Combined assembly of long and short sequencing reads improve the efficiency of exploring the soil metagenome.

BACKGROUND: Advances in DNA sequencing technologies have transformed our capacity to perform life science research, decipher the dynamics of complex soil microbial communities and exploit them for plant disease management. However, soil is a complex conglomerate, which makes functional metagenomics studies very challenging. RESULTS: Metagenomes were assembled by long-read (PacBio, PB), short-read (Illumina, IL), and mixture of PB and IL (PI) sequencing of soil DNA samples were compared. Ortholog analyses and functional annotation revealed that the PI approach significantly increased the contig length of the metagenomic sequences compared to IL and enlarged the gene pool compared to PB. The PI approach also offered comparable or higher species abundance than either PB or IL alone, and showed significant advantages for studying natural product biosynthetic genes in the soil microbiomes. CONCLUSION: Our results provide an effective strategy for combining long and short-read DNA sequencing data to explore and distill the maximum information out of soil metagenomics.

Dynamics of Verticillium dahliae race 1 population under managed agricultural ecosystems.

BACKGROUND: Plant pathogens and their hosts undergo adaptive changes in managed agricultural ecosystems, by overcoming host resistance, but the underlying genetic adaptations are difficult to determine in natural settings. Verticillium dahliae is a fungal pathogen that causes Verticillium wilt on many economically important crops including lettuce. We assessed the dynamics of changes in the V. dahliae genome under selection in a long-term field experiment. RESULTS: In this study, a field was fumigated before the Verticillium dahliae race 1 strain (VdLs.16) was introduced. A derivative 145-strain population was collected over a 6-year period from this field in which a seggregating population of lettuce derived from Vr1/vr1 parents were evaluated. We de novo sequenced the parental genome of VdLs.16 strain and resequenced the derivative strains to analyze the genetic variations that accumulate over time in the field cropped with lettuce. Population genomics analyses identified 2769 single-nucleotide polymorphisms (SNPs) and 750 insertion/deletions (In-Dels) in the 145 isolates compared with the parental genome. Sequence divergence was identified in the coding sequence regions of 378 genes and in the putative promoter regions of 604 genes. Five-hundred and nine SNPs/In-Dels were identified as fixed. The SNPs and In-Dels were significantly enriched in the transposon-rich, gene-sparse regions, and in those genes with functional roles in signaling and transcriptional regulation. CONCLUSIONS: Under the managed ecosystem continuously cropped to lettuce, the local adaptation of V. dahliae evolves at a whole genome scale to accumulate SNPs/In-Dels nonrandomly in hypervariable regions that encode components of signal transduction and transcriptional regulation.

Genome Sequence Data of MAT1-1 and MAT1-2 Idiomorphs from Verticillium dahliae.

Though Verticillium dahliae is an asexually reproducing fungus, it is considered heterothallic owing to the presence of only one of the two mating-type idiomorphs (MAT1-1 or MAT1-2) in individual isolates. But sexual reproduction has never been observed either in nature or in the laboratory. All of the genomic information in the literature thus far has therefore come from studies on isolates carrying only the MAT1-2 idiomorph. Herein, we sequenced and compared high-quality reference genomes of MAT1-1 strain S011 and MAT1-2 strain S023 obtained from the same sunflower field. The two genomic sequences displayed high synteny, and encoded similar number genes, a similarity especially notable among pathogenicity-related genes. Homolog analysis between these two genomes revealed that 80% of encoded genes are highly conserved (95% identity and coverage), but only 20% of the single copy genes were identical. These novel genome resources will support the analysis of the structure and function of the two idiomorphs and provide valuable tools to elucidate the evolution and potential mechanisms of sexual reproduction in V. dahliae.

Genome Sequence of Verticillium dahliae Race 1 Isolate VdLs.16 From Lettuce.

Verticillium dahliae is a widespread fungal pathogen that causes Verticillium wilt on many economically important crops and ornamentals worldwide. Populations of V. dahliae have been divided into two distinct races based upon differential host responses in tomato and lettuce. Recently, the contemporary race 2 isolates were further divided into an additional race in tomato. Herein, we provide a high-quality reference genome for the race 1 strain VdLs.16 isolated from lettuce in California, U.S.A. This resource will contribute to ongoing research that aims to elucidate the genetic basis of V. dahliae pathogenicity and population genomic diversity.

Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae.

Verticillium dahliae is a broad host-range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved. Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed. Population genomics previously revealed that G-LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G-LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N-lauroylethanolamine (N-acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase. The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.

Genome-wide analysis of the gene families of resistance gene analogues in cotton and their response to Verticillium wilt.

BACKGROUND: Gossypium raimondii is a Verticillium wilt-resistant cotton species whose genome encodes numerous disease resistance genes that play important roles in the defence against pathogens. However, the characteristics of resistance gene analogues (RGAs) and Verticillium dahliae response loci (VdRLs) have not been investigated on a global scale. In this study, the characteristics of RGA genes were systematically analysed using bioinformatics-driven methods. Moreover, the potential VdRLs involved in the defence response to Verticillium wilt were identified by RNA-seq and correlations with known resistance QTLs. RESULTS: The G. raimondii genome encodes 1004 RGA genes, and most of these genes cluster in homology groups based on high levels of similarity. Interestingly, nearly half of the RGA genes occurred in 26 RGA-gene-rich clusters (Rgrcs). The homology analysis showed that sequence exchanges and tandem duplications frequently occurred within Rgrcs, and segmental duplications took place among the different Rgrcs. An RNA-seq analysis showed that the RGA genes play roles in cotton defence responses, forming 26 VdRLs inside in the Rgrcs after being inoculated with V. dahliae. A correlation analysis found that 12 VdRLs were adjacent to the known Verticillium wilt resistance QTLs, and that 5 were rich in NB-ARC domain-containing disease resistance genes. CONCLUSIONS: The cotton genome contains numerous RGA genes, and nearly half of them are located in clusters, which evolved by sequence exchanges, tandem duplications and segmental duplications. In the Rgrcs, 26 loci were induced by the V. dahliae inoculation, and 12 are in the vicinity of known Verticillium wilt resistance QTLs.

Extracellular vesicles of Bifidobacterium longum reverse the acquired carboplatin resistance in ovarian cancer cells via p53 phosphorylation on Ser15.

We previously found that the relative abundance of Bifidobacterium was increased after chemotherapy; however, the role of Bifidobacterium longum in chemotherapeutic drug resistance in ovarian cancer (OVC) remains unclear. This study aimed to understand the potential effects and mechanism of B. longum extracellular vesicles (B. longum-EVs) on carboplatin (CBP) resistance in OVC. Eight normal and 11 ovarian tissues were collected and the expression of B. longum genomic DNA and its association with acquired CBP resistance in OVC patients was determined. After isolating EVs by ultracentrifugation from B. longum (ATCC 15707), CBP-resistant A2780 cells were treated with PBS, CBP, B. longum-EVs, or CBP + B. longum-EVs, and subsequently analyzed by CCK-8, Edu staining, Annexin V/PI double staining, wound healing, and Transwell assays to detect cell viability, proliferation, apoptosis, migration, and invasion, respectively. MRP1, ATP7A, ATP7B, and p53 expression as well as p53 phosphorylation were measured by western blot analysis. S15A mutation of p53 was assessed to examine the potential role of p53 Ser15 phosphorylation in CBP-resistant OVC. B. longum levels were elevated and positively associated with CBP resistance in OVC patients. Only high concentrations of B. longum-EVs attenuated A2780 cell proliferation, apoptosis, migration, and invasion. B. longum-EVs exposure significantly enhanced the sensitivity of CBP-resistant A2780 cells to CBP and decreased the expression of drug resistance-related proteins. The effect of B. longum-EVs on reversing CBP resistance was completely inhibited by S15A mutation of p53. B. longum-EVs enhanced the sensitivity of OVC cells to CBP through p53 phosphorylation on Ser15.

Functional Genomics and Comparative Lineage-Specific Region Analyses Reveal Novel Insights into Race Divergence in Verticillium dahliae.

Verticillium dahliae is a widespread soilborne fungus that causes Verticillium wilt on numerous economically important plant species. In tomato, until now, three races have been characterized based on the response of differential cultivars to V. dahliae, but the genetic basis of race divergence in V. dahliae remains undetermined. To investigate the genetic basis of race divergence, we sequenced the genomes of two race 2 strains and four race 3 strains for comparative analyses with two known race 1 genomes. The genetic basis of race divergence was described by the pathogenicity-related genes among the three races, orthologue analyses, and genomic structural variations. Global comparative genomics showed that chromosomal rearrangements are not the only source of race divergence and that race 3 should be split into two genotypes based on orthologue clustering. Lineage-specific regions (LSRs), frequently observed between genomes of the three races, encode several predicted secreted proteins that potentially function as suppressors of immunity triggered by known effectors. These likely contribute to the virulence of the three races. Two genes in particular that can act as markers for race 2 and race 3 (VdR2e and VdR3e, respectively) contribute to virulence on tomato, and the latter acts as an avirulence factor of race 3. We elucidated the genetic basis of race divergence through global comparative genomics and identified secreted proteins in LSRs that could potentially play critical roles in the differential virulence among the races in V. dahliae. IMPORTANCE Deciphering the gene-for-gene relationships during host-pathogen interactions is the basis of modern plant resistance breeding. In the Verticillium dahliae-tomato pathosystem, two races (races 1 and 2) and their corresponding avirulence (Avr) genes have been identified, but strains that lack these two Avr genes exist in nature. In this system, race 3 has been described, but the corresponding Avr gene has not been identified. We de novo-sequenced genomes of six strains and identified secreted proteins within the lineage-specific regions (LSRs) distributed among the genomes of the three races that could potentially function as manipulators of host immunity. One of the LSR genes, VdR3e, was confirmed as the Avr gene for race 3. The results indicate that differences in transcriptional regulation may contribute to race differentiation. This is the first study to describe these differences and elucidate roles of secreted proteins in LSRs that play roles in race differentiation.

Improved antitumor activity of cisplatin combined with Ganoderma lucidum polysaccharides in U14 cervical carcinoma-bearing mice.

Research on anticervical cancer is urgently required to enhance clinical outcomes. As a main anticancer drug for cervical carcinoma, cisplatin (CIS) has been used for a lot of years in clinical therapy. However, serious adverse effects including nephrotoxicity and neurotoxicity limit its long-term treatment. Our main goal of this study is to investigate the improvement of Ganoderma lucidum polysaccharides (GPS) on CIS-induced antitumor effect of in U14 cervical carcinoma-bearing mice. The results showed that GPS + CIS could not only inhibit the growth of the tumor but also improve the spleen and thymus indexes. Moreover, little toxicological effects were observed on hepatic function and renal function in GPS + CIS treated mice bearing U14 tumor cells. Further analysis of the tumor inhibition mechanism indicated that the number of apoptotic tumor cells increased significantly, the expression of Bax increased and the expression of Bcl-2 decreased dramatically in cervical cancer sections after oral administration of GPS + CIS for 14 days. This GPS/CIS combined therapy represents intriguing therapeutic strategy for U14 cervical carcinoma providing not only superior efficacy but also a higher safety level.

Comparative genomic, transcriptomic and secretomic profiling of Penicillium oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106, and identification of two novel regulatory genes of cellulase and xylanase gene expression.

BACKGROUND: The filamentous fungus Penicillium oxalicum is a potential alternative to Trichoderma reesei for industrial production of a complete cellulolytic enzyme system for a bio-refinery. Comparative omics approaches can support rational genetic engineering and/or breeding of filamentous fungi with improved cellulase production capacity. In this study, comparative genomic, transcriptomic and secretomic profiling of P. oxalicum HP7-1 and its cellulase and xylanase hyper-producing mutant EU2106 were employed to screen for novel regulators of cellulase and xylanase gene expression. RESULTS: The 30.62 Mb P. oxalicum HP7-1 genome was sequenced, and 9834 protein-coding genes were annotated. Re-sequencing of the mutant EU2106 genome identified 274 single nucleotide variations and 12 insertion/deletions. Comparative genomic, transcriptomic and secretomic profiling of HP7-1 and EU2106 revealed four candidate regulators of cellulase and xylanase gene expression. Deletion of these candidate genes and measurement of the enzymatic activity of the resultant mutants confirmed the identity of three regulatory genes. POX02484 and POX08522, encoding a putative Zn(II)2Cys6 DNA-binding domain and forkhead protein, respectively, were found to be novel, while PoxClrB is an ortholog of ClrB, a key transcriptional regulator of cellulolytic enzyme gene expression in filamentous fungi. ΔPOX02484 and ΔPOX08522 mutants exhibited significantly reduced ß-glucosidase activity, increased carboxymethylcellulose cellulase and xylanase activities, and altered transcription level of cellulase and xylanase genes compared with the parent strain ΔPoxKu70, with Avicel as the sole carbon source. CONCLUSIONS: Two novel genes, POX02484 and POX08522, were found and characterized to regulate the expression of cellulase and xylanase genes in P. oxalicum. These findings are important for engineering filamentous fungi to improve cellulase and xylanase production.