Hydrolysis of Gluten-Derived Celiac Disease-Triggering Peptides across a Broad pH Range by RmuAP1: A Novel Aspartic Peptidase Isolated from Rhodotorula mucilaginosa.

An aspartate peptidase with proteolytic activity toward gluten was identified from an isolated red yeast Rhodotorula mucilaginosa strain. This peptidase consists of 425 amino acids, comprising an N-terminal signal peptide, a propeptide, and a C-terminal catalytic domain. The catalytic domain, termed RmuAP1CD, could be secreted by the recombinant oleaginous yeast Yarrowia lipolytica, whose genome contains the expression cassette for RmuAP1CD. RmuAP1CD exhibited optimum activity at pH 2.5 when acting on bovine serum albumin. Moreover, it facilitated the hydrolysis of gluten-derived immunogenic peptides (GIPs), which are responsible for triggering celiac disease symptoms, across a pH range of 3.0-6.0. The preferred cleavage sites are P-Q-Q-↓-P-Q in the 26-mer and P-Q-L-↓-P-Y in the 33-mer GIPs. Conversely, porcine pepsin cannot hydrolyze these two GIPs. The ability of RmuAP1CD to degrade GIPs under acidic conditions of the stomach indicates its potential as a viable oral enzyme therapy for celiac disease.

Sessile Trichomes Play Major Roles in Prey Digestion and Absorption, While Stalked Trichomes Function in Prey Predation in Byblis guehoi.

Carnivorous plants in the genus Byblis obtain nutrients by secreting viscous glue drops and enzymes that trap and digest small organisms. Here, we used B. guehoi to test the long-held theory that different trichomes play different roles in carnivorous plants. In the leaves of B. guehoi, we observed a 1:2.5:14 ratio of long-stalked, short-stalked, and sessile trichomes. We demonstrated that the stalked trichomes play major roles in the production of glue droplets, while the sessile trichomes secrete digestive enzymes, namely proteases and phosphatases. In addition to absorbing digested small molecules via channels/transporters, several carnivorous plants employ a more efficient system: endocytosis of large protein molecules. By feeding B. guehoi fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) to monitor protein transport, we found that sessile trichomes exhibited more endocytosis than long- and short-stalked trichomes. The uptaken FITC-BSA was delivered to the neighboring short epidermal cells in the same row as the sessile trichomes, then to the underlying mesophyll cells; however, no signals were detected in the parallel rows of long epidermis cells. The FITC control could be taken up by sessile trichomes but not transported out. Our study shows that B. guehoi has developed a well-organized system to maximize its food supply, consisting of stalked trichomes for prey predation and sessile trichomes for prey digestion. Moreover, the finding that sessile trichomes transfer large, endocytosed protein molecules to the underlying mesophyll, and putatively to the vascular tissues, but not laterally to the terminally differentiated epidermis, indicates that the nutrient transport system has evolved to maximize efficiency.

The Bph45 Gene Confers Resistance against Brown Planthopper in Rice by Reducing the Production of Limonene.

Brown planthopper (BPH), a monophagous phloem feeder, consumes a large amount of photoassimilates in rice and causes wilting. A near-isogenic line 'TNG71-Bph45' was developed from the Oryza sativa japonica variety 'Tainung 71 (TNG71) carrying a dominant BPH-resistance locus derived from Oryza nivara (IRGC 102165) near the centromere of chromosome 4. We compared the NIL (TNG71-Bph45) and the recurrent parent to explore how the Bph45 gene confers BPH resistance. We found that TNG71-Bph45 is less attractive to BPH at least partially because it produces less limonene. Chiral analysis revealed that the major form of limonene in both rice lines was the L-form. However, both L- and D-limonene attracted BPH when applied exogenously to TNG71-Bph45 rice. The transcript amounts of limonene synthase were significantly higher in TNG71 than in TNG71-Bph45 and were induced by BPH infestation only in the former. Introgression of the Bph45 gene into another japonica variety, Tainan 11, also resulted in a low limonene content. Moreover, several dominantly acting BPH resistance genes introduced into the BPH-sensitive IR24 line compromised its limonene-producing ability and concurrently decreased its attractiveness to BPH. These observations suggest that reducing limonene production may be a common resistance strategy against BPH in rice.

Efficient Hydrolysis of Gluten-Derived Celiac Disease-Triggering Immunogenic Peptides by a Bacterial Serine Protease from Burkholderia gladioli.

Celiac disease is an autoimmune disorder triggered by toxic peptides derived from incompletely digested glutens in the stomach. Peptidases that can digest the toxic peptides may formulate an oral enzyme therapy to improve the patients' health condition. Bga1903 is a serine endopeptidase secreted by Burkholderia gladioli. The preproprotein of Bga1903 consists of an N-terminal signal peptide, a propeptide region, and an enzymatic domain that belongs to the S8 subfamily. Bga1903 could be secreted into the culture medium when it was expressed in E. coli. The purified Bga1903 is capable of hydrolyzing the gluten-derived toxic peptides, such as the 33- and 26-mer peptides, with the preference for the peptide bonds at the carbonyl site of glutamine (P1 position). The kinetic assay of Bga1903 toward the chromogenic substrate Z-HPQ-pNA at 37 °C, pH 7.0, suggests that the values of Km and kcat are 0.44 ± 0.1 mM and 17.8 ± 0.4 s-1, respectively. The addition of Bga1903 in the wort during the fermentation step of beer could help in making gluten-free beer. In summary, Bga1903 is usable to reduce the gluten content in processed foods and represents a good candidate for protein engineering/modification aimed to efficiently digest the gluten at the gastric condition.

Fungicidal Activity of Volatile Organic Compounds Emitted by Burkholderia gladioli Strain BBB-01.

A Burkholderia gladioli strain, named BBB-01, was isolated from rice shoots based on the confrontation plate assay activity against several plant pathogenic fungi. The genome of this bacterial strain consists of two circular chromosomes and one plasmid with 8,201,484 base pairs in total. Pangenome analysis of 23 B. gladioli strains suggests that B. gladioli BBB-01 has the closest evolutionary relationship to B. gladioli pv. gladioli and B. gladioli pv. agaricicola. B. gladioli BBB-01 emitted dimethyl disulfide and 2,5-dimethylfuran when it was cultivated in lysogeny broth and potato dextrose broth, respectively. Dimethyl disulfide is a well-known pesticide, while the bioactivity of 2,5-dimethylfuran has not been reported. In this study, the inhibition activity of the vapor of these two compounds was examined against phytopathogenic fungi, including Magnaporthe oryzae, Gibberella fujikuroi, Sarocladium oryzae, Phellinus noxius and Colletotrichumfructicola, and human pathogen Candida albicans. In general, 2,5-dimethylfuran is more potent than dimethyl disulfide in suppressing the growth of the tested fungi, suggesting that 2,5-dimethylfuran is a potential fumigant to control plant fungal disease.

Proliferating Cell Nuclear Antigen Suppresses RNA Replication of Bamboo Mosaic Virus through an Interaction with the Viral Genome.

Bamboo mosaic virus (BaMV), a member of the Potexvirus genus, has a monopartite positive-strand RNA genome on which five open reading frames (ORFs) are organized. ORF1 encodes a 155-kDa nonstructural protein (REPBaMV) that plays a core function in replication/transcription of the viral genome. To find out cellular factors modulating the replication efficiency of BaMV, a putative REPBaMV-associated protein complex from Nicotiana benthamiana leaf was isolated on an SDS-PAGE gel, and a few proteins preferentially associated with REPBaMV were identified by tandem mass spectrometry. Among them, proliferating cell nuclear antigen (PCNA) was particularly noted. Overexpression of PCNA strongly suppressed the accumulation of BaMV coat protein and RNAs in leaf protoplasts. In addition, PCNA exhibited an inhibitory effect on BaMV polymerase activity. A pulldown assay confirmed a binding capability of PCNA toward BaMV genomic RNA. Mutations at D41 or F114 residues, which are critical for PCNA to function in nuclear DNA replication and repair, disabled PCNA from binding BaMV genomic RNA as well as suppressing BaMV replication. This suggests that PCNA bound to the viral RNA may interfere with the formation of a potent replication complex or block the replication process. Interestingly, BaMV is almost invisible in the newly emerging leaves where PCNA is actively expressed. Accordingly, PCNA is probably one of the factors restricting the proliferation of BaMV in young leaves. Foxtail mosaic virus and Potato virus X were also suppressed by PCNA in the protoplast experiment, suggesting a general inhibitory effect of PCNA on the replication of potexviruses.IMPORTANCE Knowing the dynamic interplay between plant RNA viruses and their host is a basic step toward first understanding how the viruses survive the plant defense mechanisms and second gaining knowledge of pathogenic control in the field. This study found that plant proliferating cell nuclear antigen (PCNA) imposes a strong inhibition on the replication of several potexviruses, including Bamboo mosaic virus, Foxtail mosaic virus, and Potato virus X Based on the tests on Bamboo mosaic virus, PCNA is able to bind the viral genomic RNA, and this binding is a prerequisite for the protein to suppress the virus replication. This study also suggests that PCNA plays an important role in restricting the proliferation of potexviruses in the rapidly dividing tissues of plants.

Increased Expression of 9-Cis-Epoxycarotenoid Dioxygenase, PtNCED1, Associated With Inhibited Seed Germination in a Terrestrial Orchid, Phaius tankervilliae.

The phytohormone abscisic acid (ABA) is involved in regulating seed dormancy and germination. A crucial step of ABA biosynthesis in higher plants is the oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED). Seed development in orchids is unusual because the embryos are minute in size, without obvious histodifferentiation, and lack endosperm. To understand the regulation of ABA biosynthesis in orchid seeds, we isolated and characterized a full-length cDNA encoding an NCED homolog, PtNCED1, from developing seeds of an ornamental orchid, Phaius tankervilliae. Germination percentage was high at 90 days after pollination (DAP), when a globular embryo proper with a degenerating suspensor was evident. After 90 DAP, seed maturation was accompanied by a decrease in water content and a concomitant increase in ABA content and PtNCED1 mRNA level along with a marked decrease in germination percentage. Mature seeds pretreated with NaOCl solution lowered ABA content and improved seed germination. Moreover, after seed germination, developing protocorms could respond to dehydration stress. Dehydration of protocorms stimulated an increase in PtNCED1 level along with ABA content. Our results provide evidence of the involvement of PtNCED1 in regulating endogenous ABA content in developing seeds and protocorms. The accumulation of endogenous ABA content in orchid seeds may have a critical role in seed dormancy and the protocorm response to water stress after seed germination.

OIP30, a RuvB-like DNA helicase 2, is a potential substrate for the pollen-predominant OsCPK25/26 in rice.

Calcium ions are a well-known essential component for pollen germination and tube elongation. Several calcium-dependent protein kinases (CDPKs) are expressed predominantly in mature pollen grains and play a critical role in pollen. However, none of their interacting proteins or downstream substrates has been identified. Using yeast two-hybrid screening, we isolated OsCPK25/26-interacting protein 30 (OIP30), which is also predominantly expressed in pollen. OIP30 encodes a RuvB-like DNA helicase 2 (RuvBL2) that is well conserved in eukaryotic species from yeast to human. Yeast and Drosophila defective in RuvBL2 are non-viable. The interaction between OsCPK26 and OIP30 was confirmed by far-Western blot and pull-down experiments. OIP30 was phosphorylated in a calcium-dependent manner by OsCPK26 but not OsCPK2, which is highly similar to OsCPK26 in sequence and expression profile. OIP30 unwound partial duplex DNA with a 3' to 5' directionality by ATP hydrolysis. Concurrently, the ATPase activity of OIP30 depended on single-stranded DNA. OsCPK26 phosphorylated OIP30 and enhanced both its helicase and ATPase activity about 3-fold. OIP30 may be the potential downstream substrate for OsCPK25/26 in pollen. This report characterizes a RuvBL in plants and links its activities with its upstream regulator.

Characterization of Oncidium 'Gower Ramsey' transcriptomes using 454 GS-FLX pyrosequencing and their application to the identification of genes associated with flowering time.

Oncidium 'Gower Ramsey' is a valuable and successful commercial orchid for the floriculture industry in Taiwan. However, no genome reference for entire sequences of the transcribed genes currently exists for Oncidium orchids, to facilitate the development of molecular biological studies and the breeding of these orchids. In this study, we generated Oncidium cDNA libraries for six different organs: leaves, pseudobulbs, young inflorescences, inflorescences, flower buds and mature flowers. We utilized 454-pyrosequencing technology to perform high-throughput deep sequencing of the Oncidium transcriptome, yielding >0.9 million reads with an average length of 328 bp, for a total of 301 million bases. De novo assembly of the sequences yielded 50,908 contig sequences with an average length of 493 bp from 796,463 reads and 120,219 singletons. The assembled sequences were annotated using BLAST, and a total of 12,757 and 13,931 unigene transcripts from the Arabidopsis and rice genomes were matched by TBLASTX, respectively. A Gene Ontology (GO) analysis of the annotated Oncidium contigs revealed that the majority of sequenced genes were associated with 'unknown molecular function', 'cellular process' and 'intracellular components'. Furthermore, a complete flowering-associated expressed sequence that included most of the genes in the photoperiod pathway and the 15 CONSTANS-LIKE (COL) homologs with the conserved CCT domain was obtained in this collection. These data revealed that the Oncidium expressed sequence tag (EST) database generated in this study has sufficient coverage to be used as a tool to investigate the flowering pathway and various other biological pathways in orchids. An OncidiumOrchidGenomeBase (OOGB) website has been constructed and is publicly available online (http://predictor.nchu.edu.tw/oogb/).

Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation.

Membrane-associated ATPase constitutes an essential element common to all secretion machineries in Gram-negative bacteria. How ATP hydrolysis by these ATPases is coupled to secretion process remains unclear. Here we identified R286 as a key residue in the type II secretion system (T2SS) ATPase XpsE of Xanthomonas campestris that plays a pivotal role in coupling ATP hydrolysis to protein translocation. Mutation of R286 to alanine made XpsE hydrolyse ATP at a rate five times that of the wild-type XpsE. Yet the mutant XpsE(R286A) is non-functional in protein secretion via T2SS. Detailed analyses indicated that the mutant XpsE(R286A) lost the ability co-ordinating the N- and C-domain of XpsE. Without significantly influencing XpsE binding affinity with ATP or its oligomerization, R286A mutation however, caused XpsE lose the ability to associate with the cytoplasmic membrane via XpsL(N). As a consequence, ATP hydrolysis by XpsE was uncoupled from protein secretion. Because R286 is highly conserved among members of the secretion NTPase superfamily, we speculate that its equivalent in other homologues may also play a critical energy coupling role for T2SS, type IV pilus assembly and type IV secretion system.

XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL.

GspE belongs to a secretion NTPase superfamily, members of which are involved in type II/IV secretion, type IV pilus biogenesis and DNA transport in conjugation or natural transformation. Predicted to be a cytoplasmic protein, GspE has nonetheless been shown to be membrane-associated by interacting with the N-terminal cytoplasmic domain of GspL. By taking biochemical and genetic approaches, we observed that ATP binding triggers oligomerization of Xanthomonas campestris XpsE (a GspE homolog) as well as its association with the N-terminal domain of XpsL (a GspL homolog). While isolated XpsE exhibits very low intrinsic ATPase activity, association with XpsL appears to stimulate ATP hydrolysis. Mutation at a conserved lysine residue in the XpsE Walker A motif causes reduction in its ATPase activity without significantly influencing its interaction with XpsL, congruent with the notion that XpsE-XpsL association precedes ATP hydrolysis. For the first time, functional significance of ATP binding to GspE in type II secretion system is clearly demonstrated. The implications may also be applicable to type IV pilus biogenesis.

Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis.

The major pseudopilin XpsG is an essential component of type II secretion apparatus of Xanthomonas campestris pv. campestris. Along with other ancillary pseudopilins, it forms a pilus-like structure spanning between cytoplasmic and outer membranes. Associations of pseudopilins with non-pseudopilin members of type II secretion apparatus were not well documented, probably due to their dynamic or unstable nature. In this study, by treating intact cells with a cleavable cross-linker dithiobis(succinimidylpropionate) (DSP), followed by metal chelating chromatography and immunoblotting on secretion-positive strains of X. campestris pv. campestris, we discovered associations of XpsGh with XpsN (GspC), as well as XpsD. These associations were detectable in a strain missing all components, but XpsO, of the type II secretion apparatus. However, chromosomal non-polar mutation in each gene exerted different effects upon the association between the other two. The XpsGh/XpsD association is undetectable in xpsN mutant; however, it was restored to a limited extent by overproducing XpsD protein. The XpsGh/XpsN association is unaltered by a lack of XpsD protein or an elevation of its abundance. Co-immune precipitation between XpsN and XpsD, while being independent of XpsG, was nonetheless enhanced by raising XpsG protein level. These observations agree with the proposition that the type II secretion apparatus in a cell may exist as an integrated multiprotein complex with all components working in concert. Moreover, in functional machinery, the association of the major pseudopilin XpsG with secretin XpsD appears strongly dependent on the existence of XpsN, the GspC protein.

Functional dissection of the XpsN (GspC) protein of the Xanthomonas campestris pv. campestris type II secretion machinery.

Type II secretion machinery is composed of 12 to 15 proteins for translocating extracellular proteins across the outer membrane. XpsL, XpsM, and XpsN are components of such machinery in the plant pathogen Xanthomonas campestris pv. campestris. All are bitopic cytoplasmic-membrane proteins, each with a large C-terminal periplasmic domain. They have been demonstrated to form a dissociable ternary complex. By analyzing the C-terminally truncated XpsN and PhoA fusions, we discovered that truncation of the C-terminal 103 residues produced a functional protein, albeit present below detectable levels. Furthermore, just the first 46 residues, encompassing the membrane-spanning sequence (residues 10 to 32), are sufficient to keep XpsL and XpsM at normal abundance. XpsN46(His6), synthesized in Escherichia coli, is able to associate in a membrane-mixing experiment with the XpsL-XpsM complex preassembled in X. campestris pv. campestris. The XpsN N-terminal 46 residues are apparently sufficient not only for maintaining XpsL and XpsM at normal levels but also for their stable association. The membrane-spanning sequence of XpsN was not replaceable by that of TetA. However, coimmunoprecipitation with XpsL and XpsM was observed for XpsN97::PhoA, but not XpsN46::PhoA. Only XpsN97::PhoA is dominant negative. Single alanine substitutions for three charged residues within the region between residues 47 and 97 made the protein nonfunctional. In addition, the R78A mutant XpsN protein was pulled down by XpsL-XpsM(His6) immobilized on an Ni-nitrilotriacetic acid column to a lesser extent than the wild-type XpsN. Therefore, in addition to the N-terminal 46 residues, the region between residues 47 and 97 of XpsN probably also plays an important role in interaction with XpsL-XpsM.

OSTF1: a HD-GL2 family homeobox gene is developmentally regulated during early embryogenesis in rice.

In many eukaryotic organisms, homeobox genes are important regulators that specify the cell fate and body plan in early embryogenesis. In this study, a gene designated OSTF1 (Oryza sativa transcription factor 1) encoding a homeodomain protein in rice was isolated and characterized. The encoded OSTF1, although sharing only approximately 51% sequence identity with other HD-GL2 members, contains four characteristic motifs (an N-terminal acidic region, a homeodomain, a truncated leucine zipper, and a START domain). OSTF1 was detected as a single copy gene in rice. The transcripts were absent in young panicle or mature spikelet before anthesis, but appeared very early in the pollinated grain with a transient profile. In vegetative tissues examined, expression was only detectable in root. In situ hybridization analysis on developing grains revealed that OSTF1 was strongly and uniformly expressed in the embryo at the globular stage and preferentially localized to the protoderm at 3-6 d after pollination. Expression was also detectable in the integument and throughout the endosperm. Although OSTF1 is not closely related to the remaining HD-GL2 members in sequences, this gene exhibits an analogous epidermis-preferential expression pattern.

A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus.

The cytoplasmic membrane proteins XpsL, XpsM and XpsN are components required for type II secretion in Xanthomonas campestris pv. campestris. We performed metal-chelating chromatography to partially purify the His(6)-tagged XpsM (XpsMh)-containing complex. Immunoblot analysis revealed that both XpsL and XpsN co-eluted with XpsMh. The co-fractionated XpsL and XpsN proteins co-immune precipitated with each other, suggesting the existence of an XpsL-XpsM-XpsN complex. Ternary complex formation does not require other Xps protein components of the type II secretion apparatus. Further purification upon size-exclusion chromatography revealed that XpsN is prone to dissociate from the complex. Reassociation of XpsN with the XpsL-XpsMh complex immobilized on a nickel column is more effective than with XpsMh alone. Membrane-mixing experiments suggested that the XpsL-XpsMh complex and XpsN probably dissociate and reassociate in the membrane vesicles. Comparison of the half-lives of the XpsL-XpsMh-XpsN and XpsL-XpsMh complexes revealed that XpsL dissociates from the latter at a faster rate than from the former. Dissociation and reassociation between XpsL and XpsM were also demonstrated with membrane-mixing experiments. A dynamic model is proposed for the XpsL-XpsM-XpsN complex.

XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes.

GspG, -H, -I, -J and -K proteins are members of the pseudopilin family. They are the components required for the type II secretion pathway, which translocates proteins across the outer membrane of Gram-negative bacteria to the extracellular milieu. They were predicted to form a pilus-like structure, and this has been shown for PulG of Klebsiella oxytoca by using electron microscopy. In the present study, we performed biochemical analyses of the XpsG protein of Xanthomonas campestris pv. campestris and observed that it is a pillar-like structure spanning the cytoplasmic and outer membranes. Subcellular fractionation revealed a soluble form (SF) of XpsG, in addition to the membrane form. Chromatographic analysis of SF XpsG in the absence of a detergent indicated that it is part of a large complex (>440 kDa). In vitro studies indicated that XpsG is prone to aggregate in the absence of a detergent. We isolated and characterized a non-functional mutant defective in forming the large complex. It did not interfere with the function of wild-type XpsG and was not detectable in the SF. Moreover, unlike wild-type XpsG, which was distributed in both the cytoplasmic and outer membranes, it appeared only in the cytoplasmic membrane. When wild-type XpsG was co-expressed with His6-tagged XpsH but not with untagged XpsH, SF XpsG bound to nickel and co-eluted with XpsH. This result suggests the presence of other pseudopilin components in the XpsG-containing large-sized molecules.