The Plasmodiophora brassicae Golgi-localized UPF0016 protein PbGDT1 mediates calcium but not manganese transport in yeast and Nicotiana benthamiana.

Manganese and calcium homeostasis and signalling, in eukaryotic organisms, are regulated through membrane located pumps, channels and exchangers, including the Mn2+/Ca2+ uncharacterized protein family 0016 (UPF0016). Here we show that Plasmodiophora brassicae PbGDT1 is a member of the UPF0016 and an ortholog of Saccharomyces cerevisiae Gdt1p (GCR Dependent Translation Factor 1) protein involved in manganese homeostasis as well as the calcium mediated stress response in yeast. PbGDT1 complemented the ScGdt1p and ScPMR1 (Ca2+ ATPase) double null mutant under elevated calcium stress but not under elevated manganese conditions. In both yeast and Nicotiana benthamiana, PbGDT1 localizes to the Golgi apparatus, with additional ER association in N. benthamiana. Expression of PbGDT1 in N. benthamiana, suppresses BAX-triggered cell death, further highlighting the importance of calcium homeostasis in maintaining cell physiology and integrity in a stress environment.

Evolutionary divergence in embryo and seed coat development of U's Triangle Brassica species illustrated by a spatiotemporal transcriptome atlas.

The economically valuable Brassica species include the six related members of U's Triangle. Despite the agronomic and economic importance of these Brassicas, the impacts of evolution and relatively recent domestication events on the genetic landscape of seed development have not been comprehensively examined in these species. Here we present a 3D transcriptome atlas for the six species of U's Triangle, producing a unique resource that captures gene expression data for the major subcompartments of the seed, from the unfertilized ovule to the mature embryo and seed coat. This comprehensive dataset for seed development in tetraploid and ancestral diploid Brassicas provides new insights into evolutionary divergence and expression bias at the gene and subgenome levels during the domestication of these valued crop species. Comparisons of gene expression associated with regulatory networks and metabolic pathways operating in the embryo and seed coat during seed development reveal differences in storage reserve accumulation and fatty acid metabolism among the six Brassica species. This study illustrates the genetic underpinnings of seed traits and the selective pressures placed on seed production, providing an immense resource for continued investigation of Brassica polyploid biology, genomics and evolution.

A clubroot pathogen effector targets cruciferous cysteine proteases to suppress plant immunity.

Plant pathogen effector proteins are key to pathogen virulence. In susceptible host Brassicas, the clubroot pathogen, Plasmodiophora brassicae, induces the production of nutrient-sink root galls, at the site of infection. Among a list of 32 P. brassiae effector candidates previously reported by our group, we identified SSPbP53 as a putative apoplastic cystatin-like protein highly expressed during the secondary infection. Here we found that SSPbP53 encoding gene is conserved among several P. brassicae pathotypes and that SSPbP53 is an apoplastic protein able to directly interact with and inhibit cruciferous papain-like cysteine proteases (PLCPs), specifically Arabidopsis XYLEM CYSTEINE PEPTIDASE 1 (AtXCP1). The severity of clubroot disease is greatly reduced in the Arabidopsis xcp1 null mutant (AtΔxcp1) after infection with P. brassicae resting spores, indicating that the interaction of P. brassicae SSPbP53 with XCP1 is important to clubroot susceptibility. SSPbP53 is the first cystatin-like effector identified and characterized for a plant pathogenic protist.

Endomembrane-Targeting Plasmodiophora brassicae Effectors Modulate PAMP Triggered Immune Responses in Plants.

Plasmodiophora brassicae is a devastating obligate, intracellular, biotrophic pathogen that causes clubroot disease in crucifer plants. Disease progression is regulated by effector proteins secreted by P. brassicae. Twelve P. brassicae putative effectors (PbPEs), expressed at various stages of disease development [0, 2, 5, 7, 14, 21, and 28 days post inoculation (DPI)] in Arabidopsis and localizing to the plant endomembrane system, were studied for their roles in pathogenesis. Of the 12 PbPEs, seven showed an inhibitory effect on programmed cell death (PCD) as triggered by the PCD inducers, PiINF1 (Phytophthora infestans Infestin 1) and PiNPP1 (P. infestans necrosis causing protein). Showing the strongest level of PCD suppression, PbPE15, a member of the 2-oxoglutarate (2OG) and Fe (II)-dependent oxygenase superfamily and with gene expression during later stages of infection, appears to have a role in tumorigenesis as well as defense signaling in plants. PbPE13 produced an enhanced PiINF1-induced PCD response. Transient expression, in Nicotiana benthamiana leaves of these PbPEs minus the signal peptide (SP) (Δsp PbPEGFPs), showed localization to the endomembrane system, targeting the endoplasmic reticulum (ER), Golgi bodies and nucleo-cytoplasm, suggesting roles in manipulating plant cell secretion and vesicle trafficking. Δsp PbPE13GFP localized to plasma membrane (PM) lipid rafts with an association to plasmodesmata, suggesting a role at the cell-to-cell communication junction. Membrane relocalization of Δsp PbPE13GFP, triggered by flagellin N-terminus of Pseudomonas aeruginosa (flg22 - known to elicit a PAMP triggered immune response in plants), supports its involvement in raft-mediated immune signaling. This study is an important step in deciphering P. brassicae effector roles in the disruption of plant immunity to clubroot disease.

Alternative splicing dynamics and evolutionary divergence during embryogenesis in wheat species.

Among polyploid species with complex genomic architecture, variations in the regulation of alternative splicing (AS) provide opportunities for transcriptional and proteomic plasticity and the potential for generating trait diversities. However, the evolution of AS and its influence on grain development in diploid grass and valuable polyploid wheat crops are poorly understood. To address this knowledge gap, we developed a pipeline for the analysis of alternatively spliced transcript isoforms, which takes the high sequence similarity among polyploid wheat subgenomes into account. Through analysis of synteny and detection of collinearity of homoeologous subgenomes, conserved and specific AS events across five wheat and grass species were identified. A global analysis of the regulation of AS in diploid grass and polyploid wheat grains revealed diversity in AS events not only between the endosperm, pericarp and embryo overdevelopment, but also between subgenomes. Analysis of AS in homoeologous triads of polyploid wheats revealed evolutionary divergence between gene-level and transcript-level regulation of embryogenesis. Evolutionary age analysis indicated that the generation of novel transcript isoforms has occurred in young genes at a more rapid rate than in ancient genes. These findings, together with the development of comprehensive AS resources for wheat and grass species, advance understanding of the evolution of regulatory features of AS during embryogenesis and grain development in wheat.

Receptor-Like Kinases BAK1 and SOBIR1 Are Required for Necrotizing Activity of a Novel Group of Sclerotinia sclerotiorum Necrosis-Inducing Effectors.

Sclerotinia sclerotiorum is a characteristic necrotrophic plant pathogen and is dependent on the induction of host cell death for nutrient acquisition. To identify necrosis-inducing effectors, the genome of S. sclerotiorum was scanned for genes encoding small, secreted, cysteine-rich proteins. These potential effectors were tested for their ability to induce necrosis in Nicotiana benthamiana via Agrobacterium-mediated expression and for cellular localization in host cells. Six novel proteins were discovered, of which all but one required a signal peptide for export to the apoplast for necrotizing activity. Virus-induced gene silencing revealed that the five necrosis-inducing effectors with a requirement for secretion also required the plant co-receptor-like kinases Brassinosteroid Insensitive 1-Associated Receptor Kinase 1 (BAK1) and Suppressor of BAK1-Interacting Receptor-like Kinase 1 (SOBIR1) for the induction of necrosis. S. sclerotiorum necrosis-inducing effector 2 (SsNE2) represented a new class of necrosis-inducing proteins as orthologs were identified in several other phytopathogenic fungi that were also capable of inducing necrosis. Substitution of conserved cysteine residues with alanine reduced, but did not abolish, the necrotizing activity of SsNE2 and full-length protein was required for function as peptides spanning the entire protein were unable to induce necrosis. These results illustrate the importance of necrosis-inducing effectors for S. sclerotiorum virulence and the role of host extracellular receptor(s) in effector-triggered susceptibility to this pathogen.

The Transcriptional Landscape of Polyploid Wheats and Their Diploid Ancestors during Embryogenesis and Grain Development.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticum turgidum (var durum), which putatively arose from diploid ancestors Triticum urartu, Aegilops speltoides, and Aegilops tauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

Live cell imaging of Plasmodiophora brassicae-host plant interactions based on a two-step axenic culture system.

Plasmodiophora brassicae, a parasitic protist, induces club-shaped tumor-like growth of host Brassicas roots and hypocotyls after infection. Due to its soil-borne nature and intracellular, biotrophic parasitism the infection biology and early pathogenesis remains in doubt. In this study, we have established a new protocol, based on a two-step axenic culture of P. brassicae with its host tissues, for easy and in planta observation of cellular interactions between P. brassicae and host plants: first, coculture of P. brassicae with infected canola root tissues, on growth-medium plates, enables the propagation of P. brassicae that serves as pure inoculum for pathogenicity assays, and second, the pure inoculum is subsequently used for pathogenicity tests on both canola and Arabidopsis seedlings grown on medium plates in Petri dishes. During the first axenic culture, we established a staining protocol by which the pathogen was fluorescently labeled with Nile red and calcofluor white, thus allowing in planta observation of pathogen development. In the pathogenicity assays, our results showed that axenic cultures of P. brassicae, in calli, remains fully virulent and completes its life cycle in both canola and Arabidopsis roots grown in Petri dishes. Combining visualization of fluorescent probe-labeled P. brassicae structures with fluorescent protein tagging of Arabidopsis cellular components, further revealed dynamic responses of host cells at the early stages of P. brassicae infection. Thus, established protocols for in planta detection of P. brassicae structures and the live cell imaging of P. brassicae-Arabidopsis interactions provide a novel strategy for improving our detailed knowledge of P. brassicae infection in host tissues.

Effects of eIFiso4G1 mutation on seed oil biosynthesis.

Fatty acid biosynthesis is a primary metabolic pathway that occurs in plastids, whereas the formation of glycerolipid molecules for the majority of cellular membrane systems and the deposition of storage lipid in seeds takes place in the cytosolic compartment. In this report, we present a study of an Arabidopsis mutant, ar21, with a novel seed fatty acid phenotype showing higher contents of eicosanoic acid (20:1) and oleic acid (18:1) and a reduced level of α-linolenic acid (18:3). A combination of map-based cloning and whole-genome sequencing identified the genetic basis underlying the fatty acid phenotype as a lesion in the plant-specific eukaryotic translation initiation factor eIFiso4G1. Transcriptome analysis on developing seeds revealed a reduced level of plastid-encoded genes. Specifically, decreases in both transcript and protein levels of an enzyme involved in fatty acid biosynthesis, the ß-subunit of the plastidic heteromeric acetyl-CoA carboxylase (htACCase) encoded by accD, were evident in the mutant. Biochemical assays showed that the developing seeds of the mutant possessed a decreased htACCase activity in the plastid but an elevated activity of homomeric acetyl-CoA carboxylase (hmACCase). These results suggested that the increased 20:1 was attributable at least in part to the enhanced cytosolic hmACCase activity. We also detected a significant repression of FATTY ACID DESATURASE 3 (FAD3) during seed development, which correlated with a decreased 18:3 level in seed oil. Together, our study on a mutant of eIFiso4G1 uncovered multifaceted interactions between the cytosolic and plastidic compartments in seed lipid biosynthesis that impact major seed oil traits.

Multifaceted Roles of the Ras Guanine-Nucleotide Exchange Factor ChRgf in Development, Pathogenesis, and Stress Responses of Colletotrichum higginsianum.

The infection process of Colletotrichum higginsianum, which causes a disease of crucifers, involves several key steps: conidial germination, appressorial formation, appressorial penetration, and invasive growth in host tissues. In this study, the ChRgf gene encoding a Ras guanine-nucleotide exchange factor protein was identified by screening T-DNA insertion mutants generated from Agrobacterium tumefaciens-mediated transformation that were unable to cause disease on the host Arabidopsis thaliana. Targeted gene deletion of ChRgf resulted in a null mutant (ΔChrgf-42) with defects in vegetative growth, hyphal morphology, and conidiation, and poor surface attachment and low germination on hydrophobic surfaces; however, there were no apparent differences in appressorial turgor pressure between the wild type and the mutant. The conidia of the mutant were unable to geminate on attached Arabidopsis leaves and did not cause any disease symptoms. Intracellular cyclic adenosine monophosphate levels in the ΔChrgf mutant were lower than that of the wild type. Our results suggest that ChRgf is a key regulator in response to salt and osmotic stresses in C. higginsianum, and indicate that it is involved in fungal pathogenicity. This gene seems to act as an important modulator upstream of several distinct signaling pathways that are involved in regulating vegetative growth, conidiation, infection-related structure development, and stress responses of C. higginsianum.

Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens.

The rapid reorganization and polarization of actin filaments (AFs) toward the pathogen penetration site is one of the earliest cellular responses, yet the regulatory mechanism of AF dynamics is poorly understood. Using live-cell imaging in Arabidopsis, we show that polarization coupled with AF bundling involves precise spatiotemporal control at the site of attempted penetration by the nonadapted barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We further show that the Bgh-triggered AF mobility and organelle aggregation are predominately driven by the myosin motor proteins. Inactivation of myosins by pharmacological inhibitors prevents bulk aggregation of organelles and blocks recruitment of lignin-like compounds to the penetration site and deposition of callose and defensive protein, PENETRATION 1 (PEN1) into the apoplastic papillae, resulting in attenuation of penetration resistance. Using gene knockout analysis, we demonstrate that highly expressed myosins XI, especially myosin XI-K, are the primary contributors to cell wall-mediated penetration resistance. Moreover, the quadruple myosin knockout mutant xi-1 xi-2 xi-i xi-k displays impaired trafficking pathway responsible for the accumulation of PEN1 at the cell periphery. Strikingly, this mutant shows not only increased penetration rate but also enhanced overall disease susceptibility to both adapted and nonadapted fungal pathogens. Our findings establish myosins XI as key regulators of plant antifungal immunity.

Overexpression of a novel biotrophy-specific Colletotrichum truncatum effector, CtNUDIX, in hemibiotrophic fungal phytopathogens causes incompatibility with their host plants.

The hemibiotrophic fungus Colletotrichum truncatum causes anthracnose disease on lentils and a few other grain legumes. It shows initial symptomless intracellular growth, where colonized host cells remain viable (biotrophy), and then switches to necrotrophic growth, killing the colonized host plant tissues. Here, we report a novel effector gene, CtNUDIX, from C. truncatum that is exclusively expressed during the late biotrophic phase (before the switch to necrotrophy) and elicits a hypersensitive response (HR)-like cell death in tobacco leaves transiently expressing the effector. CtNUDIX homologs, which contain a signal peptide and a Nudix hydrolase domain, may be unique to hemibiotrophic fungal and fungus-like plant pathogens. CtNUDIX lacking a signal peptide or a Nudix motif failed to induce cell death in tobacco. Expression of CtNUDIX:eGFP in tobacco suggested that the fusion protein might act on the host cell plasma membrane. Overexpression of CtNUDIX in C. truncatum and the rice blast pathogen, Magnaporthe oryzae, resulted in incompatibility with the hosts lentil and barley, respectively, by causing an HR-like response in infected host cells associated with the biotrophic invasive hyphae. These results suggest that C. truncatum and possibly M. oryzae elicit cell death to signal the transition from biotrophy to necrotrophy.

EST mining identifies proteins putatively secreted by the anthracnose pathogen Colletotrichum truncatum.

BACKGROUND: Colletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. This pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. Transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. C. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. Some of them might be transition specific. RESULTS: A directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of C. truncatum and 5000 expressed sequence tags (ESTs) with an average read of > 600 bp from the 5-prime end were generated. Nearly 39% of the ESTs were predicted to encode proteins of fungal origin and among these, 162 ESTs were predicted to contain N-terminal signal peptides (SPs) in their deduced open reading frames (ORFs). The 162 sequences could be assembled into 122 tentative unigenes comprising 32 contigs and 90 singletons. Sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins (CEAPs), candidate effectors and other proteins. Eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble (lack of transmembrane domain), cysteine-rich proteins with a putative SP. For a selected subset of CEAPs and candidate effectors, semiquantitative RT-PCR showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. Using potato virus X (PVX) based transient expression assays, we showed that one of the candidate effectors, i. e. contig 8 that encodes a cerato-platanin (CP) domain containing protein, unlike CP proteins from other fungal pathogens was unable to elicit a hypersensitive response (HR). CONCLUSIONS: The current study catalogues proteins putatively secreted at the in planta biotrophy-necrotrophy transition of C. truncatum. Some of these proteins may have a role in establishing compatible interaction with the host plant.

GP120-specific exosome-targeted T cell-based vaccine capable of stimulating DC- and CD4(+) T-independent CTL responses.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutics. In this study, we generated ovalbumin (OVA)-pulsed and pcDNAgp120-transfected dendritic cell (DC)-released exosomes (EXOova and EXOgp120) and ConA-stimulated C57BL/6 CD8(+) T cells. OVA- and Gp120-Texo vaccines were generated from CD8(+) T cells with uptake of EXOova and EXOgp120, respectively. We demonstrate that OVA-Texo stimulates in vitro and in vivo OVA-specific CD4(+) and CD8(+) cytotoxic T lymphocyte (CTL) responses leading to long-term immunity against OVA-expressing BL6-10(OVA) melanoma. Interestingly, CD8(+) T cell responses are DC and CD4(+) T cell independent. Importantly, Gp120-Texo also stimulates Gp120-specific CTL responses and long-term immunity against Gp120-expressing B16 melanoma. Therefore, this novel HIV-1-specific EXO-targeted Gp120-Texo vaccine may be useful in induction of efficient CTL responses in AIDS patients with DC dysfunction and CD4(+) T cell deficiency.

Membrane-bound HSP70-engineered myeloma cell-derived exosomes stimulate more efficient CD8(+) CTL- and NK-mediated antitumour immunity than exosomes released from heat-shocked tumour cells expressing cytoplasmic HSP70.

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour-derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T-cell immune responses. To enhance EXO-based antitumour immunity, we generated an engineered myeloma cell line J558(HSP) expressing endogenous P1A tumour antigen and transgenic form of membrane-bound HSP70 and heat-shocked J558(HS) expressing cytoplasmic HSP70, and purified EXO(HSP) and EXO(HS) from J558(HSP) and J558(HS) tumour cell culture supernatants by ultracentrifugation. We found that EXO(HSP) were able to more efficiently stimulate maturation of DCs with up-regulation of Ia(b) , CD40, CD80 and inflammatory cytokines than EXO(HS) after overnight incubation of immature bone-marrow-derived DCs (5 × 106 cells) with EXO (100 µg), respectively. We also i.v. immunized BALB/c mice with EXO (30 µg/mouse) and assessed P1A-specific T-cell responses after immunization. We demonstrate that EXO(HSP) are able to stimulate type 1 CD4(+) helper T (Th1) cell responses, and more efficient P1A-specific CD8(+) cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXO(HS) . In addition, we further elucidate that EXO(HSP) -stimulated antitumour immunity is mediated by both P1A-specific CD8(+) CTL and non-P1A-specific natural killer (NK) responses. Therefore, membrane-bound HSP70-expressing tumour cell-released EXO may represent a more effective EXO-based vaccine in induction of antitumour immunity.

Tumor apoptotic bodies inhibit CTL responses and antitumor immunity via membrane-bound transforming growth factor-beta1 inducing CD8+ T-cell anergy and CD4+ Tr1 cell responses.

Tumor cell apoptosis induced by radiation therapy results in apoptotic tumor cells and apparition of membrane blebs termed apoptotic bodies (APB). The immune responses induced by apoptotic tumor cells have been extensively studied. However, the role of APB in modulation of tumor immune responses is elusive. In this study, we induced apoptosis in 90% ovabumin-expressing EG7 tumor cells by in vitro irradiation (9,000 rad) of tumor cells with a subsequent cell culture for 9 hours. APB purified from irradiation-induced apoptotic EG7 cell culture supernatant by differential ultracentrifugation were vesicles with 50 to 90 nm in diameter and expressed apoptosis-specific Annexin V, 14-3-3, and Histone H3. We then investigated its potential modulation in DC(OVA)-induced T-cell responses and antitumor immunity. We found that EG7-derived APB were tolerogenic and capable of suppressing DC(OVA)-stimulated CD8+ CTL responses and antitumor immunity via its induction of CD8+ T-cell anergy and type 1 regulatory CD4+ T-cell responses. Analysis of apoptotic tumor cells and APB revealed the expression of membrane-bound transforming growth factor (TGF)-beta1 associated with irradiation-induced apoptosis formation, which is a result from activation of transcriptional factor NF-AT specific for TGF-beta1 promoters. Our data further elucidate that it is the membrane-bound TGF-beta1 expression on APB that contributes to its in vitro antiproliferative effect as shown by using neutralizing TGF-beta1-specific antibody. Administration of anti-TGF-beta1 antibody in vivo also blocked APB-mediated immune suppression of CD8+ CTL responses and antitumor immunity. Therefore, our study may have great impact in designing a combined radiation therapy with immunotherapy of cancer.

CD4+ Th-APC with acquired peptide/MHC class I and II complexes stimulate type 1 helper CD4+ and central memory CD8+ T cell responses.

T cell-T cell Ag presentation is increasingly attracting attention. We previously showed that the in vitro OVA-pulsed dendritic cell (DC(OVA))-activated CD4(+) Th cells acquired OVA peptide/MHC (pMHC) class I and costimulatory molecules such as CD54 and CD80 from DC(OVA) and acted as CD4(+) Th-APC capable of stimulating OVA-specific CD8(+) CTL responses. In this study, we further applied the OVA-specific TCR-transgenic OT I and OT II mice with deficiency of various cytokines or costimulatory molecule genes useful for studying the molecular mechanisms underlying in Th-APC's stimulatory effect. We demonstrated that DC(OVA)-stimulated OT II CD4(+) Th-APC also acquired costimulatory molecules such as CD40, OX40L, and 4-1BBL and the functional pMHC II complexes by DC(OVA) activation. CD4(+) Th-APC with acquired pMHC II and I were capable of stimulating CD4(+) Th1 and central memory CD8(+)44(+)CD62L(high)IL-7R(+) T cell responses leading to antitumor immunity against OVA-expressing mouse B16 melanoma. Their stimulatory effect on CD8(+) CTL responses and antitumor immunity is mediated by IL-2 secretion, CD40L, and CD80 signaling and is specifically targeted to CD8(+) T cells in vivo via acquired pMHC I. In addition, CD4(+) Th-APC expressing OVA-specific TCR, FasL, and perforin were able to kill DC(OVA) and neighboring Th-APC expressing endogenous and acquired pMHC II. Taken together, we show that CD4(+) Th-APC can modulate immune responses by stimulating CD4(+) Th1 and central memory CD8(+) T cell responses and eliminating DC(OVA) and neighboring Th-APC. Therefore, our findings may have great impacts in not only the antitumor immunity, but also the regulatory T cell-dependent immune tolerance in vivo.

Novel exosome-targeted CD4+ T cell vaccine counteracting CD4+25+ regulatory T cell-mediated immune suppression and stimulating efficient central memory CD8+ CTL responses.

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXO(OVA)) express exosomal peptide/MHC class I and costimulatory molecules. These EXO(OVA)-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXO(OVA)-uptaken (targeted)CD4+ aT cell's stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.

Bidirectional membrane molecule transfer between dendritic and T cells.

The acquisition of dendritic cell (DC) molecules by T cells has been previously reported. However, it remains unclear whether the transfer is only mono- or bidirectional. In this study, we incubated CMFDA-labeled ovalbumin (OVA)-pulsed DC2.4 (DC2.4(OVA)) cells with Dil-labeled OT II CD4(+) T cells and analyzed the potential bidirectional molecule transfer. We also assessed the distribution of internalized membrane using two engineered DC2.4/Ia(b)GFP and MF4/TCRCFP DC lines. Our findings showed that membrane molecule transfer is bidirectional. CD4(+) T cells acquired Ia(b), CD11c, CD40, and CD80 from DC2.4(OVA) cells, and conversely DC2.4(OVA) cells took up CD4, CD25, CD69, and T cell receptor from T cells. The internalized molecules acquired by T cells and DCs mostly localized in endosomes and lysosomes, respectively. Taken together, this study demonstrated a novel phenomenon of bidirectional membrane molecule transfer between DCs and T cells.

Transcriptional regulation of genes involved in the pathways of biosynthesis and supply of methyl units in response to powdery mildew attack and abiotic stresses in wheat.

From a library of 3,000 expression sequence tags (ESTs), derived from the epidermis of a diploid wheat (Triticum monococcum) inoculated with Blumeria graminis f. sp. tritici (Bgt), we cloned 23 cDNAs representing 12 genes that are involved in the pathways of biosynthesis and supply of methyl units. We studied the transcription of these genes to investigate how the methyl units are generated and regulated in response to Bgt infection and abiotic stresses in wheat. Expression of 5, 10-methylene-tetrahydrofolate reductase, methionine synthase, S-adenosylmethionine synthetase, and S-adenosylhomocystein hydrolase transcripts were highly induced at an early stage of infection. This induction was specific to the epidermis and linked to host cell wall apposition (CWA) formation, suggesting that the pathways for generation of methyl units are transcriptionally activated for the host defense response. Levels of S-adenosylmethionine decarboxylase, caffeic acid 3-O-methyltransferase, 1-aminocyclopropane-1-carboxylate oxidase mRNA, but not phosphoethanolamine N-methyltransferase and nicotianamine synthase mRNA, were up-regulated after infection and showed similar expression patterns to genes involved in the pathways of generation of methyl units, revealing possible routes of methyl transfer towards polyamine, lignin and ethylene biosynthesis rather than glycine betaine and nicotianamine in response to Bgt attack. After imposing various abiotic stresses, genes involved in the pathways of generation and supply of methyl units were also up-regulated differentially, suggesting that the generation of sufficient methyl units at an early stage might be crucial to the mitigation of multiple stresses.