Regulatory Mechanisms and Evolutionary Insights of Phytoalexin Biosynthesis in Rice.

Transcriptomic analysis of UV-treated rice leaves reveals UV-induced phytoalexin biosynthetic pathways and their regulatory networks in rice

NextGen microbial natural products discovery.

Plants frequently possess operon-like gene clusters for specialized metabolism. Cultivated rice, Oryza sativa, produces antimicrobial diterpene phytoalexins represented by phytocassanes and momilactones, and the majority of their biosynthetic genes are clustered on chromosomes 2 and 4, respectively. These labdane-related diterpene phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate or syn-copalyl diphosphate. The two gene clusters consist of genes encoding diterpene synthases and chemical-modification enzymes including P450s. In contrast, genes for the biosynthesis of gibberellins, which are labdane-related phytohormones, are scattered throughout the rice genome similar to other plant genomes. The mechanism of operon-like gene cluster formation remains undefined despite previous studies in other plant species. Here we show an evolutionary insight into the rice gene clusters by a comparison with wild Oryza species. Comparative genomics and biochemical studies using wild rice species from the AA genome lineage, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis and the progenitor of Asian cultivated rice Oryza rufipogon indicate that gene clustering for biosynthesis of momilactones and phytocassanes had already been accomplished before the domestication of rice. Similar studies using the species Oryza punctata from the BB genome lineage, the distant FF genome lineage species Oryza brachyantha and an outgroup species Leersia perrieri suggest that the phytocassane biosynthetic gene cluster was present in the common ancestor of the Oryza species despite the different locations, directions and numbers of their member genes. However, the momilactone biosynthetic gene cluster evolved within Oryza before the divergence of the BB genome via assembly of ancestral genes.

When plants sense the attack of microbial organisms, they initiate a series of defense responses. One of the most important defense components is the production of phytoalexins that are newly synthesized as anti-microbial secondary metabolites; however, knowledge about the signaling components regulating phytoalexin biosynthesis is limited. Mitogen-activated protein kinase (MAPK) cascades are key components in the defense signaling evoked by recognition of microbe-associated molecular patterns (MAMPs) that regulate several defense responses including phytoalexin biosynthesis. In Arabidopsis, biosynthesis of an indole-derived phytoalexin, camalexin, is regulated by MAPK cascades including AtMPK3, AtMPK4 and AtMPK6. Recently, we characterized a novel MAPK cascade in rice (OsMKK4-OsMPK3/OsMPK6) that induces production of diterpenoid phytoalexins by regulating the expression of their biosynthetic genes. Downstream signals of MAPK cascades are thought to be mediated by several transcription factors. To date, AtWRKY33 and OsTGAP1 have been identified as transcriptional activators of phytoalexin biosynthesis in Arabidopsis and rice. Here, we discuss and compare the regulatory mechanisms for phytoalexin biosynthesis through MAPK cascades and transcription factors in Arabidopsis and rice.

BackgroundDirect cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-sized gene clusters for heterologous as well as homologous expression.ResultsA versatile Escherichia coli-Streptomyces shuttle bacterial artificial chromosomal (BAC) conjugation vector, pSBAC, was used along with a cluster tandem integration approach to carry out homologous and heterologous overexpression of a large 80-kb polyketide biosynthetic pathway gene cluster of tautomycetin (TMC), which is a protein phosphatase PP1/PP2A inhibitor and T cell-specific immunosuppressant. Unique XbaI restriction sites were precisely inserted at both border regions of the TMC biosynthetic gene cluster within the chromosome of TMC-producing Streptomyces sp. CK4412, followed by site-specific recombination of pSBAC into the flanking region of the TMC gene cluster. The entire TMC gene cluster was then rescued as a single giant recombinant pSBAC by XbaI digestion of the chromosomal DNA as well as subsequent self-ligation. Next, the recombinant pSBAC construct containing the entire TMC cluster in E. coli was directly conjugated into model Streptomyces strains, resulting in rapid and enhanced TMC production. Moreover, introduction of the TMC cluster-containing pSBAC into wild-type Streptomyces sp. CK4412 as well as a recombinant S. coelicolor strain resulted in a chromosomal tandem repeat of the entire TMC cluster with 14-fold and 5.4-fold enhanced TMC productivities, respectively.ConclusionsThe 80-kb TMC biosynthetic gene cluster was isolated in a single integration vector, pSBAC. Introduction of TMC biosynthetic gene cluster in TMC non-producing strains has resulted in similar amount of TMC production yield. Moreover, over-expression of TMC biosynthetic gene cluster in original producing strain and recombinant S. coelicolor dramatically increased TMC production. Thus, this strategy can be employed to develop a custom overexpression scheme of entire metabolite pathway clusters present in actinomycetes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0325-2) contains supplementary material, which is available to authorized users.

In actinomycetes, the onset of secondary metabolite biosynthesis is often triggered by the quorum-sensing signal gamma-butyrolactones (GBLs) via specific binding to their cognate receptors. However, the presence of multiple putative GBL receptor homologues in the genome suggests the existence of an alternative regulatory mechanism. Here, in the model streptomycete Streptomyces coelicolor, ScbR2 (SCO6286, a homologue of GBL receptor) is shown not to bind the endogenous GBL molecule SCB1, hence designated "pseudo" GBL receptor. Intriguingly, it could bind the endogenous antibiotics actinorhodin and undecylprodigiosin as ligands, leading to the derepression of KasO, an activator of a cryptic type I polyketide synthase gene cluster. Likewise, JadR2 is also a putative GBL receptor homologue in Streptomyces venezuelae, the producer of chloramphenicol and cryptic antibiotic jadomycin. It is shown to coordinate their biosynthesis via direct repression of JadR1, which activates jadomycin biosynthesis while repressing chloramphenicol biosynthesis directly. Like ScbR2, JadR2 could also bind these two disparate antibiotics, and the interactions lead to the derepression of jadR1. The antibiotic responding activities of these pseudo GBL receptors were further demonstrated in vivo using the lux reporter system. Overall, these results suggest that pseudo GBL receptors play a novel role to coordinate antibiotic biosynthesis by binding and responding to antibiotics signals. Such an antibiotic-mediated regulatory mechanism could be a general strategy to coordinate antibiotic biosynthesis in the producing bacteria.

Fungi produce a wide variety of compounds, especially those that exhibit biological activity. Such compounds may aid the survival of fungi in the environment or may contribute to host infection for pathogenic species. Regarding dermatophytes, which affect a large number of patients worldwide, studies on metabolites that exhibit biological activity are scarce. In this study, to gain insight into the interaction with skin microbiota at the site of infection, we searched for compounds that exhibit antibacterial activity among the metabolites of Trichophyton rubrum. We rediscovered viomellein, a red pigment, as a potent antibacterial compound and identified its biosynthetic gene (vio) cluster by RNA-sequencing and gene deletion analyses. Sequential reconstruction of the vio cluster genes in Aspergillus oryzae revealed the biosynthetic pathway for viomellein via nor-toralactone, semivioxanthin, and vioxanthin production. The vio gene cluster is widely conserved among dermatophytes and is also present in some Aspergillus and Penicillium species. Consistent with the results, viomellein and its structural analogs, xanthomegnin, and vioxanthin, were shown to be produced by most dermatophyte species. These results suggest that dermatophytes can produce diverse naphthopyranone compounds, some of which have strong growth inhibitory effects against bacteria. This study provides a previously unknown molecular entity for antibiotic production by dermatophytes and provides insight into the interaction between commensal bacteria and dermatophytes.IMPORTANCEDermatophytes are widespread human pathogens in the world, but the mechanisms of infection have been little studied. Although bacterial density at the site of infection is abundant, interaction between dermatophytes and the bacterial community has not yet been studied. Here, to understand the infection ecology of dermatophytes, we searched for antimicrobial substances that would be effective against the dermal bacterial community. We discovered viomellein, which exhibits strong antibacterial activity against gram-positive bacteria such as Staphylococcus aureus, and its biosynthetic genes are shared not only by dermatophytes but also by other fungi. Since many dermatophytes showed the ability to produce viomellein, it is likely that this is the initial infection strategy of dermatophytes, which has been a mystery for long.

BackgroundUltra violet radiation leads to accumulation of phytoalexins (PA) in rice (Oryza sativa) which are typically accumulated when the plants are infected with rice blast pathogen Magnaporthe oryzae. Although extensive works have been done in elucidating phytoalexin biosynthesis, UV stress signal transduction leading to accumulations of rice phytoalexin is largely unknown.ResultsIn the present study, the involvement of mitogen activated protein kinase (MAPK) cascade has been shown in UV induced regulation of genes in phytoalexin biosynthesis in rice. UV induced activation of MAPK and expression of PA biosynthesis genes were shown to be inhibited with staurosporin and MAPK inhibitors. Transcript regulation studies and kinase assays indicated involvement of OsMKK6 in the process. Transgenic rice overexpressing constitutive active OsMKK6EE exhibited higher expression of genes of PA biosynthesis pathway upon UV stress and also upon infection with M. oryzae.ConclusionThese results suggest a key role of OsMKK6 in regulation of UV responsive expression of genes of PA biosynthesis in rice. This study will help to elucidate the intricate signalling components of UV leading to phytoalexins biosynthesis in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-6-35) contains supplementary material, which is available to authorized users.

Streptomyces bacteria are known for their prolific production of secondary metabolites, many of which have been widely used in human medicine, agriculture and animal health. To guide the effective prioritization of specific biosynthetic gene clusters (BGCs) for drug development and targeting the most prolific producer strains, knowledge about phylogenetic relationships of Streptomyces species, genome-wide diversity and distribution patterns of BGCs is critical. We used genomic and phylogenetic methods to elucidate the diversity of major classes of BGCs in 1,110 publicly available Streptomyces genomes. Genome mining of Streptomyces reveals high diversity of BGCs and variable distribution patterns in the Streptomyces phylogeny, even among very closely related strains. The most common BGCs are non-ribosomal peptide synthetases, type 1 polyketide synthases, terpenes, and lantipeptides. We also found that numerous Streptomyces species harbor BGCs known to encode antitumor compounds. We observed that strains that are considered the same species can vary tremendously in the BGCs they carry, suggesting that strain-level genome sequencing can uncover high levels of BGC diversity and potentially useful derivatives of any one compound. These findings suggest that a strain-level strategy for exploring secondary metabolites for clinical use provides an alternative or complementary approach to discovering novel pharmaceutical compounds from microbes.

Rice plants synthesize a unique group of diterpenoid phytoalexins (DPs) that exhibit broad-spectrum antimicrobial activities and are biosynthesized by enzymes encoded by three biosynthetic gene clusters. However, the regulatory mechanisms of their biosynthesis remain unclear. Here, the regulatory roles of the transcription factor OsWRKY10 and its interacting VQ motif-containing protein OsVQ8 in DPs biosynthesis and disease resistance were investigated via genetic and biochemical analyses. Their CRISPR/Cas9-mediated knockout and over-expressing (OE) lines, as well as crossed lines WRKY10OE/vq8, were generated. OsVQ8 phosphorylation by mitogen-activated protein kinase (MAPK) cascades was examined. We found that OsWRKY10 co-expresses with and activates a specific set of genes involved in DPs biosynthesis, thereby enhancing DPs accumulation and disease resistance against both fungal blast and bacterial blight. We demonstrate that OsWRKY10 interacts with the VQ motif-containing protein OsVQ8, modulating DPs biosynthesis through OsVQ8 phosphorylation by the activated OsMKK4-OsMPK6 cascade upon perception of pathogen-associated molecular patterns. Our findings highlight how the interaction between OsVQ8 and OsWRKY10 serves as a molecular switch to regulate gene clusters and the entire pathway of DPs biosynthesis in rice and provides valuable insights for genetic engineering aimed at enhancing phytoalexin production and broad-spectrum disease resistance in staple food crops.

The aim of the work was to analyse biosynthetic gene clusters (BGC) of Bacillus velezensis ONU 553 based on bioinformatics approach. Methods. Identification of species was processed with tools of TYGS server; EzBioCloud was used to calculate ANI. Analysis of biosynthetic gene, bacteriocin, and antibiotic resistance gene clusters using antiSMASH, Bagel4, respectively. The results. It is shown that the results of identification, phylogenetic analysis and DNA-DNA hybridization (DDH) carried out in silico proved that the strain Bacillus velezensis ONU 553 belongs to the operational group B. amyloliquefaciens (OGBa). Sequences identified as possible phages and CpG-islands were found in the genome of our strain. 12 biosynthetic gene clusters (BGC) were identified using antiSMASH. One new cluster capable of synthesizing a new metabolite was identified (region 11). The presence of two clusters of bacteriocins in the genome of Bacillus velezensis ONU 553, which are assigned to uberolysin/carnocyclin and the antimicrobial peptide LCI based on the identification of the core gene, is shown. Conclusions. The preliminary identification of the Bacillus velezensis ONU 553 strain as a representative of the Bacillus velezensis strain of the B. amyloliquefaciens group (OGBa) was confirmed. The presence of gene clusters of secondary metabolites responsible for the synthesis of surfactins, polyene antibiotics, antimicrobial peptides, macrolide antibiotics and bacteriocins was shown. The obtained results indicate that the Bacillus velezensis ONU 553 strain is promising for use in the field of "Blue Biotechnology" for the development of new drugs with antimicrobial and antifungal activity.

Phytoalexin production by amino acid conjugates of jasmonic acid through induction of naringenin-7- O-methyltransferase, a key enzyme on phytoalexin biosynthesis in rice ( Oryza sativa L.)

Wild and cultivated rice show a significant difference in anthocyanin biosynthesis in the leaf. The regulation system of anthocyanin biosynthesis in rice leaf and the causal mechanism of the difference in this biosynthesis between wild and cultivated rice remain largely unknown. In this study, a genome-wide association study and transcriptome analysis were performed to identify the determinant factors and dissect the regulatory system for anthocyanin biosynthesis in rice leaves. OsC1, OsRb and OsDFR were identified as the determinants of anthocyanin biosynthesis in rice leaves. Artificial selection of certain null mutations of OsC1 and OsRb was the main causal mechanism underlying the loss of anthocyanin pigmentation in most cultivated rice. OsP1 and the MYB-bHLH-WD40 complexes regulate anthocyanin biosynthetic genes in rice leaves with partial functional overlap. OsP1 specifically activates upstream biosynthetic genes (OsCHS, OsCHI and OsF3'H) for anthocyanin biosynthesis, whereas the ternary MYB-bHLH-WD40 complex activates all anthocyanin biosynthetic genes including OsCHS, OsCHI, OsF3'H, OsF3H, OsDFR and OsANS. OsC1 and OsRb are tissue-specific regulators that do not influence anthocyanin biosynthesis in the pericarp. Our results reveal the determinant factors, regulatory system and domestication of anthocyanin biosynthesis in rice leaves, and show the potential of engineering anthocyanin biosynthesis in rice.

Simple SummaryBiosurfactants are amphiphilic molecules produced by microorganisms with a hydrophilic and a hydrophobic group, able to reduce surface tension. These molecules are largely used in the environmental, food, pharmaceutical, medical, and cleaning industries, among others. Serratia strains are ubiquitous microorganisms with the ability to produce biosurfactants, such as serrawettins. These extracellular lipopeptides are described as biocides against many bacteria and fungi. This work used comparative genomics to determine the distribution and organization of the serrawettins W1 and W2 biosynthetic gene clusters in all the 84 publicly available genomes of the Serratia genus. Here, the serrawettin W1 gene clusters’ organization is reported for the first time. The serrawettin W1 biosynthetic gene swrW and serrawettin W2 biosynthetic gene swrA were present in 17 and 11 Serratia genomes, respectively. The same genes in the biosynthetic clusters frame the swrW and swrA biosynthetic genes. This work identified four genes common to all serrawettin gene clusters, highlighting their key potential in the serrawettins biosynthetic process.Serratia strains are ubiquitous microorganisms with the ability to produce serratomolides, such as serrawettins. These extracellular lipopeptides are described as biocides against many bacteria and fungi and may have a nematicidal activity against phytopathogenic nematodes. Serrawettins W1 and W2 from different strains have different structures that might be correlated with distinct genomic organizations. This work used comparative genomics to determine the distribution and the organization of the serrawettins biosynthetic gene clusters in all the 84 publicly available genomes of the Serratia genus. The serrawettin W1 and W2 gene clusters’ organization was established using antiSMASH software and compared with single and short data previously described for YD25TSerratia. Here, the serrawettin W1 gene clusters’ organization is reported for the first time. The serrawettin W1 biosynthetic gene swrW was present in 17 Serratia genomes. Eighty different coding sequence (CDS) were assigned to the W1 gene cluster, 13 being common to all clusters. The serrawettin W2 swrA gene was present in 11 Serratia genomes. The W2 gene clusters included 68 CDS with 24 present in all the clusters. The genomic analysis showed the swrA gene constitutes five modules, four with three domains and one with four domains, while the swrW gene constitutes one module with four domains. This work identified four genes common to all serrawettin gene clusters, highlighting their essential potential in the serrawettins biosynthetic process.

C.R.HarwoodS.M.CuttingMolecular Biological Methods for Bacillus1990John Wiley & Sons Ltd.627 Seiten, Preis: £ 75,00