Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens

Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host/ecological niche can influence shaping the P450 content of an organism. The present study initiates our understanding of P450 family patterns in basidiomycete biotrophic plant pathogens.     

Comparison of Cytochrome P450 Genes from Six Plant Genomes

Plants depend on cytochrome P450 (CYP) enzymes for nearly every aspect of their biology. In several sequenced angiosperms, CYP genes constitute up to 1% of the protein coding genes. The angiosperm sequence diversity is encapsulated by 59 CYP families, of which 52 families form a widely distributed core set. In the 20 years since the first plant P450 was sequenced, 3,387 P450 sequences have been identified and annotated in plant databases. As no new angiosperm CYP families have been discovered since 2004, it is now apparent that the sampling of CYP diversity is beginning to plateau. This review presents a comparison of 1,415 cytochrome P450 sequences from the six sequenced genomes of Vitis vinifera (grape), Carica papaya (papaya), Populus trichocarpa (poplar), Oryza sativa (rice), Arabidopsis thaliana (Arabidopsis or mouse ear’s cress) and Physcomitrella patens (moss). An evolutionary analysis is presented that tracks land plant P450 innovation over time from the most ancient and conserved sequences to the newest dicot-specific families. The earliest or oldest P450 families are devoted to the essential biochemistries of sterol and carotenoid synthesis. The next evolutionary radiation of P450 families appears to mediate crucial adaptations to a land environment. And, the newest CYP families appear to have driven the diversity of angiosperms in mediating the synthesis of pigments, odorants, flavors and order-/genus-specific secondary metabolites. Family-by-family comparisons allow the visualization of plant genome plasticity by whole genome duplications and massive gene family expansions via tandem duplications. Molecular evidence of human domestication is quite apparent in the repeated P450 gene duplications occurring in the grape genome.     

Rapid Birth–Death Evolution Specific to Xenobiotic Cytochrome P450 Genes in Vertebrates

Genes vary greatly in their long-term phylogenetic stability and there exists no general explanation for these differences. The cytochrome P450 (CYP450) gene superfamily is well suited to investigating this problem because it is large and well studied, and it includes both stable and unstable genes. CYP450 genes encode oxidase enzymes that function in metabolism of endogenous small molecules and in detoxification of xenobiotic compounds. Both types of enzymes have been intensively studied. My analysis of ten nearly complete vertebrate genomes indicates that each genome contains 50–80 CYP450 genes, which are about evenly divided between phylogenetically stable and unstable genes. The stable genes are characterized by few or no gene duplications or losses in species ranging from bony fish to mammals, whereas unstable genes are characterized by frequent gene duplications and losses (birth–death evolution) even among closely related species. All of the CYP450 genes that encode enzymes with known endogenous substrates are phylogenetically stable. In contrast, most of the unstable genes encode enzymes that function as xenobiotic detoxifiers. Nearly all unstable CYP450 genes in the mouse and human genomes reside in a few dense gene clusters, forming unstable gene islands that arose by recurrent local gene duplication. Evidence for positive selection in amino acid sequence is restricted to these unstable CYP450 genes, and sites of selection are associated with substrate-binding regions in the protein structure. These results can be explained by a general model in which phylogenetically stable genes have core functions in development and physiology, whereas unstable genes have accessory functions associated with unstable environmental interactions such as toxin and pathogen exposure. Unstable gene islands in vertebrates share some functional properties with bacterial genomic islands, though they arise by local gene duplication rather than horizontal gene transfer.     

Structural organization,classification and phylogenetic relationship of cytochrome P450 genes in Citrus clementina and Citrus sinensis

The genus Citrus is an important fruit crop and nutritional source for the good health of humans. Cytochrome P450s represent about 1 % of the proteome and mediate diverse biochemical reactions pertaining to both primary and secondary metabolism. Analysis of Citrus genomic resources identified 296 plant cytochrome P450s (CYP) coding genes in Citrus clementina, 272 in double haploid (dh) Citrus sinensis, and 202 in C. sinensis. In C. clementina and dh C. sinensis, CYP genes are distributed into nine clans. In the three genomes, single intron containing CYP genes are predominant in the A-type families. Among non-A-type CYP families, multiple intron containing genes are predominant. More number of genes in CYP A-type families over non-A-type families is attributed to rapid evolution of A-type genes facilitated by their gene organization. Further, complex gene organization of non-A-type genes with the presence of multiple introns might have contributed to the slower evolvement of paralogs. Majority of introns (1,660) from three genomes showed canonical GT-AG splice sites. However, 33 introns showed non-conventional GC… PyAG splice sites and functionality of these splice sites is confirmed by the ESTs lacking this intron. Across the families, gene organization is conserved between the three genomes. In dh C. sinensis, 22 genes were identified to have alternate splicing. Examination of scaffolds in C. clementina revealed that majority of the Citrus CYP genes are solitary and a few of them are in clusters of 3–8 genes. PCR amplification of C. sinensis genomic DNA with gene-specific primers failed to amplify out-grouped genes Ccl-CYP706A16 and Ccl-CYP706B1, confirming that they are specific to C. clementina. Differential number of CYP genes observed between C. clementina and C. sinensis is attributed to the extent of variability between their parents representing ancestral taxa.     

Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica

Background

Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results

The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion

Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.     

Structural organization and classification of cytochrome P450 genes in flax (Linum usitatissimum L.)

Flax CYPome analysis resulted in the identification of 334 putative cytochrome P450 (CYP450) genes in the cultivated flax genome. Classification of flax CYP450 genes based on the sequence similarity with Arabidopsis orthologs and CYP450 nomenclature, revealed 10 clans representing 44 families and 98 subfamilies. CYP80, CYP83, CYP92, CYP702, CYP705, CYP708, CYP728, CYP729, CYP733 and CYP736 families are absent in the flax genome. The subfamily members exhibited conserved sequences, length of exons and phasing of introns. Similarity search of the genomic resources of wild flax species Linum bienne with CYP450 coding sequences of the cultivated flax, revealed the presence of 127 CYP450 gene orthologs, indicating amplification of novel CYP450 genes in the cultivated flax. Seven families CYP73, 74, 75, 76, 77, 84 and 709, coding for enzymes associated with phenylpropanoid/fatty acid metabolism, showed extensive gene amplification in the flax. About 59% of the flax CYP450 genes were present in the EST libraries.     

Cloning and Expression of Multiple Cytochrome P450 Genes: Induction by Fipronil in Workers of the Red Imported Fire Ant (Solenopsis invicta Buren)

Both exogenous and endogenous compounds can induce the expression of cytochrome P450 genes. The insect cytochrome P450 genes related to insecticide resistance are likely to be expressed as the “first line of defense” when challenged with insecticides. In this study, four cytochrome P450 genes, SinvCYP6B1, SinvCYP6A1, SinvCYP4C1, and SinvCYP4G15, were firstly isolated from workers of the red imported fire ant (Solenopsis invicta) through rapid amplification of cDNA ends (RACE) and sequenced. The fipronil induction profiles of the four cytochrome P450 genes and the two previously isolated CYP4AB1 and CYP4AB2 were characterized in workers. The results revealed that the expression of SinvCYP6B1, SinvCYP6A1, CYP4AB2, and SinvCYP4G15, increased 1.4-fold and 1.3-fold more than those of acetone control, respectively, after 24 h exposure to fipronil at concentrations of 0.25 μg mL⁻¹ (median lethal dose) and 0.56 μg mL⁻¹ (90% lethal dose), while no significant induction of the expression of CYP4AB1 and SinvCYP4C1 was detected. Among these genes, SinvCYP6B1 was the most significantly induced, and its maximum expression was 3.6-fold higher than that in acetone control. These results might suggest that multiple cytochrome P450 genes are co-up-regulated in workers of the fire ant through induction mechanism when challenged with fipronil. These findings indicated that cytochrome P450 genes play an important role in the detoxification of insecticides and provide a theoretical basis for the mechanisms of insecticide metabolism in the fire ant.     

Spontaneous transposition of HzSINE1 into CYP321A2 is undetectable in the field populations of Helicoverpa zea

The enrichment of transposable elements (TEs) within allelochemical- and insecticide-metabolizing P450 alleles in Helicoverpa zea enables these P450s to gain TE-introduced adaptive variations otherwise not readily available to cope with the ever-changing and diverse xenobiotic stress factors in varying cropping systems. The critical role of each TE-inserted P450 allele depends on whether the inserted P450 allele is more adaptive than its TE-free counterpart or not. Previous study has reported a HzSINE1-inserted CYP321A2 allele in a laboratory strain of H. zea reared with xenobiotic-free artificial diets. Here we show that the HzSINE1-inserted CYP321A2 allele transcribes into two HzSINE1 sequence-containing mutant mRNA isoforms of different length that encode an identical C terminus-truncated and heme-binding region deleted non-functional P450. Nonetheless, HzSINE1 insertion does not disrupt the regulatory functional aspect of CYP321A2 since this allele is constitutively expressed and highly inducible by the allelochemicals xanthotoxin, quercetin and chlorogenic acid. Furthermore, while the HzSINE1-inserted CYP321A2 allele is fixed in the laboratory strain, the insertion is purged in the bifenthrin-resistant strain and the Georgia field population of H. zea. To sum up, the HzSINE1-inserted CYP321A2 allele represents an allelochemical-inducible non-functional P450 allele that is selected against in the field populations frequently encountering toxic plant allelochemicals and synthetic insecticides. However, such an insertion can reach fixation under the xenobiotic-free laboratory rearing conditions most likely due to random genetic drift across multiple generations.     

Phylogenetic analysis of Bacillus P450 monooxygenases and evaluation of their activity towards steroids

Cytochrome P450 (P450) open reading frames (ORFs) identified in genome sequences of Bacillus species are potential resources for new oxidation biocatalysts. Phylogenetic analysis of 29 Bacillus P450 ORFs revealed that the P450s consist of a limited number of P450 families, CYP102, CYP106, CYP107, CYP109, CYP134, CYP152, and CYP197. Previously, we identified the catalytic activities of three P450s of Bacillus subtilis towards steroids by rapid substrate screening using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Here, we further applied this method to evaluate the activity of Bacillus cereus P450s towards steroids. Five P450 genes were cloned from B. cereus ATCC 10987 based on its genomic sequence and were expressed in Escherichia coli. These P450s were reacted with a mixture of 30 compounds that mainly included steroids, and the reaction mixtures were analyzed using FT-ICR/MS. We found that BCE_2659 (CYP106) catalyzed the monooxygenation of methyltestosterone, progesterone, 11-ketoprogesterone, medroxyprogesterone acetate, and chlormadinone acetate. BCE_2654 (CYP107) monooxygenated testosterone enanthate, and BCE_3250 (CYP109) monooxygenated testosterone and compactin. Based on the phylogenetic relationship and the known substrate specificities including ones identified in this study, we discuss the catalytic potential of Bacillus P450s towards steroids.     

Cytochrome P450 Genes from the Sacred Lotus Genome

Cytochrome P450 monooxygenases (P450s) in the sacred lotus (Nelumbo nucifera) genome have been identified and named according to systematic P450 nomenclatures. Comparisons of these sequences with those in the papaya and grape CYPomes have indicated that gene blooms exist in the CYP89, CYP94, CYP96 and CYP714 families and that less dramatic expansions exist in the CYP71 and CYP72 families. Expansions in the CYP94 and CYP96 families may be associated with generation of the extremely hydrophobic leaf surfaces associated with the “lotus effect” in this water-adapted species, since these families are known to hydroxylate fatty acids and alkanes in the wax biosynthetic pathways of other plant species. Evolution of the CYP719 and CYP80 families may be associated with production of a number of benzylisoquinoline and aporphine alkaloids. Structures for anonaine and roemerine, two of the most abundant aporphine alkaloids in lotus leaves and seeds, contain methylenedioxy bridges that are known to be generated by members of the CYP719 family. With only one CYP719A22 gene existing in the lotus genome, it is likely that it is involved in making aporphine alkaloids. The fact that CYP719 has not previously been seen in angiosperm phylogeny below the order of Ranunculales suggests that its presence in lotus (in the Proteales) presents an evolutionary terminus prior to its loss in more recent eudicot species. With several CYP80 family genes existing in the lotus genome, there are multiple candidates for those involved in conducting benzylisoquinoline alkaloid synthesis.     

Characterization of two NADPH: Cytochrome P450 reductases from cotton (Gossypium hirsutum)

Cytochrome P450 monooxygenases (P450s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics, and their activities rely on a partner enzyme, cytochrome P450 reductase (CPR, E.C.1.6.2.4). Two CPR cDNAs, GhCPR1 and GhCPR2, were isolated from cotton (Gossypium hirsutum). They are 71% identical to each other at the amino acid sequence level and belong to the Class I and II of dicotyledonous CPRs, respectively. The recombinant enzymes reduced cytochrome c, ferricyanide and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner, and supported the activity of CYP73A25, a cinnamate 4-hydroxylase of cotton. Both GhCPR genes were widely expressed in cotton tissues, with a reduced expression level of GhCPR2 in the glandless cotton cultivar. Expression of GhCPR2, but not GhCPR1, was inducible by mechanical wounding and elicitation, indicating that the GhCPR2 is more related to defense reactions, including biosynthesis of secondary metabolites.     

Identification and characterization of six cytochrome P450 genes belonging to CYP4 and CYP6 gene families in the silkworm,Bombyx mori

It was predicted that the genome of silkworm, Bombyx mori, has at least 79 P450 genes; however, P450 genes that are related to the catabolism of exogenous compounds were not reported. In this study we cloned two CYP4 (named CPY4M5 and CYP4M9) and four CYP6 (named CYP6AB5, CYP6AE9, CYP6AE22 and CYP6AU1) genes by using both bioinformatics and RT-PCR approaches. Sequence analysis showed that these genes contained conserved P450 gene sequence regions and one conserved intron. CYP4M5 and CYP4M9 genes were clustered together in a mode of “head-to-tail” possibly due to gene duplication. Blast analysis showed that these P450 genes shared significant similarity with CYP4 and CYP6 genes that are involved in the catabolism and detoxification of exogenous compounds in other insect species. RT-PCR results showed that these P450 genes were highly expressed in the midgut and fat body of B. mori. As the instar age increased, these P450 genes exhibit different expression patterns. When B. mori was exposed to 1.75 × 10^?5 % of cypermethrin, 3.5 × 10^?6 % of cypermethrin and 0.1 % of rutin, expression of CYP6AB5 was increased by 2.3-fold, 2.2-fold and 1.9-fold, respectively. Exposure of B. mori to 0.1 % quercetin does not change the expression of CYP6AB5. In contrast, expression of the other five P450 genes was inhibited after exposed to these compounds.     

Regulation of expression of cytochromes P450 family 1 in cell cultures at different stages of tumor transformation

Xenobiotic lipophilic compounds, including carcinogenic substances and antitumor agents, are metabolized by isoforms of cytochrome P450 (CYP). The constitutive and induced expression of genes for CYP in tumors is known to decrease as compared to that in homologous normal tissue; this determines to a significant degree the higher resistance of tumors to the effects of some cytostatics. To reveal the tumor transformation stage at which changes in the level of CYP of family 1 take place, we compared the levels of mRNA expression of the genes for CYP1A1, CYP1B1, and that of intracellular proteins regulating the CYP synthesis: Ah-receptor, ARNT, and AHRR. We studied embryonic fibroblast-like cells, the same cells immortalized either by Rauscher virus or spontaneously after passage of a crisis, as well as the cells of three transformed clones (K1, K2, and K8) obtained by action of benzo(a)pyrene on the cells immortalized by Rauscher virus. The constitutive level of expression of the studied genes was revealed in all cell cultures. Benz(a)anthracene induction increased the mRNA expression level for all inducible genes (CYP1A1, 1B1, and AHRR) in the initial culture immortalized by Rauscher virus and in the transformed clone K2. In the culture of the spontaneously immortalized cells and in the transformed clone K1 there was induction only of the CYP1B1 gene. In the cell culture of transformed clone K8, induction of all the inducible genes did not occur. This absence of induction was not caused by hyper expression of the AHRR-protein which blocks induction. The results indicate that the ability of immortalized cells to induce CYP isoforms is determined not only by the property of immortality, but also by how this property was produced. It was also shown that transformed clones, in spite of their common origin, differ from each other in their induction of CYP1 isoforms. The same level of mRNA expression of the genes Ah-receptor and ARNT occurred in cells in which induction of all inducible genes took place, and in those in which induction of all inducible genes did not take place, indicating that, apart from the known participants in the transduction of the induction signal, some other, thus far unknown, factors also take part.     

Expression of Xenobiotic Metabolizing Cytochrome P450 Genes in a Spinosad-Resistant Musca domestica L. Strain

Background

Spinosad is important in pest management strategies of multiple insect pests. However, spinosad resistance is emerging in various pest species. Resistance has in some species been associated with alterations of the target-site receptor, but in others P450s seems to be involved. We test the possible importance of nine cytochrome P450 genes in the spinosad-resistant housefly strain 791spin and investigate the influence of spinosad on P450 expression in four other housefly strains.

Results

Significant differences in P450 expression of the nine P450 genes in the four strains after spinosad treatment were identified in 40% of cases, most of these as induction. The highly expressed CYP4G2 was induced 6.6-fold in the insecticide susceptible WHO-SRS females, but decreased 2-fold in resistant 791spin males. CYP6G4 was constitutively higher expressed in the resistant strain compared to the susceptible strain. Furthermore, CYP6G4 gene expression was increased in susceptible WHO-SRS flies by spinosad while the expression level did not alter significantly in resistant fly strains. Expression of CYP6A1 and male CYP6D3 was constitutively higher in the resistant strain compared to the susceptible. However, in both cases male expression was higher than female expression.

Conclusion

CYP4G2, CYP6A1, CYP6D3 and CYP6G4 have expressions patterns approaching the expectations of a hypothesized sex specific spinosad resistance gene. CYP4G2 fit requirements of a spinosad resistance gene best, making it the most likely candidate. The overall high expression level of CYP4G2 throughout the strains also indicates importance of this gene. However, the data on 791spin are not conclusive concerning spinosad resistance and small contributions from multiple P450s with different enzymatic capabilities could be speculated to do the job in 791spin. Differential expression of P450s between sexes is more a rule than an exception. Noteworthy differences between spinosad influenced expression of P450 genes between a field population and established laboratory strains were shown.     

Characterization of multiple <Emphasis Type="Italic">CYP9A</Emphasis> genes in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed, focusing mainly on gene duplication. All four CYP9A subfamily members from the silkworm, Bombyx mori, were cloned by RT-PCR and designated CYP9A19–CYP9A22 by the P450 Nomenclature Committee. They each contain an open reading frame of 1,593 bp in length and encode a putative polypeptide of 531 amino acids. Both nucleic acid and amino acid sequences share very high identities with one another. The typical motifs of insect cytochrome P450, including the heme-binding region, helix-C, helix-I, helix-K, and PERF, show high sequence conservation among the multiple proteins. Alignment with their cDNA sequences revealed that these paralogues share identical gene structures, each comprising ten exons and nine introns of variable sizes. The locations of their introns (all nine introns follow the GT–AG rule) are absolutely conserved. CYP9A19, CYP9A20, and CYP9A21 form a tandem cluster on chromosome 17, whereas CYP9A22 is separated from the cluster by four tandem alcohol-dehydrogenase-like genes. Their phylogenetic relationships and structural comparisons indicated that these paralogues arose as the results of gene duplication events. RT-PCR detected their mRNAs in different “first line of defense” tissues, as well as in several other organs, suggesting diverse functions. Tissue-selective expression also indicates their functional divergence. The identified CYP9A genes have not yet been found outside the Lepidoptera, and are probably unique to the Lepidoptera. They show high sequence and structural similarities to each other, indicating that the Lepidoptera-specific P450s may be of functional importance. This analysis constitutes the first report of the clustering, spatial organization, and functional divergence of P450 in the silkworm.