Plant biology

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Plant biology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in plant biology. Current Opinion in Plant Biology 2000, 3:1–9 Contents (chosen by) 1 Growth and development (Scheres and Höfte) 2 Genome studies and molecular genetics (Lemieux and

Grossniklaus) Plant biotechnology (Dunwell) Physiology and metabolism (Hill and Sweetlove) Plant–microbe interactions (Metraux) Cell signalling (McAinsh and Palme) Gene regulation (Weisshaar) Cell biology (Berger)

4 4 5 6 8 8 • ••

of special interest of outstanding interest

Growth and development Selected by Ben Scheres* and Herman Höfte† *Utrecht University, Utrecht, The Netherlands †INRA, Versailles, France

Co-ordinated polar localisation of auxin efflux carrier PIN1 by GNOM ARF GEF. Steinmann T, Geldner N, Grebe M, Mangold S, Jackson CL, Paris S, Gälweiler L, Palme K, Jürgens G: Science 1999, 286:316-318. •• Significance: Arabidopsis gnom mutants display defects in the establishment of a polar axis. Biochemical evidence in this paper implicates the GNOM protein in membrane trafficking. The failure of gnom mutants to localize the PIN FORMED 1 (PIN1) transmembrane protein, a component of an auxin efflux carrier, in a consistent polar fashion may explain how gnom polarity defects result from altered auxin distribution. Findings: The polar localization of PIN1 was coordinated between cells in wild-type but not gnom mutant embryos. The GNOM protein displayed brefeldin A (BFA)-sensitive guanosine-exchange activity on mammalian ADP ribosylation factor G protein (ARF), consistent with a role in protein trafficking, and GNOM co-purified with membrane fractions in a BFA-dependent fashion. BFA-disrupted polar PIN1 protein localization in emerging lateral roots, suggesting that ARF guanosinenucleotide exchange factors (GEFs), like GNOM, are continuously required for the polar localization of auxin transporters. An epigenetic mutation responsible for natural variation in floral symmetry. Cubas P, Vincent C, Coen E: Nature 1999, 401:157-161. AND

Control of organ asymmetry in flowers of Antirrhinum. Luo D, Carpenter R, Copsey C, Vincent C, Clark J, Coen E: Cell 1999, 99:367-376. • Significance: The snapdragon cycloidea (cyc) gene is involved in the establishment of floral asymmetry. In the first

paper, it is shown that a naturally occurring radial-symmetric floral mutant of toadflax (Linaria vulgaris) is caused by the transcriptional silencing of a cyc homologue. Thus, epigenetic mutations may play a significant role in natural morphological variation. In the second paper, it is shown that the snapdragon cyc gene cooperates with the homologous dichotoma (dich) gene to establish dorsal fates, and that the dich gene specifies a dorsal sub-domain. Findings: Wild-type flowers of toadflax display adaxial–abaxial (dorsoventral) asymmetry. Natural mutants, which are radially symmetrical, show methylation-dependent RFLPs within a cyc homologue, Lcyc. Lcyc is expressed in adaxial regions of wildtype flowers but is absent from mutant flowers. Nevertheless, sequencing revealed no nucleotide differences between the wild-type and the mutant. The snapdragon dich gene encodes a protein with 77% homology to cyc. It is expressed, like cyc, in the dorsal domain, but becomes restricted to the most dorsal part of this domain. Ectopic expression of cyc dorsalizes lateral and ventral petals in the backpetals mutant, but these petals do not acquire the dich-associated dorsal-most characteristics. These findings support the idea that cyc and dich specify distinct dorsal subdomains. Distinct mechanisms promote polarity establishment in carpels of Arabidopsis. Eshed Y, Baum SF, Bowman JL: Cell 1999, 99:199-209. •• Significance: At least three Arabidopsis YABBY genes are involved in the specification of abaxial cell fate in above-ground organs. This function was not, however, evident for the founding member of this family of transcription factor-encoding genes, CRABS CLAW (CRC), which is required for proper carpel development. In this paper, a role for CRC in adaxial–abaxial polarity is revealed through the analysis of a genetic enhancer, the GYMNOS (GYM) gene. Findings: Arabidopsis gym ; crc mutants display ectopic ovule formation and other phenotypes that are indicative of adaxialization of carpels. gym mutations alone delay maturation of the carpels and other tissues, and so CRC and GYM are redundantly required for abaxialization. The additional observation that CRC over-expression promotes abaxialization is consistent with these findings. GYM encodes a member of the SWI2/SNF1 chromatin-remodeling factor family and is expressed in all dividing cells. GYM seems to prevent carpel adaxialization in a crc background by repression of AINTEGUMENTA, which is thought to be required for primordium outgrowth and thus required for the formation of ectopic adaxial ovules in crc mutants. A plant regulator controlling development of symbiotic root nodules. Schauser L, Roussis A, Stiller J, Stougaard J: Nature 1999, 402:191-195. •• Significance: This is the first example of the cloning of a central regulator of symbiotic nitrogen-fixing root nodules. This paper also demonstrates the feasibility of the use of transposon mutagenesis to clone genes from Lotus japonicus. Findings: Nodule-less (nin, or nodule inception) mutants were identified in an Ac transposon-mutagenised Lotus population.

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These mutants still recognise bacteria, as indicated by the curling of the root hairs, but are unable to produce infection threads or to initiate nodule primordia. The NIN gene encodes a putative transcription factor of the bZIP or bHLH/Z-type similar to the Chlamydomonas gene MID, which controls mating-type in nitrogen-limiting conditions. NIN mRNA is expressed at a low level in roots and is upregulated in growing and mature nodules. A species of small antisense RNA in posttranscriptional gene silencing in plants. Hamilton AJ, Baulcombe DC: Science 1999, 286:950-952. AND

The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A, Mello CC: Cell 1999, 99:123132. •• Significance: Posttranscriptional gene silencing (PTGS) is a sequence-specific defence mechanism that can target viral and cellular mRNAs. In the first paper, it is shown convincingly that transgene- or virus-induced PTGS in plants is correlated with the presence of antisense RNA of a uniform length of about 25 nt that is complementary to the targeted mRNA. This antisense RNA is probably de novo synthesised. It is large enough to be a specificity determinant and short enough to migrate through plasmodesmata thereby signalling from cell to cell. In the second paper, mutants defective in RNA interference (RNAi) were identified in C. elegans. RNAi is a PTGS-like phenomenon induced by dsRNA. One of the genes, RDE-1 encodes a member of the piwi/sting/argonaute/zwille/eIF2C gene-family that is conserved between plants and animals. These observations lead to the following interesting possibilities: first, PTGS in plants and RNAi in animals may involve similar molecular mechanisms, and second, the inability to maintain stem cells in the meristem of zll mutants, and related defects in ago, may be a direct consequence of a defect in PTGS. This evidence supports the attractive idea that PTGS may also play a role in cell-to-cell signalling during plant development. Findings: Three types of transgene-induced PTGS and one virus-induced PTGS were analysed in different Solanaceous species. A 25 nt antisense RNA was found in each case. A screen for mutants that are resistant to dsRNA-induced RNAi in C. elegans identified mutants in two loci that showed a strong resistance to RNAi, but had no other developmental defects. Among the RDE-1 homologs, STING in Drosophila is also thought to be involved in PTGS. PIWI and ZLL are required to maintain the status of stem cells in Drosophila and the Arabidopsis meristem, respectively. Whether ago and zwi also show defects in PTGS, and whether PTGS is the basis of these developmental defects, remains to be determined. Imprinting of the MEDEA polycomb gene in the Arabidopsis endosperm. Kinoshita T, Yadegari R, Harada JJ, Goldberg RB, Fischer RL: Plant Cell 1999, 11:1945-1952. • Significance: This paper confirms that paternal alleles of MEDEA (MEA), a gene required for normal embryo and endosperm development, are specifically silenced in the Arabidopsis endosperm but not in the embryo and other tissues. This supports the parental conflict theory, which predicts that a gene that suppresses endosperm development and restricts nutrient flow to the embryo (as would be the case for MEA) is preferentially expressed by a maternal allele.

Findings: Loss-of-function mutants for the polycomb proteinencoding MEA gene show an abortion of the embryo and excessive endosperm proliferation. It has been shown previously that only the maternal allele determines the phenotype, irrespective of the paternal allele. Using a reverse transcription PCR strategy, the expression of paternal and maternal MEA alleles was followed in the progeny of crosses between two ecotypes.

Genome studies and molecular genetics Selected by Bertrand Lemieux* and Ueli Grossniklaus† *University of Delaware, Newark, Delaware, USA †Friedrich Miescher Institute, CH–4002, Basel, Switzerland

Identification of candidate coding region single nucleotide polymorphisms in 165 human genes using assembled expressed sequence tags. Garg K, Green P, Nickerson DA: Genome Res 1999, 9:1087-1092. • Significance: This analysis reveals that assembled expressed sequence tags (ESTs) from multiple libraries may provide a rich source of comparative sequences to search for single nucleotide polymorphisms (cSNPs) within coding regions in the human genome. Similar information should be present in plant EST databases. Findings: The authors generated contigs that represent the complete coding sequences of 850 known human genes to scan them for single nucleotide substitutions. A total of 200, one candidate single nucleotide polymorphisms were found in the coding sequences (cSNPs) of 165 of these genes. The authors estimate that the average number of differences between any pair of chromosomes is 3 per 10 000 bp in coding regions of the genome. Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome. Rabinowicz PD, Schutz K, Dedhia N, Yordan C, Parnell LD, Stein L, McCombie WR, Martienssen RA: Nat Genet 1999, 23:305-308. •• Significance: The use of restrictive libraries in plant genome shotgun sequencing should allow significant representation of genes, reducing the number of reactions required. This report also demonstrates that repeats are the primary targets of DNA methylation in maize. Findings: The maize genome is composed of a relatively small number of genes and a large fraction of repetitive DNA. The bulk of this repetitive DNA originates from transposable and retrotransposable elements. The authors report that maize repeat sequences can be largely excluded from genomic shotgun libraries by the selection of an appropriate host strain. The authors hypothesize that this coding sequence enrichment is due to the sensitivity of methylated sequences to bacterial restriction—modification systems. Indeed, it has been suggested that most of these repeat elements in the maize genome are heavily methylated relative to gene-coding sequences. The authors suggest that genetic filtration of libraries is possible when the insert size of the clones is less than the average size of genes. A genetic strategy to eliminate self-activator baits prior to high-throughput yeast two-hybrid screens. Walhout AJ, Vidal M: Genome Res 1999, 9:1128-1134. • Significance: The development of a more robust yeast twohybrid system is essential for the creation of reliable protein

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interaction maps and may provide essential clues to protein functions. Findings: The authors report a novel genetic strategy that eliminates self-activator baits prior to screening using the yeast two-hybrid system. In the two-hybrid system, interaction between two proteins, expressed as fusions with a DNA binding domain and an activation domain, respectively, reconstitutes a functional transcription factor. This factor activates the expression of a reporter gene, leading to a selectable phenotype. Unfortunately, a high percentage of cloned sequences encode polypeptides that, when fused to a DNA binding domain, can activate transcription in the absence of any twohybrid-interacting partner protein. These self-activator baits are an inherent problem of large-scale two-hybrid system based screens for protein—protein interactions. The strategy developed by the authors is based on a negative-growth selection that eliminates the latter. A species of small antisense RNA in posttranscriptional gene silencing in plants. Hamilton AJ, Baulcombe DC: Science 1999, 286:950-952. •• Significance: The presence of these 25 nucleotide (nt) antisense RNAs could be used as a means of detecting plants that will undergo posttranscriptional gene silencing (PTGS). Findings: The authors report that three types of transgeneinduced PTGS and one example of virus-induced PTGS in plants seem to be associated with 25 nt antisense RNA molecules. These molecules were detected in transgenic plants containing an endogenous gene, 1-aminocyclopropane-1-carboxylate oxidase, under the control of the 35S promoter. The small RNA molecules were also involved in two cases of cosuppression and in transgenic plants carrying 35S-glucuronidase (GUS) transgenes. Considering that in some examples of PTGS silencing is initiated in a localized region of the plant, it has been suggested that a signal molecule is produced at the site of initiation and mediates a systemic spread of silencing to other tissues. This systemic PTGS was tested with a green fluorescent protein (GFP) transgene. It too, was associated with 25 nt GFP antisense RNA. Finally, the 25 nt RNA was detected in virus-infected plants four days after inoculation of Nicotiana benthamiana and continued to accumulate for at least another six days in the inoculated leaf. This RNA was not detected in mock-inoculated leaves. As the presence of these RNA molecules required either transgene sense-transcription or RNA virus-replication to be present, the authors conclude that these molecules are probably synthesized from an RNA template and that they may convey specificity to PTGS. Genome rearrangements by nonlinear transposons in maize. Zhang J, Peterson T: Genetics 1999, 153:1403-1410. • Significance: Transposons are considered important mediators of genome reorganization as they induce chromosomal rearrangements such as deletions, duplications, inversions and reciprocal translocations. Although McClintock described the occurrence of chromosomal rearrangements involving Ds that were dependent on Ac activity almost 50 years ago, only the chromosome breakage induced by ‘double Ds’ elements has been investigated at the molecular level. This paper shows that a transposition event, which involved transposon ends located on different DNA molecules (nonlinear transposition), created a large chromosomal rearrangement. Findings: Chromosomal rearrangements derived from an unstable allele of the maize P1 (pericarp color) gene were iso-

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lated from a twinned sector on a maize ear. The progenitor allele contained a full-length Ac transposon and an Ac terminal fragment (fractured Ac, fAc). The two rearranged alleles contained a deficiency and the corresponding inverted duplication, respectively. Molecular analysis of the rearrangement breakpoints showed that the derived alleles were generated by a single transposition event involving the ends of Ac and fAc that were located on sister chromatids. Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity. Vielle-Calzada J-P, Thomas J, Spillane C, Coluccio A, Hoeppner MA, Grossniklaus U: Genes Dev 1999, 13:2971-2982. AND

Imprinting of the MEDEA Polycomb gene in the Arabidopsis endosperm. Kinoshita T, Yadegari R, Harada JJ, Goldberg RB, Fischer RL: Plant Cell 1999, 11:1945-1952. •• Significance: MEDEA (MEA) is one of a few genes that display a gametophytic maternal effect. Seeds derived from a female gametophyte carrying a mutant mea allele abort irrespective of the paternal contribution. These papers report that the mea maternal effect is due to the regulation of MEA by genomic imprinting. The first paper also identifies a zygotically required transacting factor of imprinting, DDM1 (DECREASE IN DNA METHYLATION1). As DDM1 encodes a putative chromatin-remodelling factor and genome-wide methylation levels drop by 70% in its absence, methylation and/or chromatin structure are likely to be interrelated with genomic imprinting in Arabidopsis. Findings: MEA is expressed in the female gametophyte prior to fertilization, and at high levels in the embryo and endosperm during seed development, indicating that MEA is expressed both maternally and zygotically. The zygotic expression of MEA was investigated, using a combination of highly sensitive in situ hybridization, which allows the visualization of active transcription sites at the mea locus, and allele-specific reverse transcription PCR. Maternally inherited MEA alleles were found to be transcribed after fertilization, whereas the paternally inherited alleles are silenced. During the early stages of development, this epigenetic regulation occurs in both the embryo and the endosperm. Later during seed development, the paternally inherited allele is detectable in the embryo but usually not in the endosperm suggesting that the imprint at the mea locus is differentially stable in embryo and endosperm. Finally, it was shown that seeds that inherit a mutant mea copy from the female parent can survive in the absence of DDM1 activity suggesting that zygotic MEA activity is sufficient to ensure normal seed development. DDM1 was shown to be required for the maintenance of the imprint at the mea locus but not for its establishment. The structure and paramutagenicity of the R-marbled haplotype of Zea mays. Panavas T, Weir J, Walker EL: Genetics 1999, 153:979-991. • Significance: Paramutation is an epigenetic phenomenon that leads to meiotically heritable silencing of an allele in particular heterozygous combinations. For instance, a paramutable allele of the r (red color) locus such as R-r, becomes heritably silenced after exposure to the paramutagenic allele R-mb (Rmarbled). This paper reports the structure and paramutation properties of the R-mb allele. Importantly, transposons at R-mb do not play a significant role in paramutagenicity as has been previously suggested.

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Findings: Molecular studies showed that the R-mb allele is comprised of three r1 genes arranged as direct repeats. Crossover derivatives with fewer copy numbers were isolated and tested for their paramutagenic strength. Paramutagenicity correlated directly with r1 copy number as Kermicle had previously reported for another paramutagenic allele, R-stippled. A novel transposon, Shooter, was found to be responsible for the marbled phenotype (excisions from the distal-most r1 gene) but its removal does not affect paramutagenic strength. Finally, no Doppia elements were found at R-mb suggesting that transposons are not required for paramutagenicity, a possible role that has previously been attributed to Doppia.

Plant biotechnology Selected by Jim Dunwell University of Reading, Reading, Berkshire, UK

Biosynthetic origin of conjugated double bonds: production of fatty acids of high-value drying oils in transgenic soybean embryos. Cahoon EB, Carlson TJ, Ripp KG, Schweiger BJ, Cook GA, Hall SE, Kinney AJ: Proc Natl Acad Sci USA 1999, 96:12935-12940. • Significance: This new method describes how transgenic soybean can be used to produce a high-value commercial product for use in paints, varnishes and inks. Findings: Genes encoding ∆12-oleic acid desaturase were isolated from Momordica charantia and Impatiens balsamina, and introduced into transgenic soybean. Embryos from the transgenic plants accumulated α-eleostearic and α-parinaric acid at levels up to 17% (wt/wt) of total fatty acids; neither of these compounds were found in non-transformed plants. Green fluorescent protein as a marker for expression of second gene in transgenic plants. Harper BK, Mabon SA, Leffel SM, Halfhill MD, Richards HA, Moyer KA, Stewart CN: Nat Biotechnol 1999, 17:1125-1129. • Significance: This improved method may be of value in monitoring the presence and expression of transgenes in field crops. Findings: The authors used a green fluorescent protein (GFP) gene linked to an insecticidal Bt cry1Ac gene to produce transgenic tobacco and oilseed rape. Under field conditions, the amount of GFP fluorescence predicts the expression level of the Bt gene. Plants expressing the GFP gene showed no evidence of any yield penalty. These results suggest that the easily assayed GFP system could be used to predict the expression level of any linked gene that is otherwise is difficult or inconvenient to assay.

Physiology and metabolism Selected by Steven Hill and Lee Sweetlove University of Oxford, Oxford, UK

Sucrose synthase activity does not restrict glycolysis in roots of transgenic potato plants under hypoxic conditions. Biemelt S, Hajirezaei MR, Melzer M, Albrecht G, Sonnewald U: Planta 1999, 210:41-49. • Significance: The role of sucrose synthase activity in hypoxia tolerance is explored. Findings: The response of potato roots to hypoxia included induction of both the enzymes required for ethanolic fermentation and sucrose synthase activity. In order to further investigate the role of sucrose synthase the response of transgenic plants with reduced activity of this enzyme was also studied. There was no apparent effect of low sucrose synthase activity on

glycolysis under hypoxia, and ATP contents were unaltered. The growth rate of the transgenic plants after their return to air was slower than the wild-type, suggesting that sucrose synthase is important for recovery from hypoxia. Increased resistance to oxidative stress in transgenic tobacco plants overexpressing bacterial serine acetyltransferase. Blaszczyk A, Brodzik R, Sirko A: Plant J 1999, 20:237-243. • Significance: This work reveals an important control step in sulphur metabolism. Findings: Transgenic tobacco plants that had elevated serine acetyltransferase activity were produced. These plants contained increased concentrations of cysteine and glutathione, that provide evidence of a role for serine acetyltransferase in controlling sulphur assimilation rate. These transgenic plants show increased resistance to hydrogen peroxide treatment, which implies that glutathione concentration is important in responses to oxidative stress. The nitrate and ammonium nitrate supply have a major influence on the response of photosynthesis, carbon metabolism, nitrogen metabolism and growth to elevated carbon dioxide in tobacco. Geiger M, Haake V, Ludewig F, Sonnewald U, Stitt M: Plant Cell Environ 1999, 22:11771199. • Significance: This paper is an important advance in our understanding of plant responses to elevated CO2. Findings: The effect of elevated CO2 on the growth and metabolism of tobacco plants was studied over a range of nitrogen supplies. Many of the effects of elevated CO2 are altered when the nitrogen status of the plant is varied. In particular, it is demonstrated that photosynthetic acclimation to elevated CO2 results, at least in part, from nitrogen limitation rather than from an excess of carbohydrate. A defect in β-oxidation causes abnormal inflorescence development in Arabidopsis. Richmond RA, Bleecker AB: Plant Cell 1999, 11:1911-1923. •• Significance: This work reveals the role of β-oxidation of fatty acids in the regulation of inflorescence development. Findings: The abnormal inflorescence 1 (aim1) mutation of Arabidopsis was isolated on the basis of its morphological phenotype: after the transition to flowering the apical meristem becomes disorganised and produces abnormal flowers that have reduced fertility. The AIM1 gene was cloned by T-DNA tagging and shows substantial homology to genes encoding the multifunctional protein that catalyses the β-oxidation of fatty acids. The AIM1 protein, produced in E. coli was shown to have enoyl-CoA hydratase activity. Further evidence of the role of AIM1 in β-oxidation is the altered fatty acid composition of the leaves of aim1 plants, and the insensitivity of aim1 plants to the herbicide 2,4-diphenoxybutyric acid, whose action is dependent on β-oxidation of fatty acids. The effect of elevated concentrations of fructose-2,6-bisphosphate on carbon metabolism during deacidification in the crassulacean acid metabolism plant Kalanchöe daigremontiana. Truesdale MR, Toldi O, Scott P: Plant Physiol 1999, 121:957-964. •• Significance: This is the first use of a transgenic approach to investigate the metabolism of a crassulacean acid metabolism (CAM) plant.

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Findings: Transgenic Kalanchöe daigremontiana plants that overexpressed a cDNA encoding 6-phosphofructo-2-kinase from rat liver were produced to investigate the role of fructose2,6-bisphosphate (F26BP) in the regulation of deacidification. The transgenic plants had elevated F26BP contents that led to increased starch synthesis and decreased sucrose synthesis during photosynthesis. The rate of malate turnover was, however, unaltered in the transgenic plants. These results suggest that F26BP regulates photosynthetic partitioning in a similar way in both CAM and C3 plants, but does not influence the CAM cycle.

Plant–microbe interactions Selected by Jean-Pierre Metraux University of Fribourg, Fribourg, Switzerland

A novel class of eukaryotic zinc-binding proteins is required for disease resistance signaling in barley and development in C. elegans. Shirasu K, Lahaye T, Tan MW, Zhou F, Azevedo C, Schulze-Lefert P: Cell 1999, 99:355-366. •• Significance: A component of a zinc-binding protein was characterized that operates in resistance gene signaling as well as in development. Findings: Barley rar1 mutants are compromised in their pathogen-induced accumulation of H2O2 and cell death. Rar1 is a convergence point in the signaling of resistance to the powdery mildew fungus that is triggered by a number of race-specific (R) resistance genes. Rar1 functions upstream of H2O2, preceeding cell death at the site of infection. Map-based cloning of Rar1 revealed a gene that encodes a 25-kDa protein which includes two copies of a 60-amino acid cystein and histidine-rich domain (CHORD). This autonomous Zn2+-binding domain is conserved in tandem organisation in protozoa, metazoa and plants. Silencing of a CHORD-containing gene in nematodes leads to semisterility and embryo lethality, suggesting an essential function of the wild-type protein in nematode development. These exciting results indicate that R signaling in plants relies on a gene that has very important common elements across phyla.

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Osbourn AE: Proc Natl Acad Sci USA 1999, 96:1292312928. •• Significance: For the first time, direct evidence is presented to show that triterpenoid secondary metabolites that are constitutively present in healthy oat plants play a major contribution in pathogen resistance. Findings: Plants accumulate a large variety of antimicrobial secondary metabolites (phytoanticipins), and their relation to plant defense against pathogens has been the subject of debate for a long time. It was tacitly assumed that they represent a preformed chemical barrier to fungal attack. The authors of this paper have isolated mutants of the diploid oat Avena strigosa that are deficient in the oat phytoanticipin avenacin. These sad (saponin-deficient) mutants are susceptible to a variety of fungal pathogens and it is shown that this susceptibility results directly from their saponin deficiency. It appears that saponins, a class of widespread phytoanticipins, are likely to represent a significant barrier to phytopathogenic fungi. Arabidopsis thaliana PAD4 encodes a lipase-like gene that is important for salicylic acid signaling. Jirage D, Tootle TL, Reuber TL, Frost LN, Feys BJ, Parker JE, Ausubel FM, Glazebrook J: Proc Natl Acad Sci USA 1999, 96:1358313588. •• Significance: The control of multiple expression of salicylic acid-dependent defense responses is mediated by a lipase-like gene. Findings: Previously isolated Arabidopsis pad4 mutants show a reduced accumulation of the phytoalexin camalexin, salicylic acid (SA) and the pathogenesis-related protein PR-1 after infection with the pathogenic bacterium Pseudomonas syrinage pv maculicola. PAD4 was isolated by positional cloning and the predicted amino acid sequence shows similarity to eukaryotic lipases and esterases. PAD4 transcripts were found to accumulate after P. syringae infection independently of the regulatory protein NPR1. In contrast, PAD4 expression induced by SA shows NPR1-dependency. PAD4 is postulated to participate in a positive regulatory loop leading to increased SA levels that activate SA-dependent defense responses.

A species of small antisense RNA in posttranscriptional gene silencing in plants. Hamilton AJ, Baulcombe DC: Science 1999, 286:950-952. •• Significance: A long sought-after antisense RNA that could account for posttranscriptional gene silencing was discovered in silenced plant tissues. It takes the form of a 25 nt antisense RNA. Findings: Posttranscriptional gene silencing (PTGS) is a plant defense mechanism that acts against viral DNA or DNA introduced by transformation. PTGS is explained by the formation of an antisense RNA that binds with the target RNA, thereby inhibiting its translation or promoting its degradation. Thus far, no such antisense RNA has ever been observed in cells undergoing PTGS. In virus-infected plants or in plants expressing either a homologous or a heterologous gene, a 25 nt antisense RNA could be detected whenever PTGS occurred. This small RNA may be a specific determinant of PTGS. It is small enough to move through plasmodesmata and may, therefore, be the elusive signal of the systemic spread of silencing through plant tissues.

Developmental regulated mechanisms affect the ability of a fungal pathogen to infect and colonize tobacco leaves. Hugot K, Aimé S, Conrod S, Poupet A, Galiana E: Plant J 1999, 20:163-170. • Significance: Two sets of disease responses control the growth of a fungal pathogen during floral development. Findings: During flowering, tobacco plants develop resistance to the fungus Phytophthora parasitica. Firstly, the effectiveness of infecting fungi is restrained through the accumulation of cytotoxic products in the intercellular fluids of leaves. These products inhibit the germination of fungal spores in vitro. Secondly, the growth of fungal hyphae in the plant tissue is restricted by a mechanism that is similar to that induced during systemic acquired resistance; resistance correlates with the apoplastic accumulation of pathogenesis-related protein PR-1 and requires SA. These observations demonstrate how the transition to floral development leads to the activation of at least two regulatory pathways that are involved in the induction of disease resistance mechanisms.

Compromised disease resistance in saponin-deficient plants. Papadopoulou K, Melton RE, Leggett M, Daniels MJ,

Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by

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salicylic acid and the NIM1 gene. Dong H, Delaney TP, Bauer DW, Beer SV: Plant J 1999, 20:207-215. • Significance: The induction of systemic, acquired resistance by the bacterial elicitor harpin depends on salicylic acid, but not on jasmonic acid or the ethylene-dependent signal transduction pathways. Findings: Harpin is the first characterized bacterial protein that induces resistance to a broad spectrum of pathogens in various plants. Harpin, and other known inducers of resistance responses, were compared in wild-type Arabidopsis plants and in plants impaired in their responses to salicylic acid (SA), jasmonic acid (JA) and ethylene (Eth). Systemic, induced resistance against Peronospora parasitica and Pseudomonas syringae pv tomato can be induced by localized applications of harpin. This resistance is accompanied by the induction of the pathogenesis-related proteins (PRs) PR-1 and PR-2. Neither systemic acquired resistance (SAR) nor PRs are expressed after harpin treatment in NahG plants, which overexpress a SAdegrading enzyme, or in npr1 mutants that are defective in the response to SA. Mutants that are affected in their response to JA and Eth, however, develop harpin-induced SAR as do wildtype plants. Harpin induces SAR by a SA-dependent mechanism and the action of harpin is upstream of SA. Magnaporthe grisea Pth11 is a novel plasma membrane protein that mediates appressorium differentiation in response to inductive substrate clues. De Zwaan TM, Carroll AM, Valent B, Sweigard JA: Plant Cell 1999, 11:2013-2030. AND

Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea. Dixon KP, Xu JR, Smirnoff N, Talbot NJ: Plant Cell 1999, 11:2045-2058. •• Significance: Two novel genes are described that code for proteins involved in surface recognition and in osmoregulation, respectively, in the rice pathogen Magnaporthe grisea. Findings: The first paper describes a novel mutant of Magnaporthe grisea that is deficient in its appressorium formation. The pth11 mutant is affected in pathogenicity and specifically compromised in the formation of appressoria on inductive surfaces. The authors isolated the PTH11 gene and found that it encodes a transmembrane protein that localizes to the cell membrane and the vacuole. The pth11 mutant phenotype could be suppressed by exogenously applied signals but in a surprisingly complex fashion. Exogenous cAMP, but not diacylgylycerol, restores pathogenicity, whereas either signal restores appressorium formation. In the second paper, a mitogen-activated protein kinase gene (OSM1) was isolated. This gene is involved in the regulation of hyperosmotic stress by promoting the accumulation of arabitol, which is responsible for turgor maintenance in M. grisea. Null mutants of OSM1, obtained by gene replacement, are sensitive to osmotic stress. These mutants show morphological defects when grown under hyperosmotic conditions and have a reduced capacity to accumulate arabitol. Curiously, osm1 mutants have unaltered glycerol accumulation and turgor generation in the appressorium, and their pathogenicity is unaffected. These findings indicate that different signal transduction pathways regulate turgor maintenance during hyperosmotic stress and appressorium-mediated plant penetration. Both papers highlight the complexity of the signal transduction cascades that mediate the functionality of appressorium in this major rice pathogen.

Identification of a locus in Arabidopsis controlling both the expression of rhizobacteria-mediated induced systemic resistance (ISR) and basal resistance against Pseudomonas syringae pv tomato. Ton J, Pieterse CMJ, van Loon LC: Mol Plant–Microbe Interact 1999, 12:911-918. • Significance: Rhizobacteria-mediated systemic induced resistance requires a single dominant gene that operates in basal resistance against Pseudomonas syringae. Findings: Induced systemic resistance (ISR) can be elicited by non-pathogenic rhizobacterial strains of Pseudomonas fluorescens. ISR is phenotypically similar to systemic acquired resistance (SAR), which is induced by pathogen infection. The author screened ten ecotypes of Arabidopsis for their potential to express ISR and SAR against the foliar pathogen Pseudomonas syringae pv tomato (Pst) and found that all of the ecotypes expressed SAR and ISR, with the exception of ecotypes RLD and Wassilewskija (Ws) that expressed only SAR. The non-responsiveness of RLD and Ws was associated with their high susceptibility to Pst infection. Crosses were made between RLD or Ws and the responsive ecotypes Columbia or Landsberg erecta. Analyses of the progeny in the F2 and F3 generations indicated that ISR responsiveness and basal resistance to Pst are monogenically inherited and genetically linked. Complementation analyses show that both RLD and Ws are affected at the same locus. This locus maps to chromosome 3, between markers B4 and GL1. Thus, a single dominant gene functioning in basal resistance to Pst is required for ISR. Naturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells. Goverse A, Rouppe van der Voort J, Rouppe van der Voort C, Kavelaars A, Smant G, Schots A, Bakker J, Helder J: Mol Plant–Microbe Interact 1999, 12:872-881. • Significance: The first report showing that nematode secretions can increase proliferation of both plant and mammalian cells. Findings: The biological relevance of nematode secretion was investigated in the potato cyst nematode Globodera rostochiensis. Secretions induced the proliferation of tobacco leaf protoplasts in a medium containing the auxin naphthyl acetic acid and the kinetin 6-benzylaminopurine. Using this bioassay, the mitogenic activity could be shown to reside in a low-molecular-weight peptide (<3 kDa). This peptide is not related to auxin or cytokinin, and it does not affect the sensitivity of the protoplasts to these hormones. Together with the mitogen phytohemagglutinin, this peptide can affect the proliferation of peripheral blood mononuclear cells. The activation of cell proliferation in a non-host cell system suggests that a general signal transduction mechanism is activated by a nematode-secreted oligopeptide. This mitogenic peptide might induce cell cycle reactivation required for the development of feeding cells in nematode-infected roots.

Cell signalling Selected by Martin McAinsh Department of Biology, Lancaster University, Lancaster, UK

Phytochrome signalling is mediated through nucleoside diphosphate kinase 2. Choi G, Yi H, Lee J, Kwon Y-K, Soh MS, Shin B, Luka Z, Hahn T-R, Song P-S: Nature 1999, 401:610613.

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• Significance: Identification of nucleoside diphosphate kinase 2 (NDPK2) as a component of phytochrome signalling in plants. Findings: The authors have used yeast two-hybrid screening to identify NDPK2 as a protein that interacts with phytochrome A. The authors show that NDPK2 interacts preferentially with the red-light-activated form of phytochrome causing a significant increase in NDPK2 activity through a red-light-dependent decrease in Km. Localisation studies show that NDPK2 and the phytochromes share the same subcellular space; this is essential for their interaction. Importantly, a ndpk2 mutant that lacks NDPK2 shows a partial defect in its responses, including cotyledon opening and greening, to both red and far-red light. These data highlight NDPK2 as a signalling component that interacts directly with phytochrome. Different circadian oscillators control Ca2+ fluxes and Lhcb gene expression. Sai J, Johnson CH: Proc Natl Acad Sci USA 1999, 96:11659-11663. • Significance: New evidence for separate circadian pacemakers controlling molecular events in plants. Findings: The authors show that circadian oscillations of cytosolic free calcium concentration ([Ca2+]cyt) and the rate of transcription of the light-harvesting complex (Lhc)b gene exhibit free-running rhythms, with different periods in constant conditions when monitored in the double reporter strain, LAQ, of tobacco. Crucially, the oscillations of Lhcb-promoter activity persisted in undifferentiated calli in the absence of [Ca2+]cyt oscillations. The authors therefore conclude that there is no functional link between the circadian pacemakers underlying these two rhythms in tobacco. Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells. Allen GJ, Kuchitsu K, Chu SP, Murata Y, Schroeder JI: Plant Cell 1999, 11:1785-1798. •• Significance: This paper reports advances in positioning the ABI1 and ABI2 protein phosphatases in the abscisic acid (ABA) signal transduction pathway that leads to stomatal closure in Arabidopsis. Findings: The authors studied the effects of ABA on guard cell cytosolic free calcium concentration ([Ca2+]t), plasma membrane anion channel activity and stomatal aperture in the abi1 and abi2 ABA-insensitive mutants of Arabidopsis. ABAinduced increases in [Ca2+]cyt are significantly smaller in abi1 and abi2 guard cells than in wild-type guard cells. In both mutants, however, anion currents and stomatal closure are stimulated by increases in guard cell [Ca2+]cyt. The abi1 and abi2 mutations can, therefore, be bypassed by increasing [Ca2+]cyt. These findings place the ABI1 and ABI2 phosphatases upstream of, or close to, ABA-induced increases in [Ca2+]cyt. Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity. Jacob T, Ritchie S, Assmann SM, Gilroy S: Proc Natl Acad Sci USA 1999, 96:12192-12197. • Significance: A new insight into the mechanisms of abscisic acid (ABA) signal transduction in stomatal guard cells. Findings: The authors report that ABA stimulates an increase in phospholipase D (PLD) activity and phosphatidic acid (PtdOH) production in guard cells, and that 1-butanol, an inhibitor of PtdOH production by PLD activity, inhibits the ABAinduced production of PtdOH and the response of stomata to ABA. PtdOH mimics the effects of ABA on stomatal aperture

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and the guard cell inward K+ channel, appearing to act via a pathway that is parallel to that mediated by cyclic ADP-ribose. These findings indicate a role for PLD in ABA signal transduction through the production of PtdOH. Selected by Klaus Palme Max-Delbrueck-Laboratorium in der Max-Planck-Gesellschaft, Köln, Germany

Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Apse MP, Aharon GS, Snedden WA, Blumwald E: Science 1999, 285:1256-1258. •• Significance: Salt tolerant Arabidopsis was engineered by overexpressing a single vacuolar Na+/H+ antiport encoding gene. This strategy opens the possibility of generating a wide range of salt tolerant crop plants. Findings: AtNHX1 was identified from the Arabidopsis genome sequencing project on the basis of its similarity to the yeast Nhx1 gene product. Overexpression of AtNHX1 in transgenic Arabidopsis revealed that the protein was located in the vacuole membrane, and that the transgenic plants accumulated much more Na+ than wild-type plants. Growth of transgenic Arabidopsis plants in soil containing as much as 200 mM Na+ was possible. The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth. Rate DN, Cuenca JV, Bowman GR, Guttman DS, Greenberg JT: Plant Cell 1999,11:1695-1708. • Significance: The dominant gain-of-function mutation accelerated cell death (acd6) is suppressed by removing the signal molecule salicylic acid (SA), suggesting a role for SA in cell growth modulation. Findings: The acd6 mutant was isolated in a screen for genes involved in regulating pathogen defenses. One plant was identified that showed reduced symptoms of the virulent pathogen P.s. pv maculicola. Acd6 plants show increased resistance to Pseudomonas syringae, but have a reduced stature and patches of dead and enlarged cells after infection by this pathogen. In plants carrying the nahG transgene, which encodes a SA hydroxylase, these disease responses are suppressed. Moreover, treatment of the acd6-nahG plants with a synthetic inducer of the SA pathway, BTH, results in hyperactivation of all acd6 phenotypes causing tumor-like abnormal plant growth. A more direct role for SA in cell growth control is therefore indicated. ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling. Gosti F, Beaudoin N, Serizet C, Webb AAR, Vartanian N, Giraudat J: Plant Cell 1999, 11:1897-1909. AND

Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells. Allen GJ, Kuchitsu K, Chu SP, Murata Y, Schroeder JI: Plant Cell 1999, 11:1785-1798. •• Significance: Both articles present significant insight into the mechanisms of abscisic acid (ABA) action. Findings: Abscisic acid is a key regulator of seed maturation and germination, and mediates adaptive responses to environmental stresses. Gosti et al. isolated several intragenic, recessive revertant alleles of the dominant abi1-1 mutation. All suppressor mutations were missense mutations in conserved regions of the PP2C domain of ABI1 that caused a

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phosphatase deficient ABI1 protein. Loss of PP2C activity in the abi-1 mutant increased its responsiveness to ABA suggesting that wild-type ABI1 is a negative regulator of ABA responses. The second report by Allen et al. sheds light on the role of abi1-1 and abi2-1 phosphatase mutations in [Ca2+]cytsignalling in guard cells. The authors beautifully demonstrate that both mutations impair early ABA-signalling events upstream of, or close to, ABA-induced [Ca2+]cyt elevations. They further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by increasing the [Ca2+]cyt experimentally. The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptorlike kinase. Jeong S, Trotochaud AE, Clark SE: Plant Cell 1999, 11:1925-1933. • Significance: The authors show that CLAVATA2 (CLV2) is required for CLAVATA1 (CLV1) protein accumulation. Findings: CLV2 was cloned using a T-DNA insertion mutant. The CLV2 gene encodes a 720-amino-acid protein with a signal peptide and characteristic leucine-rich repeat motifs. The single copy gene was found to be expressed in many different tissues, but predominantly in apices. On the basis of results obtained by protein gel blotting, Jeong and co-workers conclude that CLV2 is required for the normal accumulation of CLV1 and its assembly into protein complexes. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchinson TJ: Science 1999, 286:971-974. • Significance: This paper describes a novel phenotype-based screening strategy for dissecting complex processes in cells. Findings: Mayer and colleagues performed an ambitious screen to identify small, cell-permeable compounds that inhibit mitosis by blocking the function of essential spindle proteins other than tubulin. Using an antibody against a mitosis-specific phosphorylation site on the nucleolar protein nucleolin they performed a whole-cell, high-throughput immuno(blot)detection assay to identify compounds that increase the phosphorylation of nucleolin. In a second assay, the effects of 139 compounds (from a library of 16,320 compounds) on microtubules, actin and chromatin in fixed cells were examined using fluorescence microscopy. The approach resulted in the identification of a novel drug, named monastrol, which inhibits the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. Fluorescent proteins from nonbioluminiscent Anthozoa species. Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zraisky AG, Markelov ML, Lukyanov SA: Nat Biotechnol 1999, 17:969-973. •• Significance: This paper describes a breakthrough in the search for novel fluorescent proteins with altered bioluminiscence characteristics. Findings: Six cDNAs encoding new fluorescent proteins were isolated from fluorescent, but nonbioluminiscent corals of the Indian and Pacific oceans using degenerate primers for the well known Aequorea green fluorescent protein (GFP). Two of these proteins have spectral characteristics that are dramatically different from GFP, emitting at yellow and red wavelengths. All of the proteins show the same β-scan fold observed in GFP providing a basis for further rational improvement of the new

proteins. The availability of the red fluorescent protein (RFP) from Clontech will soon allow the expected benefits of RFP for plant research to be realised.

Gene regulation Selected by Bernd Weisshaar Max-Planck-Institut fur Züchtungsforschung, Köln, Germany

IFL1, a gene regulating interfascicular fiber differentiation in Arabidopsis, encodes a homeodomain–leucine zipper protein. Zhong R, Ye Z-H: Plant Cell 1999, 11:2139-2152. • Significance: Identification of the homeodomain-leucine zipper (HD-ZIP) factor IFL1 as a component in vascular tissue differentiation. Findings: In ifl1-mutant plants the formation of normal interfascicular fibers in stems is prevented and abberant vascular bundels are formed. The IFL1 (INTERFASCICULAR FIBERLESS 1) gene was identified by positional cloning. A number of mutant alleles were characterised, and the IFL1 protein was found to be localised to the nucelus. Analyses of transgenic plants containing IFL1 promoter::GUS (uidA ORF) fusions showed that the IFL1 gene is active in the tissue affected in ifl1-mutant plants. ACGT-containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent. Hobo T, Asada M, Kowyama Y, Hattori T: Plant J 1999, 19:679689. • Significance: A detailed analysis of the abscisic acid (ABA) responsive region of the rice Em (OsEM) gene is presented. The EM gene encodes an abundant protein that is found in mature embryos of developing seeds in many plants. Findings: Functional assays using rice protoplasts were used to dissect the ABA-responsive region of the OsEM gene, which consists of an ACGT-containing element (ACE) and an additional cis-acting sequence (designated CE3). The results show that the same cis-acting sequences are involved in regulation by VIVIPAROUS1 and ABA, and that the ACE and CE3 sequences are functionally equivalent cis-acting elements.

Cell biology Selected by Frederic Berger Reproduction et Developement des Plantes, Lyon, France

Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF. Steinmann T, Geldner N, Grebe M, Mangold S, Jackson CL, Paris S, Gälweiler L, Palme K, Jürgens G: Science 1999, 286:316-318. •• Significance: This work demonstrates how a highly specialised vesicle-trafficking pathway is involved in the polarisation of the plant embryo. Findings: Functional complementation and GDP/GTP exchange assays are used to demonstrate that GNOM is a brefeldin-A (BFA)-sensitive ARF GEF, a class of compounds involved in vesicle trafficking. GNOM is immunolocalised in the membrane-rich and cytoplasmic fractions, and BFA treatments reduce the relative size of the cytosolic fraction. The growth of gnom-mutated cells in suspension culture does not differ from that of wild-type cells, showing that GNOM is not a general regulator of vesicle transport. Embryos with gnom mutations, however, do not show the polar localisation of the auxin efflux carrier PIN1 as is observed in the wild-type. PIN1 is also localised in a polar manner in wild-type embryos during the early development of the lateral roots; this is disrupted by BFA treatment which probably interferes with GNOM function.

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Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchinson TJ: Science 1999, 286:971-974. • Significance: A specific inhibitor of mitosis is isolated using a screening strategy that could be used for other biological functions. Findings: A screening strategy is developed that identifies the effect of small molecules on cell cycle related processes. The strategy is based on successive in vitro and in vivo assays for the effect of molecules on cell cycle events. The authors are able to distinguish 52 microtubule-destabilising agents, one microtubule-stabilising agent and five molecules with a specific effect on mitosis from a total of 16 320 compounds analysed. Monastrol, a dihydropyrimidine derivative, modifies normal bipolar mitotic spindles into monopolar spindles, in which centrosomes occupy the centre of the cell and chromosomes are located at the periphery. This agent was shown to inhibit the function of a specific kinesin. Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localised calcium influx. Li H, Lin Y, Heath RM, Zhu MX, Yang Z: Plant Cell 1999, 11:17311742. • Significance: This study provides a clear link between the localisation of a small GTP-binding protein of the Rho family (Rop1At) and the maintenance of the polar tip-growth involving Ca2+ gradients in pollen tubes. Findings: Isotropic growth of pollen tubes results from the expression, and also the overexpression, of a constitutively active form of Rop1At. The loss of polar tip growth is correlated with the abnormal localisation of a Rop1At−green-fluorescentprotein fusion. The expression of a dominant negative Rop1At signal causes an inhibition of pollen tube growth that can be rescued by high extracellular Ca2+ concentration. The link between localised Rop1At and the Ca2+ gradient at the tip of

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the pollen tube is confirmed by the inhibition of Rop1At following injections of antibodies against it. MAF1, a novel plant protein interacting with matrix attachment region binding MFP1, is located at the nuclear envelope. Gindulis F, Peffer NJ, Meier I: Plant Cell 1999, 11:1755-1767. • Significance: The characterisation of a novel protein involved in nuclear architecture. Findings: Yeast two-hybrid and in vitro binding assays using the matrix attachment region filament-like protein 1 (MFP1) allow the identification of the MFP1-associated-factor-1 (MAF1). MAF1−green-fluorescent-protein fusion and immunolocalisation show that MAF1 is located at the nuclear envelope and is a component of the nuclear matrix. This protein shows no significant homology to known proteins and is ubiquitously expressed. A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein. Dorokhov YL, Mäkinen K, Frolova OY, Saarinen J, Kalkkinen N, Atabekov JG, Saarma M: FEBS Lett 1999, 461:223-228. • Significance: This study gives insight into the potential role of an ubiquitous cell-wall protein in viral movement through the plasmodesmata. Findings: Cell-wall polypeptides from tobacco are identified as potential binding sites for tobamovirus movement proteins in in vitro assays. One of the cell wall proteins identified is purified using an affinity-chromatographic approach, and the resulting sequence data show its strong homology with a pectin methylesterase from tomato. Although no activity of the tobacco protein was observed in vitro and no work was carried out in vivo, this study provides potential candidate proteins for plasmodesmata function.