Dirk Prüfer

Tomato Ve disease resistance genes encode cell surface-like receptors

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLφ or YXXφ endocytosis signals (φ is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.

The potato leafroll virus 17K movement protein is phosphorylated by a membrane-associated protein kinase from potato with biochemical features of protein kinase C

The 17 kDa protein (pr17), the phloem‐limited movement protein (MP) of potato leafroll luteovirus (PLRV), is associated with membranous structures and localized to plasmodesmata [Tacke et al. (1993) Virology 197, 274–282; Schmitz, J. (1995) Ph.D. Thesis, University of Cologne]. In planta the protein is predominantly present in its phosphorylated form, but it is rapidly dephosphorylated during isolation under native conditions. In an effort to examine the nature of the protein kinase(s) involved in the phosphorylation reaction, pr17 deletion mutants were expressed as fusion proteins in a bacterial expression vector system and tested for their ability to be phosphorylated by potato membrane preparations as well as by commercially available kinases. A fusion protein containing the nucleic acid‐binding, basic, C‐proximal domain (pr17C1) was identified to be phosphorylated by a Ca2+‐ and phospholipid‐dependent, membrane‐associated protein kinase. This protein kinase activity was inhibited by the addition of (19–36) protein kinase C (PKC) inhibitory peptide, known to be a highly specific inhibitor of mammalian PKC. Moreover, also the mammalian PKC from rat was able to phosphorylate pr17 in vitro. The results suggest that phosphorylation of pr17 takes place at membranous structures, possibly at the deltoid plasmodesmata connecting the sieve cell‐companion cell complex of the phloem, by the activity of PKC‐related, membrane‐associated protein kinase activity.

Ribosomal frameshifting in plants : a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by −1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta‐glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a −1 frameshift occurs at a frequency of approximately 1%. Site‐directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem‐loop structure. Part of this stem‐loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid‐binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template‐binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.

Laticifer specific cis-prenyltransferase silencing affects the rubber, triterpene and inulin content of Taraxacum brevicorniculatum

Certain Taraxacum species, such as Taraxacum koksaghyz and Taraxacum brevicorniculatum, produce large amounts of high-quality natural rubber in their latex, the milky cytoplasm of specialized cells known as laticifers. This high-molecular mass biopolymer consists mainly of poly(cis-1,4-isoprene) and is deposited in rubber particles by particle-bound enzymes that carry out the stereospecific condensation of isopentenyl diphosphate units. The polymer configuration suggests that the chain-elongating enzyme (rubber transferase; EC 2.5.1.20) is a cis-prenyltransferase (CPT). Here, we present a comprehensive analysis of transgenic T. brevicorniculatum plants in which the expression of three recently isolated CPTs known to be associated with rubber particles (TbCPT1 to -3) was heavily depleted by laticifer-specific RNA interference (RNAi). Analysis of the CPT-RNAi plants by nuclear magnetic resonance, size-exclusion chromatography, and gas chromatography-mass spectrometry indicated a significant reduction in rubber biosynthesis and a corresponding 50% increase in the levels of triterpenes and the main storage carbohydrate, inulin. Transmission electron microscopy revealed that the laticifers in CPT-RNAi plants contained fewer and smaller rubber particles than wild-type laticifers. We also observed lower activity of hydroxymethylglutaryl-coenzyme A reductase, the key enzyme in the mevalonate pathway, reflecting homeostatic control of the isopentenyl diphosphate pool. To our knowledge, this is the first in planta demonstration of latex-specific CPT activity in rubber biosynthesis.

Polyphenoloxidase Silencing Affects Latex Coagulation in Taraxacum Species

Latex is the milky sap that is found in many different plants. It is produced by specialized cells known as laticifers and can comprise a mixture of proteins, carbohydrates, oils, secondary metabolites, and rubber that may help to prevent herbivory and protect wound sites against infection. The wound-induced browning of latex suggests that it contains one or more phenol-oxidizing enzymes. Here, we present a comprehensive analysis of the major latex proteins from two dandelion species, Taraxacum officinale and Taraxacum kok-saghyz, and enzymatic studies showing that polyphenoloxidase (PPO) is responsible for latex browning. Electrophoretic analysis and amino-terminal sequencing of the most abundant proteins in the aqueous latex fraction revealed the presence of three PPO-related proteins generated by the proteolytic cleavage of a single precursor (pre-PPO). The laticifer-specific pre-PPO protein contains a transit peptide that can target reporter proteins into chloroplasts when constitutively expressed in dandelion protoplasts, perhaps indicating the presence of structures similar to plastids in laticifers, which lack genuine chloroplasts. Silencing the PPO gene by constitutive RNA interference in transgenic plants reduced PPO activity compared with wild-type controls, allowing T. kok-saghyz RNA interference lines to expel four to five times more latex than controls. Latex fluidity analysis in silenced plants showed a strong correlation between residual PPO activity and the coagulation rate, indicating that laticifer-specific PPO plays a major role in latex coagulation and wound sealing in dandelions. In contrast, very little PPO activity is found in the latex of the rubber tree Hevea brasiliensis, suggesting functional divergence of latex proteins during plant evolution.

Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz

BackgroundNatural rubber is a biopolymer with exceptional qualities that cannot be completely replaced using synthetic alternatives. Although several key enzymes in the rubber biosynthetic pathway have been isolated, mainly from plants such as Hevea brasiliensis, Ficus spec. and the desert shrub Parthenium argentatum, there have been no in planta functional studies, e.g. by RNA interference, due to the absence of efficient and reproducible protocols for genetic engineering. In contrast, the Russian dandelion Taraxacum koksaghyz, which has long been considered as a potential alternative source of low-cost natural rubber, has a rapid life cycle and can be genetically transformed using a simple and reliable procedure. However, there is very little molecular data available for either the rubber polymer itself or its biosynthesis in T. koksaghyz.ResultsWe established a method for the purification of rubber particles - the active sites of rubber biosynthesis - from T. koksaghyz latex. Photon correlation spectroscopy and transmission electron microscopy revealed an average particle size of 320 nm, and 13C nuclear magnetic resonance (NMR) spectroscopy confirmed that isolated rubber particles contain poly(cis-1,4-isoprene) with a purity >95%. Size exclusion chromatography indicated that the weight average molecular mass (w) of T. koksaghyz natural rubber is 4,000-5,000 kDa. Rubber particles showed rubber transferase activity of 0.2 pmol min-1 mg-1. Ex vivo rubber biosynthesis experiments resulted in a skewed unimodal distribution of [1-14C]isopentenyl pyrophosphate (IPP) incorporation at a w of 2,500 kDa. Characterization of recently isolated cis-prenyltransferases (CPTs) from T. koksaghyz revealed that these enzymes are associated with rubber particles and are able to produce long-chain polyprenols in yeast.ConclusionsT. koksaghyz rubber particles are similar to those described for H. brasiliensis. They contain very pure, high molecular mass poly(cis-1,4-isoprene) and the chain elongation process can be studied ex vivo. Because of their localization on rubber particles and their activity in yeast, we propose that the recently described T. koksaghyz CPTs are the major rubber chain elongating enzymes in this species. T. koksaghyz is amenable to genetic analysis and modification, and therefore could be used as a model species for the investigation and comparison of rubber biosynthesis.

An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein

Potato leafroll polerovirus (PLRV) genomic RNA acts as a polycistronic mRNA for the production of proteins P0, P1, and P2 translated from the 5′-proximal half of the genome. Within the P1 coding region we identified a 5-kDa replication-associated protein 1 (Rap1) essential for viral multiplication. An internal ribosome entry site (IRES) with unusual structure and location was identified that regulates Rap1 translation. Core structural elements for internal ribosome entry include a conserved AUG codon and a downstream GGAGAGAGAGG motif with inverted symmetry. Reporter gene expression in potato protoplasts confirmed the internal ribosome entry function. Unlike known IRES motifs, the PLRV IRES is located completely within the coding region of Rap1 at the center of the PLRV genome.

Proteins from the FLOWERING LOCUS T‐like subclade of the PEBP family act antagonistically to regulate floral initiation in tobacco

Flowering is an important agronomic trait that often depends on the integration of photoperiod, vernalization, gibberellin and/or autonomous signaling pathways by regulatory proteins such as FLOWERING LOCUS T (FT), a member of the phosphatidylethanolamine-binding protein (PEBP) family. Six PEBP family proteins control flowering in the model plant Arabidopsis thaliana, and their regulatory functions are well established, but variation in the number and structural diversity of PEBPs in different species means their precise functions must be determined on a case-by-case basis. We isolated four novel FT-like genes from Nicotiana tabacum (tobacco), and determined their expression profiles in wild-type plants and their overexpression phenotypes in transgenic plants. We found that all four genes were expressed in leaves under short-day conditions, and at least NtFT3 expression was restricted to phloem companion cells. We also found that the NtFT1, NtFT2 and NtFT3 proteins are floral inhibitors (atypical for FT-like proteins), whereas only NtFT4 is a floral inducer. We were unable to detect the expression of these genes under long-day conditions, suggesting that all four tobacco FT-like proteins may control flowering in response to short days. Phylogenetic analysis of PEBP family proteins and their functions in different solanaceous species confirmed that gene duplication and divergence within the FT-like clade has led to the evolution of antagonistic regulators that may help to fine-tune floral initiation in response to environmental cues.

Molecular Cloning and Characterization of Rubber Biosynthetic Genes from Taraxacum koksaghyz

Rubber biosynthesis requires the action of specific enzymes known as cis-prenyltransferases (CPTs). These enzymes are responsible for the sequential addition of isopentenyl pyrophosphate units to the growing polyisoprene chain, a biochemical reaction thought to be stimulated by the presence of small rubber particle proteins (SRPPs). We have cloned, characterized, and analyzed the expression of three CPT genes (TkCPT1–3) and five SRPP genes (TkSRPP1–5) from the rubber-producing plant Taraxacum koksaghyz. The deduced TkCPT amino acid sequences showed significant levels of sequence identity with Hevea brasiliensis CPTs. We also found no obvious differences between SRPPs from T. koksaghyz, another rubber producer, and a non-rubber plant. The roles of the individual TkCPTs and TkSRRPs in rubber biosynthesis are discussed.

Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem

The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits.