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The GRAS protein family encodes a family of plant-specific transcription factors that have diverse and significant functions in regulating plant development and anthocyanin biosynthesis. Our study ...

As one of the major groups of small post-translationally modified peptides, the CLV3/EMBRYO SURROUNDING REGION-RELATED (CLE)-like (CLEL) peptide family has been reported to regulate root growth, lateral root development and plant gravitropic responses in Arabidopsis thaliana. In this study, we identified 12 CLEL genes in Populus trichocarpa and performed a comprehensive bioinformatics analysis on these genes. Among them, five P. trichocarpa CLELs (PtrCLELs) were revised with new gene models. All of these PtrCLEL proteins were structurally similar to the A. thaliana CLELs (AtCLELs), including an N-terminal signal peptide, a conserved C-terminal 13-amino-acid CLEL motif and a variable intermediate region. In silico and quantitative real-time PCR analyses showed that PtrCLELs were widely expressed in various tissues, including roots, leaves, buds and stems. Exogenous application of chemically synthesized PtrCLEL peptides resulted in wavy or curly roots and reduced lateral root formation in A. thaliana. Moreover, germinating Populus deltoides seedlings on a growth medium containing these peptides caused the roots to thicken and to form abnormal lateral roots, in many cases in clusters. Anatomical and histological changes in thickened roots were further investigated by treating Populus 717 cuttings with the PtrCLEL10 peptide. We observed that root thickening was mainly due to an increased number of cells in the epidermis, hypodermis and cortex. The results of our study suggested that PtrCLEL and AtCLEL genes encode proteins with similar protein structures, sequences of peptide motif and peptide activities on developing roots. The activities of PtrCLEL peptides in root development were species-dependent.

Pear is a major fruit tree crop distributed worldwide, yet its breeding is a very time-consuming process. To facilitate molecular breeding and gene identification, here we have performed genome-wide association studies (GWAS) on eleven fruit traits. We identify 37 loci associated with eight fruit quality traits and five loci associated with three fruit phenological traits. Scans for selective sweeps indicate that traits including fruit stone cell content, organic acid and sugar contents might have been under continuous selection during breeding improvement. One candidate gene, PbrSTONE, identified in GWAS, has been functionally verified to be involved in the regulation of stone cell formation, one of the most important fruit quality traits in pear. Our study provides insights into the complex fruit related biology and identifies genes controlling important traits in pear through GWAS, which extends the genetic resources and basis for facilitating molecular breeding in perennial trees., Studies of fruit quality traits in pears are lagging behind the other major fruit trees. Here, the authors conduct GWAS of fruit quality and phenological traits in a panel of 312 sand pear accessions using SNPs called from resequencing data, and reveal the involvement of a lignin formation-related protein in regulating stone cell development.

BackgroundThe NBS disease-related gene family coordinates the inherent immune system in plants in response to pathogen infections. Previous studies have identified NBS-encoding genes inPyrus bretschneideri(‘Dangshansuli’, an Asian pear) andPyrus communis(‘Bartlett’, a European pear) genomes, but the patterns of genetic variation and selection pressure on these genes during pear domestication have remained unsolved.ResultsIn this study, 338 and 412 NBS-encoding genes were identified from Asian and European pear genomes. This difference between the two pear species was the result of proximal duplications. About 15.79% orthologous gene pairs had Ka/Ks ratio more than one, indicating two pear species undergo strong positive selection after the divergence of Asian and European pear. We identified 21 and 15 NBS-encoding genes under fire blight and black spot disease-related QTL, respectively, suggesting their importance in disease resistance. Domestication caused decreased nucleotide diversity across NBS genes in Asian cultivars (cultivated 6.23E-03; wild 6.47E-03), but opposite trend (cultivated 6.48E-03; wild 5.91E-03) appeared in European pears. Many NBS-encoding coding regions showed Ka/Ks ratio of greater than 1, indicating the role of positive selection in shaping diversity of NBS-encoding genes in pear. Furthermore, we detected 295 and 122 significantly different SNPs between wild and domesticated accessions in Asian and European pear populations. Two NBS genes (Pbr025269.1andPbr019876.1) with significantly different SNPs showed >5x upregulation between wild and cultivated pear accessions, and > 2x upregulation inPyrus calleryanaafter inoculation withAlternaria alternata. We propose that positively selected and significantly different SNPs of an NBS-encoding gene (Pbr025269.1) regulate gene expression differences in the wild and cultivated groups, which may affect resistance in pear againstA. alternata.ConclusionProximal duplication mainly led to the different number of NBS-encoding genes inP. bretschneideriandP. communisgenomes. The patterns of genetic diversity and positive selection pressure differed between Asian and European pear populations, most likely due to their independent domestication events. This analysis helps us understand the evolution, diversity, and selection pressure in the NBS-encoding gene family in Asian and European populations, and provides opportunities to study mechanisms of disease resistance in pear.

Background Structural variations (SVs) have been reported to play an important role in genetic diversity and trait regulation. Many computer algorithms detecting SVs have recently been developed, but the use of multiple algorithms to detect high-confidence SVs has not been studied. The most suitable sequencing depth for detecting SVs in pear is also not known. Results In this study, a pipeline to detect SVs using next-generation and long-read sequencing data was constructed. The performances of seven types of SV detection software using next-generation sequencing (NGS) data and two types of software using long-read sequencing data (SVIM and Sniffles), which are based on different algorithms, were compared. Of the nine software packages evaluated, SVIM identified the most SVs, and Sniffles detected SVs with the highest accuracy (> 90%). When the results from multiple SV detection tools were combined, the SVs identified by both MetaSV and IMR/DENOM, which use NGS data, were more accurate than those identified by both SVIM and Sniffles, with mean accuracies of 98.7 and 96.5%, respectively. The software packages using long-read sequencing data required fewer CPU cores and less memory and ran faster than those using NGS data. In addition, according to the performances of assembly-based algorithms using NGS data, we found that a sequencing depth of 50× is appropriate for detecting SVs in the pear genome. Conclusion This study provides strong evidence that more than one SV detection software package, each based on a different algorithm, should be used to detect SVs with higher confidence, and that long-read sequencing data are better than NGS data for SV detection. The SV detection pipeline that we have established will facilitate the study of diversity in other crops.

Seedless fruits are highly marketable because they are easier to eat than fruits with seeds. ‘Shijiwuhe’ is a seedless pear cultivar that is a mutant derived from an F1 hybridization population (‘Bartlett’ x ‘Yali’). Little is known about the key genes controlling seedless pear fruit. In this study, field experiments revealed that seedless ‘Shijiwuhe’ pear was not due to parthenocarpy, and that it was self-incompatible. Single nucleotide polymorphisms (SNPs), small insertions and deletions (InDels) and structural variations (SVs) were characterized using DNA sequencing data between ‘Shijiwuhe’ and parental cultivars. A total of 1498 genes were found to be affected by SV and over 50% of SVs were located in promoter regions. Transcriptome analysis was conducted at three time points (4, 8, and 12 days after cross-pollination) during early fruit development of ‘Shijiwuhe’, ‘Bartlett’, and ‘Yali’. In total, 1438 differentially expressed genes (DEGs) were found between ‘Shijiwuhe’ and parental cultivars ‘Bartlett’ and ‘Yali’. We found 1193 SVs that caused differential expression of genes at 4 DACP. Among them, over 100 genes were in pathways related to seed nutrition and energy storage and 41 candidate genes encoded several important transcription factors, such as MYB, WRKY, NAC, and bHLH, which might play important roles in seed development. The qRT-PCR results also confirmed that the candidate genes with SVs showed differential expression between ‘Shijiwuhe’ pear and ‘Bartlett’ or ‘Yali’. This study, which combined field experiments, SV detection, and transcriptome analysis might provide an effective way to predict the candidate genes regulating the seedless trait and important gene resources for genetic improvement of pear.

We have previously reported the successful adaptation of human hookworm Necator americanus in the golden hamster, Mesocricetus auratus. This animal model was used to test a battery of hookworm (N. americanus and Ancylostoma caninum) recombinant antigens as potential vaccine antigens. Hamsters immunized a leading vaccine candidate N. americanus-Ancylostoma secreted protein 2 (Na-ASP-2) and challenged with N. americanus infective larvae (L3), resulted in 30-46.2% worm reduction over the course of three vaccine trials, relative to adjuvant controls. In addition, significant reduction of worm burdens was also observed in the hamsters immunized with adult hookworm antigens A. caninum aspartic protease 1 (Ac-APR-1); A. caninum-glutathione-S transferase 1 (Ac-GST-1) and Necator cysteine proteases 2 (Na-CP-2) (44.4%, 50.6%, and 29.3%, respectively). Our data on the worm burden reductions afforded by these hookworm antigens approximate the level of protection reported previously from dogs challenged with A. caninum L3, and provide additional evidence to support these hookworm antigens as vaccine candidates for human hookworm infection. The hamster model of N. americanus provides useful information for the selection of antigens to be tested in downstream vaccine development.

Ac-MTP-2 is an astacin-like metalloprotease secreted by adult Ancylostoma caninum hookworms. Ac-mtp-2 cDNA was cloned by immunoscreening a cDNA library with antisera prepared against adult A. caninum excretory/secretory (ES) products. The full-length Ac-mtp-2 contains 850 bp cDNA encoding a 233 amino acid open reading frame (ORF) with 32% amino acid identity to Ce-NSP-4, a pharyngeal cell-derived secreted metalloprotease of the nematode Caenorhabditis elegans. The predicted ORF contained a conserved Met-turn sequence (SXMHY), but only a partial zinc-binding signature sequence (GXXXEHXRXER instead of HEXXHXXGXXHEXXRXDR) found in other astacins. However, by both gelatin gel electrophoresis and azocasein digestion, the recombinant Ac-MTP-2 exhibited proteolytic activity that was inhibited by the zinc chelator 1,10-phenanthroline and Ac-TMP, a putative tissue inhibitor of metalloprotease that was previously shown to be a highly abundant component of adult A. caninum ES products. By RT-PCR, Western blot Ac-MTP-2 was found only expressed in adult hookworms and secreted in the adult ES products. Immunolocalization with antisera shows that Ac-MTP-2 is located to the esophageal glands (confirming its role as a secretory protein), as well as to the parasite uterus. It is hypothesized that Ac-MTP-2 functions in the extracorporeal digestion of the intestinal mucosal plug lodged in the buccal capsule of the adult parasite.

cDNAs encoding 2 Ancylostoma-secreted proteins (ASPs), Ancylostoma ceylanicum (Ay)-ASP-1 and Ay-ASP-2, were cloned from infective third-stage larvae (L3) of the hookworm A. ceylanicum and were expressed as soluble recombinant fusion proteins secreted by the yeast Pichia pastoris. The recombinant fusion proteins were purified, adjuvant formulated, and injected intramuscularly into hamsters. Hamsters vaccinated either by oral vaccination with irradiated L3 (irL3) or by injections of the adjuvants alone served as positive and negative controls, respectively. Anti-ASP-1 and anti-ASP-2 antibody titers exceeded 1 : 100000. Each vaccinated hamster was challenged orally with 100 L3. Two groups of vaccinated hamsters (i.e., those vaccinated with either irL3 or ASP-2 formulated with Quil A) exhibited significant reductions in adult hookworm burdens, compared with control hamsters. The hookworms recovered from the hamsters vaccinated with ASP-2 plus Quil A were reduced in length. Splenomegaly, which was observed in control hamsters, was not seen in hamsters vaccinated with either irL3 or ASP-2 formulated with Quil A. These results indicate that ASP-2 is a promising molecule for the development of a hookworm vaccine.

We report the cloning and expression of Ac -GST-1, a novel glutathione S -transferase from the adult hookworm Ancylostoma caninum , and its possible role in parasite blood feeding and as a vaccine target. The predicted Ac -GST-1 open reading frame contains 207 amino acids (mass, 24 kDa) and exhibited up to 65% amino acid identity with other nematode GSTs. mRNA encoding Ac -GST-1 was detected in adults, eggs, and larval stages, but the protein was detected only in adult hookworm somatic extracts and excretory/secretory products. Using antiserum to the recombinant protein, Ac -GST-1 was immunolocalized to the parasite hypodermis and muscle tissue and weakly to the intestine. Recombinant Ac -GST-1 was enzymatically active, as determined by conjugation of glutathione to a model substrate, and exhibited a novel high-affinity binding site for hematin. The possible role of Ac -GST-1 in parasite heme detoxification during hemoglobin digestion or heme uptake prompted interest in evaluating it as a potential vaccine antigen. Vaccination of dogs with Ac -GST-1 resulted in a 39.4% reduction in the mean worm burden and 32.3% reduction in egg counts compared to control dogs following larval challenge, although the reductions were not statistically significant. However, hamsters vaccinated with Ac -GST-1 exhibited statistically significant worm reduction (53.7%) following challenge with heterologous Necator americanus larvae. These studies suggest that Ac -GST-1 is a possible drug and vaccine target for hookworm infection.

Syrian Golden hamsters were vaccinated with the recombinant fusion proteins Ay-ASP-2 and Ay-MTP-1 from the infective larvae of the hookworm Ancylostoma ceylanicum. Vaccines comprised each antigen alone or the combination of the two proteins. All vaccinated group developed high antibody titers (>1:40,000); coadministration of a second antigen did not significantly affect the magnitude of the antibody response. Following challenge, hamsters vaccinated with each single antigen exhibited reductions in worm burden (32% and 28% to Ay-ASP-2 and Ay-MTP-1, respectively) and fecal egg counts (56% and 43%, respectively). A vaccine cocktail, containing both antigens further reduced worm burden (36%) and fecal egg counts (59%) (p

The Ancylostoma-secreted proteins are a family of nematode-specific cysteine-rich secreted proteins belonging to the pathogenesis-related protein superfamily. Previously we reported that third stage infective larvae of Ancylostoma caninum produce two different Ancylostoma-secreted proteins, a single and double-domain Ancylostoma-secreted protein, designated as Ancylostoma-secreted protein-1 and Ancylostoma-secreted protein-2, respectively. Here we report that adult A. caninum hookworms produce and release four additional Ancylostoma-secreted proteins (Ancylostoma-secreted protein-3-6). Using antiserum against adult excretory/secretory products, Ancylostoma-secreted protein cDNAs were isolated from cDNA expression libraries. Immunolocalisation experiments using specific antisera indicated that the single-domain Ac-Ancylostoma-secreted protein-3 is located in the adult pharyngeal and oesophageal glands. Ac-Ancylostoma-secreted protein-4, Ancylostoma-secreted protein-5 and Ancylostoma-secreted protein-6 are composed of two pathogenesis-related protein domains linked in tandem as a heterodimorphic repeat. Ac-Ancylostoma-secreted protein-4 is localised to the cuticular surface of the adult hookworm, whereas Ac-Ancylostoma-secreted protein-5 was found in the intestinal brush border membrane, and Ancylostoma-secreted protein-6 in the cephalic and excretory glands. All of the adult Ancylostoma-secreted proteins were identified in excretory/secretory products of adult hookworms by Western blotting and are presumably released by the parasite. None of the adult Ancylostoma-secreted proteins were detected by immunoblotting in L3 extracts, although mRNAs of Ac-Ancylostoma-secreted protein-3 and Ac-Ancylostoma-secreted protein-4 were present in the larval stage. The functions of the adult Ancylostoma-secreted proteins are unknown, although the secretion of multiple family members by the adult suggests an important role in the establishment or maintenance of the parasitic relationship.

Antibody against adult Ancylostoma caninum excretory-secretory (ES) products was used to immunoscreen a cDNA expression library leading to the isolation of cDNAs encoding putative hookworm fatty-acid and retinol-binding proteins. Ac-far-1 and Ac-far-2 cDNAs encode open reading frames corresponding to approximately 20kDa proteins with 91 percent amino acid identity. Ac-FAR-1 and Ac-FAR-2 exhibit clear similarities to other FARs of parasitic nematodes, most closely to two of the FAR proteins of Caenorhabditis elegans (Ce-FAR-1 and Ce-FAR-2). By reverse transcriptase polymerase chain reaction (RT-PCR) assay, Ac-far-1 mRNA was detected in both adult and third-stage larvae of A. caninum. However, the respective proteins were detectable by immunoblot only in adult hookworm ES products and adult extracts. Using fluorescence-based binding assays, bacterial recombinant Ac-FAR-1 was found to bind fatty acids and retinol (Vitamin A) with dissociation constants in the micromolar region. Circular dichroism spectra indicated that Ac-FAR-1 possesses a high level of alpha-helix, similar to Ov-FAR-1 from Onchocerca volvulus. This is the first demonstration of a functional FAR secreted by adult hookworms and provides further evidence that FAR proteins secreted by parasitic nematodes are crucial to parasitism.