Reprogramming the translatome during daily light transitions as affected by cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1/C2.

Dark-light and light-dark transitions during the day are switching points of leaf metabolism that strongly affect the regulatory state of the cells, and this change is hypothesized to affect the translatome. The cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1 and GAPC2 function in glycolysis, and carbohydrate and energy metabolism, but GAPC1/C2 also shows moonlighting functions in gene expression and post-transcriptional regulation. In this study we examined the rapid reprogramming of the translatome that occurs within 10 min at the end of the night and the end of the day in wild-type (WT) Arabidopsis and a gapc1/c2 double-knockdown mutant. Metabolite profiling compared to the WT showed that gapc1/c2 knockdown led to increases in a set of metabolites at the start of day, particularly intermediates of the citric acid cycle and linked pathways. Differences in metabolite changes were also detected at the end of the day. Only small sets of transcripts changed in the total RNA pool; however, RNA-sequencing revealed major alterations in polysome-associated transcripts at the light-transition points. The most pronounced difference between the WT and gapc1/c2 was seen in the reorganization of the translatome at the start of the night. Our results are in line with the proposed hypothesis that GAPC1/C2 play a role in the control of the translatome during light/dark transitions.

Coat protein of rice stripe virus enhances autophagy activity through interaction with cytosolic glyceraldehyde-3-phosphate dehydrogenases, a negative regulator of plant autophagy.

Viral infection commonly induces autophagy, leading to antiviral responses or conversely, promoting viral infection or replication. In this study, using the experimental plant Nicotiana benthamiana, we demonstrated that the rice stripe virus (RSV) coat protein (CP) enhanced autophagic activity through interaction with cytosolic glyceraldehyde-3-phosphate dehydrogenase 2 (GAPC2), a negative regulator of plant autophagy that binds to an autophagy key factor, autophagy-related protein 3 (ATG3). Competitive pull-down and co-immunoprecipitation (Co-IP)assays showed that RSV CP activated autophagy by disrupting the interaction between GAPC2 and ATG3. An RSV CP mutant that was unable to bind GAPC2 failed to disrupt the interaction between GAPC2 and ATG3 and therefore lost its ability to induce autophagy. RSV CP enhanced the autophagic degradation of a viral movement protein (MP) encoded by a heterologous virus, citrus leaf blotch virus (CLBV). However, the autophagic degradation of RSV-encoded MP and RNA-silencing suppressor (NS3) proteins was inhibited in the presence of CP, suggesting that RSV CP can protect MP and NS3 against autophagic degradation. Moreover, in the presence of MP, RSV CP could induce the autophagic degradation of a remorin protein (NbREM1), which negatively regulates RSV infection through the inhibition of viral cell-to-cell movement. Overall, our results suggest that RSV CP induces a selective autophagy to suppress the antiviral factors while protecting RSV-encoded viral proteins against autophagic degradation through an as-yet-unknown mechanism. This study showed that RSV CP plays dual roles in the autophagy-related interaction between plants and viruses.

A Specific Protective Mechanism Against Chloroplast Photo-Reactive Oxygen Species in Phosphate-Starved Rice Plants.

Phosphorus (Pi) starvation prevents a good match between light energy absorption and photosynthetic carbon metabolism, generating photo-reactive oxygen species (photo-ROS) in chloroplasts. Plants have evolved to withstand photo-oxidative stress, but the key regulatory mechanism underlying it remains unclear. In rice (Oryza sativa), DEEP GREEN PANICLE1 (DGP1) is robustly up-regulated in response to Pi deficiency. DGP1 decreases the DNA-binding capacities of the transcriptional activators GLK1/2 on the photosynthetic genes involved in chlorophyll biosynthesis, light harvesting, and electron transport. This Pi-starvation-induced mechanism dampens both electron transport rates through photosystem I and II (ETRI and ETRII) and thus mitigates the electron-excessive stress in mesophyll cells. Meanwhile, DGP1 hijacks glycolytic enzymes GAPC1/2/3, redirecting glucose metabolism toward the pentose phosphate pathway with superfluous NADPH production. Phenotypically, light irradiation induces O2 - production in Pi-starved WT leaves but is observably accelerated in dgp1 mutant and impaired in GAPCsRNAi and glk1glk2 lines. Interestingly, overexpressed DGP1 in rice caused hyposensitivity to ROS-inducers (catechin and methyl viologen), but the dgp1 mutant shows a similar inhibitory phenotype with the WT seedlings. Overall, the DGP1 gene serves as a specific antagonizer against photo-ROS in Pi-starved rice plants, which coordinates light-absorbing and anti-oxidative systems by orchestrating transcriptional and metabolic regulations, respectively.

'Candidatus Liberibacter asiaticus' secretory protein SDE3 inhibits host autophagy to promote Huanglongbing disease in citrus.

Antimicrobial acroautophagy/autophagy plays a vital role in degrading intracellular pathogens or microbial molecules in host-microbe interactions. However, microbes evolved various mechanisms to hijack or modulate autophagy to escape elimination. Vector-transmitted phloem-limited bacteria, Candidatus Liberibacter (Ca. Liberibacter) species, cause Huanglongbing (HLB), one of the most catastrophic citrus diseases worldwide, yet contributions of autophagy to HLB disease proliferation remain poorly defined. Here, we report the identification of a virulence effector in "Ca. Liberibacter asiaticus" (Las), SDE3, which is highly conserved among the "Ca. Liberibacter". SDE3 expression not only promotes the disease development of HLB and canker in sweet orange (Citrus sinensis) plants but also facilitates Phytophthora and viral infections in Arabidopsis, and Nicotiana benthamiana (N. benthamiana). SDE3 directly associates with citrus cytosolic glyceraldehyde-3-phosphate dehydrogenases (CsGAPCs), which negatively regulates plant immunity. Overexpression of CsGAPCs and SDE3 significantly inhibits autophagy in citrus, Arabidopsis, and N. benthamiana. Intriguingly, SDE3 undermines autophagy-mediated immunity by the specific degradation of CsATG8 family proteins in a CsGAPC1-dependent manner. CsATG8 degradation is largely rescued by treatment with an inhibitor of the late autophagic pathway, E64d. Furthermore, ectopic expression of CsATG8s enhances Phytophthora resistance. Collectively, these results suggest that SDE3-CsGAPC interactions modulate CsATG8-mediated autophagy to enhance Las progression in citrus.Abbreviations: ACP: asian citrus psyllid; ACD2: ACCELERATED CELL DEATH 2; ATG: autophagy related; Ca. Liberibacter: Candidatus Liberibacter; CaMV: cauliflower mosaic virus; CMV: cucumber mosaic virus; Cs: Citrus sinensis; EV: empty vector; GAPC: cytosolic glyceraldehyde-3-phosphate dehydrogenase; HLB: huanglongbing; H2O2: hydrogen peroxide; Las: liberibacter asiaticus; Laf: liberibacter africanus; Lam: liberibacter americanus; Pst: Pseudomonas syringae pv. tomato; PVX: potato virus X; ROS: reactive oxygen species; SDE3: sec-delivered effector 3; TEM: transmission electron microscopy; VIVE : virus-induced virulence effector; WT: wild-type; Xcc: Xanthomonas citri subsp. citri.

[Prokaryotic expression of the GapC protein of Streptococcus uberis and prediction, identification of its B-cell epitopes].

The GapC protein of Streptococcus uberis located on the surface of bacteria is a protein with glyceraldehyde-3-phosphate dehydrogenase activity. It participates in cellular processes and exhibits a variety of biological activities. In addition, it has good antigenicity. The aim of this study was to predict the possible B-cell epitopes of the GapC protein and verify the immunogenicity of candidate epitope peptides. The gapC gene of S. uberis isolate RF5-1 was cloned into a recombinant expression plasmid pET-28a-GapC and inducibly expressed. The purified protein was used to immunize experimental rabbits to produce anti-GapC polyclonal antibodies. The three-dimensional structure and three-dimensional location of the GapC B-cell epitopes and the homology comparison of the GapC protein and its B-cell epitopes were carried out using bioinformatics softwares. The results showed that the 44-kDa GapC protein had a good immunological reactivity. Six linear and 3 conformational dominant B-cell epitopes against the GapC protein were selected and synthesized. Three dimensional analysis indicated that the selected peptides have better antigen epitope formation potential. Rabbit anti-GapC polyclonal antibodies were generated after immunized with the purified GapC protein, and the polyclonal antibodies were used to identify the epitope peptide by an indirect ELISA. The ELISA results showed that all of the 9 epitope peptides could react with anti-GapC polyclonal antibodies with varying titers. Among them, the epitope polypeptide 266AANDSYGYTEDPIVSSD282 reacted with the polyclonal antibodies significantly stronger than with other epitope peptides. This study laid an experimental foundation for in-depth understanding of the immunological properties and utilizing effective epitopes of the GapC protein of S. uberis.

Snakin-2 interacts with cytosolic glyceraldehyde-3-phosphate dehydrogenase 1 to inhibit sprout growth in potato tubers.

The potato tuber is the main nutrient supply and reproductive organ; however, tuber sprouting can reduce its commercial value. Snakin-2 (StSN2) was first reported as an antimicrobial peptide that positively regulates potato disease resistance. Our recent study suggested StSN2 overexpression inhibited sprout growth, while the sprouting process was accelerated in StSN2 RNAi lines. Cytoplasmic glyceraldehyde-3- phosphate dehydrogenase 1 (StGAPC1) was identified as a candidate protein that interacts with StSN2 by coimmunoprecipitation/mass spectrometry (CoIP/MS) experiments. Here, we report that the expression levels of StSN2 and StGAPC1 decreased during sprouting compared with dormancy. Coexpression of StSN2 and StGAPC1 in bud eyes and apical buds was verified by immunofluorescence analysis of paraffin sections. In addition, interaction of StSN2 and StGAPC1 was confirmed by yeast two-hybrid, coimmunoprecipitation and split luciferase complementation assays. Overexpression of StGAPC1 depressed sprout growth, which is similar to the function of StSN2, and StSN2- and StGAPC1-overexpressing lines showed decreased glucose, fructose and galactose content. The interaction of StSN2 and StGAPC1 enhanced StGAPC1 activity and decreased its oxidative modification to inhibit sprout growth. Our results suggest that StSN2 plays a regulatory role in tuber sprout growth through interaction with StGAPC1.

Prokaryotic expression of the GapC protein of Streptococcus uberis and prediction, identification of its B-cell epitopes / 生物工程学报

The GapC protein of Streptococcus uberis located on the surface of bacteria is a protein with glyceraldehyde-3-phosphate dehydrogenase activity. It participates in cellular processes and exhibits a variety of biological activities. In addition, it has good antigenicity. The aim of this study was to predict the possible B-cell epitopes of the GapC protein and verify the immunogenicity of candidate epitope peptides. The gapC gene of S. uberis isolate RF5-1 was cloned into a recombinant expression plasmid pET-28a-GapC and inducibly expressed. The purified protein was used to immunize experimental rabbits to produce anti-GapC polyclonal antibodies. The three-dimensional structure and three-dimensional location of the GapC B-cell epitopes and the homology comparison of the GapC protein and its B-cell epitopes were carried out using bioinformatics softwares. The results showed that the 44-kDa GapC protein had a good immunological reactivity. Six linear and 3 conformational dominant B-cell epitopes against the GapC protein were selected and synthesized. Three dimensional analysis indicated that the selected peptides have better antigen epitope formation potential. Rabbit anti-GapC polyclonal antibodies were generated after immunized with the purified GapC protein, and the polyclonal antibodies were used to identify the epitope peptide by an indirect ELISA. The ELISA results showed that all of the 9 epitope peptides could react with anti-GapC polyclonal antibodies with varying titers. Among them, the epitope polypeptide 266AANDSYGYTEDPIVSSD282 reacted with the polyclonal antibodies significantly stronger than with other epitope peptides. This study laid an experimental foundation for in-depth understanding of the immunological properties and utilizing effective epitopes of the GapC protein of S. uberis.

Analysis of SI-Related BoGAPDH Family Genes and Response of BoGAPC to SI Signal in Brassica oleracea L.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is not only involved in carbohydrate metabolism, but also plays an important role in stress resistance. However, it has not been reported in Brassica oleracea. In this study, we performed a genome-wide identification of BoGAPDH in B. oleracea and performed cloning and expression analysis of one of the differentially expressed genes, BoGAPC. A total of 16 members of the BoGAPDH family were identified in B. oleracea, which were conserved, distributed unevenly on chromosomes and had tandem repeat genes. Most of the genes were down-regulated during self-pollination, and the highest expression was found in stigmas and sepals. Different transcriptome data showed that BoGAPDH genes were differentially expressed under stress, which was consistent with the results of qRT-PCR. We cloned and analyzed the differentially expressed gene BoGAPC and found that it was in the down-regulated mode 1 h after self-pollination, and the expression was the highest in the stigma, which was consistent with the result of GUS staining. The promoter region of the gene not only has stress response elements and plant hormone response elements, but also has a variety of specific elements for regulating floral organ development. Subcellular localization indicates that the BoGAPC protein is located in the cytoplasm and belongs to the active protein in the cytoplasm. The results of prokaryotic expression showed that the size of the BoGAPC protein was about 37 kDa, which was consistent with the expected results, indicating that the protein was induced in prokaryotic cells. The results of yeast two-hybrid and GST pull-down showed that the SRK kinase domain interacted with the BoGAPC protein. The above results suggest that the BoGAPDH family of B. oleracea plays an important role in the process of plant stress resistance, and the BoGAPC gene may be involved in the process of self-incompatibility in B. oleracea, which may respond to SI by encoding proteins directly interacting with SRK.

The Recombinant Expression Proteins FnBP and ClfA From Staphylococcus aureus in Addition to GapC and Sip From Streptococcus agalactiae Can Protect BALB/c Mice From Bacterial Infection.

Dairy cow mastitis is a serious disease that is mainly caused by intramammary infection with Staphylococcus aureus and Streptococcus agalactiae [group B streptococcus (GBS)]. FnBP and ClfA are the virulence factors of S. aureus, while GapC is the respective factor for S. agalactiae. Sip is a highly immunogenic protein, and it is conserved in all GBS serotypes. In this study, we analyzed the abovementioned four genes prepared a FnBP+ClfA chimeric protein (FC), a GapC+Sip chimeric protein (GS), and a FnBP+ClfA+GapC+Sip chimeric protein (FCGS) based on the antigenic sites to evaluate their use in vaccine development. After expression and purification of the recombinant proteins in Escherichia coli, BALB/c mice were immunized with them to examine resistance effects. The total lethal and half lethal doses of S. aureus and S. agalactiae were then measured, and the immunoprotective effects of the fusion proteins were evaluated. The FC and FCGS chimeric proteins could induce mice to produce high levels of antibodies, and bacterial loads were significantly reduced in the spleens and livers after challenge. After immunization with FCGS, the recipients resisted the attacks of both S. aureus and S. agalactiae, indicating the potential of the fusion protein as a mastitis vaccine.

Evaluation of different antigenic preparations against necrotic enteritis in broiler birds using a novel Clostridium perfringens type G strain.

OBJECTIVE: Keeping in view, the constraints faced by the Indian broiler industry with lack of a suitable vaccine against Necrotic Enteritis (NE), a study has been proposed to explore the prevalence and detail characterization of C. perfringens type G in NE suspected broiler chicken in the process of suitable vaccine development. METHODS: Intestinal scrapings/faecal contents of NE suspected broiler chickens were screened to establish the prevalence of C.perfringens type G in broiler birds. A most pathogenic, highly resistant type G isolate of C. perfringens, bearing both tpeL and gapC gene was selected for preparation of three different vaccine formulations, and to evaluate their immunogenic potential in broiler birds. RESULTS: Screening of clinical samples of NE suspected broiler birds revealed C. perfringens type G, bearing gapC gene in 51.22% samples, of which 47.62% revealed tpeL gene. Seven of the tpeLpos type G isolates were comparatively more pathogenic for mice, of which, one exhibited multidrug resistance towards ciprofloxacin, norfloxacin, tetracycline and levofloxacin. The sonicated supernatant (SS) prepared from the selected tpeL and gapC positive isolate could maintain a significantly higher protective IgG response than toxoid and bacterin preparation from the 21st to 28thday of age in immunized birds. CONCLUSION: The additional TpeL toxin in C. perfringens type G has been proved to be an additional key biological factor in the pathogenesis of NE in broiler chickens. Considering the release of more immunogenic proteins, the SS proved to be a better immunogenic preparation against NE with a multiple immunization dose.

Immunogenicity of multi-epitope vaccines composed of epitopes from Streptococcus dysgalactiae GapC.

Glyceraldehyde-3-phosphate dehydrogenase C (GapC) of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce a protective immune response against S. dysgalactiae infection. To investigate the immune response and protective efficacy induced by epitope-vaccines against S. dysgalactiae infection, we constructed epitope-vaccines GTB1, GB1B2, and GTB1B2 using a T cell epitope (GapC63-77, abbreviated as GT) and two B cell epitopes (GapC30-36, abbreviated as GB1, and GapC97-103, abbreviated as GB2), which were identified in GapC1-150 of S. dysgalactiae in tandem by a GSGSGS linker. BALB/c mice were immunized via an intramuscular injection with the epitope vaccines. The levels of the cytokines, IFN-γ, IL-4, and IL-17, secreted by splenic lymphocytes and the antibody levels in the sera of the immunized mice were detected by ELISA. The immunized mice were subsequently challenged with S. dysgalactiae, and the bacterial colonization in the immunized-mouse organs was examined using the plate counting method. The results showed that the level of the cytokines induced by GTB1B2 was lower than that induced by GapC1-150, but higher than that induced by other epitope vaccines. The level of IgG induced by GTB1B2 was lower than that induced by GapC1-150, but higher than the levels induced by other epitope vaccines. The bacterial colonization numbers in the organs of the mice immunized with GTB1B2 were higher those of the mice immunized with GapC1-150, but significantly lower than those from the mice immunized with other epitope-vaccines. Our results demonstrated that the T cell and B cell epitopes in the epitope-vaccines worked synergistically against bacterial challenge. The multi-epitope vaccine, GTB1B2, could induce stronger cellular and humoral immune responses, and provide a better protective effect against S. dysgalactiae infection.

Low-copy nuclear markers in Isoëtes (Isoëtaceae) identified with transcriptomes.

PREMISE OF THE STUDY: Few genetic markers provide phylogenetic information in closely related species of Isoëtes (Isoëtaceae). We describe the development of primers for several putative low-copy nuclear markers to resolve the phylogeny of Isoëtes, particularly in the southeastern United States. METHODS AND RESULTS: We identified regions of interest in Isoëtes transcriptomes based on low-copy genes in other plants. Primers were designed for these regions and tested with 16 taxa of Isoëtes and one species of Lycopodium. Parts of the pgiC, gapC, and IBR3 gene regions show phylogenetic signal within the North American and Mediterranean clades of Isoëtes. CONCLUSIONS: Transcriptome data prove useful for identification and primer design of low-copy genes. Three new markers show potential for inferring phylogenies in regional clades of Isoëtes, and possibly across the entire genus.

Over-expression of SINAL7 increases biomass and drought tolerance, and also delays senescence in Arabidopsis.

The seven in absentia like 7 gene (At5g37890, SINAL7) from Arabidopsis thaliana encodes a RING finger protein belonging to the SINA superfamily that possesses E3 ubiquitin-ligase activity. SINAL7 has the ability to self-ubiquitinate and to mono-ubiquitinate glyceraldehyde-3-P dehydrogenase 1 (GAPC1), suggesting a role for both proteins in a hypothetical signaling pathway in Arabidopsis. In this study, the in vivo effects of SINAL7 on plant physiology were examined by over-expressing SINAL7 in transgenic Arabidopsis plants. Phenotypic and gene expression analyses suggest the involvement of SINAL7 in the regulation of several vegetative parameters, essentially those that affect the aerial parts of the plants. Over-expression of SINAL7 resulted in an increase in the concentrations of hexoses and sucrose, with a concommitant increase in plant biomass, particularly in the number of rosette leaves and stem thickness. Interestingly, using the CAB1 (chlorophyll ab binding protein 1) gene as a marker revealed a delay in the onset of senescence. Transgenic plants also displayed a remarkable level of drought resistance, indicating the complexity of the response to SINAL7 over-expression.

Molecular identification of GAPDHs in cassava highlights the antagonism of MeGAPCs and MeATG8s in plant disease resistance against cassava bacterial blight.

KEY MESSAGE: MeGAPCs were identified as negative regulators of plant disease resistance, and the interaction of MeGAPCs and MeATG8s was highlighted in plant defense response. As an important enzyme of glycolysis metabolic pathway, glyceraldehyde-3-P dehydrogenase (GAPDH) plays important roles in plant development, abiotic stress and immune responses. Cassava (Manihot esculenta) is most important tropical crop and one of the major food crops, however, no information is available about GAPDH gene family in cassava. In this study, 14 MeGAPDHs including 6 cytosol GAPDHs (MeGAPCs) were identified from cassava, and the transcripts of 14 MeGAPDHs in response to Xanthomonas axonopodis pv manihotis (Xam) indicated their possible involvement in immune responses. Further investigation showed that MeGAPCs are negative regulators of disease resistance against Xam. Through transient expression in Nicotiana benthamiana, we found that overexpression of MeGAPCs led to decreased disease resistance against Xam. On the contrary, MeGAPCs-silenced cassava plants through virus-induced gene silencing (VIGS) conferred improved disease resistance. Notably, MeGAPCs physically interacted with autophagy-related protein 8b (MeATG8b) and MeATG8e and inhibited autophagic activity. Moreover, MeATG8b and MeATG8e negatively regulated the activities of NAD-dependent MeGAPDHs, and are involved in MeGAPCs-mediated disease resistance. Taken together, this study highlights the involvement of MeGAPCs in plant disease resistance, through interacting with MeATG8b and MeATG8e.

Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein.

The GapC protein of Staphylococcus aureus (S. aureus) is a surface protein that is highly conserved among Staphylococcus strains, and it can induce protective humoral immune responses. However, B-cell epitopes in S. aureus GapC have not been reported. In this study, we generated a monoclonal antibody (mAb2A9) targeting S. aureus GapC. Through a passive immunity test, mAb2A9 was shown to partially protect mice against S. aureus infection. We screened the motif 236PVATGSLTE243 that is recognized by mAb2A9 using a phage-display system. The motif sequence exactly matched amino acids 236-243 of the S. aureus GapC protein. Then, we identified the key amino acids in the motif using site-directed mutagenesis. Site-directed mutagenesis revealed that residues P236, G240, L242, and T243 formed the core of the 236PVATGSLT243 motif. In addition, this epitope was proven to be located on the surface of S. aureus, and it induced a protective humoral immune response against S. aureus infection in immunized mice. Overall, our results characterized a conserved B-cell epitope, which will be an attractive target for designing effective epitope-based vaccines against S. aureus infection.

Fates of Evolutionarily Distinct, Plastid-type Glyceraldehyde 3-phosphate Dehydrogenase Genes in Kareniacean Dinoflagellates.

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome-one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte-derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid-type glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte-derived tertiary plastids: "gapC1h" acquired from the haptophyte endosymbiont and "gapC1p" inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid-type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT-derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme.

AtCAP2 is crucial for lytic vacuole biogenesis during germination by positively regulating vacuolar protein trafficking.

Protein trafficking is a fundamental mechanism of subcellular organization and contributes to organellar biogenesis. AtCAP2 is an Arabidopsis homolog of the Mesembryanthemum crystallinum calcium-dependent protein kinase 1 adaptor protein 2 (McCAP2), a member of the syntaxin superfamily. Here, we show that AtCAP2 plays an important role in the conversion to the lytic vacuole (LV) during early plant development. The AtCAP2 loss-of-function mutant atcap2-1 displayed delays in protein storage vacuole (PSV) protein degradation, PSV fusion, LV acidification, and biosynthesis of several vacuolar proteins during germination. At the mature stage, atcap2-1 plants accumulated vacuolar proteins in the prevacuolar compartment (PVC) instead of the LV. In wild-type plants, AtCAP2 localizes to the PVC as a peripheral membrane protein and in the PVC compartment recruits glyceraldehyde-3-phosphate dehydrogenase C2 (GAPC2) to the PVC. We propose that AtCAP2 contributes to LV biogenesis during early plant development by supporting the trafficking of specific proteins involved in the PSV-to-LV transition and LV acidification during early stages of plant development.

Cytosolic glyceraldehyde-3-phosphate dehydrogenases play crucial roles in controlling cold-induced sweetening and apical dominance of potato (Solanum tuberosum L.) tubers.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme that functions in producing energy and supplying intermediates for cellular metabolism. Recent researches indicate that GAPDHs have multiple functions beside glycolysis. However, little information is available for functions of GAPDHs in potato. Here, we identified 4 putative cytosolic GAPDH genes in potato genome and demonstrated that the StGAPC1, StGAPC2, and StGAPC3, which are constitutively expressed in potato tissues and cold inducible in tubers, encode active cytosolic GAPDHs. Cosuppression of these 3 GAPC genes resulted in low tuber GAPDH activity, consequently the accumulation of reducing sugars in cold stored tubers by altering the tuber metabolite pool sizes favoring the sucrose pathway. Furthermore, GAPCs-silenced tubers exhibited a loss of apical dominance dependent on cell death of tuber apical bud meristem (TAB-meristem). It was also confirmed that StGAPC1, StGAPC2, and StGAPC3 interacted with the autophagy-related protein 3 (ATG3), implying that the occurrence of cell death in TAB-meristem could be induced by ATG3 associated events. Collectively, the present research evidences first that the GAPC genes play crucial roles in diverse physiological and developmental processes in potato tubers.

Isolation, pathogenicity and characterization of a novel bacterial pathogen Streptococcus uberis from diseased mandarin fish Siniperca chuatsi.

In recent years, mandarin fish had a high mortality rate associated with abnormal swimming, exophthalmia, corneal opacity and eye hemorrhage on a fish farm located at Foshan city, Guangdong province, China. Three isolates of Gram-positive, chain-forming cocci were recovered from moribund fish, and designated as SS131025-1, SS131025-2, and SS131025-3. These isolates were identified as Streptococcus uberis according to their morphologic and physio-biochemical characteristics as well as phylogenetic analysis based on their 16S rRNA and GapC gene sequences. The pathogenicity of S. uberis to mandarin fish was determined by challenge experiments. Results of artificial challenge showed S. uberis infected healthy mandarin fish and lead to death by eyeball injection or immersion route, and the LD50 of SS131025-1 with eyeball injection was 2.0 × 106.42 CFU per fish. Moreover extracellular product (ECP) of the isolated S.uberis induced CPB cell apoptosis and cause death of mandarin fish. In addition, these S. uberis strains could also infect tilapia, but not grass carp and crucian carp, and grew in brain-heart infusion broth with an optimal temperature of 37 °C, pH of 7.0, and salinity of 0%. Antibiotic sensitivity testing indicated that these isolates were susceptible to rifampicin and furazolidone but resistant to 20 kinds of antibiotics. Histopathologically, infection with S. uberis could cause serious pathological changes in brain tissues such as vacuoles in matrix, swollen mitochondria with lysis of cristae and disintegration, and lots of coccus was observed both under electron and light microscope. These results shed some light on the pathogenicity of the isolates and how to prevent and control S. uberis infection in mandarin fish.

Immunogenicity of amino acids 1-150 of Streptococcus GapC displayed on the surface of Escherichia coli.

Streptococcus is one of the main pathogens that cause bovine mastitis. They includes into S.agalactiae, S.dysgalactiae, and S.uberis. The GapC protein is a virulence factor that is expressed on the surface of Streptococcus species. GapC is highly antigenic and immunization with GapC confers cross-protection against all three species. Our previous data showed that amino acids 1-150 of GapC (GapC1-150) of S. dysgalactiae conferred similar immunoprotection compared to full-length GapC. Thus, the present study aimed to construct a recombinant Escherichia coli XL1-Blue strain that displayed GapC1-150 on its surface, and to investigate the immunogenicity of the surface-localized GapC1-150. To do so, the ompA gene of the E. coli XL1-Blue strain was replaced with the lpp'-ompA-gapC11-150 or lpp'-ompA genes by λ Red recombination, the former of which fused GapC1-150 to an Lpp lipoprotein signal peptide and amino acids 1-159 of OmpA; the recombinant strains were named XL1-Blue/LOG76 and XL1-Blue/LO11, respectively. GapC1-150 was confirmed to localize to the surface of the XL1-Blue/LOG76 strain by an indirect enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell sorter analysis, and laser-scanning confocal microscopy. Then, ICR mice were immunized intramuscularly with the XL1-Blue/LOG76 or XL1-Blue/LO11 strains, or recombinant GapC1-150. The sera of the immunized mice were collected and the anti-GapC1-150 antibody levels were detected by ELISA. Lymphocytes secreting interleukin (IL)-4 and interferon-γ were detected by an enzyme-linked ImmunoSpot assay, as was the level of IL-17A level in the supernatant of cultured splenic lymphocytes. The mice immunized with the XL1-Blue/LOG76 strain or GapC1-150 exhibited better cellular and humoral immunity. Lastly, the immunized mice were challenged with S. uberis, S. dysgalactiae, and S. agalactiae strains, and mice that were immunized with the XL1-Blue/LOG76 strain were better protected than those that were immunized with the XL1-Blue/LO11 strain. These results indicate that it is feasible to display GapC1-150 on the E. coli surface as a vaccine against Streptococcus species.