Erkki Truve

Arabidopsis thaliana myosin XIK is involved in root hair as well as trichome morphogenesis on stems and leaves

Summary.Myosins form a large superfamily of molecular motors that move along actin filaments. The functions of myosins in plant cells are thought to be related to various processes: cell division, movement of mitochondria and chloroplasts, cytoplasmic streaming, rearrangement of transvacuolar strands, and statolith positioning. Class VIII and XI myosins are represented in the Arabidopsis thaliana genome by 4 and 13 potential genes, respectively. The roles of individual class XI myosins and their cellular targets in A. thaliana are still unclear. In this work we implemented a reverse genetic approach to analyse the loss-of-function mutants of XIK, a representative of class XI myosins in A. thaliana. Three different T-DNA insertion mutants in the myosin XIK gene showed similar phenotypes: impaired growth of root hair cells, twisted shape of stem trichomes, and irregular size, branch positioning, and branch expansion of leaf trichomes. Morphometric analysis of mutant seedlings showed that the average length of root hairs was reduced up to 50% in comparison with wild-type root hairs, suggesting an involvement of the class XI myosin XIK in tip growth. On leaves, the proportion of trichomes with short branches was doubleed in mutant plants, and the mutant trichomes possessed a mildly twisted shape. Therefore, we concluded that myosin XIK is involved also in the elongation of stalks and branches of trichomes.

Phenotypes and Functional Effects Caused by Various Viral RNA Silencing Suppressors in Transgenic Nicotiana benthamiana and N. tabacum

RNA silencing suppressor genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. These suppressors were P1 of Rice yellow mottle virus (RYMV), P1 of Cocksfoot mottle virus, P19 of Tomato bushy stunt virus, P25 of Potato virus X, HcPro of Potato virus Y (strain N), 2b of Cucumber mosaic virus (strain Kin), and AC2 of African cassava mosaic virus (ACMV). HcPro caused the most severe phenotypes in both Nicotiana spp. AC2 also produced severe effects in N. tabacum but a much milder phenotype in N. benthamiana, although both HcPro and AC2 affected the leaf tissues of the two Nicotiana spp. in similar ways, causing hyperplasia and hypoplasia, respectively. P1-RYMV caused high lethality in the N. benthamiana plants but only mild effects in the N. tabacum plants. Phenotypic alterations produced by the other transgenes were minor in both species. Interestingly, the suppressors had very different effects on crucifer-infecting Tobamovirus (crTMV) infections. AC2 enhanced both spread and brightness of the crTMV-green fluorescent protein (GFP) lesions, whereas 2b and both P1 suppressors enhanced spread but not brightness of these lesions. P19 promoted spread of the infection into new foci within the infiltrated leaf, whereas HcPro and P25 suppressed the spread of crTMV-GFP lesions.

Myosin inhibitors block accumulation movement of chloroplasts in Arabidopsis thaliana leaf cells

Summary.Chloroplasts alter their distribution within plant cells depending on the external light conditions. Myosin inhibitors 2,3-butanedione monoxime (BDM), N-ethylmaleimide (NEM), and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) were used to study the possible role of myosins in chloroplast photorelocation in Arabidopsis thaliana mesophyll cells. None of these agents had an effect on the chloroplast high-fluence-rate avoidance movement but all of the three myosin inhibitors blocked the accumulation movement of chloroplasts after a high-fluence-rate irradiation of the leaves. The results suggest that myosins have a role in A. thaliana chloroplast photorelocation.

MPB2C, a Microtubule-Associated Plant Factor, Is Required for Microtubular Accumulation of Tobacco Mosaic Virus Movement Protein in Plants

Movement protein binding 2C (MPB2C) is a plant endogenous microtubule-associated protein previously identified as an interaction partner of tobacco (Nicotiana tabacum) mosaic virus movement protein (TMV-MP). In this work, the role of MPB2C in cell-to-cell transport of TMV-MP, viral spread of TMV, and subcellular localization of TMV-MP was examined. To this end, plants with reduced MPB2C levels were generated by a gene-silencing strategy. Local and systemic spread of TMV and cell-to-cell movement of TMV-MP were unimpaired in MPB2C-silenced plants as compared to nonsilenced plants, indicating that MPB2C is not required for intercellular transport of TMV-MP itself or spread of TMV. However, a clear change in subcellular distribution of TMV-MP characterized by a nearly complete loss of microtubular localization was observed in MPB2C-silenced plants. This result shows that the MPB2C is a central player in determining the complex subcellular localization of TMV-MP, in particular its microtubular accumulation, a phenomenon that has been frequently observed and whose role is still under discussion. Clearly, MPB2C mediated accumulation of TMV-MP at microtubules is not required for intercellular spread but may be a means to withdraw the TMV-MP from the cell-to-cell transport pathway.

Myosins XI-K, XI-1, and XI-2 are required for development of pavement cells, trichomes, and stigmatic papillae in Arabidopsis

BackgroundThe positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport.ResultsSimultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils.ConclusionsWe conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes.

Effects of viral silencing suppressors on tobacco ringspot virus infection in two Nicotiana species

This study investigated the effects of silencing suppressors derived from six different viruses (P1, P19, P25, HcPro, AC2 and 2b), expressed in transgenic Nicotiana tabacum and Nicotiana benthamiana plants, on the infection pattern of tobacco ringspot virus (TRSV) potato calico strain. In N. benthamiana, this virus produced an initial infection with severe systemic symptoms, but the infection was strongly reduced within a few weeks as the plant recovered from the infection. P25 and HcPro silencing suppressors effectively prevented recovery in this host, allowing continuous accumulation of the viral RNA as well as of the virus-specific small interfering RNAs, in the systemically infected leaves. In the P1-, P19-, AC2- or 2b-expressing transgenic N. benthamiana, the recovery was not complete. Susceptibility of N. tabacum to this virus was temperature sensitive. At lower temperatures, up to 25 degrees C, the plants became systemically infected, but at higher temperatures, the infections were limited to the inoculated leaves. In these preventative conditions, all silencing suppressor transgenes (except P25, which was expressed at very low levels) allowed the establishment of systemic infections. Very strong and consistent systemic infections were observed in HcPro- and AC2-expressing plants.

AtRLI2 is an endogenous suppressor of RNA silencing.

RNA silencing is a mechanism involved in gene regulation during development and anti-viral defense in plants and animals. Although many viral suppressors of this mechanism have been described up to now, this is not the case for endogenous suppressors. We have identified a novel endogenous suppressor in plants: RNase L inhibitor (RLI) of Arabidopsis thaliana. RLI is a very conserved protein among eukaryotes and archaea. It was first known as component of the interferon-induced mammalian 2′–5′ oligoadenylate (2–5A) anti-viral pathway. This protein is in several organisms responsible for essential functions, which are not related to the 2–5A pathway, like ribosome biogenesis and translation initiation. Arabidopsis has two RLI paralogs. We have described in detail the expression pattern of one of these paralogs (AtRLI2), which is ubiquitously expressed in all plant organs during different developmental stages. Infiltrating Nicotiana benthamiana green fluorescent protein (GFP)-transgenic line with Agrobacterium strains harboring GFP and AtRLI2, we proved that AtRLI2 suppresses silencing at the local and at the systemic level, reducing drastically the amount of GFP small interfering RNAs.

Sobemoviruses possess a common CfMV-like genomic organization.

Summary.Based on structural differences in the ORF2 region, the sobemoviruses have been subdivided into southern cowpea mosaic virus (SCPMV)-like and cocksfoot mottle virus (CfMV)-like types of genome organization. However, nearly identical amino acid sequences are encoded by these subgroups in different reading frames of ORF2, suggesting that insertion or deletion of appropriate nucleotides could restore similar genomic organizations for these viruses. We resequenced the regions of inconsistency for isolates of four SCPMV-like viruses: lucerne transient streak virus, ryegrass mottle virus, southern bean mosaic virus, and SCPMV. A comparison of nucleic acid composition of these sequences with previously published ones revealed crucial differences that established a common CfMV-like genomic organization for these sobemoviruses.

RNA-binding activities of cocksfoot mottle sobemovirus proteins.

Cocksfoot mottle virus (CfMV) has a positive-sense ssRNA genome containing four open reading frames (ORFs). ORF1 encoded protein (P1) is the putative movement protein; the product of ORF2a (P2a) contains VPg and the motifs characteristic of serine proteases. P2b, encoded by ORF2b, is the putative RNA-dependent RNA polymerase. P3, the coat protein, is encoded by ORF3. CfMV P1, P2a, P2b, and P3, containing a six histidine tag at the amino terminus, were expressed in Escherichia coli, purified and their RNA-binding activities were analysed. The northwestern blot assay showed that His-tagged P1, P2a, P2b, and P3 were able to interact with ssRNA transcripts in a sequence-nonspecific manner. The filter-binding assay confirmed the ssRNA-binding capacity of recombinant P1, P2a, and P3. The RNA-binding activities of His-tagged P3 and native coat protein were similar. P1 and P2a binding to ssRNA decreased markedly by increasing NaCl concentrations. In contrast, P3 had the RNA-binding optimum at 100-200 mM NaCl. We discuss the possible amino acid motifs involved in the RNA-binding of CfMV proteins.

P1 Protein of Cocksfoot Mottle Virus is Indispensable for the Systemic Spread of the Virus

Cocksfoot mottle sobemovirus (CfMV) encodes a non-conserved protein P1 from the 5′ ORF1 of genomic RNA. The functions of CfMV P1 are unknown. In the current study we show that P1-deficient CfMV can replicate both in oat leaves and barley suspension culture cells but can not infect oat plants systemically. However, the absence of P1 reduces the efficiency of virus accumulation considerably. The infectivity of the mutant virus restores as a result of the spontaneous transversion. CfMV P1:EGFP shows a very limited cell-to-cell movement in leaf epidermal cells. In Sf9 insect cells CfMV P1 localizes in the fraction of membranes and inclusions but not in soluble cytoplasmic protein fraction.