The type-1 ribosome-inactivating protein OsRIP1 triggers caspase-independent apoptotic-like death in HeLa cells.

Ribosome-inactivating proteins (RIPs) are capable of removing a specific adenine from 28S ribosomal RNA, thus inhibiting protein biosynthesis in an irreversible manner. In this study, recombinant OsRIP1, a type 1 RIP from rice (Oryza sativa L.), was investigated for its anti-proliferative properties. Human cervical cancer HeLa cells were incubated in the presence of OsRIP1 for 24-72 h. OsRIP1 treatment yielded an anti-proliferation response of the HeLa cells and resulted in apoptotic-like blebbing of the plasma membrane without causing DNA fragmentation. OsRIP1 labeled with FITC accumulated at the cell surface. Pull-down assays identified ASPP1 (Apoptosis-Stimulating Protein of p53 1) and IFITM3 (interferon-induced transmembrane protein 3) as potential interaction partners for OsRIP1. Transcript levels for several critical genes related to different signaling pathways were quantified by RT-qPCR. OsRIP1 provoked HeLa cells to undergo caspase-independent cell death, associated with a significant transcriptional upregulation of the apoptotic gene PUMA, interferon regulatory factor 1 (IRF1) and the autophagy-related marker LC3. No changes in caspase activities were observed. Together, these data suggest that apoptotic-like events were involved in OsRIP1-driven caspase-independent cell death that might trigger the IRF1 signaling pathway and LC3-mediated autophagy.

Effect of RIP Overexpression on Abiotic Stress Tolerance and Development of Rice.

Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can inhibit protein translation by depurinating rRNA. Most plant RIPs are synthesized with a leader sequence that sequesters the proteins to a cell compartment away from the host ribosomes. However, several rice RIPs lack these signal peptides suggesting they reside in the cytosol in close proximity to the plant ribosomes. This paper aims to elucidate the physiological function of two nucleocytoplasmic RIPs from rice, in particular, the type 1 RIP referred to as OsRIP1 and a presumed type 3 RIP called nuRIP. Transgenic rice lines overexpressing these RIPs were constructed and studied for developmental effects resulting from this overexpression under greenhouse conditions. In addition, the performance of transgenic seedlings in response to drought, salt, abscisic acid and methyl jasmonate treatment was investigated. Results suggest that both RIPs can affect methyl jasmonate mediated stress responses.

OsEUL Lectin Gene Expression in Rice: Stress Regulation, Subcellular Localization and Tissue Specificity.

The Euonymus lectin (EUL) family is a unique group of carbohydrate-binding proteins that is omnipresent in plants. Sequences encoding EUL-related lectins have been retrieved from all completely sequenced plant genomes. The rice (Oryza sativa) genome contains 5 functional EUL genes referred to as OsEULS2, OsEULS3, OsEULD1a, OsEULD1b, and OsEULD2. In this study we focused on the tissue specific expression, stress inducibility and subcellular localization of the rice EULs. Even though the EUL domain sequence is highly conserved among the rice EULs (at least 80% sequence similarity) different biotic and abiotic stress treatments yielded unique responses for the different EULs. Transcript levels for OsEULs were differentially affected by drought and salt stress, ABA treatment, pathogen infection or insect infestation. Analysis of promoter activity revealed differential expression and tissue specificity for the 5 OsEUL genes, with most expression observed in the vascular system of roots and shoots, as well as in the root tips and seeds. At cell level, all OsEULs are located in the nucleus whereas OsEULD1b and OsEULD2 also locate to the cytoplasm. This paper contributes to the functional characterization of the EULs and provides insight in the biological importance of this family of proteins for rice.

Involvement of OsRIP1, a ribosome-inactivating protein from rice, in plant defense against Nilaparvata lugens.

Rice is the most important staple food in the world, but rice production is challenged by several biotic stress factors like viruses, bacteria, fungi and pest insects. One of the most notorious pest insects is Nilaparvata lugens, commonly known as the brown planthopper, which feeds on rice phloem sap and can cause serious damage to rice fields. In order to protect themselves, plants express a wide array of defense proteins such as ribosome-inactivating proteins (RIPs). This study shows that the expression of 'OsRIP1' is highly induced in rice plants infested with N. lugens, with transcript levels more than 100-fold upregulated in infested plants compared to non-infested plants. Furthermore, recombinant OsRIP1 was toxic for brown planthoppers when administered through liquid artificial diet. OsRIP1 inactivated insect ribosomes in vitro, suggesting that its toxicity relates to the enzymatic activity of OsRIP1. Over-expression of OsRIP1 in transgenic rice plants did not affect the performance of insects reared on these plants, most likely due to insufficient concentrations of OsRIP1 in the phloem. The data obtained in this research indicate that OsRIP1 can play a role in plant defense against herbivorous insects.

Structure and Activity of a Cytosolic Ribosome-Inactivating Protein from Rice.

Ribosome-inactivating proteins (RIPs) are cytotoxic enzymes that inhibit protein translation by depurinating ribosomal RNA. Although most plant RIPs are synthesized with leader sequences that sequester them away from the host ribosomes, several RIPs from cereals lack these signal peptides and therefore probably reside in the cytosol near the plant ribosomes. More than 30 RIP genes have been identified in the rice (Oryza sativa spp. japonica) genome, many of them lacking a signal peptide. This paper focuses on a presumed cytosolic type-1 RIP from rice, referred to as OsRIP1. Using 3D modeling it is shown that OsRIP1 structurally resembles other cereal RIPs and has an active site that meets the requirements for activity. Furthermore, localization studies indicate that OsRIP1-eGFP fusion proteins reside in the nucleocytoplasmic space when expressed in epidermal cells of Nicotiana benthamiana or Arabidopsis thaliana suspension cells. Finally, OsRIP1 was recombinantly produced in Escherichia coli and was demonstrated to possess catalytic activity. Interestingly, this recombinant RIP inactivates wheat ribosomes far less efficiently than rabbit ribosomes in an in vitro system. These findings raise some interesting questions concerning the mode of action and physiological role of OsRIP1. This is the first time a RIP from rice is investigated at protein level and is shown to possess biological activity.

Extensive Evolution of Cereal Ribosome-Inactivating Proteins Translates into Unique Structural Features, Activation Mechanisms, and Physiological Roles.

Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can depurinate rRNAs thereby inhibiting protein translation. Although these proteins have also been detected in bacteria, fungi, and even some insects, they are especially prevalent in the plant kingdom. This review focuses on the RIPs from cereals. Studies on the taxonomical distribution and evolution of plant RIPs suggest that cereal RIPs have evolved at an enhanced rate giving rise to a large and heterogeneous RIP gene family. Furthermore, several cereal RIP genes are characterized by a unique domain architecture and the lack of a signal peptide. This advanced evolution of cereal RIPs translates into distinct structures, activation mechanisms, and physiological roles. Several cereal RIPs are characterized by activation mechanisms that include the proteolytic removal of internal peptides from the N-glycosidase domain, a feature not documented for non-cereal RIPs. Besides their role in defense against pathogenic fungi or herbivorous insects, cereal RIPs are also involved in endogenous functions such as adaptation to abiotic stress, storage, induction of senescence, and reprogramming of the translational machinery. The unique properties of cereal RIPs are discussed in this review paper.

Cell cycle-dependent O-GlcNAc modification of tobacco histones and their interaction with the tobacco lectin.

The Nicotiana tabacum agglutinin or Nictaba is a nucleocytoplasmic lectin that is expressed in tobacco after the plants have been exposed to jasmonate treatment or insect herbivory. Nictaba specifically recognizes GlcNAc residues. Recently, it was shown that Nictaba is interacting in vitro with the core histone proteins from calf thymus. Assuming that plant histones - similar to their animal counterparts - undergo O-GlcNAcylation, this interaction presumably occurs through binding of the lectin to the O-GlcNAc modification present on the histones. Hereupon, the question was raised whether this modification also occurs in plants and if it is cell cycle dependent. To this end, histones were purified from tobacco BY-2 suspension cells and the presence of O-GlcNAc modifications was checked. Concomitantly, O-GlcNAcylation of histone proteins was studied. Our data show that similar to animal histones plant histones are modified by O-GlcNAc in a cell cycle-dependent fashion. In addition, the interaction between Nictaba and tobacco histones was confirmed using lectin chromatography and far Western blot analysis. Collectively these findings suggest that Nictaba can act as a modulator of gene transcription through its interaction with core histones.