A leucine-rich repeat region is conserved in pollen extensin-like (Pex) proteins in monocots and dicots

We previously isolated a pollen-specific gene encoding a pollen tube wall-associated glycoprotein with a globular domain and an extensin domain from maize (mPex1). To evaluate which protein domains might be important for function, we isolated a second monocot gene (mPex2) and a dicot gene (tPex). Each gene encodes a signal sequence, an N-terminal globular domain comprised of a variable region, a leucine-rich repeat (LRR) with an adjacent cysteine-rich region, a transition region and an extensin-like C-terminal domain. The LRRs of the maize and tomato Pex proteins are highly conserved. Although the extensin domains in the maize and tomato proteins vary in length and in amino acid sequence, they are likely to be structurally conserved. Additional putative Pex gene sequences were identified by either GenBank search (Arabidopsis) or PCR (sorghum and potato); all encode conserved LRRs. The presence of a conserved LRR in the known and potential Pex proteins strongly suggests that this motif is involved in the binding of a specific ligand during pollen tube growth. Gene expression studies using RNA and protein blotting as well as promoter-reporter gene fusions in transient and stable transformation indicate that the tomato Pex gene is pollen-specific.     

The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots

Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity. The genomic region spanning nucleotides 5999–7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana. In A. sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive. In A. thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique. The transgene was inherited and active in progeny plants of both A. sativa and A. thaliana.     

BRCTx is a novel, highly conserved RAD18-interacting protein

The BRCT domain is a highly conserved module found in many proteins that participate in DNA damage checkpoint regulation, DNA repair, and cell cycle control. Here we describe the cloning, characterization, and targeted mutagenesis of Brctx, a novel gene with a BRCT motif. Brctx was found to be expressed ubiquitously in adult tissues and during development, with the highest levels found in testis. Brctx-deficient mice develop normally, show no pathological abnormalities, and are fertile. BRCTx binds to the C terminus of hRAD18 in yeast two-hybrid and immunoprecipitation assays and colocalizes with this protein in the nucleus. Despite this, Brctx-deficient murine embryonic fibroblasts (MEFs) do not show overt sensitivity to DNA-damaging agents. MEFs from Brctx-deficient embryos grow at a similar rate to wild-type MEF CD4/CD8 expressions, and the cell cycle parameters of thymocytes from wild-type and Brctx knockout animals are indistinguishable. Intriguingly, the BRCT domain of BRCTx is responsible for mediating its localization to the nucleus and centrosome in interphase cells. We conclude that, although highly conserved, Brctx is not essential for the above-mentioned processes and may be redundant.     

Differences in meristems between monocots and dicots and susceptibility to attack by gall-inducing insects

We used comparative methods that account for the phylogenetic correlations among species to test hypotheses about the community of gall-inducing insects on dicotyledonous and monocotyledonous plants and woody and herbaceous angiosperms in the UK. We found that the species richness of gall-inducing insects on dicots was greater than on monocots and that the odds of a dicot having an associated gall-inducing insect is 42% higher than for a monocot. Woody angiosperms have higher species richness of associated gall-inducing insects than do herbaceous angiosperms. Furthermore, using a Monte Carlo analysis we found that attacks by gall-inducing insects on monocot families were phylogenetically clustered in the order Poales, particularly within the grass family Poaceae. We suggest that the higher risk of attack on dicots and higher species richness of gall-inducing insects on woody angiosperms, which are exclusively dicots, arises because of differences in the abundance or susceptibility of dicot meristems to attack by gall-inducing insects. Architectural and anatomical differences between monocots and dicots that give rise to differences in meristem abundance and anatomy appear to play an important role in determining the occurrence and richness of associated gall-inducing insects on host plants.     

A novel kinesin-like protein with a calmodulin-binding domain

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with ³⁵S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca²⁺-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCK1 is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca²⁺/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.     

A calmodulin-regulated protein kinase linked to neuron survival is a substrate for the calmodulin-regulated death-associated protein kinase

Death-associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase and a drug-discovery target for neurodegenerative diseases. However, a protein substrate relevant to neuronal death had not been described. We identified human brain CaM-regulated protein kinase kinase (CaMKK), an enzyme key to neuronal survival, as the first relevant substrate protein by using a focused proteomics- and informatics-based approach that can be generalized to protein kinase open reading frames identified in genome projects without prior knowledge of biochemical context. First, DAPK-interacting proteins were detected in yeast two-hybrid screens and in immunoprecipitates of brain extracts. Second, potential phosphorylation site sequences in yeast two-hybrid hits were identified on the basis of our previous results from positional-scanning synthetic-peptide substrate libraries and molecular modeling. Third, reconstitution assays using purified components demonstrated that DAPK phosphorylates CaMKK with a stoichiometry of nearly 1 mol of phosphate per mole of CaMKK and a K(m) value of 3 microM. Fourth, S511 was identified as the phosphorylation site by peptide mapping using mass spectrometry, site-directed mutagenesis, and Western blot analysis with a site-directed antisera targeting the phosphorylated sequence. Fifth, a potential mechanism of action was identified on the basis of the location of S511 near the CaM recognition domain of CaMKK and demonstrated by attenuation of CaM-stimulated CaMKK autophosphorylation after DAPK phosphorylation. The results raise the possibility of a CaM-regulated protein kinase cascade as a key mechanism in acute neurodegeneration amenable to therapeutic targeting.     

Isolation of plasma-membrane-bound calcium/calmodulin-regulated protein kinase from pea using Western blotting

Membranes isolated from pea buds contain protein-kinase activity which is greatly activated by low concentrations of calcium ions. This paper describes a simple purification of this enzyme with a novel means of detecting enzyme activity by Western blotting. The purified enzyme appears to autophosphorylate primarily on serine residues, is activated by bovine calmodulin and additional evidence from phase partitioning indicate most of this enzyme to be located in the plasma membrane.Abbreviations PAGE polyacrylamide-gel electrophoresis - SDS sodium dodecyl sulphate     

Diversification of genes encoding granule-bound starch synthase in monocots and dicots is marked by multiple genome-wide duplication events

Starch is one of the major components of cereals, tubers, and fruits. Genes encoding granule-bound starch synthase (GBSS), which is responsible for amylose synthesis, have been extensively studied in cereals but little is known about them in fruits. Due to their low copy gene number, GBSS genes have been used to study plant phylogenetic and evolutionary relationships. In this study, GBSS genes have been isolated and characterized in three fruit trees, including apple, peach, and orange. Moreover, a comprehensive evolutionary study of GBSS genes has also been conducted between both monocots and eudicots. Results have revealed that genomic structures of GBSS genes in plants are conserved, suggesting they all have evolved from a common ancestor. In addition, the GBSS gene in an ancestral angiosperm must have undergone genome duplication ～251 million years ago (MYA) to generate two families, GBSSI and GBSSII. Both GBSSI and GBSSII are found in monocots; however, GBSSI is absent in eudicots. The ancestral GBSSII must have undergone further divergence when monocots and eudicots split ～165 MYA. This is consistent with expression profiles of GBSS genes, wherein these profiles are more similar to those of GBSSII in eudicots than to those of GBSSI genes in monocots. In dicots, GBSSII must have undergone further divergence when rosids and asterids split from each other ～126 MYA. Taken together, these findings suggest that it is GBSSII rather than GBSSI of monocots that have orthologous relationships with GBSS genes of eudicots. Moreover, diversification of GBSS genes is mainly associated with genome-wide duplication events throughout the evolutionary course of history of monocots and eudicots.     

A novel alpha-helical protein,specific to and highly conserved in plants,is associated with the nuclear matrix fraction

A cDNA for a novel plant protein was isolated from tomato. Nuclear Matrix Protein 1 (NMP1) is a ubiquitously expressed 36 kDa protein, which has no homologues in animals and fungi, but is highly conserved among flowering and non-flowering plants, including gymnosperms, moss, and the liverwort Marchantia polymorpha. NMP1 is predominantly alpha-helical with multiple stretches of short amphipathic regions. Cell fractionation, immunofluorescence, and GFP localization experiments showed that NMP1 is located both in the cytoplasm and nucleus and that the nuclear fraction is associated with the nuclear matrix. NMP1 is a candidate for a plant-specific structural protein with a function both in the nucleus and cytoplasm.     

A merozoite receptor protein from Plasmodium knowlesi is highly conserved and distributed throughout Plasmodium

The 66-kDa merozoite surface antigen (PK66) of Plasmodium knowlesi, a simian malaria, possesses vaccine-related properties that are thought to originate from a receptor-like role in parasite invasion of erythrocytes. We report the complete sequence of PK66 which allowed the demonstration that highly conserved analogues exist throughout Plasmodium including a recently reported gene from P. falciparum (Peterson, M. G., Marshall, V. M., Smythe, J. A., Crewther, P. E., Lew, A., Silva, A., Anders, R. F., and Kemp, D. J. (1989) Mol. Cell. Biol. 9, 3151-3155). These analogues are highly promising vaccination candidates. The distribution of PK66 changes after schizont rupture in a coordinate manner associated with merozoite invasion. The protein is concentrated at the apical end prior to rupture, following which it can distribute itself entirely across the surface of the free merozoite. During invasion, immunofluorescence studies suggest that, PK66 is excluded from the erythrocyte at, and behind, the invasion interface.     

A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.     

The rice/maize pathogen Cochliobolus spp. infect and reproduce on Arabidopsis revealing differences in defensive phytohormone function between monocots and dicots

The fungal genus Cochliobolus describes necrotrophic pathogens that give rise to significant losses on rice, wheat, and maize. Revealing plant mechanisms of non‐host resistance (NHR) against Cochliobolus will help to uncover strategies that can be exploited in engineered cereals. Therefore, we developed a heterogeneous pathosystem and studied the ability of Cochliobolus to infect dicotyledons. We report here that C. miyabeanus and C. heterostrophus infect Arabidopsis accessions and produce functional conidia, thereby demonstrating the ability to accept Brassica spp. as host plants. Some ecotypes exhibited a high susceptibility, whereas others hindered the necrotrophic disease progression of the Cochliobolus strains. Natural variation in NHR among the tested Arabidopsis accessions can advance the identification of genetic loci that prime the plant’s defence repertoire. We found that applied phytotoxin‐containing conidial fluid extracts of C. miyabeanus caused necrotic lesions on rice leaves but provoked only minor irritations on Arabidopsis. This result implies that C. miyabeanus phytotoxins are insufficiently adapted to promote dicot colonization, which corresponds to a retarded infection progression. Previous studies on rice demonstrated that ethylene (ET) promotes C. miyabeanus infection, whereas salicylic acid (SA) and jasmonic acid (JA) exert a minor function. However, in Arabidopsis, we revealed that the genetic disruption of the ET and JA signalling pathways compromises basal resistance against Cochliobolus, whereas SA biosynthesis mutants showed a reduced susceptibility. Our results refer to the synergistic action of ET/JA and indicate distinct defence systems between Arabidopsis and rice to confine Cochliobolus propagation. Moreover, this heterogeneous pathosystem may help to reveal mechanisms of NHR and associated defensive genes against Cochliobolus infection.     

The nucleolar RNA-binding protein B-36 is highly conserved among plants

The nucleolar protein B-36 is an RNA-associated protein which has a number of properties in common with pre-mRNA-binding proteins (hnRNP proteins). Like the hnRNP proteins, B-36 appears to be evolutionarily conserved among various eukaryotes (protists and several animal species). The conservation of B-36 throughout the plant kingdom has been investigated using a panel of nine monoclonal antibodies previously shown to recognize a minimum of four different epitopes in Physarum B-36, the protein used to generate the monoclonal antibodies. Two of the epitopes (I and III) are widely conserved in 34 kDa proteins (presumably B-36 homologues) from the various species tested (Chlamydomonas, moss, fern, oat, onion, carrot, and bean). Using immunofluorescence localization in moss and carrot protoplasts, the cross-reacting proteins were shown to be restricted to the nucleolus, further confirming their probable homology to B-36. Epitopes I and III are also unique to the B-36 homologues as demonstrated by the failure of any other bands to cross-react. Another epitope (IV) was specifically recognized in the plant B-36 homologues but exhibited greatly reduced affinity for the monoclonal antibody relative to Physarum B-36. The remaining epitope (II), unlike the others, exhibited variable conservation in the plant B-36 homologues and, in addition, was present in several other seemingly unrelated proteins. Finally, several of the plant species exhibited two cross-reacting variants at roughly the 34 kDa position and in at least one of these cases a single monoclonal antibody was able to distinguish between the two variants, a result indicating that the variants do have bona fide structural differences.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1

cDNA coding for protein phosphatase 2A (PP2A) has been isolated from Drosophila head and eye imaginal disc libraries. Drosophila PP2A mRNA is expressed throughout development, but is most abundant in the early embryo. The cDNA hybridises to a single site on the left arm of the second chromosome at position 28D2-4. The deduced amino acid sequence (309 residues) of Drosophila PP2A shows 94% identity with either rabbit PP2A alpha or PP2A beta, indicating that PP2A may be the most conserved of all known enzymes.     

The Egg apparatus 1 gene from maize is a member of a large gene family found in both monocots and dicots

The maize ZmEA1 protein was recently postulated to be involved in short-range pollen tube guidance from the embryo sac. To date, EA1-like sequences had only been identified in monocot species. Using a more conserved C-terminal motif found in the monocot species, numerous ZmEA1-like sequences were retrieved in EST databases from dicot species, as well as from unannotated genomic sequences of Arabidopsis thaliana. RT-PCR analyses were produced for these unannotated genes and showed that these were indeed expressed genes. Further structural and phylogenetic analyses revealed that all members of the EA1-like (EAL) gene family shared a conserved 27–29 amino acid motif, termed the EA box near the C-terminal end, and appear to be secretory proteins. Therefore, the EA box proteins defines a new class of small secretory proteins, some of which being possibly involved in pollen tube guidance. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle.

Microtubule-associated motor proteins are thought to be involved in spindle formation and chromosome movements in mitosis/meiosis. We have molecularly cloned cDNAs for a gene that codes for a novel member of the kinesin family of proteins. Nucleotide sequencing reveals that the predicted gene product is a 73 kDa protein and is related to some extent to the Drosophila node gene product, which is involved in chromosomal segregation during meiosis. A sequence similar to the microtubule binding motor domain of kinesin is present in the N-terminal half of the protein, and its ability to bind to microtubules is demonstrated. Furthermore we show that its C-terminal half contains a putative nuclear localization signal similar to that of Jun and is able to bind to DNA. Accordingly, the protein was termed Kid (kinesin-like DNA binding protein). Indirect immunofluorescence studies show that Kid colocalizes with mitotic chromosomes and that it is enriched in the kinetochore at anaphase. Thus, we propose that Kid might play a role(s) in regulating the chromosomal movement along microtubules during mitosis.