Claudine M. Carvalho

A New Branch of Endoplasmic Reticulum Stress Signaling and the Osmotic Signal Converge on Plant-specific Asparagine-rich Proteins to Promote Cell Death

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.

Localization and domain characterization of Arabidopsis golgin candidates

Golgins are large coiled-coil proteins that play a role in tethering of vesicles to Golgi membranes and in maintaining the overall structure of the Golgi apparatus. Six Arabidopsis proteins with the structural characteristics of golgins were isolated and shown to locate to Golgi stacks when fused to GFP. Two of these golgin candidates (GC1 and GC2) possess C-terminal transmembrane (TM) domains with similarity to the TM domain of human golgin-84. The C-termini of two others (GC3/GDAP1 and GC4) contain conserved GRAB and GA1 domains that are also found in yeast Rud3p and human GMAP210. GC5 shares similarity with yeast Sgm1p and human TMF and GC6 with yeast Uso1p and human p115. When fused to GFP, the C-terminal domains of AtCASP and GC1 to GC6 localized to the Golgi, showing that they contain Golgi localization motifs. The N-termini, on the other hand, label the cytosol or nucleus. Immuno-gold labelling and co-expression with the cis Golgi Q-SNARE Memb11 resulted in a more detailed picture of the sub-Golgi location of some of these putative golgins. Using two independent assays it is further demonstrated that the interaction between GC5, the TMF homologue, and the Rab6 homologues is conserved in plants.

Regulated Nuclear Trafficking of rpL10A Mediated by NIK1 Represents a Defense Strategy of Plant Cells against Virus

The NSP-interacting kinase (NIK) receptor-mediated defense pathway has been identified recently as a virulence target of the geminivirus nuclear shuttle protein (NSP). However, the NIK1–NSP interaction does not fit into the elicitor–receptor model of resistance, and hence the molecular mechanism that links this antiviral response to receptor activation remains obscure. Here, we identified a ribosomal protein, rpL10A, as a specific partner and substrate of NIK1 that functions as an immediate downstream effector of NIK1-mediated response. Phosphorylation of cytosolic rpL10A by NIK1 redirects the protein to the nucleus where it may act to modulate viral infection. While ectopic expression of normal NIK1 or a hyperactive NIK1 mutant promotes the accumulation of phosphorylated rpL10A within the nuclei, an inactive NIK1 mutant fails to redirect the protein to the nuclei of co-transfected cells. Likewise, a mutant rpL10A defective for NIK1 phosphorylation is not redirected to the nucleus. Furthermore, loss of rpL10A function enhances susceptibility to geminivirus infection, resembling the phenotype of nik1 null alleles. We also provide evidence that geminivirus infection directly interferes with NIK1-mediated nuclear relocalization of rpL10A as a counterdefensive measure. However, the NIK1-mediated defense signaling neither activates RNA silencing nor promotes a hypersensitive response but inhibits plant growth and development. Although the virulence function of the particular geminivirus NSP studied here overcomes this layer of defense in Arabidopsis, the NIK1-mediated signaling response may be involved in restricting the host range of other viruses.

Fluorescence lifetime imaging of interactions between Golgi tethering factors and small GTPases in plants.

Peripheral tethering factors bind to small GTPases in order to obtain their correct location within the Golgi apparatus. Using fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) we visualized interactions between Arabidopsis homologues of tethering factors and small GTPases at the Golgi stacks in planta. Co‐expression of the coiled‐coil proteins AtGRIP and golgin candidate 5 (GC5) [TATA element modulatory factor (TMF)] and the putative post‐Golgi tethering factor AtVPS52 fused to green fluorescent protein (GFP) with mRFP (monomeric red fluorescent protein) fusions to the small GTPases AtRab‐H1b, AtRab‐H1c and AtARL1 resulted in reduced GFP lifetimes compared to the control proteins. Interestingly, we observed differences in GFP quenching between the different protein combinations as well as selective quenching of GFP‐AtVPS52‐labelled structures. The data presented here indicate that the FRET‐FLIM technique should prove invaluable in assessing protein interactions in living plant cells at the organelle level.

Golgi membrane dynamics after induction of a dominant-negative mutant Sar1 GTPase in tobacco

An inducible system has been established in Nicotiana tabacum plants allowing controlled expression of Sar1-GTP and thus the investigation of protein dynamics after inhibition of endoplasmic reticulum (ER) to Golgi transport. Complete Golgi disassembly and redistribution of Golgi markers into the ER was observed within 18-24h after induction. At the ultrastructural level Sar1-GTP expression led to a decrease in Golgi stack size followed by Golgi fragmentation and accumulation of vesicle remnants. Induction of Sar1-GTP resulted in redistribution of the green fluorescent protein (GFP)-tagged Arabidopsis golgins AtCASP and GC1 (golgin candidate 1, an Arabidopsis golgin 84 isoform) into the ER or cytoplasm, respectively. Additionally, both fusion proteins were observed in punctate structures, which co-located with a yellow fluorescent protein (YFP)-tagged version of Sar1-GTP. The Sar1-GTP-inducible system is compared with constitutive Sar1-GTP expression and brefeldin A treatment, and its potential for the study of the composition of ER exit sites and early cis-Golgi structures is discussed.

A novel nucleocytoplasmic traffic GTPase identified as a functional target of the bipartite geminivirus nuclear shuttle protein

SUMMARY In contrast to the accumulated data on nuclear transport mechanisms of macromolecules, little is known concerning the regulated release of nuclear-exported complexes and their subsequent trans-cytoplasmic movement. The bipartite begomovirus nuclear shuttle protein (NSP) facilitates the nuclear export of viral DNA and cooperates with the movement protein (MP) to transport viral DNA across the plant cell wall. Here, we identified a cellular NSP-interacting GTPase (NIG) with biochemical properties consistent with a nucleocytoplasmic transport role. We show that NIG is a cytosolic GTP-binding protein that accumulates around the nuclear envelope and possesses intrinsic GTPase activity. NIG interacts with NSP in vitro and in vivo (under transient expression), and redirects the viral protein from the nucleus to the cytoplasm. We propose that NIG acts as a positive contributor to geminivirus infection by modulating NSP nucleocytoplasmic shuttling and hence facilitating MP-NSP interaction in the cortical cytoplasm. In support of this, overexpression of NIG in Arabidopsis enhances susceptibility to geminivirus infection. In addition to highlighting the relevance of NIG as a cellular co-factor for NSP function, our findings also have implications for general nucleocytoplasmic trafficking of cellular macromolecules.

Conserved threonine residues within the A-loop of the receptor NIK differentially regulate the kinase function required for antiviral signaling.

NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation.

Molecular variability of cowpea mild mottle virus infecting soybean in Brazil

Molecular variability was assessed for 18 isolates of cowpea mild mottle virus (CPMMV, genus Carlavirus, family Betaflexiviridae) found infecting soybean in various Brazilian states (Bahia, Goiás, Maranhão, Mato Grosso, Minas Gerais, Pará) in 2001 and 2010. A variety of symptoms was expressed in soybean cv. CD206, ranging from mild (crinkle/blistering leaves, mosaic and vein clearing) to severe (bud blight, dwarfing, leaf and stem necrosis). Recombination analysis revealed only one CPMMV isolate to be recombinant. Pairwise comparisons and phylogenetic analysis were performed for partial genomes (ORF 2 to the 3’ terminus) and for each ORF individually (ORFs 2 to 6), showing the isolates to be distinct. The topology of the phylogenetic tree could be related to symptoms, but not to the year of collection or geographical origin. Additionally, the phylogenetic analysis supported the existence of two distinct strains of the virus, designated CPMMV-BR1 and CPMMV-BR2, with molecular variations between them.

NSP-Interacting GTPase: A cytosolic protein as cofactor for nuclear shuttle proteins

Despite the significant progress in the identification of essential components of the nuclear transport machinery, some events of this process are still unclear. Particularly, functional information about the release of nuclear-exported macromolecules at the cytoplasmic side of the nuclear pore complex and their subsequent trans-cytoplasmic movement is lacking. Recently, we identified a cytoplasmic GTPase, designated NIG (NSP-interacting GTPase), which may play a relevant role in these processes. NIG interacts in vivo with the geminivirus NSP and promotes the translocation of the viral protein from the nucleus to the cytoplasm where it is redirected to the cell surface to interact with the viral movement protein, MP. Here we position the NIG function into the mechanistic model for the intracellular trafficking of viral DNA and discuss the putative role of NIG in general cellular nucleocytoplasmic transport of nucleic acid-protein complexes. Addendum to: Carvalho CM, Fontenelle MR, Florentino LH, Santos AA, Zerbini FM, Fontes EPB. A novel nucleocytoplasmic traffic GTPase identified as a functional target of the bipartite geminivirus nuclear shuttle protein. Plant J 2008; DOI: 10.1111/j.1365-313X.2008.03556.x.

Distinct repressing modules on the distal region of the SBP2 promoter contribute to its vascular tissue-specific expression in different vegetative organs

The Glycine max sucrose binding protein (GmSBP2) promoter directs vascular tissue-specific expression of reporter genes in transgenic tobacco. Here we showed that an SBP2-GFP fusion protein under the control of the GmSBP2 promoter accumulates in the vascular tissues of vegetative organs, which is consistent with the proposed involvement of SBP in sucrose transport-dependent physiological processes. Through gain-of-function experiments we confirmed that the tissue-specific determinants of the SBP2 promoter reside in the distal cis-regulatory domain A, CRD-A (position −2000 to −700) that is organized into a modular configuration to suppress promoter activity in tissues other than vascular tissues. The four analyzed CRD-A sub-modules, designates Frag II (−1785/−1508), Frag III (−1507/−1237), Frag IV (−1236/−971) and Frag V (−970/−700), act independently to alter the constitutive pattern of −92pSBP2-mediated GUS expression in different organs. Frag V fused to −92pSBP2-GUS restored the tissue-specific pattern of the full-length promoter in the shoot apex, but not in other organs. Likewise, Frag IV confined GUS expression to the vascular bundle of leaves, whereas Frag II mediated vascular specific expression in roots. Strong stem expression-repressing elements were located at positions −1485 to −1212, as Frag III limited GUS expression to the inner phloem. We have also mapped a procambium silencer to the consensus sequence CAGTTnCaAccACATTcCT which is located in both distal and proximal upstream modules. Fusion of either repressing element-containing module to the constitutive −92pSBP2 promoter suppresses GUS expression in the elongation zone of roots. Together our results demonstrate the unusual aspect of distal sequences negatively controlling tissue-specificity of a plant promoter.