Guiling Sun

Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431

BackgroundPaclitaxel (Taxol™) is an important anticancer drug with a unique mode of action. The biosynthesis of paclitaxel had been considered restricted to the Taxus species until it was discovered in Taxomyces andreanae, an endophytic fungus of T. brevifolia. Subsequently, paclitaxel was found in hazel (Corylus avellana L.) and in several other endophytic fungi. The distribution of paclitaxel in plants and endophytic fungi and the reported sequence homology of key genes in paclitaxel biosynthesis between plant and fungi species raises the question about whether the origin of this pathway in these two physically associated groups could have been facilitated by horizontal gene transfer.ResultsThe ability of the endophytic fungus of hazel Penicillium aurantiogriseum NRRL 62431 to independently synthesize paclitaxel was established by liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. The genome of Penicillium aurantiogriseum NRRL 62431 was sequenced and gene candidates that may be involved in paclitaxel biosynthesis were identified by comparison with the 13 known paclitaxel biosynthetic genes in Taxus. We found that paclitaxel biosynthetic gene candidates in P. aurantiogriseum NRRL 62431 have evolved independently and that horizontal gene transfer between this endophytic fungus and its plant host is unlikely.ConclusionsOur findings shed new light on how paclitaxel-producing endophytic fungi synthesize paclitaxel, and will facilitate metabolic engineering for the industrial production of paclitaxel from fungi.

Deep Sequencing Reveals Transcriptome Re-Programming of Taxus × media Cells to the Elicitation with Methyl Jasmonate

Background Plant cell culture represents an alternative source for producing high-value secondary metabolites including paclitaxel (Taxol®), which is mainly produced in Taxus and has been widely used in cancer chemotherapy. The phytohormone methyl jasmonate (MeJA) can significantly increase the production of paclitaxel, which is induced in plants as a secondary metabolite possibly in defense against herbivores and pathogens. In cell culture, MeJA also elicits the accumulation of paclitaxel; however, the mechanism is still largely unknown. Methodology/Principal Findings To obtain insight into the global regulation mechanism of MeJA in the steady state of paclitaxel production (7 days after MeJA addition), especially on paclitaxel biosynthesis, we sequenced the transcriptomes of MeJA-treated and untreated Taxus × media cells and obtained ∼ 32.5 M high quality reads, from which 40,348 unique sequences were obtained by de novo assembly. Expression level analysis indicated that a large number of genes were associated with transcriptional regulation, DNA and histone modification, and MeJA signaling network. All the 29 known genes involved in the biosynthesis of terpenoid backbone and paclitaxel were found with 18 genes showing increased transcript abundance following elicitation of MeJA. The significantly up-regulated changes of 9 genes in paclitaxel biosynthesis were validated by qRT-PCR assays. According to the expression changes and the previously proposed enzyme functions, multiple candidates for the unknown steps in paclitaxel biosynthesis were identified. We also found some genes putatively involved in the transport and degradation of paclitaxel. Potential target prediction of miRNAs indicated that miRNAs may play an important role in the gene expression regulation following the elicitation of MeJA. Conclusions/Significance Our results shed new light on the global regulation mechanism by which MeJA regulates the physiology of Taxus cells and is helpful to understand how MeJA elicits other plant species besides Taxus.

Root parasitic plant Orobanche aegyptiaca and shoot parasitic plant Cuscuta australis obtained Brassicaceae-specific strictosidine synthase-like genes by horizontal gene transfer

BackgroundBesides gene duplication and de novo gene generation, horizontal gene transfer (HGT) is another important way of acquiring new genes. HGT may endow the recipients with novel phenotypic traits that are important for species evolution and adaption to new ecological niches. Parasitic systems expectedly allow the occurrence of HGT at relatively high frequencies due to their long-term physical contact. In plants, a number of HGT events have been reported between the organelles of parasites and the hosts, but HGT between host and parasite nuclear genomes has rarely been found.ResultsA thorough transcriptome screening revealed that a strictosidine synthase-like (SSL) gene in the root parasitic plant Orobanche aegyptiaca and the shoot parasitic plant Cuscuta australis showed much higher sequence similarities with those in Brassicaceae than with those in their close relatives, suggesting independent gene horizontal transfer events from Brassicaceae to these parasites. These findings were strongly supported by phylogenetic analysis and their identical unique amino acid residues and deletions. Intriguingly, the nucleus-located SSL genes in Brassicaceae belonged to a new member of SSL gene family, which were originated from gene duplication. The presence of introns indicated that the transfer occurred directly by DNA integration in both parasites. Furthermore, positive selection was detected in the foreign SSL gene in O. aegyptiaca but not in C. australis. The expression of the foreign SSL genes in these two parasitic plants was detected in multiple development stages and tissues, and the foreign SSL gene was induced after wounding treatment in C. australis stems. These data imply that the foreign genes may still retain certain functions in the recipient species.ConclusionsOur study strongly supports that parasitic plants can gain novel nuclear genes from distantly related host species by HGT and the foreign genes may execute certain functions in the new hosts.

Scopoletin is a phytoalexin against Alternaria alternata in wild tobacco dependent on jasmonate signalling

Summary This study demonstrates that wild tobacco Nicotiana attenuata plants accumulate the phytoalexin scopoletin to defend against the necrotrophic fungus Alternaria alternata in a JA signalling-dependent manner.

Genome-wide identification of calcium-dependent protein kinases in soybean and analyses of their transcriptional responses to insect herbivory and drought stress

Calcium-dependent protein kinases (CDPKs) are plant-specific calcium sensors that play important roles in various aspects of plant physiology. Here, we investigated phylogenic relationships, chromosomal locations, gene structures, and tissue-specific, herbivory- and drought-induced expression profiles of soybean (Glycine max) GmCDPKs. Fifty GmCDPK genes were identified, which phylogenetically grouped into 4 distinct clusters and distributed across 13 sub-clusters. Individual classes of GmCDPKs harbor highly conserved mRNA splicing sites, and their exon numbers and lengths were consistent with the phylogenetic relationships, suggesting that at least 13 ancestral CDPK genes had emerged before the split of monocots and eudicots. Gene expression analysis indicated that several GmCDPKs were tissue-specific expressed. GmCDPKs’ transcript levels changed after wounding, exhibited specific expression patterns after simulated Spodoptera exigua feeding or soybean aphid (Aphis glycines) herbivory, and were largely independent of the phytohormones jasmonic acid and salicylic acid. The most pronounced transcriptional responses were detected after drought and abscisic acid treatments with more than half of all GmCDPKs being upregulated, suggesting their important roles during abiotic stress responses in soybean. Our data provide an important foundation for further functional dissection of GmCDPKs, especially in the context of soybean-insect interactions and drought stress adaptation.

Oral secretions from Mythimna separata insects specifically induce defense responses in maize as revealed by high-dimensional biological data

Attack from insect herbivores poses a major threat to plant survival, and accordingly, plants have evolved sophisticated defence systems. Maize is cultivated as a staple crop worldwide, and insect feeding causes large production losses. Despite its importance in agriculture, little is known about how maize reacts to insect herbivory. Taking advantage of advances in sequencing and mass spectrometry technology, we studied the response of maize to mechanical wounding and simulated Mythimna separata (a specialist insect) herbivory by applying its oral secretions (OS) to wounds. In comparison to the responses induced by mechanical wounding, OS elicited larger and longer-lasting changes in the maize transcriptome, proteome, metabolome and phytohormones. Specifically, many genes, proteins and metabolites were uniquely induced or repressed by OS. Nearly 290 transcription factor genes from 39 families were involved in OS-induced responses, and among these, more transcription factor genes were specifically regulated by OS than by wounding. This study provides a large-scale omics dataset for understanding maize response to chewing insects and highlights the essential role of OS in plant-insect interactions.

Baseline Survey of Root-Associated Microbes of Taxus chinensis (Pilger) Rehd

Taxol (paclitaxel) a diterpenoid is one of the most effective anticancer drugs identified. Biosynthesis of taxol was considered restricted to the Taxus genera until Stierle et al. discovered that an endophytic fungus isolated from Taxus brevifolia could independently synthesize taxol. Little is known about the mechanism of taxol biosynthesis in microbes, but it has been speculated that its biosynthesis may differ from plants. The microbiome from the roots of Taxus chinensis have been extensively investigated with culture-dependent methods to identify taxol synthesizing microbes, but not using culture independent methods.,Using bar-coded high-throughput sequencing in combination with a metagenomics approach, we surveyed the microbial diversity and gene composition of the root-associated microbiomefrom Taxus chinensis (Pilger) Rehd. High-throughput amplicon sequencing revealed 187 fungal OTUs which is higher than any previously reported fungal number identified with the culture-dependent method, suggesting that T. chinensis roots harbor novel and diverse fungi. Some operational taxonomic units (OTU) identified were identical to reported microbe strains possessing the ability to synthesis taxol and several genes previously associated with taxol biosynthesis were identified through metagenomics analysis.

Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells.

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC-MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.

Stem parasitic plant Cuscuta australis (dodder) transfers herbivory-induced signals among plants

Significance Cuscuta spp. (i.e., dodders) are plant parasites that connect to the vasculature of their host plants to extract water, nutrients, and even macromolecules. Knowledge of ecologically meaningful communications between host plants and Cuscuta, or between Cuscuta bridge-connected hosts, has remained obscure until now. Here we show that herbivore attack on one of the Cuscuta bridge-connected plants induces gene expression and increases the activity of trypsin proteinase inhibitors, and thus elevates the resistance to insects in other undamaged but Cuscuta-connected plants. This Cuscuta-mediated interplant signaling is rapid, conserved, far-reaching, and partly requires the plant hormone jasmonic acid. Although Cuscuta parasites can negatively influence their host plants, under certain circumstances, they may also provide ecologically relevant information-based benefits. Cuscuta spp. (i.e., dodders) are stem parasites that naturally graft to their host plants to extract water and nutrients; multiple adjacent hosts are often parasitized by one or more Cuscuta plants simultaneously, forming connected plant clusters. Metabolites, proteins, and mRNAs are known to be transferred from hosts to Cuscuta, and Cuscuta bridges even facilitate host-to-host virus movement. Whether Cuscuta bridges transmit ecologically meaningful signals remains unknown. Here we show that, when host plants are connected by Cuscuta bridges, systemic herbivory signals are transmitted from attacked plants to unattacked plants, as revealed by the large transcriptomic changes in the attacked local leaves, undamaged systemic leaves of the attacked plants, and leaves of unattacked but connected hosts. The interplant signaling is largely dependent on the jasmonic acid pathway of the damaged local plants, and can be found among conspecific or heterospecific hosts of different families. Importantly, herbivore attack of one host plant elevates defensive metabolites in the other systemic Cuscuta bridge-connected hosts, resulting in enhanced resistance against insects even in several consecutively Cuscuta-connected host plants over long distances (> 100 cm). By facilitating plant-to-plant signaling, Cuscuta provides an information-based means of countering the resource-based fitness costs to their hosts.

Two hAT transposon genes were transferred from Brassicaceae to broomrapes and are actively expressed in some recipients

A growing body of evidence is pointing to an important role of horizontal gene transfer (HGT) in the evolution of higher plants. However, reports of HGTs of transposable elements (TEs) in plants are still scarce, and only one case is known of a class II transposon horizontally transferred between grasses. To investigate possible TE transfers in dicots, we performed transcriptome screening in the obligate root parasite Phelipanche aegyptiaca (Orobanchaceae), data-mining in the draft genome assemblies of four other Orobanchaceae, gene cloning, gene annotation in species with genomic information, and a molecular phylogenetic analysis. We discovered that the broomrape genera Phelipanche and Orobanche acquired two related nuclear genes (christened BO transposase genes), a new group of the hAT superfamily of class II transposons, from Asian Sisymbrieae or a closely related tribe of Brassicaceae, by HGT. The collinearity of the flanking genes, lack of a classic border structure, and low expression levels suggest that BO transposase genes cannot transpose in Brassicaceae, whereas they are highly expressed in P. aegyptiaca.