A novel approach for multi-domain and multi-gene family identification provides insights into evolutionary dynamics of disease resistance genes in core eudicot plants

Background

Recent advances in DNA sequencing techniques resulted in more than forty sequenced plant genomes representing a diverse set of taxa of agricultural, energy, medicinal and ecological importance. However, gene family curation is often only inferred from DNA sequence homology and lacks insights into evolutionary processes contributing to gene family dynamics. In a comparative genomics framework, we integrated multiple lines of evidence provided by gene synteny, sequence homology and protein-based Hidden Markov Modelling to extract homologous super-clusters composed of multi-domain resistance (R)-proteins of the NB-LRR type (for NUCLEOTIDE BINDING/LEUCINE-RICH REPEATS), that are involved in plant innate immunity.

Results

To assess the diversity of R-proteins within and between species, we screened twelve eudicot plant genomes including six major crops and found a total of 2,363 NB-LRR genes. Our curated R-proteins set shows a 50% average for tandem duplicates and a 22% fraction of gene copies retained from ancient polyploidy events (ohnologs). We provide evidence for strong positive selection and show significant differences in molecular evolution rates (Ka/Ks-ratio) among tandem- (mean = 1.59), ohnolog (mean = 1.36) and singleton (mean = 1.22) R-gene duplicates. To foster the process of gene-edited plant breeding, we report species-specific presence/absence of all 140 NB-LRR genes present in the model plant Arabidopsis and describe four distinct clusters of NB-LRR “gatekeeper” loci sharing syntenic orthologs across all analyzed genomes.

Conclusion

By curating a near-complete set of multi-domain R-protein clusters in an eudicot-wide scale, our analysis offers significant insight into evolutionary dynamics underlying diversification of the plant innate immune system. Furthermore, our methods provide a blueprint for future efforts to identify and more rapidly clone functional NB-LRR genes from any plant species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-966) contains supplementary material, which is available to authorized users.     

Maturation of Human Herpesvirus 6A Glycoprotein O Requires Coexpression of Glycoprotein H and Glycoprotein L

Glycoprotein O (gO) is conserved among betaherpesviruses, but little is known about the maturation process of gO in human herpesvirus 6 (HHV-6). We found that HHV-6 gO maturation was accompanied by cleavage of its carboxyl terminus and required coexpression of gH and gL, which promoted the export of gO out of the endoplasmic reticulum (ER). Finally, we also found that gO was not required for HHV-6A growth in T cells.     

The C9orf72-SMCR8-WDR41 complex is a GAP for small GTPases

ABSTRACT

Massive expansions of the hexanucleotide in C9orf72 are the primary genetic origins of familial amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). Current studies have found that this repeat sequence participates in the disease process by producing neurotoxic substances and reducing the level of C9orf72 protein; however, the progress in the functional study of C9orf72 is slow. Recently, a stable complex, consisting of C9orf72, SMCR8, and WDR41, has been implicated in regulating membrane trafficking and macroautophagy. We reported the cryo-electron microscopy (cryo-EM) structure of the C9orf72-SMCR8-WDR41 complex (CSW complex), unveiling that the CSW complex is a dimer of heterotrimers. Intriguingly, in the heterotrimer of the C9orf72-SMCR8-WDR41, C9orf72 interacts with SMCR8 in a manner similar to the FLCN-FNIP2 complex. Nevertheless, WDR41 is connected to the DENN domain of SMCR8 through its N-terminal β-strand and C-terminal helix but does not directly interact with C9orf72. Notably, the C9orf72-SMCR8 complex was demonstrated to act as a GAP for RAB8A and RAB11A in vitro.     

ApoB-54.8, a truncated apolipoprotein found primarily in VLDL, is associated with a nonsense mutation in the apoB gene and hypobetalipoproteinemia

A new, large kindred with hypobetalipoproteinemia and a previously undescribed truncated form of apolipoprotein B (apoB) has been identified. The asymptomatic, Caucasian male proband (CK, aged 37 years) has total plasma cholesterol, triglyceride, low density lipoprotein-(LDL) cholesterol, high density lipoprotein- (HDL) cholesterol, and apoB concentrations of 108, 131, 32, 50, and 16 mg/dl, respectively. Plasma samples of 11 family members spanning three generations, which had less than 5th percentile concentrations of LDL-cholesterol, contained three apoB bands detected on immunoblots: the normal apoB-100 and apoB-48 and an unusual band of apparent molecular mass of 299,356 +/- 9580 daltons (approximately 54% the molecular weight of apoB-100). Additional immunoblotting experiments using several different anti-apoB monoclonal antibodies showed that the carboxyl terminal of apoB-100 had been deleted somewhere between amino acid residues 2148-2488. A segment of genomic DNA from the proband was amplified by polymerase chain reaction (PCR) between nucleotides 7491-7791 of Exon 26 of the apoB gene. The DNA segment was cloned into pGEM3Zf(-) and sequenced. A C----T transition was found at nucleotide 7665, resulting in a premature stop codon at amino acid residue 2486 corresponding to apoB-54.8. These results were confirmed by direct sequencing of PCR products from three apoB-54.8 positive and three apoB-54.8 negative kindred members. Allele-specific oligonucleotides were used to identify other affected family members. Cosegregation of apoB-54.8 with the C----T transition occurred in all cases. Based on haplotypes constructed from restriction fragment length polymorphism, variable number of tandem repeats, and 5' insertion/deletion analyses and from the presence or absence of apoB-54.8, it was possible to assign a single allele of apoB to the mutation throughout the family. In contrast with other shorter truncations such as apoB-31, apoB-40, and apoB-46, which are found with particles in the HDL density range, and apoB-89 that is found primarily with LDL, apoB-54.8 was found primarily in very low density lipoproteins, much less in LDL, and was virtually absent in HDL. This suggests that the length of the truncation may significantly affect the metabolism of the associated lipoprotein particles.     

Efficient production of triacylglycerols rich in docosahexaenoic acid (DHA) by osmo-heterotrophic marine protists

Thraustochytrids have recently emerged as a promising source for docosahexaenoic acid (DHA) production due to their high growth rate and oil content. In this study, two thraustochytrid isolates, Aurantiochytrium sp. PKU#SW7 and Thraustochytriidae sp. PKU#Mn16 were used for DHA production. Following growth parameters were optimized to maximize DHA production: temperature, pH, salinity, and glucose concentration. Both isolates achieved the highest DHA yield at the cultivation temperature of 28 °C, pH 6, 100 % seawater, and 2 % glucose. A DHA yield of 1.395 g/l and 1.426 g/l was achieved under the optimized culture conditions. Further investigation revealed that both isolates possess simple fatty acids profiles with palmitic acid and DHA as their dominant constituents, accounting for ～79 % of total fatty acids. To date, very few studies have focused on the DHA distribution in various lipid fractions which is an important factor for identifying strains with a potential for industrial DHA production. In the present study, the lipids profiles of each strain both revealed that the majority of DHA was distributed in neutral lipids (NLs), and the DHA distribution in NLs of PKU#SW7 was exclusively in the form of triacylglycerols (TAGs) which suggest that PKU#SW7 could be utilized as an alternative source of DHA for dietary supplements. The fermentation process established for both strains also indicating that Aurantiochytrium sp. PKU#SW7 was more suitable for cultivation in fermenter. In addition, the high percentage of saturated fatty acids produced by the two thraustochytrids indicates their potential application in biodiesel production. Overall, our findings suggest that two thraustochytrid isolates are suitable candidates for biotechnological applications.     

Quantitative response of cell growth and Tuber polysaccharides biosynthesis by medicinal mushroom Chinese truffle Tuber sinense to metal ion in culture medium

During the submerged fermentation of medicinal mushroom Chinese truffle Tuber sinense, there was no significant effect of metal ion on the cell growth and the production of intracellular polysaccharides, while metal ion and its concentration significantly affected the production of extracellular polysaccharides (EPS). By using the approach of "one-variable-at-a-time", 50 mM Mg2+ was identified to be the most favorable for EPS production, and the next was 10 mM K+. A mathematical model, constructed by response surface methodology combination with full factorial design, was applied to study the synergic effect of Mg2+ and K+. EPS production reached its peak value of 5.86 g/L under their optimal combination of 30 mM Mg2+ and 5 mM K+ predicted by the model, which was higher by 130.7% compared with the basal fermentation medium without metal ion. The validation experiment showed the experimental values agreed with the predicted values well. EPS production obtained in this work was the highest reported in the culture of T. sinense.     

Protein Kinase D Promotes Airway Epithelial Barrier Dysfunction and Permeability through Down-regulation of Claudin-1

At the interface between host and external environment, the airway epithelium serves as a major protective barrier. In the present study we show that protein kinase D (PKD) plays an important role in the formation and integrity of the airway epithelial barrier. Either inhibition of PKD activity or silencing of PKD increased transepithelial electrical resistance (TEER), resulting in a tighter epithelial barrier. Among the three PKD isoforms, PKD3 knockdown was the most efficient one to increase TEER in polarized airway epithelial monolayers. In contrast, overexpression of PKD3 wild type, but not PKD3 kinase-inactive mutant, disrupted the formation of apical intercellular junctions and their reassembly, impaired the development of TEER, and increased paracellular permeability to sodium fluorescein in airway epithelial monolayers. We further found that overexpression of PKD, in particular PKD3, markedly suppressed the mRNA and protein levels of claudin-1 but had only minor effects on the expression of other tight junctional proteins (claudin-3, claudin-4, claudin-5, occludin, and ZO-1) and adherent junctional proteins (E-cadherin and β-catenin). Immunofluorescence study revealed that claudin-1 level was markedly reduced and almost disappeared from intercellular contacts in PKD3-overexpressed epithelial monolayers and that claudin-4 was also restricted from intercellular contacts and tended to accumulate in the cell cytosolic compartments. Last, we found that claudin-1 knockdown prevented TEER elevation by PKD inhibition or silencing in airway epithelial monolayers. These novel findings indicate that PKD negatively regulates human airway epithelial barrier formation and integrity through down-regulation of claudin-1, which is a key component of tight junctions.     

Functional analyses of a putative plasma membrane Na+/H+ antiporter gene isolated from salt tolerant Helianthus tuberosus

Jerusalem artichokes (Helianthus tuberosus L.) can tolerate relatively higher salinity, drought and heat stress. In this paper, we report the cloning of a Salt Overly Sensitive 1 (SOS1) gene encoding a plasma membrane Na⁺/H⁺ antiporter from a highly salt-tolerant genotype of H. tuberosus, NY1, named HtSOS1 and characterization of its function in yeast and rice. The amino acid sequence of HtSOS1 showed 83.4 % identity with the previously isolated SOS1 gene from the Chrysanthemum crassum. The mRNA level in the leaves of H. tuberosus was significantly up-regulated by presence of high concentrations of NaCl. Localization analysis using rice protoplast expression showed that the protein encoded by HtSOS1 was located in the plasma membrane. HtSOS1 partially suppressed the salt sensitive phenotypes of a salt sensitive yeast strain. In comparison with wild type (Oryza sativa L., ssp. Japonica. cv. Nipponbare), the transgenic rice expressed with HtSOS1 could exclude more Na⁺ and accumulate more K⁺. Expression of HtSOS1 decreased Na⁺ content much larger in the shoot than in the roots, resulting in more water content in the transgenic rice than WT. These data suggested that HtSOS1 may be useful in transgenic approaches to improving the salinity tolerance of glycophyte.