Biosynthesis of Antibiotic Leucinostatins in Bio-control Fungus Purpureocillium lilacinum and Their Inhibition on Phytophthora Revealed by Genome Mining

Purpureocillium lilacinum of Ophiocordycipitaceae is one of the most promising and commercialized agents for controlling plant parasitic nematodes, as well as other insects and plant pathogens. However, how the fungus functions at the molecular level remains unknown. Here, we sequenced two isolates (PLBJ-1 and PLFJ-1) of P. lilacinum from different places Beijing and Fujian. Genomic analysis showed high synteny of the two isolates, and the phylogenetic analysis indicated they were most related to the insect pathogen Tolypocladium inflatum. A comparison with other species revealed that this fungus was enriched in carbohydrate-active enzymes (CAZymes), proteases and pathogenesis related genes. Whole genome search revealed a rich repertoire of secondary metabolites (SMs) encoding genes. The non-ribosomal peptide synthetase LcsA, which is comprised of ten C-A-PCP modules, was identified as the core biosynthetic gene of lipopeptide leucinostatins, which was specific to P. lilacinum and T. ophioglossoides, as confirmed by phylogenetic analysis. Furthermore, gene expression level was analyzed when PLBJ-1 was grown in leucinostatin-inducing and non-inducing medium, and 20 genes involved in the biosynthesis of leucionostatins were identified. Disruption mutants allowed us to propose a putative biosynthetic pathway of leucinostatin A. Moreover, overexpression of the transcription factor lcsF increased the production (1.5-fold) of leucinostatins A and B compared to wild type. Bioassays explored a new bioactivity of leucinostatins and P. lilacinum: inhibiting the growth of Phytophthora infestans and P. capsici. These results contribute to our understanding of the biosynthetic mechanism of leucinostatins and may allow us to utilize P. lilacinum better as bio-control agent.


Introduction
Plant parasitic nematodes with wide host ranges cause enormous crop and economic losses amounting to $157 billion annually worldwide [1,2].Biological control by fungi has become increasingly popular due to nematicides' risks of environmental toxicity and adverse effects on human health [3].One of the most promising and commercialized agents, Purpureocillium lilacinum, has been evaluated to assess its bio-control activity against plant nematodes in a number of studies [2,4].In particular, P. lilacinum has been reported to effectively control such species as the cotton aphid Aphis gossypii [5], the greenhouse whitefly Trialeurodes vaporariorum, the glasshouse red spider mite Tetranychus urticae [6], and the leaf-cutting ant Acromyrmex lundii [7].
The genus Purpureocillium was recently proposed for of Ophiocordycipitaceae, based on the internal transcribed spacer (ITS) and translation elongation factor 1-α (TEF) sequences of P. lilacinum, although it was originally classified in the genus Paecilomyces [8].P. lilacinum is commonly isolated from soil, plant roots, nematodes and insects, and it occasionally infects people.This fungus employs flexible lifestyles, including soil-saprobes, plant-endophytes and nematode pathogens.Opportunistic infection occurs when nematode eggs encounter P. lilacinum; therefore, parasitism can be a mechanism for nematode bio-control (Fig 1A).It has now been confirmed that a serine protease [9], a cuticle-degrading protease [10] and chitinase [11] play important roles in infection by degrading nematode eggshells.
Recently, the production of SMs has been shown to be a mechanism that kills nematodes.For example, culture filtrates of P. lilacinum, in which leucinostatins were produced, caused strong mortality and inhibited nematode reproduction [12].In addition to leucinostatins, a few other SMs have been isolated from P. lilacinum.The novel pyridone alkaloid paecilomide, an acetylcholinesterase inhibitor, was produced when this fungus was co-cultured with Salmonella typhimurium [13].Two xanthone-anthraquinone heterodimers, acremoxanthone C and acremonidin A, were isolated in the course of a search for calmodulin ligands [14].
The leucinostatins (Fig 1B) are a family of lipopeptide antibiotics isolated from P. lilacinum [15], Paecilomyces marquandii [16][17][18] and Acremonium sp.[19].Leucinostatin A contains nine amino acid residues, including the unusual amino acid 4-methyl-L-proline (MePro), 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHyMeOA), hydroxyleucine (HyLeu), αaminoisobutyric acid (AIB), β-Ala, and a 4-methylhex-2-enoic acid at the N-terminus as well as an N1,N1-dimethylpropane-1,2-diamine (DPD) at the C-terminus.Twenty-four different structures have been described in the leucinostatin series [20].Leucinostatin A significantly suppressed prostate cancer growth in a coculture system in which prostate stromal cells stimulated the growth of DU-145 human prostate cancer cells through insulin-like growth factor I [21].When screening for antitrypanosomal compounds among several peptide antibiotics, leucinostatins showed the most potent activity against trypanosomes.Trypanosome infection causes human African trypanosomiasis, which is one of the world's most neglected diseases lacking satisfactory drugs [22].Furthermore, leucinostatins have displayed broad bioactivity against bacteria and fungi.These antibiotics' functions are based on their ability to inhibit ATP synthesis in the mitochondria as well as different phosphorylation pathways [23].These findings drew our attention to the relationships between the bio-control function of P. lilacinum and leucinostatins.Furthermore, genetic and molecular information regarding the biosynthesis of this family of lipopeptide antibiotics, of which little was known to date, could contribute to increasing its production and screening for more efficient derivative compounds.
Genome sequences have shed light on the mechanism of the endoparasitic lifestyle or nematode control beyond biological research.During the preparation of our manuscript, the genome sequence of P. lilacinum was published [24].Two other plant nematode endoparasitic fungi, Pochonia chlamydosporia [25] and Hirsutella minnesotensis [26], were recently sequenced.Genome sequencing revealed that P. chlamydosporia encoded a wide array of hydrolytic enzymes and transporters expressed at the mRNA level, which supported its multitrophic lifestyle, and H. minnesotensis, which mainly invades juvenile stage cyst nematodes, putatively conducted its parasitic process through lectins, secreted proteases and SMs.Thus, the genome sequence of P. lilacinum provides an opportunity to better understand its mechanism in controlling plant nematodes, and it would be useful to enhance its capabilities as a bio-control agent.At the same time, the genome sequence has the potential to solve the biosynthetic puzzle of leucinostatins as well as to detect novel genes and metabolites that might be of value in agriculture and medicine.
Here, we present the results of genome sequencing of the PLBJ-1 and PLFJ-1 strains of the bio-control agent P. lilacinum, and we increased our knowledge of its bio-control capabilities by comparing the sequences of P. lilacinum with those of other fungi.The genome revealed a repertoire of SM-encoding genes that illustrated the potential for using this fungus to discover natural products.Furthermore, we identified the leucinostatin gene cluster (lcs cluster) and proposed a hypothetical pathway for biosynthesis through genetic manipulation.In the course of screening for new activities of leucinostatins, we found that they inhibited the most notorious oomycetes P. infestans, which causes potato late blight and results in global yield losses of 16% [27].

Results
General structure of the P. lilacinum genome Two P. lilacinum isolates, PLBJ-1 and PLFJ-1, were sequenced to ensure the accuracy of the genome information and the subsequent analysis.PLBJ-1 and PLFJ-1 were assembled into 144 and 163 scaffolds, respectively, with total sizes of 38.14 and 38.53 Mb, while the published TERIBC I was assembled into 301 scaffolds with a total size of 38.82 Mb (Table 1).The comparative genome sizes of related fungi species are listed in S1 Table .A total of 11,773 and 11,763 gene models were predicted in both genomes, respectively, parallel to other ascomycetes fungi (S1 Table ).BLASTN analysis was performed between the two genomes and demonstrated that 83.56% of the PLBJ-1 genome and 82.79% of the PLFJ-1 genome shared high synteny (Fig 2A).According to the syntenic relationship of PLBJ-1 and PLFJ-1, we reconstructed 10 super-scaffolds (S2 Table ), which illustrated the physical ubieties of the assembled scaffolds; e.g., scaffold 00006, scaffold 00016 and scaffold 00015 in PLFJ-1 were combined into a superscaffold (Fig 2B).The overall syntenic relationship of PLBJ-1 and TERIBC 1 showed that 76.52% of the PLBJ-1 genome and 75.12% of the TERIBC 1 genome shared high synteny (S1 Fig).Approximately 6.07% of the repeat sequences that included transposon elements (TEs) (~4.37%) and tandem repeats (~1.70%) were identified in PLBJ-1.The Class I Strains PLBJ-1 and PLFJ-1 of P. lilacinum are obtained in this study, strain TERIBC 1 was sequenced in [24] recently.(retrotransposons) and Class II (DNA transposons) TEs occupied ~1.80% and ~0.76% of the genome, respectively.The PLFJ-1 isolate harbored a similar number of repeat sequences (6.00%).The distribution of the TE families was similar in the two isolates, with the exception of certain families, e.g., I, Gypsy, Penelope, Tc1-Mariner and hAT (S3 Table ).In total, the two isolates of P. lilacinum contained a larger number of retrotransposons than DNA transposons.P. lilacinum exhibited expansion of repeat content comparable to other ascomycetes fungi, with the exception of H. minnesotensis, Ophiocordyceps sinensis and Fusarium oxysporum (fol), in which the repeat sequences accounted for more than one quarter of the genome (S1 Table ).
In TERIBC 1, approximately 1.68% of the genome sequence was identified as repeat content.
Among the predicted genes of PLBJ-1, 90.4% were supported by RNASeq data from mycelia cultured in PDB.Both strains exhibited a consistent KOG pattern.Except for the category "General function prediction only", which was ambiguously sorted to a certain group, the most abundant KOG categories were "Signal transduction mechanisms", "Posttranslational modification, protein turnover, chaperones", "Lipid transport and metabolism", and "Intracellular trafficking, secretion, and vesicular transport" (S2 Fig) .A signal peptide analysis showed that 1,410 genes of PLBJ-1 and 1,448 genes of PLFJ-1 encoded putatively secreted proteins.
CAZymes that cleave and build polysaccharides could be required when P. lilacinum degraded the structural polysaccharide armor of nematode eggshells, such as chitin, during the course of its parasitism.The protease could stop the development of nematode eggs and drastically alter the eggshell structures when applied individually or in combination with chitinases [28,29].A detailed examination of the CAZymes and proteases of P. lilacinum was performed and compared with other fungi, including nematode parasitic fungi (P.chlamydosporia and H. minnesotensis), nematode-trapping fungi (Arthrobotrys oligospora and Monacrosporium haptotylum), entomopathogenic fungi (T.inflatum, Beauveria bassiana, Cordyceps militaris, Metarhizium robertsii, and O. sinensis), a mycoparasitic fungus (T.ophioglossoides), a saprotrophic fungus (T.reesei) and a plant pathogenic fungus (F.oxysporum).We identified 53 families containing 239 genes in PLBJ-1 and 55 families containing 253 genes in PLFJ-1 that encoded glycoside hydrolases (GH), which was more than the other fungi (an average of 213) (S4 Table ).The most abundant family in PLBJ-1 and PLFJ-1 was GH18, which was represented by 32 and 41 chitinases, respectively, that degrade the chitin present in the chitin protein complex of the nematode eggshell [30].Consistent with GHs, PLBJ-1 and PLFJ-1 contained relatively more carbohydrate-binding modules (CBMs) (59 and 64, respectively) (S5 Table ), which were frequently appended to the enzymes involved in polysaccharide depolymerization.A series of carbohydrate esterase (CE)-encoding genes were also detected in the P. lilacinum genomes (33 and 32, respectively), including the most abundant sterol esterases (CE10) and cutinases (CE5), which are virulence factors of some plant pathogens [31] (S6 Table ).Another major class of CAZymes, the glycosyltransferases (GT), establish natural glycosidic linkages across a broad range of small and macromolecules, and they were represented in the PLBJ-1 genome with 115 members in 32 families and in the PLFJ-1 genome with 124 members in 32 families (S7 Table ).These enzymes' classification demonstrated that they exhibited less variability in ascomycetes than did GHs, a trend that was maintained in a previous analysis [32].The P. lilacinum genome contained more proteases (430 and 443, respectively) than other fungi (an average of 396).The largest category of proteases encoded in PLBJ-1 and PLFJ-1 were serine proteases (194 and 198, respectively) (S8 Table), 76 and 81 of which were secreted proteins, respectively.Among the serine proteases, we identified 34 subtilisins (S8) and ten serine carboxyproteases (S10) in the PLBJ-1 genome (36 and 11, respectively, in PLFJ-1), which were reported to be involved in infection and the lethal activity of nematodes [28,33].The metalloprotease (108 in PLBJ-1 and 109 in PLFJ-1) and cysteine protease (66 in PLBJ-1 and 68 in PLFJ-1) families also accounted for a significant proportion of the proteases.
A whole genome analysis was conducted against the pathogen-host interaction (PHI) gene database to identify potential virulence-associated genes, under the assumption that the homologue of an experimentally validated pathogenic gene suggested that it played a pathogenic role [34].We demonstrated that 2,844 (24.1%) and 2,892 (24.6%) proteins of PLBJ-1 and PLFJ-1, respectively, showed sequence similarity to those in the PHI database.Among these proteins, 299 and 317 proteins of PLBJ-1 and PLFJ-1, respectively, were classified as putatively secreted proteins.The KOG functional class distribution of genes related to PHI showed a similar pattern to the whole genome KOG analysis (S2 Fig) .The PHI database search yielded 195 CAZymes in PLBJ-1 and 217 in PLFJ-1, 28 and 36 of which were chitinases (GH18), respectively.Of the proteases, 125 in PLBJ-1 and 132 in PLFJ-1 were pathogenic genes according to the PHI database, of which 64 and 72 were identified as secreted proteins, respectively, and these proteins were more likely to function during the infection process [35].

Phylogenomic relationship and orthologous analysis
A phylogenomic tree was constructed based on 855 single-copy orthologues of P. lilacinum and 34 other filamentous fungi, with Saccharomyces cerevisiae as the outgroup.The results verified that P. lilacinum belongs to Ophiocordycipitaceae, as described by Jennifer Luangsa-ard [8], and it formed a clade with T. inflatum, T. ophioglossoides, O. sinensis [36], O. unilateralis [37] and H. minnesotensis (Fig 3A).The inferred phylogeny illustrated that T. inflatum and T. ophioglossoides were most closely related to P. lilacinum, and they diverged after their split with O. sinensis, H. minnesotensis and O. unilateralis.This phylogeny also reinforced the previous analysis that found that the split between Cordycipitaceae (including B. bassiana and C. militaris) and Clavicipitaceae (including P. chlamydosporium and M. anisopliae) occurred before Ophiocordycipitaceae diverged from Clavicipitaceae (Fig 3A).The three nematode parasitic fungi P. chlamydosporium, H. minnesotensis and P. lilacinum clustered with insect pathogens, indicating that nematode and insect pathogens might share a common ancestor.
A comparative genomic analysis was performed between P. lilacinum and other nematoderelated fungi (the nematode parasites P. chlamydosporia and H. minnesotensis and the nematode-trapping fungi A. oligospora and M. haptotylum).A total of 17,995 orthologous clusters consisting of 76,151 proteins were identified, of which 4,652 clusters containing 35,972 proteins were mapped to all four of the fungi types (Fig 3B).On the whole, the nematode-trapping fungi, which capture nematodes through an entirely different mechanism compared to P. lilacinum [26], possessed the largest number of unique gene clusters, although they had a more distant phylogenetic relationship with the other fungi in Hypocreales (Fig 3B).P. lilacinum contained a large number (3651) of species-specific clusters, while P. lilacinum shared 7,700, 6,673 and 5,253 clusters with P. chlamydosporia, H. minnesotensis and the nematode-trapping fungi, respectively.

Analysis of paralogous gene families
Lineage-specific expansions could provide material for the evolution of a specific functional system or adaptation in eukaryotes [38].To study gene family expansions in P. lilacinum, a comparative genomic analysis of 15 fungal species (PLBJ-1, PLFJ-1, P. chlamydosporia strain 123, P. chlamydosporia strain 170, H. minnesotensis, A. oligospora, M. haptotylum, T. inflatum, B. bassiana, C. militaris, M. robertsii, O. sinensis, T. ophioglossoides, T. reesei, and F. oxysporum) was performed.In total, 1,963 gene families with more than one gene expansion were identified in both PLBJ-1 and PLFJ-1, of which 1,761 gene families were only present in P. lilacinum, and some gene families with significant expansion are listed in S9 Table.However, most families were annotated as reverse transcriptases and transposases, and the others were related to transporters or lyases.When the nematode parasitic fungi P. chlamydosporium and H. minnesotensis were considered, 2,936 orthologous clusters showed expansion in the five isolates.The largest paralogous expansion contained protein families associated with SMs, such as cytochrome P450s, oxidoreductases, and transporters.In addition, these families also contained transcription factors, glycosyl hydrolases, the hAT family, the majority of which are listed in S10 Table.

SMs analysis based on the sequence of the P. lilacinum genome
To evaluate the capability of P. lilacinum to produce SMs, we searched the genome of PLBJ-1 and PLFJ-1 for biosynthetic genes encoding the four classes of the main SM-associated synthetases, including polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), terpene synthase (TS) and dimethylallyl tryptophan synthase (DMATS) [26].A uniform SM profile with parallel categories and numbers was presented in the two genomes (S11 Table ).In total, 13 PKSs, 10 NRPSs, two PKS-like enzymes, 10 NRPS-like enzymes, one DMATS, 4 TSs and one PKS-NRPS hybrid were identified in the PLBJ-1 genome, as described in S11 Table .Compared to sequenced species in Ophiocordycipitaceae, the number of SMs in P. lilacinum (41) was similar to the 45 SMs in T. ophioglossoides, 39 SMs in O. unilateralis, more than 30 SMs in Ophiocordyceps sinensis, fewer than 55 SMs in T. inflatum [39], and 101 SMs in the nematode endoparasitic fungus H. minnesotensis [26].These core backbone genes were dispersed among 39 clusters with other enzymes, such as transcriptional regulators, P450s and transporters, as predicted by antiSMASH (antibiotics and Secondary Metabolite Analysis SHell) [40] (S11 Table ).According to the BLAST results from the NCBI NR database, no homologues of functionally characterized SMs were detected.Among them, we detected the expression of 29 core genes with FPKM (fragments per kilobase of transcript per million mapped fragments) values > 0.5, using an RNA-seq analysis of PLBJ-1 cultured in PDB medium for 8 days.
A phylogenetic tree was constructed based on the KS domain amino acid sequence of the PKSs in P. lilacinum and the products of known PKSs, which were divided into three main clades: non-reducing (NR) PKSs, partially reducing (PR) PKSs and highly reducing (HR) PKSs (S3 Fig) .VFPBJ_05021, VFPBJ_09342, VFPBJ_09755, and VFPBJ_10843 were predicted as NR PKS-encoding genes, and they shared the highest homology with the non-reducing biosynthetic genes, such as citrinin [41] and griseofulvin [42].VFPBJ_00212, VFPBJ_02527, VFPBJ_02532, VFPBJ_03442, VFPBJ_05962, VFPBJ _06473, VFPBJ _07567 and VFPBJ_09314 were distributed in the HR PKS clade in close relationship with HR polyketides, such as fumonisin synthase Fum1p [43].The phylogenetic analysis was consistent with the domain structure analysis of degree of reduction, in which the HR PKS contained the reductive domains KR (keto-reductase), ER (enoyl reductase) and DH (dehydratase), while the NR PKS did not contain these domains (S3 Fig, S11 Table).VFPBJ_05021 and VFPBJ_09342 were grouped with the antibiotics griseofulvin and citrinin with a bootstrap value of 100%, and they shared a common domain structure.This finding suggested that griseofulvin/citrinin or structurally related compounds could be produced by P. lilacinum.However, we did not detect these compounds when P. lilacinum was cultured in PDB for 8 days.
Among the 10 NRPSs, six contained one module or an incomplete module, which could encode products with one amino acid.Four NRPSs were multi-module enzymes, which could encode products composed of more than one amino acid.To examine the potential NRPS orthologues of P. lilacinum and to detect the feasible NRPS evolutionary mechanism in the family Ophiocordycipitaceae, a genealogy was created based on the A-domains from the NRPS of fungi in Ophiocordycipitaceae and several functionally characterized products (S4 Fig) .The tree depicted an intricate evolutionary relationship for the NRPS genes.A general trend throughout the tree was that, in Ophiocordycipitaceae, many A-domains clustered with orthologues in other species than with in the same protein.Notably, the 11 A-domains of the cyclosporine synthetases from T. inflatum clustered separately (S4 Fig, node 3), indicating that other species were incapable of encoding cyclosporine and that its evolution occurred after T. inflatum diverged from these fungi in Ophiocordycipitaceae.
This phylogenetic analysis of the A-domains for P. lilacinum detected a series of homologous A-domains: four of the mono-module NRPSs had functionally uncharacterized homologues.
VFPBJ_05068 was identified as siderophore synthetase, of which three of the A-domains were grouped with homologues to form a sub-clade (S4 Fig, node 2).The three A-domains of VFP BJ_06596 were grouped with TINF2556, annotated as an ergot alkaloid in T. inflatum, while TINF2556 contained four modules.
The peptaibiotics, a class of linear NRPSs that are abundant of AIB [44], were clustered into one sub-clade (S4 Fig, node 1), mainly including the peptaibiotics from T. ophioglossoides [45], T. inflatum and P. lilacinum.The ten A-domains from VFPBJ_02539 (identified as the leucinostatin biosynthetic gene lcsA in this study), clustered with the ten A-domains from the peptaibiotic TOPH_08469 in T. ophioglossoides, with bootstrap values of 100%, and a global BLAST analysis revealed that the sequence identity of the two homologues was 65%.Neither orthologue was identified in other species of Ophiocordycipitaceae.The single A-domain of lcsA was scattered in the peptaibiotic sub-clade of the tree, while A2, A5 and A6, which activated Leu or related amino acids, were identified in the subsequent study and were grouped together with a bootstrap value of 60%, suggesting that both lineage-specific changes and module duplication contributed to the evolution of the leucinostatin metabolites.In the previous study, A4, A7 and A8 of TOPH_08469 were distributed in a sub-clade enriched in A-domains encoding AIB [45], and our study demonstrated that A4, A7 and A8 of lcsA were encoded for AIB.
In T. ophioglossoides, the TOPH_08469 gene cluster was predicted to contain 28 genes from TOPH_08452 to TOPH_08478 that were located in an ~124 kb region [45].A comparative analysis of genes surrounding lcsA and TOPH_08469 cluster revealed a high synteny (Fig 4A).VFPBJ_02521 (designed as lcsG) shared 68% sequence identity with TOPH_08452, and lcsA shared 66% sequence identity with TOPH_08469.Interestingly, no homologues of the genes next to the cluster, VFPBJ_02510 to VFPBJ_02520 and VFPBJ_02540 to VFP BJ_02550, were identified in the T. ophioglossoides genome.Within the lcs cluster, two genes, cytochrome P450 lcsI and a protein with unknown function, lcsM, did not possess homologues in the TOPH_08469 cluster, while all of the leucinostatin biosynthetic genes in T. ophioglossoides (TOPH_08452 to TOPH_08469) had homologues within the lcs cluster.These results suggested that this nearly 100 kb region might have been horizontally transferred from other fungal or bacterial species.However, leucinostatins have not been reported to be produced by T. ophioglossoides to date.

Identification of the NRPS gene lcsA, which is involved in the leucinostatin biosynthesis pathway
The lipopeptide leucinostatin A contains ten amide bonds that divide the molecule into 11 moieties, including 4-methylhex-2-enoic acid, 9 amino acid residues and DPD.The property of the mixture of the polyketide and peptide moieties in the leucinostatins indicated a PKS, NRPS or hybrid PKS-NRPS origin.It is logical to consider that a single reducing PKS encodes the 4-methylhex-2-enoic acid, and a NRPS enzyme encodes the remaining portion, as in the models for emericellamide synthesis in Aspergillus nidulans [46] and pneumocandin in Glarea lozoyensis [47].Among the multi-module NRPSs in P. lilacinum, VFPBJ_05068 contains 13 domains grouping into 3 modules, VFPBJ_06596 contains seven domains grouping into three modules, and VFPBJ_11400 contains six domains grouping into two modules.These enzymes were insufficient for the assembly of nine amino acids of leucinostatins.Thus, VFPBJ_02539 was left as the only plausible candidate.VFPBJ_02539 (LcsA) consists of 11,872 amino acids and was encoded by a gene with five introns.The domain structure of LcsA was comprised of 10 C-A-PCP modules and carried the correct number of amino acids for the assembly of leucinostatins.The NRPSpredictor2 [48] offered little insight into the substrates except that the substrates of A1 and A3 were proline and leucine, respectively (S12 Table ).Two PKSs, lcsB and lcsC, located not far upstream of lcsA, which could encode 4-methylhex-2-enoic acid, indicated that this cluster is responsible for leucinostatin production.Furthermore, lcsD (VFPBJ_02533), located between lcsA and the PKSs, was annotated as an acyl-CoA ligase, offering a conceivable route for connecting the fatty acid and peptide.
To verify the associations between the putative lcsA and leucinostatins, a gene deletion method was developed for P. lilacinum based on the previous method for Fusarium oxysporum [49], with the G418 sulfate-resistance gene neo as the selection marker.A portion of lcsA (2,613 bp, including 236 bp upstream of the ORF) was knocked out by double homologous deletion cassettes with the neo marker via PEG-mediated transformation, and the resulting G418 sulfate-resistant isolates (S5A and S5B  The boundary determination of the leucinostatin biosynthesis gene cluster Different boundaries of the lcs cluster were defined by the SMURF and antiSMASH programs (Fig 4A).Nine genes flanking lcsA from VFPPL_02532 to VFPPL_02540 spanning 62 Kb were predicted to be in the cluster by SMURF (Secondary Metabolite Unique Regions Finder) [50], while a larger cluster comprising 26 genes from VFPBJ_02521 to VFPBJ_02546, spanning 120 Kb, was predicted by antiSMASH.Therefore, it was necessary to explore the genes that were involved in the pathway using a biological approach.
Changes in the culture medium could impact the general metabolic profile of an organism, based on the "OSMAC" (one strain-many compounds) hypothesis [51].Indeed, we found that P. lilacinum produced leucinostatins A and B when cultured with our lab recipe of PDB but did not produce leucinostatins when cultured in PDB-BD (see the Materials and Methods section).This result provided clues to identify the boundary of the lcs cluster using producing versus non-producing media.qRT-PCR analysis was conducted to compare the expression patterns of genes flanking lcsA when PLBJ-1 was grown in the two types of media for 8 days.Furthermore, RNA-Seq of PLBJ-1 under leucinostatin-inducing conditions (PDB medium) was performed.
As expected, the expression level of NRPS lcsA when P. lilacinum was grown in leucinostatin-inducing medium was upregulated 95-fold, compared to those grown in non-inducing medium (Fig 4B).The genes downstream of lcsA, including the putative transporter ABC gene VFPBJ_02540, did not display a higher expression level in the leucinostatin-inducing medium, indicating that they were not involved in the leucinostatin biosynthesis pathway.Correspondingly, the RNA-Seq expression profile during leucinostatin production showed a low FPKM value of VFPBJ_02540 (2.01) (S7A Fig) , while the FPKM value of lcsA was 65.4.These results indicated that the 3' edge of the cluster was lcsA.The genes upstream of lcsA from VFPBJ_02520 to VFPBJ_02538 (lcsT) were upregulated at different levels in the leucinostatininducing medium.A 16-to 2692-fold increase in expression was observed (Fig 4B ), except for three genes, VFPBJ_02520, LcsM, and lcsQ, which showed less than 10-fold increase and low FPKM values in the transcriptional data (S7A Fig) .VFPBJ_02520 was annotated as a phosphohydrolase that appeared to be involved in nucleic acid metabolism and signal transduction, instead of secondary metabolism [52].Thus, we speculated that the 5' boundary of the cluster was VFPBJ_02521 (lcsG).To support this hypothesis, the expression patterns of the genes flanking the cluster were analyzed using qRT-PCR analysis in wild type PLBJ-1 and ΔlcsA grown in leucinostatin-inducing medium.We observed an increase in the expression of wild type P. lilacinum ranging from four-to 79-fold (S7B Fig).Thus, a series of genes from VFPBJ_02521 to VFPBJ_02539, designated as lcsA to lcsT, included the core enzymes, modifying enzymes and transporter enzymes coding for the biosynthesis of leucinostatins (Fig 4A , Table 2).

Identification of the leucinostatin biosynthetic pathway
Considering the structural similarities of leucinostatin A with emericellamide A [53] and pneumocandin [47], we reasoned that a similar biosynthetic mechanism might be required to form the skeletons of lipopeptides and peptides.As reported, a single module polyketide synthase iteratively catalyzes the formation of the linear polyketide chain; in daptomycin [54] and echinocandin B [55], acyl-CoA ligase converts the fatty acid to fatty acyl CoASH; in compound W493 B [56], a thioesterase was proposed to hydrolyze the thiol bond and shuttle the product to the first module of NRPS.To determine whether the same enzymes play critical roles in the leucinostatin biosynthesis pathway, we disrupted the PKS (lcsC), ligase (lcsD) and thioesterase (lcsE)-encoding genes in the cluster by homologous recombination (S5A

Overexpression of the transcription factor lcsF
A powerful approach to enhancing the production of leucinostatins was to express transcription factors constitutively that were used for other SMs [57].lcsF encodes a putative transcription factor with a bZIP domain structure, and it is associated with secondary metabolism [58].
To In addition to the 30-fold increase in lcsF expression, the expression of the three PKS/NRPS synthase encodinggenes (lcsB, lcsC and lcsA) were increased by ~3-to 4-fold.For O-methyltransferase (lcsG), ABC transporter (lcsH and lcsO), thioesterase (lcsE), epimerase (lcsT) and the unknown function genes lcsM and lcsS, we observed ten-fold or higher upregulation.The other genes in the cluster displayed a two-to ten-fold increase in expression.Genes adjacent to the lcs cluster, VFPBJ_02520 and VFPPL_02540, were downregulated three-fold.After the wild type and OE::lcsF P. lilacinum were grown in PDB medium with shaking for 8 days, the resulting HPLC profile showed that the titers of leucinostatins A and B were elevated by at least 50% (S9B Fig) .These results provided evidence that the pathway-specific transcription factor lcsF was capable of regulating the entire gene cluster and leucinostatin biosynthesis, further verifying the boundary of this cluster.

Antagonism against the oomycetes of P. lilacinum depending on leucinostatins
The deletion and overexpression of the genes in the lcs cluster had no apparent effects on the fungal hyphae or spore phenotypes of P. lilacinum and did not cause any growth defects.It is well known that leucinostatins are antibiotics used to combat fungi and bacteria.Here, we found that leucinostatins contributed to the inhibition of oomycetes, which had not previously been reported.The growth of P. infestans and P. capsici was inhibited in a confronting incubation with wild type P. lilacinum and OE::lcsF, while the inhibition disappeared when they were grown in a confronting incubation with ΔlcsA (Fig 6

Discussion
P. lilacinum is one of the most important endo-parasites of plant nematodes.We obtained the genome sequence of two P. lilacinum strains and compared them with other nematode parasites, nematode-trapping fungi, insect parasites, a mycoparasitic fungus, a saprotrophic fungus and a plant pathogen.This method provided insights into the life strategy and evolution of nematode endoparasites.
Major gene families (GH, protease, SMs) could corroborate each other for the three P. lilacinum strains (PLBJ-1, PLFJ-1 and the published TERIBC 1).However, TERIBC 1 was predicted to encode more CEs and fewer PHI genes in contrast with PLBJ-1 and PLFJ-1 (Table 1).The lcs cluster was also detected in the TERIBC 1 genome.The genomic sequence identity of the lcs cluster (lcsG to lcsA) between PLBJ-1 and TERIBC 1 was 98.0%, and the sequence identity was 99.0% between PLBJ-1 and PLFJ-1, with the syntenic relationships shown in S13 Fig.
Although fungi have been screened for activity as bio-control agents against P. infestans, the biological control of late blight is dominated by bacterial antagonists.Microbial compounds known as biosurfactants [59] are believed to participate in the process.For example, the cyclic lipopeptide massetolide A produced by Pseudomonas fluorescens exhibited destructive effects on the zoospores of oomycetes [60].The inhibition of P. infestans by leucinostatins provided the basis for their chemical application in agriculture and for further biological studies of the antagonist P. lilacinum on oomycetes, which had not been researched previously.Leucinostatins also demonstrated inhibition against P. capsici [61], another oomyceteous plant pathogen, while the antagonism of P. lilacinum against P. capsici seemed inferior to that against P. infestans, as shown in Fig 6.
The phylogenomic analysis revealed that P. lilacinum was a member of Ophiocordycipitaceae, Hypocreales, which includes fungi engaged in various lifestyles, and it was not related to the previously considered Paecilomyces in Sordariales.This species' closest relatives, T. inflatum and T. ophioglossoides, are insect and fungal parasites (Fig 3A ), supporting the viewpoint that parasitism might occur due to the formation of novel genes that could be acquired through horizontal transfer or gene duplication and could play specific roles during host infection [62].Moreover, these results indicated that the nematode pathogens had a strong link with insect pathogens and were distantly related to nematode-trapping fungi, as previously described in [25] and [26].The large number of hydrolytic enzymes, particularly GHs and proteases, putatively secreted proteins and pathogenesis-related proteins in P. lilacinum support its various lifestyles as it encounters diverse nutrient resources [63].Chitin and proteins comprise a significant proportion of the nematode and insect surface, the degradation of which requires serine proteases and chitinases.
The development of natural compounds from bio-control fungi have recently attracted considerable interest because the production of nematode-toxic SMs could also be a strategy for fungi to infect nematodes [64].Next-generation sequencing technologies are becoming an essential tool for identifying novel genes for metabolite biosynthesis in fungi.The genome sequence of P. lilacinum revealed the potential to produce a rich repertoire of SMs, including  [66], have been isolated from the genera Acremonium and Humicoma.This category of compounds and their derivatives have remarkable biological and medicinal activities, and their total synthesis has garnered attention worldwide.Unfortunately, there still exist limitations in the current synthesis methods [67], and metabolite regulation based on molecular biosynthesis is not available because the biosynthetic genes have not been identified.One or more non-reducing PKSs (S3 Fig) might be involved in the biosynthesis of acremoxanthone C and acremonidin A, according to their structures.
By analyzing the genes located in the leucinostatin biosynthetic cluster, combined with the HPLC-MS analysis of gene deletion mutants (Fig 5 ), we were able to propose a putative biosynthetic pathway for leucinostatins (Fig 7).This hypothetical biosynthesis initiated with the assembly of 4-methylhex-2-enoic acid by a reducing PKS.However, two reducing PKS encoding genes with 38% sequence identity are present in the cluster, and both contained KS, AT, DH, cMT, ER, KR and ACP domains.We excluded the possibility of a partnership between the two PKSs in a sequential manner or a convergent manner, as has been reported for asperfuranone [68] and azaphilone A [69], based on their structures.Moreover, in the biosynthesis of chaetoviridins and chaetomugilins from Chaetomium globosum, a PKS cazF (KS-AT-DH-cMT-ER-KR-ACP) encoded an intermediate 4-methylhex-2-enoic acid [70].The protein sequence identities between cazF and lcsB/lcsC were both 29%; thus, we could not estimate which PKS was responsible for 4-methylhex-2-enoic acid.The results of RNA-seq and qRT-PCR indicated that both genes contributed to leucinostatin synthesis.The deletion of lcsC interrupted leucinostatin biosynthesis, which confirmed that lcsC is essential for the synthesis of leucinostatins.Due to the difficult genetic manipulation, we failed to obtain lcsB deletion mutant.
The lipopeptide pathways and organizations of their clusters have some striking commonalities.We got clues from the lipopeptides echinocandin B [55], pneumocandin [47] and emericellamide [53].The polyketide residue might be transferred to the NRPS LcsA, mediated by two additional putative enzymes, acyl-CoA ligase (LcsD) and thioesterase (LcsE).The linear polyketide carboxylic acid, which was released from PKS, was converted to a CoA thioester by LcsD, and then LcsE hydrolyzed the thiol bond and shuttled the polyketide intermediate to LcsA.4-Methylhex-2-enoic acid was not detected in the culture of the ΔlcsD isolate, indicating that the triketide might be sticked in the PKS enzyme to prevent its release until the ligase is added for the reaction.
The phylogenetic analysis of the ten A-domains of LcsA revealed that LcsA_A2, LcsA_A5, LcsA_A6 and LcsA_A3 were grouped into one clade, and LcsA_A4, LcsA_A7 and LcsA_A8 were grouped into another clade (S14 Fig) .The conserved domains are believed to have evolved through module duplication, and they activate similar amino acid structures [71].In the plausible model for leucinostatin synthesis, A5 and A6 incorporated leucine, A2 incorporated AHy-MeOA, the structure of which is equal to a hydroxyl-3-pentone extending at the leucine, and A3 incorporated 3-hydroxyl leucine.A4, A7 and A8 incorporated AIB.Thus, the structural similarity of amino acids activated by conserved A-domains verified that the 4-methylhex-2-enoic acid moiety in the leucinostatins was assembled by a discrete enzyme, instead of LcsA.The C domain of the first module catalyzed the condensation of 4-methylhex-2-enoic acid and MePro carried by domain A1, followed by successive condensations of nine amino acids to trigger the elongation of the linear peptide.Next, the peptide scaffold would be released by the NAD(P)H-dependent R domain (thioester reductase) at the C-terminal region of LcsA.
In the leucinostatin biosynthetic pathway, it is intriguing that the DPD residue at the C-terminus of leucinostatin A was neither an amino acid nor a carboxy acid, which are incapable of being activated by the A-domain and converting to amino acyl adenylate [72].The DPD seems to be a modified form of amino acids, whereas the primary form of this moiety cannot be determined based on the domain sequence.However, we could deduce a possible pathway for the modification of the last amino acid according to the structure and the function of the genes in the cluster (Fig 7).Originally, an Ala was likely incorporated into the decapeptide skeleton by the A10 domain, which was attached to LcsA via a thioester bond; subsequently, the R domain released this intermediate product.The NAD(P)H-dependent R domains reductively catalyzed to produce linear aldehyde 1 by off-loading peptide thioesters, following completion of the peptide skeleton, as presented in previous studies [73,74].Linear aldehydes frequently occurred as intermediates and underwent subsequent reactions, such as macrocyclization, to yield the imine product koranimine [75]; further reduction yielded myxochelin A or transamination to form an amine myxochelin B by aminotransferase [76].Regarding the leucinostatins, we speculated that aldehyde 1 would go through a transamination reaction to form compound 2, which was accomplished by the putative aminotransferase lcsP.
In this pathway, the unhydroxylated leucine of intermediate 2 undergoes hydroxylation to form compound 3. Three putative cytochrome P450-encoding genes (lcsI, lcsK and lcsN) within the cluster alternatively might catalyze this modification.Another scenario equivalent to this pathway for leucinostatin A synthesis was that a leucine was hydroxylated prior to its incorporation into the peptide.In all likelihood, the varying extents of methylation of compound 3 catalyzed to form leucinostatins A and B. It is worth mentioning that, had the methylation reaction not occurred, compound 3 might be the ultimate precursor of leucinostatin C, which is compound in leucinostatin family isolated from P. marquandii.
The AHyMeOA in leucinostatin A activated by the A2 of lcsA was regarded as a ramification of leucine because leucine is located at this position in leucinostatins C, T, F, D and H, although these compounds were not detected in the PLBJ-1 culture.Based on its structure and the presence of redundant PKSs within the cluster, alternative PKS could be involved in synthesizing the carbon chain.In addition, the leucinostatins contained the nonproteinogenic MePro, incorporated in the synthesis of nostopeptolides in Cyanobacteria [77].A zinc-dependent dehydrogenase, nosE, and a P5C reductase, nosF, were involved in the oxidation and subsequent cyclization of leucine to form MePro, and the presence of nosE and nosF recently led to screening for novel MePro-containing peptides [78].In the pneumocandins from Zalerion arboricola, feeding experiments established that leucine was cyclized to produce 3-hydroxy-4-methylproline, whereas MePro might be an intermediate [79].It was reasonable to assume that the MePro in the leucinostatins originated from leucine cyclization.Although homologues of nosE nor nosF were not present in the lcs cluster, it was plausible that MePro biosynthesis, engaged in a separate pathway, was independent of leucinostatin synthesis.Another nonproteinogenic amino acid, β-Ala, was present in leucinostatins and activated by the A9 of lcsA.A previous study of the destruxins in Metarhizium proposed that the aspartic acid decarboxylase dtxS4 triggered the decarboxylation of aspartic acid into β-Ala, as a substrate for the assembly line [80].A genome-wide blast search for genes encoding aspartic acid decarboxylases in PLBJ-1 revealed the presence of two candidate genes, VFPBJ_01400 and VFPBJ_10476, with 68% and 61% sequence identity to dtxS4, respectively, which could have catalyzed the biosynthesis of β-Ala in leucinostatins.

Conclusions
The genomes of P. lilacinum strains PLBJ-1 and PLFJ-1 were sequenced, completely assembled, annotated, and comparatively analyzed with related fungi.Phylogenomic analysis showed that P. lilacinum was most closely related to T. inflatum and T. ophioglossoides, and the cluster of nematode parasitic fungi and insect pathogens indicated their common origin.PKS and NRPS-encoding genes were thoroughly characterized and analyzed by phylogenetic analysis, from which we found that lcsA was specific to P. lilacinum and T. ophioglossoides.Furthermore, lcsA was proved to be responsible for leucinostatin biosynthesis by homologous deletion.The boundary of the lcs cluster was identified by comparison of gene expression levels when P. lilacinum was cultured in leucinostatin-inducing and non-inducing medium as well as RNA-Seq analysis.Disruption of lcsC, lcsD and lcsE demonstrated the critical roles of PKS, acyl-AMP ligase and thioesterase in the biosynthetic pathway of leucinostatins.Overexpression of the transcription factor lcsF increased the production of leucinostatins A and B through regulated expression levels of genes in the lcs cluster.We also demonstrated that leucinostatins could enable the fungus with antagonistic activity against the oomycetes.

Genome assembly, gene prediction and RNA-seq analysis
The raw sequencing data (Illumina HiSeq 2000) from the PLBJ-1 and PLFJ-1 strains were generated by BGI-Shenzhen (China) and Berry Genomics Co., Ltd.(China), respectively.A total of 13.27 Gb bases for the PLBJ-1 strain from three libraries, with average insert sizes of 165 bp, 758 bp and 5,490 bp, were obtained, and 5.88 Gb bases for the PLFJ-1 strain from two libraries with the average insert sizes of 175 bp and 4,760 bp were obtained.Both of the genomes were assembled using ALLPATHS-LG revision 42305 [81].The repeat sequences were identified as previously described [82], based on de novo and homology methods.For the de novo method, Piler [83] and RepeatScout, version 1.0.5 [84], were used to construct the repeat sequence families; then, RepeatMasker, version 4.0.5, was used for repeat analysis.For the homology method, the sequence families from Repbase, version 19.06 [85], were used for annotation by performing RepeatMasker analysis.
For gene prediction, the Augustus algorithm, version 2.7 [86], identified 11,404 and 11,554 complete genes for the PLBJ-1 and PLFJ-1 strains, respectively, and the GeneMark-ES algorithm, version 2.3f [87], discovered 11,001 and 11,070 complete genes for the PLBJ-1 and PLFJ-1 strains, respectively.The comparison showed that 9,509 and 9,562 genes of the PLBJ-1 and PLFJ-1 strains were predicted by both Augustus and GeneMark-ES.These consensus genes were considered to be high quality predicted genes and were used in this study.The additional 2,264 and 2,201 genes of the PLBJ-1 and PLFJ-1 strains were obtained according to the method in [82].EuGene, version 4.1 [88], was used to integrate multiple sources, including transcription start sites identified by Netstart [89], homologous proteins identified from the Swiss-Prot database, version 2015-07-22, by BLAST, version 2.2.26, the assembled transcripts generated by IDBA-tran, version 1.1.1[90], and the exon junctions identified from RNA-seq by Tophat, version 2.0.13 [91].The gene expression values were presented by the expected FPKMs using Cufflinks, version 2.2.1 [92], based on the Tophat [91] analysis.
The proteases were discovered by the MEROPS batch BLAST online server [97].Proteins with sequences that matched the cytochrome P450 genes [98] with E-values 1e-50 were annotated as P450 enzymes.Candidate pathogenic factors were predicted by sequence alignment against the Pathogen Host Interactions (PHI) database, version 3.5 [99], with Evalues 1e-50.In addition, the secretomes were identified based on recognizing the signal peptide and transmembrane sequences.Proteins were considered to be secreted proteins if the signal peptides were identified by at least two methods among SignalP, version 4.0 [100], Tar-getP, version 1.1 [101], Phobious, version 101 [102], and Predisi [103], and transmembrane sequences were not identified by at least one of the methods among SignalP, Phobious and TMHMM, version 2.0c [104].

Orthologous and phylogenomic analysis
Orthologous groups of genes from P. lilacinum and the other fungi listed in S14 Table were detected by OrthoMCL, version 2.0.9 [105], and then were filtered to identify the single copy orthologues.The single copy orthologues were aligned with MUSCLE [106].The poor alignment regions of the concatenated sequences were removed using Gblock, version 0.91b [107], and then the high quality sequences were used for the maximum likelihood phylogeny analysis with the Dayhoff model implemented in the TREE-PUZZLE program [108].Bootstrap support value was calculated by analyzing 1,000 replicates.

Phylogenetic analysis of the PKS and NRPS genes
The secondary metabolite genes were discovered by performing SMURF [50] and antiSMASH [40] analyses.PKS and NRPS domain structures were characterized by antiSMASH and Pfam, or were visually identified by multiple alignments.The KS domains extracted from PKS and the A-domain from NRPS were aligned by MUSCLE [106], and then a maximum likelihood phylogeny was constructed by treeBeST (http://treesoft.sourceforge.net/treebest.shtml) using 1,000 bootstrap replicates.

qRT-PCR analysis
Three biological replicates were performed for each analysis of the relative expression levels.The cDNAs were synthesized with a TIANScript Ⅱ RT Kit (TIANGEN, China).The cDNA was analyzed by qRT-PCR using SYBR Premix Ex Taq (TAKARA, Japan) on a BIO-RAD CFX96 (BIO-RAD).The housekeeping actin gene designed from VFPBJ_07912, which was similar to the reported GU299860.1,was used for normalization.The relative expression values were calculated using the 2 -ΔΔCt method.The primers are listed in S13 Table.

Molecular genetic procedures
Polyethylene glycol-mediated protoplast transformation of PLBJ-1 was performed as previously reported [49,109], with the following modifications: the protoplast was produced by 20 gL −l Driselase (Sigma) digestion for 4 h at 31°C.The regeneration medium was PDA medium containing G418 sulfate (400 μg/L), supplemented with molasses (10 g/L), saccharose (0.6 M), yeast extract (0.3 g/L), tryptone (0.3 g/L), and casein peptone (0.3 g/L) [10].The construction of knockout and overexpression plasmids originated from pKOV21 and KSTNP, and the primers are listed in S13 Table .A quick method for isolating the fungal genomic DNA was developed to screen for a large number of transformants.Briefly, a nip of mycelia was transferred to 50 μL of NaOH (50 mM) and was incubated at 95°C for 20 min.The solution was directly used for PCR amplification after 5 μL of Tris-HCl (1 M) were added to neutralize the base.

Culture extraction and HPLC-MS profiling
Cultures of 1×10 5 conidia per mL of P. lilacinum and its mutants were grown in leucinostatininducing PDB medium at 28°C on a shaker at 150 rpm for 8 days.Culture medium (7.5 L) was extracted with the same volume of EtOAc three times (each 1 h) ultrasonically.The combined EtOAc extracts were concentrated to afford a crude extract (0.4 g), which was subjected to reversed-phase ODS column chromatography eluting with MeOH-H 2 O (from 40% to 100%) to afford 6 fractions (Fr.A-Fr.F).Fr.E (40 mg) was passed through a Sephadex LH-20 column (MeOH) and yielded mixtures of 5.0 mg of leucinostatins A and B. The structure of the mixtures was further identified by standard substance using LC-MS analysis.Approximately 200 mL of culture medium were used for comparative LC-MS analysis between PLBJ-1 and its mutants.LC-MS was performed on an Agilent Accurate-Mass-QTOF LC/MS 6520 instrument.HPLC analysis was performed on a Waters HPLC system (Waters e2695, Waters 2998, Photodiode Array Detector) using an ODS column (C18, 250 × 4.6 mm, YMC Pak, 5 μm).The ODS (50 μm) column was produced by YMC Co. Ltd. (Kyoto, Japan).The Sephadex LH-20 was purchased from GE Healthcare.Analytical HPLC was conducted with a Waters HPLC system (Waters e2695, Waters 2998, Photodiode Array Detector) using an ODS column (C18, 250 × 4.6 mm, YMC Pak, 5 μm) with a flow rate of 1 mL/min.The fresh extracts were dissolved in methanol before being separated on a linear gradient of MeOH:H 2 O (0.1% formic acid) at a flow rate of 1 mL/min.Fresh extracts of mutant strains were detected for 30 min using a linear gradient of 20% to 100% (0-20 min), 100% MeOH (20-25 min), and 20% MeOH (25-30 min).The LC-MS analysis method was consistent with analytical HPLC.

Fungus bioassay
Confronting incubation of P. lilacinum (wild type, ΔlcsA and OE::lcsF) with P. infestans was performed on rye agar medium in 9 cm Petri plates, incubated simultaneously and cultured at 28°C for 24 h and then at 18°C, the optimum temperature for P. infestans, for 9 days, while confrontation with P. capsici was performed on lab-made PDA medium, cultured at 28°C for 7 days.For the inhibitory zone experiment, freshly produced sporangia of P. infestans was suspended in sterile water at a concentration of 2×10 5 sporangia/mL.One milliliter of the suspension was smeared on 15 cm Petri plates, followed by Oxford cups with a diameter of 1 cm being placed.From 5 to 60 μg (increment 5 μg) of leucinostatins A and B dissolved in 20% methanol were added to the Oxford cup, and 20 μL of 20% methanol were used a control.Then, the Oxford cups were removed after the solution was absorbed by media.Five days later, the area was calculated by drawing circles of the inhibitory zone on metric graph paper and counting the number of square millimeters within the circle [110,111].Three biological replicates were performed.At the same time, the effects of 50 μg, 33 μg, 17 μg and 8.5 μg of leucinostatins are demonstrated in S11 Fig.

Accession numbers
The genome sequences of PLBJ-1, PLFJ-1, and P. chlamydosporium strain 170 used for comparative analysis have been deposited at GenBank under the accession numbers LSBH00000000, LSBI00000000 and LSBJ00000000, respectively.

Fig 1 .
Fig 1. Lifestyles of nematophagous P. lilacinum and the structures of leucinostatins.(A) Microscopic conidiophores and conidia (c) of P. lilacinum.Scale bar = 10 μm.The soil saprophyte (s) P. lilacinum colonizes plant roots as an endophyte (e), and the parasite (p) can occur in nematode eggs in the egg mass (em) generated after the infection with the plant nematode (n).(B) Chemical structure of leucinostatins A and B. doi:10.1371/journal.ppat.1005685.g001

doi: 10 .Fig 2 .
Fig 2. Genomic synteny of PLBJ-1 and PLFJ-1.(A) The syntenic genome sequences of PLBJ-1 and PLFJ-1 were analyzed by BLASTN, with an E-value cutoff of 1e-5.The red semicircle represents the scaffolds of PLBJ-1, while the blue semicircle represents the scaffolds of PLFJ-1.Scaffold lengths of 100 Kb were used for this analysis, and the threshold of matched blocks was 1000 bp, which are connected by lines of the same color.(B) An example of a super-scaffold inferred by syntenic analysis.doi:10.1371/journal.ppat.1005685.g002

Fig 3 .
Fig 3. Phylogenomic relationships and orthologous gene clusters.(A) Maximum likelihood phylogeny was computed from a concatenated alignment of 855 groups of single-copy orthologues.Bootstrap values are shown beside the nodes.(B) The number of gene clusters shared by P. lilacinum with other major associated ecologies.Gray = P. lilacinum isolates PLBJ-1 and PLFJ-1; blue = nematode egg parasite P. chlamydosporia isolates 123 and 170; pink = nematode parasite H. minnesotensis; and yellow = nematode-trapping fungi A. oligospora and M. haptotylum.doi:10.1371/journal.ppat.1005685.g003 Fig) were verified by diagnostic PCR, using the primers in neo and outside the knockout cassette (S5C Fig) (S13 Table).Finally, one mutant (ΔlcsA) of PLBJ-1 was isolated with correct PCR amplification products from 320 G418 sulfate-resistance mutants (S5C Fig), and the remaining isolates resulted from ectopic integration of the neo gene cassette into the genome.The wild type of P. lilacinum and the ΔlcsA mutant of PLBJ-1 were cultured in PDB medium for 8 days, and the ethyl acetate extracts were analyzed by HPLC-MS.The MS spectrum of the wild type displayed two overlapping peaks at 15.6 and 16.0 min, with m/z [M+H] + of 1218.9 and 1204.9, respectively, which were assigned to leucinostatins A and B and were absent in the ΔlcsA mutant (Fig 5, S6 Fig).A comparison with the authentic standard confirmed that the missing compounds of the ΔlcsA mutant were indeed leucinostatins A and B (Fig 5, S6 Fig).As expected, these results demonstrated the essential roles of lcsA in the biosynthesis of the leucinostatins.

Fig 4 .
Fig 4. The boundary of the lcs cluster in P. lilacinum with its homologues in T. ophioglossoides.(A) Horizontal arrows of the same color represent the orthologous genes.The sequence identity between the homologous genes from two fungi is shown by shaded areas with different colors.TO, T. ophioglossoides; PL, P. lilacinum.The bars indicate boundaries of the lcs cluster predicted by antiSMASH, SMURF, and qRT-PCR.(B) The expression ratio of the genes around lcsA when expression in PLBJ-1 cultured in leucinostatin-inducing medium was compared to expression in non-inducing medium.The ratios for different genes demonstrated an extensive range, so the breakpoint was inserted into the Y axis.doi:10.1371/journal.ppat.1005685.g004

Fig 5 .
Fig 5. HPLC profiles (UV 210 nm) of culture extracts from the wild type P. lilacinum strain and mutants when grown in PDB medium.Leucinostatins A and B were detected in the wild type isolate, while they were abolished in ΔlcsA, ΔlcsC, ΔlcsD and ΔlcsE.doi:10.1371/journal.ppat.1005685.g005 Fig) and verified the mutants by PCR amplification (S8A Fig).After culturing the fungi in PDB medium and comparing the extracts with the PLBJ-1 wild type and ΔlcsA by HPLC-MS, we showed that leucinostatins A and B disappeared in ΔlcsC, ΔlcsD and ΔlcsE, similar to ΔlcsA (Fig 5, S6 Fig).
assess the function of lcsF, we cloned it into the KSTNP vector under the control of the TrpC promoter.The resulting plasmid, KSTNP-OElcsF, was randomly integrated into the genome of wild type P. lilacinum (S8B Fig).The positive transformants were screened by G418 sulfate and were diagnosed by PCR amplification of the expression cassette (S8C Fig).Transformants with an intact overexpression cassette were cultured in leucinostatin-inducing PDB medium for 8 days.The expression level of lcsF in the mycelia was analyzed by qRT-PCR, and six of ten transformants demonstrated more than 20-fold upregulation.Finally, three transformants without changes in their physiological indices were selected for the downstream test.As expected, all 20 genes in the cluster were upregulated to some extent by lcsF, with the exception of lcsL and lcsP, which were downregulated three-and five-fold (S9A Fig).

Table 1 .
Genome feature of the three P. lilacinum isolates.

Table 2 .
Description of the genes in the leucinostatin biosynthetic cluster. doi:10.1371/journal.ppat.1005685.t002 ).Similar to ΔlcsA, P. infestans could grow normally in a confronting incubation with ΔlcsC, ΔlcsD and ΔlcsE (S10 Fig).The results indicated that leucinostatins A and B inhibited the growth of some oomycetes.A gradient inhibitory zone was explored with leucinostatins A and B in different concentrations to find quantitative evidence of inhibition against P. infestans (S11 and S12 Figs).