNA and RNA quality and yield were detected using a SmartSpec Plus (Bio-Rad, Hercules, USA).

Transcriptome sequencing and de novo assembly

The concentration, purity and integrity of the RNA samples used for next-generation sequencing were further examined using an Agilent 2100 bioanalyzer (Agilent, Santa Clara, USA). Qualified RNA samples were used to construct circular single-stranded cDNA libraries, and the libraries were then sequenced on a BGISEQ-500 sequencer (BGI, Shenzhen, China). Clean reads were obtained using the filtering software SOAPnuke to remove reads containing adaptors, reads with more than 5% unknown bases, and low-quality reads (bases with a quality value < 15 accounting for more than 20% of the bases in a read) from the raw reads. These clean reads were de novo assembled using Trinity software. Finally, nonredundant unigenes were obtained using Tgicl software. All of these steps were performed by the Beijing Genomic Institute (BGI)-Wuhan in China.

Retrieval and annotation of MSDIN and POP genes

The unigene data obtained as described above were searched for MSDIN and POP genes (Galerina unigenes were provided by Professor Ping Zhang at Hunan Normal University) by using the known amino acid sequences of the MSDIN family and POP genes from A. bisporigera and G. marginata [5, 7] as queries for the online NCBI TBLASTN tool. Then, unigenes similar to the queries were manually annotated, and the coding sequences were predicted and translated into protein sequences using DNAMAN 7.0 software.

Cloning of MSDIN and POP genes

Partial MSDIN gene sequences were amplified from Amanita genomic DNA using the following degenerate primers: forward (5′-ATGTCNGAYATYAAYGCNACNCG-3′) and reverse (5′-CCAAGCCTRAYAWRGTCMACAAC-3′), according to the method of Li et al. (2014) [13]. The PCR mixtures contained 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, each primer at 0.4 μM, 1.25 U of Taq polymerase (Comwin Biotech, Beijing, China), and 1 μL of DNA template in a total volume of 25 μL. PCR was performed with the following program: initial denaturation at 94 °C for 4 min, 35 cycles at 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s, and the reaction batches were incubated at 72 °C for 2 min for terminal elongation.

Using the known MSDIN and POP genes from A. bisporigera and G. marginata as reference models [5, 7], specific primers (shown in Table S1) were designed to obtain target products that were close to the full lengths of the genes according to the flanking sequences of the CDS. The genomic DNA and cDNA of the Amanita, Galerina and Lepiota species were used as templates, and PCR was performed as follows: initial denaturation at 94 °C for 4 min, followed by 32 cycles of denaturation at 94 °C for 30 s, 55–60 °C for 30 s (annealing temperature for each target shown in Table S1), and extension at 72 °C (30 s for an MSDIN gene, 2 min for a POP gene), and a final extension at 72 °C for 5 min.

All PCR products were detected by agarose gel electrophoresis and purified using an EasyPure Quick Gel Extraction Kit (Transgen Biotech, Beijing, China). The purified products were ligated into the pEASY®-Blunt Zero Cloning Vector (Transgen Biotech, Beijing) and transformed into competent cells. Positive clones to be sequenced were selected using Amp-resistant LB medium and were further verified by colony PCR. Finally, all of the obtained genomic and coding sequences of the genes were used to manually predict the corresponding functions and structures by using DNAMAN 7.0 software.

Phylogenetic tree construction of MSDIN and POP genes

Forty-six coding sequences (CDSs) of MSDIN toxin genes and fifty-eight CDSs of POP genes were used for phylogenetic analysis, and their source and GenBank accession numbers are presented in Table 4. These sequences were aligned by using MAFFT v7.374 [37] and then manually adjusted by using BioEdit [38]. HKY + I + G and GTR + I + G were inferred as the best-fit models for the CDSs of the MSDIN and POP genes selected according to the AIC in MrModeltest v2.3 [39]. Maximum likelihood (ML) trees with 1000 bootstrap replicates and Bayesian inferences were generated with RAxML v7 [40] and MrBayes v3.1.2 [41], respectively.

Additional file 1: Table S1. Specific PCR Primers designed for peptide toxins and POP genes.