Pathogenesis-related protein-4 (PR-4) gene family in Qingke (Hordeum vulgare L. var. nudum): genome-wide identification, structural analysis and expression profile under stresses.

BACKGROUND: Pathogenesis-related (PR) proteins are active participants of plant defense against biotic and abiotic stresses. The PR-4 family features a Barwin domain at the C-terminus, which endows the host plant with disease resistance. However, comprehensive analysis of PR-4 genes is still lacking in Qingke (Hordeum vulgare L. var. nudum). METHODS AND RESULTS: Herein, a total of four PR-4 genes were identified from the genome of Qingke through HMM profiling. Devoid of the chitin-binding domain, these 4 proteins were grouped as class II PR-4s. Phylogenic analysis revealed that 127 PR-4s from 47 species were clustered into 3 major groups, among which the four Qingke PR-4s were claded into group I. Analysis of gene structure demonstrated that no intron was found in 3 out of the 4 Qingke PR-4s, and HOVUSG0928500 was the only gene contained one intron. An array of cis-acting motifs were detected in promoters of Qingke PR-4 genes, including elements associated with hormone response, light response, stress response, growth and development processes and binding sites of transcription factors, implying their diverse role. Expression profiling confirmed that Qingke PR-4s were involved in defense response against drought, cold and powdery mildews infection, and transcription of HOVUSG1974300 and HOVUSG5705400 was differentially regulated by MeJA and SA. CONCLUSION: Findings of the study provided insights into the genetic basis of the PR-4 family genes, and would promote further investigation on protein function and utilization.

The complete plastome of Sorbaria kirilowii: genome structure, comparative analysis, and phylogenetic implications.

Sorbaria kirilowii is a deciduous perennial admired for its showy white blossoms. Though of importance for horticultural purposes, the plastomic study concerning this species is still lacking. Here, the plastome of S. kirilowii was de novo assembled using the high-throughput sequencing data. The complete plastome assembly of S. kirilowii was 160,810 bp in length, with a GC content of 36.03%. It featured a typical quadripartite structure, containing a pair of inverted repeats (IRs; 26,338 bp) separated by a large single-copy (LSC; 88,762 bp) and a small single-copy (SSC, 19,372 bp). In total, 132 genes were annotated in the plastome, including 87 protein-coding genes, 8 rRNA genes, and 37 tRNA genes. Furthermore, 63 SSRs, most of which were AT-rich, were identified in the cp genome of S. kirilowii. 71.7% of the cpSSRs were shown to be located in the intergenic regions. In addition, 49 repeats of varying sizes and types were also identified in the plastome. Through comparison, eight divergence hotspots were identified between the plastome of S. kirilowii and S. sorbifolia var. stellipila. These variable regions could potentially be developed into molecular markers for species delimitation or phylogenetics in future studies. We re-investigated the relationship among 17 Rosaceae species using the plastomic sequences, and S. kirilowii was shown to be a sister to S. sorbifolia var. stellipila. Overall, this study provides plastomic resources which could facilitate marker development and phylogenomics of Rosaceae.

Shikonin inhibits cancer cell cycling by targeting Cdc25s.

BACKGROUND: Shikonin, a natural naphthoquinone, is abundant in Chinese herb medicine Zicao (purple gromwell) and has a wide range of biological activities, especially for cancer. Shikonin and its analogues have been reported to induce cell-cycle arrest, but target information is still unclear. We hypothesized that shikonin, with a structure similar to that of quinone-type compounds, which are inhibitors of cell division cycle 25 (Cdc25) phosphatases, will have similar effects on Cdc25s. To test this hypothesis, the effects of shikonin on Cdc25s and cell-cycle progression were determined in this paper. METHODS: The in vitro effects of shikonin and its analogues on Cdc25s were detected by fluorometric assay kit. The binding mode between shikonin and Cdc25B was modelled by molecular docking. The dephosphorylating level of cyclin-dependent kinase 1 (CDK1), a natural substrate of Cdc25B, was tested by Western blotting. The effect of shikonin on cell cycle progression was investigated by flow cytometry analysis. We also tested the anti-proliferation activity of shikonin on cancer cell lines by MTT assay. Moreover, in vivo anti-proliferation activity was tested in a mouse xenograft tumour model. RESULTS: Shikonin and its analogues inhibited recombinant human Cdc25 A, B, and C phosphatase with IC50 values ranging from 2.14 ± 0.21 to 13.45 ± 1.45 µM irreversibly. The molecular modelling results showed that shikonin bound to the inhibitor binding pocket of Cdc25B with a favourable binding mode through hydrophobic interactions and hydrogen bonds. In addition, an accumulation of the tyrosine 15-phosphorylated form of CDK1 was induced by shikonin in a concentration-dependent manner in vitro and in vivo. We also confirmed that shikonin showed an anti-proliferation effect on three cancer cell lines with IC50 values ranging from 6.15 ± 0.46 to 9.56 ± 1.03 µM. Furthermore, shikonin showed a promising anti-proliferation effect on a K562 mouse xenograph tumour model. CONCLUSION: In this study, we provide evidence for how shikonin induces cell cycle arrest and functions as a Cdc25s inhibitor. It shows an anti-proliferation effect both in vitro and in vivo by mediating Cdc25s.

Illumina-based analysis of the rhizosphere microbial communities associated with healthy and wilted Lanzhou lily (Lilium davidii var. unicolor) plants grown in the field.

Lanzhou lily (Liliumdavidii var. unicolor) is the best edible lily as well as a traditional medicinal plant in China. The microbes associated with plant roots play crucial roles in plant growth and health. However, little is known about the differences of rhizosphere microbes between healthy and wilted Lanzhou lily (Lilium davidii var. unicolor) plants. The objective of this study was to compare the rhizosphere microbial community and functional diversity of healthy and wilted plants, and to identify potential biocontrol agents with significant effect. Paired end Illumina Mi-Seq sequencing of 16S rRNA and ITS gene amplicons was employed to study the bacterial and fungal communities in the rhizosphere soil of Lanzhou lily plants. BIOLOG technology was adopted to investigate the microbial functional diversity. Our results indicated that there were major differences in the rhizosphere microbial composition and functional diversity of wilted samples compared with healthy samples. Healthy Lanzhou lily plants exhibited lower rhizosphere-associated bacterial diversity than diseased plants, whereas fungi exhibited the opposite trend. The dominant phyla in both the healthy and wilted samples were Proteobacteria and Ascomycota, i.e., 34.45 and 64.01 %, respectively. The microbial functional diversity was suppressed in wilted soil samples. Besides Fusarium, the higher relative abundances of Rhizoctonia, Verticillium, Penicillium, and Ilyonectria (Neonectria) in the wilted samples suggest they may pathogenetic root rot fungi. The high relative abundances of Bacillus in Firmicutes in healthy samples may have significant roles as biological control agents against soilborne pathogens. This is the first study to find evidence of major differences between the microbial communities in the rhizospheric soil of healthy and wilted Lanzhou lily, which may be linked to the health status of plants.

In vitro study of the growth, development and pathogenicity responses of Fusarium oxysporum to phthalic acid, an autotoxin from Lanzhou lily.

Continuous monoculture of Lanzhou lily (Lilium davidii var. unicolor Cotton) results in frequent incidence of fusarium wilt caused by Fusarium oxysporum. Phthalic acid (PA), a principal autotoxin from root exudates of Lanzhou lily, is involved in soil sickness by inducing autotoxicity. The aim of this study was to evaluate the direct allelopathic effects of PA on the growth, development and pathogenicity of F. oxysporum in vitro based on an ecologically relevant soil concentration. The results showed that PA slightly but not significantly inhibited the colony growth (mycelial growth) and fungal biomass of F. oxysporum at low concentrations ranging from 0.05 to 0.5 mM, and significantly inhibited the colony growth at the highest concentration (1 mM). None of the PA concentrations tested significantly inhibited the conidial germination and sporulation of F. oxysporum in liquid medium. However, mycotoxin (fusaric acid) yield and pathogenesis-related hydrolytic enzyme (protease, pectinase, cellulase, and amylase) activities were significantly stimulated in liquid cultures of F. oxysporum containing PA at ≥ 0.25 mM. We conclude that PA at a soil level (i.e. 0.25 mM) is involved in plant-pathogen allelopathy as a stimulator of mycotoxin production and hydrolytic enzyme activities in F. oxysporum, which is possibly one of the mechanisms responsible for promoting the wilt disease of lily.

Genome-wide analysis of pathogenesis-related protein-1 (PR-1) genes from Qingke (Hordeum vulgare L. var. nudum) reveals their roles in stress responses.

Proteins that are pathogenesis-related 1 (PR-1) can accumulate to high levels when plants employ defenses, being major participants in processes critical for stress responses as well as development of many species. Yet we still lack information concerning PR-1 family members in Qingke plants (Hordeum vulgare L. var. nudum). In this work, we distinguished 20 PR-1s from the Qingke genome whose encoded proteins often featured at the N-terminus a signal peptide; all 20 PR-1s were predicted to localize either periplasmically or extracellularly. The CAP domain was confirmed as being highly conserved in all these PR-1s. Phylogeny-based inference revealed that PR-1 proteins clustered into four major clades, with the majority of Qingke PR-1s distributed in clade I (17 out 20), and the other 3 distributed in clade II. Gene structure analysis showed that 16 PR-1s did not contain any introns, whereas the other four had 1-4 introns. We identified a variety of motifs that are cis-acting in the promoter regions of PR-1s; these included those potentially involved in Qingke's light response, hormonal and stress responses, circadian control and regulation of development and growth, in addition to sites where transcription factors bind to. Expression analysis uncovered several members of PR-1 genes that were strongly and rapidly induced by powdery mildew infection, phytohormones, and cold stimulus. Altogether, our study's findings enhance what is known about genetic features of PR-1 family members in H. vulgare plants, especially Qingke, and could thereby facilitate further exploration aiming to elucidate the functioning of these proteins.

Whole-genome sequencing and evolutionary analysis of the wild edible mushroom, Morchella eohespera.

Morels (Morchella, Ascomycota) are an extremely desired group of edible mushrooms with worldwide distribution. Morchella eohespera is a typical black morel species, belonging to the Elata clade of Morchella species. The biological and genetic studies of this mushroom are rare, largely hindering the studies of molecular breeding and evolutionary aspects. In this study, we performed de novo sequencing and assembly of the M. eohespera strain m200 genome using the third-generation nanopore sequencing platform. The whole-genome size of M. eohespera was 53.81 Mb with a contig N50 of 1.93 Mb, and the GC content was 47.70%. A total of 9,189 protein-coding genes were annotated. Molecular dating showed that M. eohespera differentiated from its relative M. conica at ~19.03 Mya (million years ago) in Burdigalian. Evolutionary analysis showed that 657 gene families were contracted and 244 gene families expanded in M. eohespera versus the related morel species. The non-coding RNA prediction results showed that there were 336 tRNAs, 76 rRNAs, and 45 snRNAs in the M. eohespera genome. Interestingly, there was a high degree of repetition (20.93%) in the M. eohespera genome, and the sizes of long interspersed nuclear elements, short interspersed nuclear elements, and long terminal repeats were 0.83 Mb, 0.009 Mb, and 4.56 Mb, respectively. Additionally, selection pressure analysis identified that a total of 492 genes in the M. eohespera genome have undergone signatures of positive selection. The results of this study provide new insights into the genome evolution of M. eohespera and lay the foundation for in-depth research into the molecular biology of the genus Morchella in the future.

Change of geographic distributions of the genus Morchella species responding to climate oscillations in East Asia.

Morchella is a rare macrofungi taxon with high medicinal and edible values. Influenced by recent climate oscillations and human activities, habitat fragmentation of this genus has been critical, leading to a rapid decline of the resource of Morchella. It is thus urgent to preserve Morchella species. Based on maximum entropy model (MaxEnt), and 102 geographic distribution records of Morchella species with 10 environmental factors, we simulated the changes of potential geographic distributions under the climatic conditions of the last glacial maximum (LGM), last interglacial (LIG), in contemporary period and future (2050, 2070). We further analyzed the potential changes of geographic distributions of Morchella species in East Asia under climate change and formulated the effective conservation strategies for Morchella. The results showed that the dominant environmental factors affecting the geographic distributions of Morchella species were mean temperature of coldest quarter, annual precipitation, elevation and temperature annual range, with the mean temperature of coldest quarter having the greatest contribution. Results of the species distribution models showed that the highly suitable regions for Morchella species were mainly distributed in parts of western China under contemporary period. From the LIG to LGM and then the current to the future period, the total suitable regions of Morchella species showed a trend of firstly decrease and then increase, while the highly suitable regions showed similar change with the total suitable regions. At present, there is an urgent need to conduct in situ conservation for the resources of Morchella species in highly suitable regions in western China, and to carry out ex situ conservation in the marginal ranges of highly suitable regions and moderately suitable regions of Shaanxi, Hebei, Shandong, and other regions in China.

DNA Barcoding and Species Classification of Morchella.

True morels (Morchella) are a well-known edible fungi, with economically and medicinally important values. However, molecular identification and species taxonomy of the genus Morchella have long been controversial, due to numerous intermediate morphologies among species. In this study, we determined the identification efficiency of DNA barcoding and species classification of 260 individuals from 45 Morchella species, on the basis of multiple nuclear DNA markers. DNA barcoding analysis showed that the individual DNA fragment has a lower resolution of species identification than that of combined multiple DNA markers. ITS showed the highest level of species discrimination among the individual genetic markers. Interestingly, the combined DNA markers significantly increased the resolution of species identification. A combination of four DNA genes (EF1-α, RPB1, RPB2 and ITS) showed a higher species delimitation than that any combination of two or three markers. Phylogenetic analysis suggested that the species in genus Morchella could have been divided into two large genetic clades, the Elata Clade and Esculenta Clade lineages. The two lineages divided approximately 133.11 Mya [95% HPD interval: 82.77-197.95] in the early Cretaceous period. However, some phylogenetic species of Morchella showed inconsistent evolutionary relationships with the traditional morphological classifications, which may have resulted from incomplete lineage sorting and/or introgressive hybridization among species. These findings demonstrate that the interspecific gene introgression may have affected the species identification of true morels, and that the combined DNA markers significantly improve the resolution of species discrimination.

Genome-wide analysis of the Thaumatin-like gene family in Qingke (Hordeum vulgare L. var. nudum) uncovers candidates involved in plant defense against biotic and abiotic stresses.

Thaumatin-like proteins (TLPs) participate in the defense responses of plants as well as their growth and development processes, including seed germination. Yet the functioning of TLP family genes, in addition to key details of their encoded protein products, has not been thoroughly investigated for Qingke (Hordeum vulgare L. var. nudum). Here, a total of 36 TLP genes were identified in the genome of Qingke via HMM profiling. Of them, 25 TLPs contained a signal peptide at the N-terminus, with most proteins predicted to localize in the cytoplasm or outer membrane. Sequence alignment and motif analysis revealed that the five REDDD residues required for ß-1,3-glucanase activity were conserved in 21 of the 36 Qingke TLPs. Phylogenetically, the TLPs in plants are clustered in 10 major groups. Our analysis of gene structure did not detect an intron in 15 Qingke TLPs whereas the other 21 did contain 1-7 introns. A diverse set of cis-acting motifs were found in the promoters of the 36 TLPs, including elements related to light, hormone, and stress responses, growth and development, circadian control, and binding sites of transcription factors, thus suggesting a multifaceted role of TLPs in Qingke. Expression analyses revealed the potential involvement of TLPs in plant defense against biotic and abiotic stresses. Taken together, the findings of this study deepen our understanding of the TLP family genes in Qingke, a staple food item in Tibet, which could strengthen future investigations of protein function in barley and its improved genetic engineering.

Maximum Entropy Modeling the Distribution Area of Morchella Dill. ex Pers. Species in China under Changing Climate.

Morchella is a kind of precious edible, medicinal fungi with a series of important effects, including anti-tumor and anti-oxidation effects. Based on the data of 18 environmental variables and the distribution sites of wild Morchella species, this study used a maximum entropy (MaxEnt) model to predict the changes in the geographic distribution of Morchella species in different historical periods (the Last Glacial Maximum (LGM), Mid Holocene (MH), current, 2050s and 2070s). The results revealed that the area under the curve (AUC) values of the receiver operating characteristic curves of different periods were all relatively high (>0.83), indicating that the results of the maximum entropy model are good. Species distribution modeling showed that the major factors influencing the geographical distribution of Morchella species were the precipitation of the driest quarter (Bio17), elevation, the mean temperature of the coldest quarter (Bio11) and the annual mean temperature (Bio1). The simulation of geographic distribution suggested that the current suitable habitat of Morchella was mainly located in Yunnan, Sichuan, Gansu, Shaanxi, Xinjiang Uygur Autonomous Region (XUAR) and other provinces in China. Compared with current times, the suitable area in Northwest and Northeast China decreased in the LGM and MH periods. As for the future periods, the suitable habitats all increased under the different scenarios compared with those in contemporary times, showing a trend of expansion to Northeast and Northwest China. These results could provide a theoretical basis for the protection, rational exploitation and utilization of wild Morchella resources under scenarios of climate change.

Characterization of the complete mitochondrial genome of Morchella eohespera.

Morchella eohespera Beug, Voitk & O'Donnell is a typical black morel species. In this study, using the Nanopore sequencing platform, we characterized its whole mitochondrial (mt) genome sequence. Mt genome of M. eohespera is composed of circular DNA molecules of 243,963 bp, which encoded 102 protein-coding genes (PCGs), two ribosomal RNA genes (rRNA), and 31 transfer RNA (tRNA) genes. The base composition of M. eohespera mitogenome is as follows: A (30.40%), T (29.30%), G (20.8%), and C (19.5%). The phylogenetic analysis suggested that M. eohespera was closely related to the congeneric M. importuna.

The complete nucleotide molecular sequence of plastid genome of Zanthoxylum armatum (Rutaceae).

Zanthoxylum armatum DC. (Rutaceae) is a shrub and/or tree species with the important medicinal and economic values. In this study, the plastid genome of Z. armatum was characterized by Illumina Hiseq 2500 sequencing platform. In total, the plastid genome is 158,557 bp in length, and comprises a large single copy region of 85,752 bp, a small single copy region of 17,605 bp, and two inverted repeat regions of 27,600 bp. The complete plastid genome contains 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis suggested that Z. armatum and the congeneric Z. simulans clustered into an evolutionary clade with the high support.

Characterization of the complete plastid genome of Acer tsinglingense, an endemic tree species in China.

Acer tsinglingense is an ecologically and economically important tree species in China. In this study, we characterized its whole plastid genome sequence using the Illumina sequencing platform. The complete plastid genome size of A. tsinglingense is 156,039 bp in length, including a large single-copy [LSC] region of 85,760 bp, a small single-copy [SSC] region of 18,139 bp, and a pair of inverted repeats [IRs] of 26,070 bp. The genome contains 137 genes, including 89 protein-coding genes, 40 tRNA genes, and 8 rRNA genes. The GC contents in chloroplast genome, LSC region, SSC region, and IR region were 38.0%, 36.2%, 32.4%, and 42.9%, respectively. The phylogenetic analysis based on the plastid genomes showed that A. tsinglingense was more closely related with the congeneric A. laevigatum, A. palmatum, A. wilsonii, and A. buergerianum, these species were clustered into a monophyletic clade with high bootstrap support.

The new flagella-associated collagen-like proteins ClpB and ClpC of Bacillus amyloliquefaciens FZB42 are involved in bacterial motility.

Collagen-like proteins (CLPs) share the distinctive Gly-X-Thr repeating amino acid sequence of animal collagens, and contain N- and C-terminal domain making a collagen-like structure in Bacillus amyloliquefaciens FZB42, a plant growth-promoting rhizobacterium. Our previous study demonstrated that CLPs play important roles in biofilm construction and adherence to the surfaces on plant roots. However, bacterial localization of the CLPs remains unclear. Here, disrupted strains on all four clp genes (clpA, clpB, clpC and clpD) shown fewer filament than wild-type bacteria in extracellular matrix under scanning electron microscope (SEM). Transmission electron microscopy (TEM) was used to observe the differences on filament which associated on the cell surface, then the CLPs mutation strains showed less flagella than the wild type. Immunogold labeling determined the location that ClpB and ClpC localized on the flagella surface. In addition, western blotting analysis of crude flagella extracts suggested that the ClpB and ClpC are associated to flagella as well. The mutation strains also reduced motility of swimming on the surface of soft agar medium and changed the architectural of microcolony biofilm edge. The study suggests that collagen-like protein ClpB and ClpC, as novel proteins, associated with flagella in B. amyloliquefaciens.

Comparative Digital Gene Expression Analysis of the Arabidopsis Response to Volatiles Emitted by Bacillus amyloliquefaciens.

Some plant growth-promoting rhizobacteria (PGPR) regulated plant growth and elicited plant basal immunity by volatiles. The response mechanism to the Bacillus amyloliquefaciens volatiles in plant has not been well studied. We conducted global gene expression profiling in Arabidopsis after treatment with Bacillus amyloliquefaciens FZB42 volatiles by Illumina Digital Gene Expression (DGE) profiling of different growth stages (seedling and mature) and tissues (leaves and roots). Compared with the control, 1,507 and 820 differentially expressed genes (DEGs) were identified in leaves and roots at the seedling stage, respectively, while 1,512 and 367 DEGs were identified in leaves and roots at the mature stage. Seventeen genes with different regulatory patterns were validated using quantitative RT-PCR. Numerous DEGs were enriched for plant hormones, cell wall modifications, and protection against stress situations, which suggests that volatiles have effects on plant growth and immunity. Moreover, analyzes of transcriptome difference in tissues and growth stage using DGE profiling showed that the plant response might be tissue-specific and/or growth stage-specific. Thus, genes encoding flavonoid biosynthesis were downregulated in leaves and upregulated in roots, thereby indicating tissue-specific responses to volatiles. Genes related to photosynthesis were downregulated at the seedling stage and upregulated at the mature stage, respectively, thereby suggesting growth period-specific responses. In addition, the emission of bacterial volatiles significantly induced killing of cells of other organism pathway with up-regulated genes in leaves and the other three pathways (defense response to nematode, cell morphogenesis involved in differentiation and trichoblast differentiation) with up-regulated genes were significantly enriched in roots. Interestingly, some important alterations in the expression of growth-related genes, metabolic pathways, defense response to biotic stress and hormone-related genes were firstly founded response to FZB42 volatiles.

Collagen-like proteins (ClpA, ClpB, ClpC, and ClpD) are required for biofilm formation and adhesion to plant roots by Bacillus amyloliquefaciens FZB42.

The genes of collagen-like proteins (CLPs) have been identified in a broad range of bacteria, including some human pathogens. They are important for biofilm formation and bacterial adhesion to host cells in some human pathogenic bacteria, including several Bacillus spp. strains. Interestingly, some bacterial CLP-encoding genes (clps) have also been found in non-human pathogenic strains such as B. cereus and B. amyloliquefaciens, which are types of plant-growth promoting rhizobacteria (PGPR). In this study, we investigated a putative cluster of clps in B. amyloliquefaciens strain FZB42 and a collagen-related structural motif containing glycine-X-threonine repeats was found in the genes RBAM_007740, RBAM_007750, RBAM_007760, and RBAM_007770. Interestingly, biofilm formation was disrupted when these genes were inactivated separately. Scanning electron microscopy and hydrophobicity value detection were used to assess the bacterial cell shape morphology and cell surface architecture of clps mutant cells. The results showed that the CLPs appeared to have roles in bacterial autoaggregation, as well as adherence to the surface of abiotic materials and the roots of Arabidopsis thaliana. Thus, we suggest that the CLPs located in the outer layer of the bacterial cell (including the cell wall, outer membrane, flagella, or other associated structures) play important roles in biofilm formation and bacteria-plant interactions. This is the first study to analyze the function of a collagen-like motif-containing protein in a PGPR bacterium. Knocking out each clp gene produced distinctive morphological phenotypes, which demonstrated that each product may play specific roles in biofilm formation. Our in silico analysis suggested that these four tandemly ranked genes might not belong to an operon, but further studies are required at the molecular level to test this hypothesis. These results provide insights into the functions of clps during interactions between bacteria and plants.