Evidence for a disease-resistance pathway in rice similar to the NPR1-mediated signaling pathway in Arabidopsis

The Arabidopsis NPR1/NIM1 gene is a key regulator of systemic acquired resistance (SAR). Over-expression of NPR1 leads to enhanced resistance in Arabidopsis. To investigate the role of NPR1 in monocots, we over-expressed the Arabidopsis NPR1 in rice and challenged the transgenic plants with Xanthomonas oryzae pv. oryzae (Xoo), the rice bacterial blight pathogen. The transgenic plants displayed enhanced resistance to Xoo. RNA blot hybridization indicates that enhanced resistance requires expression of NPR1 mRNA above a threshold level in rice. To identify components mediating the resistance controlled by NPR1, we used NPR1 as bait in a yeast two-hybrid screen. We isolated four cDNA clones encoding rice NPR1 interactors (named rTGA2.1, rTGA2.2, rTGA2.3 and rLG2) belonging to the bZIP family. rTGA2.1, rTGA2.2 and rTGA2.3 share 75, 76 and 78% identity with Arabidopsis TGA2, respectively. In contrast, rLG2 shares highest identity (81%) to the maize liguleless (LG2) gene product, which is involved in establishing the leaf blade-sheath boundary. The interaction of NPR1 with the rice bZIP proteins in yeast was impaired by the npr1-1 and npr1-2 mutations, but not by the nim1-4 mutation. The NPR1-rTGA2.1 interaction was confirmed by an in vitro pull-down experiment. In gel mobility shift assays, rTGA2.1 binds to the rice RCH10 promoter and to a cis-element required sequence-specifically for salicylic acid responsiveness. This is the first demonstration that the Arabidopsis NPR1 gene can enhance disease resistance in a monocot plant. These results also suggest that monocot and dicot plants share a conserved signal transduction pathway controlling NPR1-mediated resistance.     

Rice NRR, a negative regulator of disease resistance, interacts with Arabidopsis NPR1 and rice NH1

Arabidopsis NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR), which confers lasting broad-spectrum resistance. Over-expression of Arabidopsis NPR1 or the NPR1 homolog 1 (NH1) in rice results in enhanced resistance to the pathogen Xanthomonasoryzae pv. oryzae (Xoo), suggesting the presence of a related defense pathway in rice. We investigated this pathway in rice by identifying proteins that interact with NH1. Here we report the isolation and characterization of a rice cDNA encoding a novel protein, named NRR (for negative regulator of resistance). NRR interacts with NPR1 in the NPR1-interacting domain (NI25) consisting of 25 amino acids. NRR also interacts with NH1; however, NI25 was not sufficient for a strong interaction, indicating a difference between the rice and the Arabidopsis proteins. Silencing of NRR in rice had little effect on resistance to Xoo. When constitutively over-expressed in rice, NRR affected basal resistance, age-related resistance and Xa21-mediated resistance, causing enhanced susceptibility to Xoo. This phenotype was correlated with elevated NRR mRNA and protein levels and increased Xoo growth. Over-expression of NRR suppressed the induction of defense-related genes. NRR:GFP (green fluorescent protein) protein was localized to the nucleus, indicating that NRR may act directly to suppress the activation of defense genes. The fact that NRR compromises Xa21-mediated resistance indicates cross-talk or overlap between NH1- and Xa21-mediated pathways.     

APPL Proteins FRET at the BAR: Direct Observation of APPL1 and APPL2 BAR Domain-Mediated Interactions on Cell Membranes Using FRET Microscopy

Background

Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH) domain, and a C-terminal phosphotyrosine binding (PTB) domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.

Methodology

Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET) experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP) FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.

Conclusions

All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2) and heterotypic (i.e., APPL1-APPL2) manner on curved cell membranes. Furthermore, the results of our experiments did not show photoconversion of YFP into a CFP-like species following photobleaching, supporting the use of CFP donor/YFP acceptor FRET pairs in acceptor photobleaching studies.     

Enhanced disease resistance and hypersensitivity to BTH by introduction of an NH1/OsNPR1 paralog

Non‐expresser of pathogenesis‐related genes 1 (NPR1) is the master regulator of salicylic acid‐mediated systemic acquired resistance. Over‐expression of Arabidopsis NPR1 and rice NH1 (NPR1 homolog1)/OsNPR1 in rice results in enhanced resistance. While there are four rice NPR1 paralogs in the rice genome, none have been demonstrated to function in disease resistance. To study rice NPR1 paralog 3, we introduced constructs into rice and tested for effects on resistance to infection by Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight. While over‐expression of NH3 using the maize ubiquitin‐1 promoter failed to enhance resistance, introduction of an extra copy of NH3 driven by its own promoter (nNT‐NH3) resulted in clear, enhanced resistance. Progeny analysis confirms that the enhanced resistance phenotype, measured by Xoo‐induced lesion length, is associated with the NH3 transgene. Bacterial growth curve analysis indicates that bacterial population levels are reduced 10‐fold in nNT‐NH3 lines compared to control rice lines. The transgenic plants exhibit higher sensitivity to benzothiadiazole (BTH) and 2,6‐dichloroisonicotinic acid (INA) treatment as measured by increased cell death. Expression analysis of pathogenesis‐related (PR) genes showed that nNT‐NH3 plants display greatly enhanced induction of PR genes only after treatment with BTH. Our study demonstrates an alternative method to employ a regulatory protein to enhance plant defence. This approach avoids using undesirable constitutive, high‐level expression and may prove to be more practical for engineering resistance.     

Mouse apolipoprotein B editing complex 3 (APOBEC3) is expressed in germ cells and interacts with dead-end (DND1)

Background

The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation of Dnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.

Methodology/Principal Findings

Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites.

Conclusions/Significance

The 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.     

Effects of thioglycolic acid on parthenogenetic activation of Xenopus oocytes

Background

Existing in Permanent-wave solutions (PWS), thioglycolic acid (TGA) is widely used in hairdressing industry for its contribution to hair styling. However, the toxicity of TGA, especially its reproductive toxicity, gradually calls the attention of more and more researchers.

Method

In this work, xenopus oocytes were pretreated with different concentration of TGA, and then activated by calcium ionophore A23187. During culture, the oocytes activation rates were taken note at different time after adding calcium ionophore A23187. At the end of the culture period, the nuclear status was detected under confocal microscope. In addition, some other samples were collected for Western-Blotting analysis.

Result

TGA significantly inhibited the oocytes activation rate and pronuclear formation. It may be resulted from the inhibition of the degradation of p-ERK1, Mos and CyclinB2.

Conclusion

TGA inhibits in vitro parthenogenetic activation of xenopus oocytes with inhibited the degradation of proteins involved in mitogenic-activated protein kinase (MAPK) and maturation-promoting factor (MPF) pathways.     

Salt Induces Expression of RH3.2A, Encoding an H3.2-type Histone H3 Protein in Rice (Oryza sativa L.)

Histone H3 is one of the four histones, along with H2A, H2B, and H4, which form the eukaryotic nucleosome octamer core. In this study, a new gene RH3.2A encoding an H3.2-type histone H3 protein from rice (Oryza sativa L.) was reported. RH3.2A was cloned through RT-PCR from salt-treated rice seedlings. This gene encoded a protein of 136 amino acid residues that were similar to some plant histone H3 proteins reported previously. However, the cDNA sequence of RH3.2A and other rice H3 genes were different. Alignment of RH3.2A encoding protein with other plant histone H3 proteins revealed that three amino acid residues (32, 88, and 91) were markedly different between H3.1-type and H3.2-type proteins. The mRNA expression analysis of RH3.2A revealed that RH3.2A gene was upregulated by salt stress in rice roots and ABA treatment in seedlings. The potential role of RH3.2A during salt stress was discussed.     

Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research

Background

Mice expressing fluorescent proteins in neurons are one of the most powerful tools in modern neuroscience research and are increasingly being used for in vivo studies of neurodegeneration. However, these mice are often used under the assumption that the fluorescent proteins present are biologically inert.

Methodology/Principal Findings

Here, we show that thy1-driven expression of yellow fluorescent protein (YFP) in neurons triggers multiple cell stress responses at both the mRNA and protein levels in vivo. The presence of YFP in neurons also subtly altered neuronal morphology and modified the time-course of dying-back neurodegeneration in experimental axonopathy, but not in Wallerian degeneration triggered by nerve injury.

Conclusions/Significance

We conclude that fluorescent protein expressed in thy1-YFP mice is not biologically inert, modifies molecular and cellular characteristics of neurons in vivo, and has diverse and unpredictable effects on neurodegeneration pathways.     

The Repertoire of Heterotrimeric G Proteins and RGS Proteins in Ciona intestinalis

Background

Heterotrimeric G proteins and regulators of G protein signaling (RGS) proteins are key downstream interacting partners in the G protein coupled receptor (GPCR) signaling pathway. The highly versatile GPCR transmembrane signaling system is a consequence of the coupling of a diverse set of receptors to downstream partners that include multiple subforms of G proteins and regulatory proteins including RGS proteins, among others. While the GPCR repertoire of Ciona intestinalis, representing the basal chordate is known, the repertoire of the heterotrimeric G proteins and RGS proteins is unknown.

Methodology/Principal Findings

In the present study, we performed an in-silico genome-wide search of C. intestinalis for its complement of G proteins and RGS proteins. The identification of several one-to-one orthologs of human G proteins at the levels of families, subfamilies and types and of homologs of the human RGS proteins suggests an evolutionarily conserved structure function relationship of the GPCR signaling mechanism in the chordates.

Conclusions

The C. intestinalis genome encodes a highly conserved, albeit, limited repertoire of the heterotrimeric G protein complexes with the size of subunit types comparable with that in lower eukaryotes.     

Salicylic acid binds NPR3 and NPR4 to regulate NPR1-dependent defense responses

Salicylic acid (SA) is widely recognized as a key player in plant immunity. While several proteins have been previously identified as the direct targets of SA, SA-mediated plant defense signaling mechanisms remain unclear. The Nature paper from Xinnian Dong''s group demonstrates that the NPR1 paralogues NPR3 and NPR4 directly bind SA, and this binding modulates their interaction with NPR1 and thereby degradation of this key positive regulator of SA-mediated defense, shedding important new insight into the mechanism(s) of SA-mediated, NPR1-dependent plant defense signal transduction.Salicylic Acid (SA) and its derivatives (e.g., aspirin) have long been recognized for their medicinal properties as non-steroidal anti-inflammatory drugs and as pain and fever relievers. An increasing number of studies show that SA also can delay and/or prevent the development of several cancers, cardiovascular diseases, and strokes^1,2. While several SA protein targets have been identified in mammalian cells¹, their molecular and physiological modes of action remain unclear. Thus, efforts to dissect SA''s mechanisms of action continue to rely on identifying additional protein targets. Indeed, SA was recently shown to bind and activate AMP-activated protein kinase, helping to explain some of its disease-preventing effects³.SA is naturally produced in plants, and it plays diverse roles in growth, development, and responses to abiotic stresses⁴. Additionally, SA is widely recognized as a key player in multiple layers of plant disease resistance, including basal resistance, effector-triggered immunity (ETI, also termed resistance gene-mediated resistance) and systemic acquired resistance (SAR)⁵. To decipher SA-mediated plant defense signaling mechanisms, several SA-binding proteins (SABPs) have been identified, including a catalase, cytosolic ascorbate peroxidase, chloroplastic carbonic anhydrase, and methyl salicylate esterase. Extensive study of the latter protein revealed its essential role in SAR⁵. However, despite identification of the aforementioned SABPs, SA''s signaling mechanisms remain unclear. Considering SA''s many roles in plants, these SABPs may constitute only a small portion of SA''s targets; moreover, the SA receptor remained to be found.In this context, the Nature paper from Xinnian Dong''s group⁶ represents a major step forward in our understanding of SA signaling mechanisms during plant-pathogen interactions. Dong''s group has been instrumental in characterizing the function of NPR1 (Nonexpresser of Pathogenesis-Related genes 1) in plant defense⁷. While NPR1 is a key player in one of the SA-mediated defense signaling pathways, it does not appear to be an SA receptor as it does not directly bind SA⁶. Instead, SA regulates the conversion of NPR1 from an oligomeric to a monomeric form, which leads to its nuclear translocation⁸. SA also regulates NPR1 phosphorylation, which facilitates NPR1''s recruitment to a Cullin3 (CUL3) E3 ligase and subsequently proteasome-mediated degradation⁹. Now Dong''s group has demonstrated that the NPR1 paralogues NPR3 and NPR4 are adaptor proteins for the CUL3 E3 ligase that specifically target NPR1 for degradation in an SA concentration-dependent manner⁶. Supporting their conclusion, NPR3 and NPR4 contain domains typically found in CUL3 substrate adaptors, and npr3/4 single and double mutants contain elevated levels of NPR1. Furthermore, NPR3 and NPR4 directly interact with NPR1. Strikingly, SA disrupts the NPR1-NPR4 interaction, thereby making NPR1 less susceptible to degradation, whereas SA promotes the NPR1-NPR3 interaction, which makes NPR1 more accessible for degradation (Figure 1). Since NPR4 has high affinity for SA (nanomolar range) while NPR3 has low affinity for SA (micromolar range), low SA levels should reduce NPR1 degradation, whereas high SA levels should enhance it.Open in a separate windowFigure 1NPR1 homeostasis is controlled by SA binding to NPR3/NPR4 in a concentration-dependent manner. At low SA levels (High Susceptibility, left), NPR1 is unavailable to induce defense gene since it is targeted through its binding to NPR4 for degradation in proteasomes. As SA concentration increases after infection (Basal Resistance, middle), SA binds to NPR4 disrupting its interaction with NPR1. Free NPR1 can now play its role in defense gene activation. At very high concentrations (ETI, right), SA levels are sufficient to bind to NPR3 and promote its interaction with NPR1, leading to NPR1 turnover.At the biological level, nuclear accumulation of NPR1 is required for basal defense gene expression, whereas proteasome-mediated turnover is required for ETI, and a combination of NPR1 accumulation and turnover is necessary for SAR development^6,9. The results presented by Fu et al.⁶ suggest that the interplay between NPR1, NPR3/4, and an SA concentration gradient finetunes NPR1 homeostasis and thus helps specify disease resistance. According to their working model, the enhanced susceptibility exhibited by SA-deficient plants is due to unrestricted NPR4 binding to NPR1, which depletes NPR1 due to CUL3^NPR4-mediated degradation⁶. In wild-type plants, low basal SA levels may bind to NPR4, thereby allowing some NPR1 to accumulate to confer basal resistance. Following pathogen infection, recognition of pathogen effectors by plant resistance proteins induces a high level of SA in local infected tissues; in this case, CUL3^NPR3-mediated degradation would allow fast NPR1 turnover, leading to ETI. In systemic tissues, an intermediate level of SA would enable both NPR1 accumulation and turnover, leading to SAR.Clearly, the study by Fu et al.⁶ represents a major step towards elucidating the mechanism(s) of SA perception in programming defense gene expression. However, NPR3 and NPR4 may not be SA receptors in a traditional sense. An increasing body of evidence indicates the existence of SA-dependent, but NPR1-independent defense signal transduction pathways¹⁰, in which NPR3/4 may not participate. In addition, it is unknown whether NPR3/NPR4-mediated SA perception is involved in the diverse roles that this hormone plays in growth and development, or in abiotic stress. Even for NPR1-dependent defense signal transduction, it is unclear whether NPR3/NPR4 are involved in SA''s ability to induce nuclear translocation of NPR1 and/or promote NPR1 phosphorylation to facilitate the proteasome-mediated turnover. Moreover, since SA binding did not affect the gel filtration elution profile of NPR4⁶, the mechanism through which SA binding influences the ability of NPR4 (or NPR3) to bind NPR1 is currently unknown. Thus, many aspects of SA-mediated signaling remain to be explored.     

An SK3 channel/nWASP/Abi-1 complex is involved in early neurogenesis

Background

The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell.

Principal Findings

We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners.

Conclusions

Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins.     

Arabidopsis Zinc Ribbon 3 is the ortholog of yeast mitochondrial HSP70 escort protein HEP1 and belongs to an ancient protein family in mitochondria and plastids

ZR proteins belong to a phylogenetically conserved family of small zinc-ribbon proteins in plastids and mitochondria of higher plants. The function of these proteins is so far unclear. The mitochondrial proteins share sequence similarities with mitochondrial Hsp70 escort proteins (HEP) from Saccharomyces cerevisiae (HEP1) and human. Expression of the mitochondrial ZR protein from Arabidopsis, ZR3, rescued a hep1 knockout mutant from yeast. Accordingly, ZR3 was found to physically interact with mitochondrial Hsp70 from Arabidopsis. Our findings support the idea that mitochondrial and plastidic ZR proteins from higher plants are orthologs of HEP proteins.

Structured summary of protein interactions

ZR3physically interacts with mtHSC70-2 by pull down (View interaction)ZR3physically interacts with mtHSC70-1 by pull down (View interaction)