Genome Sequencing of Ciboria shiraiana Provides Insights into the Pathogenic Mechanisms of Hypertrophy Sorosis Scleroteniosis.

Ciboria shiraiana causes hypertrophy sorosis scleroteniosis in mulberry trees, resulting in huge economic losses, and exploring its pathogenic mechanism at a genomic level is important for developing new control methods. Here, genome sequencing of C. shiraiana based on PacBio RSII and Illumina HiSeq 2500 platform as well as manual gap filling was performed. Synteny analysis with Sclerotinia sclerotiorum revealed 16 putative chromosomes corresponding to 16 chromosomes of C. shiraiana. Screening of rapid-evolution genes revealed that 97 and 2.4% of genes had undergone purifying selection and positive selection, respectively. When compared with S. sclerotiorum, fewer secreted effector proteins were found in C. shiraiana. The number of genes involved in pathogenicity, including secondary metabolites, carbohydrate active enzymes, and P450s, in the C. shiraiana genome was comparable with that of other necrotrophs but higher than that of biotrophs and saprotrophs. The growth-related genes and plant cell-wall-degradation-related genes in C. shiraiana were expressed in different developmental and infection stages, and may be potential targets for prevention and control of this pathogen. These results provide new insights into C. shiraiana pathogenic mechanisms, especially host range and necrotrophy features, and lay the foundation for further study of the underlying molecular mechanisms.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

[Biological and epidemiological characteristics of the pathogen of hypertrophy sorosis scleroteniosis, Ciboria shiraiana].

Objective: We studied the biological and the epidemiological characteristics of the pathogen of hypertrophy sorosis scleroteniosis, which is a devastating fungal disease of mulberry. Methods: We studied the asexual and sexual reproductive phase stages of C. shiraiana, including the infection ability of hyphal, dormancy of sclerotia, the structures, release, number and germination of ascospores from apothecia, as well as the phenology of sclerotial germination. Results: In C. shiraiana, hyphae had no infection ability toward the female flowers of mulberry. Sclerotia of C. shiraiana must undergo cold treatment above 6 weeks, then the dormancy-breaking sclerotia could germinate to apothecia. One to fifteen apothecia were germinated from one sclerotium, and the number of ascospores in a 1.5 cm diameter apothecia could contain up to (5.6-6.3)×107. Ascospore C. shiraiana had significantly higher germination rates in acid than in neutral and alkaline environments. From late January to middle April, sclerotia germinated to apothecia, and got the highest value in the middle of March. Conclusion: C. shiraiana is a formidable pathogen to cause epidemic disease and damage in mulberry.

Chromosome-level Genomes Reveal the Genetic Basis of Descending Dysploidy and Sex Determination in Morus Plants.

Multiple plant lineages have independently evolved sex chromosomes and variable karyotypes to maintain their sessile lifestyles through constant biological innovation. Morus notabilis, a dioecious mulberry species, has the fewest chromosomes among Morus spp., but the genetic basis of sex determination and karyotype evolution in this species has not been identified. In this study, three high-quality genome assemblies were generated for Morus spp. [including dioecious M. notabilis (male and female) and Morus yunnanensis (female)] with genome sizes of 301-329 Mb and were grouped into six pseudochromosomes. Using a combination of genomic approaches, we found that the putative ancestral karyotype of Morus species was close to 14 protochromosomes, and that several chromosome fusion events resulted in descending dysploidy (2n = 2x = 12). We also characterized a ∼ 6.2-Mb sex-determining region on chromosome 3. Four potential male-specific genes, a partially duplicatedDNA helicase gene (named MSDH) and three Ty3_Gypsy long terminal repeat retrotransposons (named MSTG1/2/3), were identified in the Y-linked area and considered to be strong candidate genes for sex determination or differentiation. Population genomic analysis showed that Guangdong accessions in China were genetically similar to Japanese accessions of mulberry. In addition, genomic areas containing selective sweeps that distinguish domesticated mulberry from wild populations in terms of flowering and disease resistance were identified. Our study provides an important genetic resource for sex identification research and molecular breeding in mulberry.

Molecular Mechanisms Underlying the Biosynthesis of Melatonin and Its Isomer in Mulberry.

Mulberry (Morus alba L.) leaves and fruit are traditional Chinese medicinal materials with anti-inflammatory, immune regulatory, antiviral and anti-diabetic properties. Melatonin performs important roles in the regulation of circadian rhythms and immune activities. We detected, identified and quantitatively analyzed the melatonin contents in leaves and mature fruit from different mulberry varieties. Melatonin and three novel isoforms were found in the Morus plants. Therefore, we conducted an expression analysis of melatonin and its isomer biosynthetic genes and in vitro enzymatic synthesis of melatonin and its isomer to clarify their biosynthetic pathway in mulberry leaves. MaASMT4 and MaASMT20, belonging to class II of the ASMT gene family, were expressed selectively in mulberry leaves, and two recombinant proteins that they expressed catalyzed the conversion of N-acetylserotonin to melatonin and one of three isomers in vitro. Unlike the ASMTs of Arabidopsis and rice, members of the three ASMT gene families in mulberry can catalyze the conversion of N-acetylserotonin to melatonin. This study provides new insights into the molecular mechanisms underlying melatonin and its isomers biosynthesis and expands our knowledge of melatonin isomer biosynthesis.

Mulberry EIL3 confers salt and drought tolerances and modulates ethylene biosynthetic gene expression.

Ethylene regulates plant abiotic stress responses and tolerances, and ethylene-insensitive3 (EIN3)/EIN3-like (EIL) proteins are the key components of ethylene signal transduction. Although the functions of EIN3/EIL proteins in response to abiotic stresses have been investigated in model plants, little is known in non-model plants, including mulberry (Morus L.), which is an economically important perennial woody plant. We functionally characterized a gene encoding an EIN3-like protein from mulberry, designated as MnEIL3. A quantitative real-time PCR analysis demonstrated that the expression of MnEIL3 could be induced in roots and shoot by salt and drought stresses. Arabidopsis overexpressing MnEIL3 exhibited an enhanced tolerance to salt and drought stresses. MnEIL3 overexpression in Arabidopsis significantly upregulated the transcript abundances of ethylene biosynthetic genes. Furthermore, MnEIL3 enhanced the activities of the MnACO1 and MnACS1 promoters, which respond to salt and drought stresses. Thus, MnEIL3 may play important roles in tolerance to abiotic stresses and the expression of ethylene biosynthetic genes.

Characterization of Stilbene Synthase Genes in Mulberry (Morus atropurpurea) and Metabolic Engineering for the Production of Resveratrol in Escherichia coli.

Stilbenes have been recognized for their beneficial physiological effects on human health. Stilbene synthase (STS) is the key enzyme of resveratrol biosynthesis and has been studied in numerous plants. Here, four MaSTS genes were isolated and identified in mulberry (Morus atropurpurea Roxb.). The expression levels of MaSTS genes and the accumulation of trans-resveratrol, trans-oxyresveratrol, and trans-mulberroside A were investigated in different plant organs. A novel coexpression system that harbored 4-coumarate:CoA ligase gene (Ma4CL) and MaSTS was established. Stress tests suggested that MaSTS genes participate in responses to salicylic acid, abscisic acid, wounding, and NaCl stresses. Additionally, overexpressed MaSTS in transgenic tobacco elevated the trans-resveratrol level and increased tolerance to drought and salinity stresses. These results revealed the major MaSTS gene, and we evaluated its function in mulberry, laying the foundation for future research on stilbene metabolic pathways in mulberry.

Isolation and characterization of a novel chalcone synthase gene family from mulberry.

Chalcone synthase (CHS) is the pivotal enzyme that catalyzes the first committed step of the phenylpropanoid pathway leading to flavonoids. Here, five CHS genes were determined in mulberry (Morus atropurpurea Roxb.). Interestingly, phylogenetic analysis tended to group three MaCHSs in the stilbene synthase (STS) family and initially annotated these as MaSTSs. A co-expression system that harbored a 4-coumarate:CoA ligase gene and one of the candidate genes was established to determine the functions of this novel gene family. The fermentation result demonstrated that MaSTS in fact encoded a CHS enzyme, and was consequently retermed MaCHS. Tissue-specific expression analysis indicated that MaCHS1/MaCHS2 was highly abundant in fruit, and MaCHS4 had significant expression in root bark, stem bark and old leaves, while MaCHS3 and MaCHS5 were more expressed in old leaves. Subcellular localization experiments showed that MaCHS was localized to the cytoplasm. Transcription levels suggested MaCHS genes were involved in a series of defense responses. Over-expression of MaCHS in transgenic tobacco modified the metabolite profile, and resulted in elevated tolerance to a series of environmental stresses. This study comprehensively evaluated the function of MaCHS genes and laid the foundation for future research on MaCHS in mulberry.

Correction: Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mulberry.

[This corrects the article DOI: 10.1371/journal.pone.0155814.].

Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mul-berry.

A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol.

Screening, cloning and expression analysis of a cellulase derived from the causative agent of hypertrophy sorosis scleroteniosis, Ciboria shiraiana.

A cellulase gene (KJ700939, CsCelA) from Ciboria shiraiana that is highly expressed during the infection of mulberry fruit was screened by quantitative real-time PCR (qRT-PCR). Using cDNA isolated from infected mulberry fruits as template, the full-length 1170-bp sequence of CsCelA was obtained, which encodes a 390-amino acid protein with a putative signal peptide of 24 amino acids. The 998-bp fragment encoding the mature peptide of CsCelA was cloned into the multiple cloning site of the pPIC9K vector and overexpressed as an active protein of 55.3kDa in the methylotrophic yeast Pichia pastoris. The specific activity of induced supernatants of the recombinant cellulase (CsCelA) was 17.44U/ml and 135U/g for freeze-dried powder. The Kmax and Vmax of CsCelA for sodium carboxymethylcellulose (CMC) were 4.6mg/ml and 107.2U/mg, respectively. The supernatant and freeze-dried powder of the recombinant cellulase exhibited stable activity from pH4.0 to 9.0, and at temperatures ranging from 30°C to 55°C. Finally, the activity of the recombinant cellulase was assessed by enzymatic hydrolysis of the cell walls of mulberry leaves. CsCelA showed an endo-cellulase mode of cleavage, as assessed by thin layer chromatography (TLC).

Characterization and expression of genes involved in the ethylene biosynthesis and signal transduction during ripening of mulberry fruit.

Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1-methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening.

Characterization and expression profiles of MaACS and MaACO genes from mulberry (Morus alba L.).

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) are encoded by multigene families and are involved in fruit ripening by catalyzing the production of ethylene throughout the development of fruit. However, there are no reports on ACS or ACO genes in mulberry, partly because of the limited molecular research background. In this study, we have obtained five ACS gene sequences and two ACO gene sequences from Morus Genome Database. Sequence alignment and phylogenetic analysis of MaACO1 and MaACO2 showed that their amino acids are conserved compared with ACO proteins from other species. MaACS1 and MaACS2 are type I, MaACS3 and MaACS4 are type II, and MaACS5 is type III, with different C-terminal sequences. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) expression analysis showed that the transcripts of MaACS genes were strongly expressed in fruit, and more weakly in other tissues. The expression of MaACO1 and MaACO2 showed different patterns in various mulberry tissues. MaACS and MaACO genes demonstrated two patterns throughout the development of mulberry fruit, and both of them were strongly up-regulated by abscisic acid (ABA) and ethephon.

Cloning, overexpression, and functional characterization of a phytase from the genus bacillus.

Bacillus strain WYCQ02 was obtained from soil samples by high-temperature screening. A 1,327-bp DNA fragment containing a 1,152-bp long open reading frame named phyC-WYCQ02 was amplified from the genomic DNA of strain WYCQ02 by PCR. The ORF encoded a polypeptide of 383 amino acid residues with a putative signal peptide of 26 amino acids. The 1,089-bp fragment encoding the mature peptide of neutral phytase and a 6 × histidine tag was cloned into the plasmid pPIC9K. The expression vector, pPIC9K-phyC, was linearized and transformed in Pichia pastoris. The molecular weight of phytase was estimated to be approximately 53 kDa by electrophoresis. The optimal temperature of the purified phytase was 55°C and the optimal pH value was between 7.0 and 8.0. After incubation at 70°C for 10 and 30 min, the relative activity was still over 80 and 62%, and over 70% of enzyme activity remained in the pH range of 5.0-10.0. There was no significant difference in enzymatic activity after incubation for 30 or 60 min in buffer with different pH values, therefore the purified phytase had some acid and alkali resistance. The phytase gene and the engineered yeast strain may have value in industrial applications.

Function analysis of anthocyanidin synthase from Morus alba L. by expression in bacteria and tobacco

Background: Flavonoids are a kind of important secondary metabolite and are commonly considered to provide protection to plants against stress and UV-B for a long time. Anthocyanidin synthase (ANS), which encodes a dioxygenase in the flavonoid pathway, catalyzes the conversion of leucoanthocyanidins to anthocyanidins, but there is no direct evidence indicating that it provides tolerance to stress in plants. Results: To investigate whether ANS can increase tolerance to abiotic stress, MaANS was isolated from mulberry fruits and transformed into tobacco. Our results suggested that the bacterially expressed MaANS protein can convert dihydroquercetin to quercetin. Overexpression of MaANS remarkably increased the accumulation of total flavonoids in transgenic lines and anthocyanins in corollas of flowers. Transgenic lines showed higher tolerance to NaCl and mannitol stress. Conclusions: These results indicated that MaANS participates in various dioxygenase activities, and it can protect plants against abiotic stress by improving the ROS-scavenging ability. Thus, this alternative approach in crop breeding can be considered in the improvement of stress tolerance by enriching flavonoid production in plants

Draft genome sequence of the mulberry tree Morus notabilis.

Human utilization of the mulberry-silkworm interaction started at least 5,000 years ago and greatly influenced world history through the Silk Road. Complementing the silkworm genome sequence, here we describe the genome of a mulberry species Morus notabilis. In the 330-Mb genome assembly, we identify 128 Mb of repetitive sequences and 29,338 genes, 60.8% of which are supported by transcriptome sequencing. Mulberry gene sequences appear to evolve ~3 times faster than other Rosales, perhaps facilitating the species' spread worldwide. The mulberry tree is among a few eudicots but several Rosales that have not preserved genome duplications in more than 100 million years; however, a neopolyploid series found in the mulberry tree and several others suggest that new duplications may confer benefits. Five predicted mulberry miRNAs are found in the haemolymph and silk glands of the silkworm, suggesting interactions at molecular levels in the plant-herbivore relationship. The identification and analyses of mulberry genes involved in diversifying selection, resistance and protease inhibitor expressed in the laticifers will accelerate the improvement of mulberry plants.

A draft sequence for the genome of the domesticated silkworm (Bombyx mori).

We report a draft sequence for the genome of the domesticated silkworm (Bombyx mori), covering 90.9% of all known silkworm genes. Our estimated gene count is 18,510, which exceeds the 13,379 genes reported for Drosophila melanogaster. Comparative analyses to fruitfly, mosquito, spider, and butterfly reveal both similarities and differences in gene content.