Analyzing plant stress granules in response to plant viruses

Plant viruses have the ability to redirect host machineries and processes to establish a productive infection. Virus-host interactions lead to the reprogramming of the plant cell cycle and transcriptional controls, inhibition of cell death pathways, interference with cell signaling and protein turnover, and suppression defense pathways. Stress granules (SGs) are structures within cells that regulate gene expression during stress response, e.g. viral infection. In mammalian cells assembly of SGs is dependent on the Ras-GAP SH3-domain–binding protein (G3BP). The C-terminal domain of the viral nonstructural protein 3 (nsP3) of Semliki Forest virus (SFV) forms a complex with mammalian G3BP and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs. The binding domain of nsP3 to HsG3BP was mapped to two tandem ‘FGDF’ repeat motifs close to the C-terminus of the viral proteins. It was speculated that plant viruses employ a similar strategy to inhibit SG function.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Vicerrectorado de Investigación y Programa de doctorado "Biotecnología Avanzada

Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Gαq subunits bound to PLC

Effects of Active Conductance Distribution over Dendrites on the Synaptic Integration in an Identified Nonspiking Interneuron

The synaptic integration in individual central neuron is critically affected by how active conductances are distributed over dendrites. It has been well known that the dendrites of central neurons are richly endowed with voltage- and ligand-regulated ion conductances. Nonspiking interneurons (NSIs), almost exclusively characteristic to arthropod central nervous systems, do not generate action potentials and hence lack voltage-regulated sodium channels, yet having a variety of voltage-regulated potassium conductances on their dendritic membrane including the one similar to the delayed-rectifier type potassium conductance. It remains unknown, however, how the active conductances are distributed over dendrites and how the synaptic integration is affected by those conductances in NSIs and other invertebrate neurons where the cell body is not included in the signal pathway from input synapses to output sites. In the present study, we quantitatively investigated the functional significance of active conductance distribution pattern in the spatio-temporal spread of synaptic potentials over dendrites of an identified NSI in the crayfish central nervous system by computer simulation. We systematically changed the distribution pattern of active conductances in the neuron's multicompartment model and examined how the synaptic potential waveform was affected by each distribution pattern. It was revealed that specific patterns of nonuniform distribution of potassium conductances were consistent, while other patterns were not, with the waveform of compound synaptic potentials recorded physiologically in the major input-output pathway of the cell, suggesting that the possibility of nonuniform distribution of potassium conductances over the dendrite cannot be excluded as well as the possibility of uniform distribution. Local synaptic circuits involving input and output synapses on the same branch or on the same side were found to be potentially affected under the condition of nonuniform distribution while operation of the major input-output pathway from the soma side to the one on the opposite side remained the same under both conditions of uniform and nonuniform distribution of potassium conductances over the NSI dendrite

Gene silencing and the abutilon mosaic virus

Geminiviren richten weltweit einen großen ökologischen und ökonomischen Schaden an. Es gibt ein gesteigertes Interesse, Pflanzenpathogenen entweder durch konservative Mittel oder durch biotechnologische Nutzung von neuentdeckten Phänomenen, wie dem Post-transcriptional Gene Silencing (PTGS) Einhalt zu gebieten. Dies ist vielversprechend, weil PTGS ein pflanzeneigenes Abwehrsystem gegen Pathogene darstellt, das ubiquitär im Pflanzenreich verbreitet ist. Verschiedene PTGS-Auslöser zur Unterdrückung von Geminivirusinfektionen sollten hergestellt und charakterisiert werden. Dazu sollten Silencing-Konstrukte gegen eine Abutilon Mosaic Virus (AbMV)-Infektion entwickelt und eingesetzt werden, um letztendlich Wirtsfaktoren zu charakterisieren, die für eine geminivirale Infektion essentiell sind, zugleich aber die Wirtspflanze nicht in ihrer Entwicklung hemmen. Die DNA des AbMV wurde dazu als Gene Silencing- und Proteinexpressionsvektor umkonstruiert und mit Hilfe des Markergens Phytoendesature (PDS) und des Reportergens Green Fluorescence Protein GFP funktionell überprüft. In die infektiöse Hüllprotein-Deletionsmutante von AbMV wurde ein cDNA-Fragment von Phytoendesaturase (PDS) inseriert. Inokulierte Nicotiana benthamiana-Pflanzen zeigten im Verlauf der Infektion den erwarteten Albino-Phänotyp, der durch PDS-Silencing entsteht. Der offene Leserahmen (ORF) von GFP wurde in die gleiche AbMV-Mutante inseriert und führte zur Expression von GFP unter der Kontrolle des Hüllprotein-Promotors sowohl in Agrobakterium-infiltriertem Gewebe, als auch in systemisch infizierten Geweben, so dass AbMV als Gene Silencing- und Proteinexpressionsvektor genutzt werden kann. Im Hefe-2-Hybrid-System konnte Dr. T. Kleinow nachweisen, dass die N-terminale Domäne des movement protein (MP) von AbMV mit dem C-terminalen Bereich des Chloroplasten-lokalisierten Hitzeschock-Proteins von 70 kDa (cpHsc70) aus Arabidopsis thaliana interagiert. Um zu überprüfen, wie sich ein cpHsc70-knock down Phänotyp auf eine AbMV-Infektion auswirkt, wurde das etablierte AbMV-basierte Silencing-Vektorsystem genutzt. Als Silencing-Phänotyp konnten entlang der Leitgewebe systemisch infizierter N. benthamiana-Blätter kleine weiße Chlorosen beobachtet werden, die auf die Degradation der Chloroplasten zurückgeführt werden konnten. In cpHsc70-gesilencten Pflanzen akkumulierte zudem signifikant weniger single-stranded DNA (ssDNA), als in den Kontroll-Proben, so dass über einen Effekt von cpHsc70-silencing auf virale ssDNA spekuliert werden kann. Eine Interaktion von cpHsc70 mit dem AbMV-Transportkomplex als Grundlage für den Transport von geminiviraler DNA über Membranen wird diskutiert. Über die Eigenschaften des AbMV Transkriptions-Transaktivator Proteins (TrAP) als Silencing-Suppressor ist noch nichts bekannt. Die Funktion als Silencing-Suppressor oder dessen Interferenz mit der Silencing-assoziierten Maschinerie sollte charakterisiert werden, um gezielt Resistenz oder Toleranz zu etablieren. Eine GFP-Expressionskassette wurde zwischen zwei virale Replikationsursprünge inseriert und stabil in eine N. benthamiana-Pflanze als Transgen integriert, so dass durch Infektion mit AbMV ein Transreplikon aus dem Transgen mobilisiert werden konnte, das GFP exprimiert. In AbMV-infizierten Pflanzenzellen konnten GFP-Signale im Leitgewebe beobachtet werden und bestätigten damit die Mobilisierung und Replikation des Transreplikons. In Infiltrationsexperimenten mit Agrobakterien, die die Expression des AbMV Replikation-assoziiertem Proteins (Rep) und TrAP vermitteln, konnte gezeigt werden, dass die Mobilisierung des Transreplikons ausschließlich Rep-induziert ist. Während die Co-Expression von Rep und TrAP, genauso wie Agrobakterium-vermittelte Transfektion mit dem infektiösen AbMV DNA A Plasmid, die Mobilisierung des Transreplikons unterdrückt. Das gut charakterisierte Silencing-Suppressor Protein p19 des Cymbidium ringspot virus, eines nicht verwandten RNA Virus, neutralisierte diesen Effekt, so dass von einer siRNA-vermittelten Unterdrückung der Transreplikation ausgegangen werden konnte. Durch Darstellung der Topoisomere der viralen DNA konnte auf verschiedene Kondensierungszustände geminiviraler Minichromosomen geschlossen werden. Für AbMV konnte festgestellt werden, dass das Chromatin mit fortschreitender Infektionsdauer kondensierter und damit möglicherweise Transkriptions-inaktiver wird. Eine Überexpression von TrAP inhibierte diesen Effekt, was auf eine chromatinmodulierende Eigenschaft hinweist, während eine Funktion als Silencing-Suppressor eher unwahrscheinlich ist.In recent years, geminiviruses have emerged as leading plant pathogens that cause severe crop losses worldwide. They infect a broad range of plant species, including tomatoes, cucurbits, cassava, maize, beans and cotton. Many attempts to generate geminivirus resistance have met with limited success. As a consequence, it is essential to develop new resistance strategies and to understand viral counterdefense mechanism. RNA interference (RNAi), also called post-transcriptional gene silencing (PTGS), is a sequence-specific RNA degradation mechanism that silences a targeted gene. It is part of the natural virus defence system, whereas transcriptional gene silencing (TGS) is thought to be a defence against transposons or other invasive DNA elements. In addition, RNAi is a powerful tool for elucidating gene functions. We have developed a system based on the bipartite geminivirus Abutilon mosaic virus (AbMV) to identify host factors essential for viral infectivity. As a proof of concept, we replaced the AbMV coat protein gene by a fragment of the Nicotiana benthamiana phytoene desaturase (PDS) gene. Extensive PDS silencing was produced during further growth of inoculated plants. To use AbMV as a versatile tool to study gene function in vivo, it is desirable to use it simultaneously as a virus-based plant expression vector for foreign proteins. Plant virus-based vectors as transient gene expression systems are an attractive alternative to conventional breeding and transformation technology. Therefore, the open reading frame (ORF) of mGFP4 was inserted in place of the coat protein, leading to GFP signals in infected cells, giving the opportunity for online monitoring of virus movement through distal parts of a plant, and studying tissue specificity. The analysis confirmed that AbMV existed in tissues no other than the internal and external phloem of vascular bundle. The C-terminal portion of Arabidopisis thaliana cpHsc70-1 was found to interact with the N-terminal region of Abutilon mosaic virus (AbMV) movement protein in a yeast two hybrid assay (Dr. T. Kleinow, pers. communication). Therefore a geminivirus-based gene silencing vector harboring the cpHsc70-fragment was constructed to silence the nuclear encoded and chloroplast-localized heat shock protein of 70 kDa (cpHSC70) in N. benthamiana plants. Systemically infected leaves showed punctated photo-bleached areas similar to those induced by phytoene desaturase (PDS) silencing indicating an interference with chloroplast stability. CpHsc70-silenced plants accumulated less viral DNA, in particular single-stranded DNA (ssDNA), than PDS-silenced or AbMV-infected plants. An involvement of cpHSC70 in geminiviral movement is discussed. An AbMV-based transreplicon was constructed to monitor infection and to identify plant tissues specificity. A green fluorescent protein (GFP) expression cassette driven by the constitutive 35S promoter of Cauliflower mosaic virus (CaMV) was embedded in a truncated AbMV DNA A partial dimer and transferred to N. benthamiana plants as a transgene. Upon AbMV infection, the transreplicon was released and resulted in GFP overexpression. Agroinfiltration of these transgenic plants with a construct that expressed the AbMV replication-associated protein Rep (or AC1) alone showed, that Rep is necessary and sufficient to induce bright GFP fluorescence in the infiltrated area. Co-expression of AbMV Rep and TrAP ( or AC2), a protein that has been identified as transcriptional transactivator as well as silencing suppressor protein for other begomoviruses, surprisingly suppressed transgene transreplication. This effect however, was neutralized by the strong p19 silencing suppressor of the unrelated, RNA-containing Cymbidium ringspot virus (CymRSV) suggesting an involvement of small interfering RNA (siRNA). In order to investigate whether TrAP has an influence on the viral chromatin condensation as a response to silencing, the distribution of topoisomers of monomeric viral circular double-stranded DNA at different stages of infection was visualized. Transreplicon and viral minichromosomes were found to exist in structures dependent on Rep and/or TrAP expression. The topoisomers distribution pattern indicated the influence of AbMV TrAP in altering viral chromatin conformation, whereas suppression of transcriptional gene silencing (TGS) can presumably be excluded

Comparison of two Turnip mosaic virus P1 proteins in their ability to co-localize with the Arabidopsis thaliana G3BP-2 protein

Turnip mosaic virus (TuMV), belonging to the genus Potyvirus (family Potyviridae), has a large host range and consists of a single-stranded positive sense RNA genome encoding 12 proteins, including the P1 protease. This protein which is separated from the polyprotein by cis cleavage at its respective C-terminus, has been attributed with different functions during potyviral infection of plants. P1 of Turnip mosaic virus (P1-TuMV) harbors an FGSF-motif and FGSL-motif at its N-terminus. This motif is predicted to be a binding site for the host Ras GTPase-activating protein-binding protein (G3BP), which is a key factor for stress granule (SG) formation in mammalian systems and often targeted by viruses to inhibit SG formation. We therefore hypothesized that P1-TuMV might interact with G3BP to control and regulate plant SGs to optimize cellular conditions for the production of viral proteins. Here, we analyzed the co-localization of the Arabidopsis thaliana G3BP-2 with the P1 of two TuMV isolates, namely UK 1 and DEU 2. Surprisingly, P1-TuMV-DEU 2 co-localized with AtG3BP-2 under abiotic stress conditions, whereas P1-TuMV-UK 1 did not. AtG3BP-2::RFP showed strong SGs formation after stress, while P1-UK 1::eGFP maintained a chloroplastic signal under stress conditions, the signal of P1-DEU 2::eGFP co-localized with that of AtG3BP-2::RFP. This indicates a specific interaction between P1-DEU 2 and the AtG3BP family which is not solely based on the canonical interaction motifs

Stromuling when stressed!

Stromules are stroma-filled tubules, extruding from the plastid and surrounded by both envelope membranes, but so far, stromules remain enigmatic structures and their function unknown. Stromules can interconnect plastids and have been found to associate with the nucleus, endoplasmic reticulum, Golgi complex, plasma membrane, mitochondria and peroxisomes. This minireview briefly summarizes markers to visualize stromules, inducers of stromules and provides new data about plant virus induced stromules

Fig 1 -

(A) Typical symptoms in a GRBV-infected “Cabernet franc” plant, (B) a model for the translational regulation of GRBV v-sense proteins [5], (C) the three-cornered alfalfa hopper (Spissistilus festinus) (courtesy of Victoria Hoyle), and (D) a coalescent tree for the complete genomes of 163 grabloviruses. PrLV, Prunus latent virus; WvLV, wild Vitis latent virus; horizontal bars: 95% highest posterior density (HPD) ranges for each node (shown with permission: Thompson, 2022).</p