Overexpression of a GRAS protein lacking the DELLA domain confers altered gibberellin responses in rice

The rice SLR1 (SLENDER RICE 1) gene encodes a DELLA protein that belongs to a subfamily of the GRAS protein superfamily and that functions as a repressor of gibberellin (GA) signaling. Based on the constitutive GA response phenotype of slr1 mutants, SLR1 has been thought to be the sole DELLA-type protein suppressing GA signals in rice. However, in rice genome databases we identified two sequences homologous to SLR1: SLR1-like1 and -2 (SLRL1 and -2). SLRL1 and SLRL2 contain regions with high similarity to the C-terminal conserved domains in SLR1, but lack the N-terminal conserved region of the DELLA proteins. The expression of SLRL1 was positively regulated by GA at the mRNA level and occurred preferentially in reproductive organs, whereas SLRL2 was moderately expressed in mature leaf organs and was not affected by GA. Transformation of SLRL1 into the slr1 mutant rescued the slender phenotype of this mutant. Moreover, overexpression of SLRL1 in normal rice plants induced a dwarf phenotype with an increased level of OsGA20ox2 gene expression and diminished the GA-induced shoot elongation, suggesting that SLRL1 acts as a repressor of GA signaling. Consistent with the fact that SLRL1 does not have a DELLA domain, which is essential for degradation of DELLA proteins, a level of SLRL1 protein was not degraded by application of gibberellic acid. However, the repressive activity of SLRL1 against GA signaling was much weaker than a truncated SLR1 lacking the DELLA domain. Based on these characteristics of SLRL1, the functional roles of SLRL1 in GA signaling in rice are discussed.     

Identification and expression of a novel family of bHLH cDNAs related to Drosophila hairy and enhancer of split.

In this report we describe the initial characterization of murine, human, and Drosophila hesr-1 (for hairy and enhancer of split related-1) a novel evolutionary conserved family of hairy/enhancer of split homologs. Hesr-1 cDNAs display features typical of hairy and enhancer of split-type bHLH proteins including a N-terminal bHLH domain a conserved orange domain immediately C-terminal to the bHLH region. Despite their similarity to known hairy/enhancer of split homologs, hesr-1 cDNAs are divergent members of the hairy and enhancer of split bHLH family since the degree of sequence identity within the bHLH and their nearest homologs are relatively low. Moreover, the tetrapeptide motif, WRPW, which is found in all hairy and enhancer of split family members, is not present in hesr-1. Rather, a variant of this motif, YRPW, is found. Analysis of embryonic murine hesr-1 expression by in situ hybridization reveals strong expression in the somitic mesoderm, the central nervous system, the kidney, the heart, nasal epithelium, and limbs indicating a role for hesr-1 in the development of these tissues. Like the enhancer of split cDNAs in Drosophila, we show that hesr-1 expression depends critically on signaling through the notch pathway in murine embryos, suggesting that aspects of hesr-1 regulation and function might also be evolutionary conserved.     

Drosophila EYA Regulates the Immune Response against DNA through an Evolutionarily Conserved Threonine Phosphatase Motif

Innate immune responses against DNA are essential to counter both pathogen infections and tissue damages. Mammalian EYAs were recently shown to play a role in regulating the innate immune responses against DNA. Here, we demonstrate that the unique Drosophila eya gene is also involved in the response specific to DNA. Haploinsufficiency of eya in mutants deficient for lysosomal DNase activity (DNaseII) reduces antimicrobial peptide gene expression, a hallmark for immune responses in flies. Like the mammalian orthologues, Drosophila EYA features a N-terminal threonine and C-terminal tyrosine phosphatase domain. Through the generation of a series of mutant EYA fly strains, we show that the threonine phosphatase domain, but not the tyrosine phosphatase domain, is responsible for the innate immune response against DNA. A similar role for the threonine phosphatase domain in mammalian EYA4 had been surmised on the basis of in vitro studies. Furthermore EYA associates with IKKβ and full-length RELISH, and the induction of the IMD pathway-dependent antimicrobial peptide gene is independent of SO. Our data provide the first in vivo demonstration for the immune function of EYA and point to their conserved immune function in response to endogenous DNA, throughout evolution.     

Rice bicoid－related cDNA sequence and its expression during early embryogenesis

INTRODUCTIONThe establishment of embryonic polarity is oneof the critical events in embryogenesis. The polar distribution of maternal mRNA can be tracedto the unfertilized egg cell. After fertilization, itstranslational products trigger the zygotic targetgenes which define the embryonic region and fUIther direct the pattern formation and body planduring embryogenesis. In Drosophila, Bicoid is oneof the important maternal genes involved in thecontrol of embryo polarity and larvae segmen…     

Pax6-dependence of Six3, Eya1 and Dach1 expression during lens and nasal placode induction

The Drosophila eyeless gene plays a central role in fly eye development and controls a subordinate regulatory network consisting of the so, eya and dac genes. All three genes have highly conserved mammalian homologs, suggesting possible conservation of this eye forming regulatory network. sine oculis (so) belongs to the so/Six gene family, and Six3 is prominently expressed in the developing mammalian eye. Eya1 and Dach1 are mammalian homologs of eya and dac, respectively, and although neither Eya1 nor Dach1 knockout mice express prenatal eye defects, possibilities exist for postnatal ocular phenotypes or for functional redundancy between related family members. To examine whether expression relationships analogous to those between ey, so, eya and dac exist in early mammalian oculogenesis, we investigated Pax6, Six3, Eya1 and Dach1 protein expression in murine lens and nasal placode development. Six3 expression in the pre-placode lens ectoderm is initially Pax6-independent, but subsequently both its expression and nuclear localization become Pax6-dependent. Six3, Dach1 and Eya1 nasal expression in pre-placode ectoderm are also initially Pax6-independent, but thereafter become Pax6-dependent. Pax6, Six3, Dach1 and Eya1 are all co-expressed in the developing ciliary marginal zone, a source of retinal stem cells in some vertebrates. An in vitro protein-protein interaction is detected between Six3 and Eya1. Collectively, these findings suggest that the Pax-Eya-Six-Dach network is at best only partly conserved during lens and nasal placode development. However, the findings do not rule out the possibility that such a regulatory network acts at later stages of oculogenesis.     

Occurrence and characterization of PEND proteins in angiosperms

The PEND protein is a DNA-binding protein in the inner envelope membrane of the developing chloroplast. It consists of a short pre-sequence, an N-terminal DNA-binding domain (cbZIP), a central repeat domain, and a C-terminal transmembrane domain. PEND homologs have been detected in various angiosperms, including Arabidopsis thaliana, Brassica napus, Medicago truncatula, cucumber and cherry. Monocot homologs have also been detected in barley and rice, but sequence conservation was low in monocots. PEND-related sequences have not been detected in non-flowering plants and algae. Green fluorescent protein fusions consisting of the N-terminal as well as full-length PEND homologs in A. thaliana and B. napus were targeted to chloroplasts, and localized to nucleoids and chloroplast periphery, respectively. Immunoblot analysis suggested that crucifer homologs were present in chloroplasts probably as a dimer, as in the case of pea. These results suggest that PEND protein is present in angiosperms, and the homologs in crucifers are functionally analogous to the PEND protein in pea.     

Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily

The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.     

Molecular characterization and expression analysis of a highly conserved rice mago nashil homolog.

Mago Nashi, a protein initially shown to be essential in the development of the Drosophila oocyte, is highly conserved among species and shows no homology to any other known cellular proteins. Here we report the nucleotide sequence of a cDNA and a partial gene that encode rice Mago Nashi protein homologs. In addition, we present the tissue-specific expression pattern of mago nashi at the level of RNA and protein. The rice Mago Nashi protein shares at least 73% amino acid identity with all known animal homologs. Genomic DNA gel blot analysis indicates that two copies of the mago nashi gene exist in the rice genome, one of which has identical intron positions to those found in an Arabidopsis homolog. mago nashi is expressed in root, leaf and developing seed tissue as determined by RNA and protein gel blot analysis. Evidence from Drosophila, Caenorhabditis elegans and human studies of Mago Nashi suggests that a major function of this protein is its involvement in RNA localization. The highly conserved amino acid sequence of all Mago Nashi protein homologs across kingdoms suggests that the plant version of this protein may similarly be involved in RNA localization.     

Characterization of cDNAs encoding p53 of Bombyx mori and Spodoptera frugiperda

Complementary DNAs encoding homologs of the tumor suppressor gene, p53, were characterized from two lepidopteran insects, Bombyx mori (Bm) and Spodoptera frugiperda (Sf). They encoded predicted proteins of 368 (41.2 kDa) (Bm) and 374 (42.5 kDa) (Sf) amino acids. The sequences shared 44% amino acid and 60% nucleotide sequence identity with each other, but exhibited less than 20% amino acid and 46% nucleotide sequence identity to Drosophila melanogaster p53. Despite the sequence diversity, conserved amino acids involved in DNA and zinc binding were present in the lepidopteran sequences. Expression of Sfp53-induced apoptosis in S. frugiperda cells, and antiserum made against recombinant Sfp53 recognized a protein whose abundance increased after treatment with DNA damaging agents.     

Isolation, characterization and evolutionary analysis of resistance gene analogs in annual ryegrass, perennial ryegrass and their hybrid

Recently, a number of disease-resistance genes related to a diverse range of pathogens were isolated from a wide variety of plant species. The majority of plant disease-resistance genes encoded a nucleotide-binding site (NBS) domain. According to the comparisons of the NBS domain of cloned R -genes, it has shown highly conserved amino acid motifs in this structure, which made it possible to isolate resistance gene analogs (RGAs) by PCR using degenerate primers. We have designed three pairs of degenerate primers based on two conserved motifs in the NBS domain of resistance proteins encoded by R -genes to amplify genomic sequences from ryegrass ( Lolium sp.). Sixteen NBS-like RGAs were isolated from turf and forage type grasses. The sequence analysis of these RGAs revealed that there existed a high similarity (up to 85%) between RGA sequences among ryegrass species and other plants. The alignment of the predicted amino acid sequences of RGAs showed that ryegrass RGAs contained four conserved motifs (P-Loop, kinase-2, kinase-3a, GLPL) present in other known plant NBS-leucine rich repeat resistance genes. These ryegrass RGAs all belonged to non-toll and interleukin-1 receptor subclass. Phylogenetic analysis of ryegrass RGAs and other cloned R -genes indicated that gene mutation was the predominant source of gene variations, and the sequence polymorphism was due to purifying selection rather than diversifying selection. We further analyzed the source of gene variation in other monocots, rice, barley, wheat, and maize based on the data published before. Our analysis indicated that the source of RGA diversity in these monocots was the same as in ryegrass. Thus, monocots were probably the same as dicots in the source of RGA diversity. Ryegrass RGAs in the present paper represented a large group of resistance gene homologs in monocots. We discussed the origin and the evolution of R -genes in grass species.     

Molecular cloning of human XPAC gene homologs from chicken, Xenopus laevis and Drosophila melanogaster.

We cloned homologs of the human Xeroderma Pigmentosum Group A complementing (XPAC) gene from chicken, Xenopus laevis and Drosophila melanogaster. A comparison of the amino acid sequences of these homologs with that of the human XPAC protein revealed that in the NH2-terminal domain there are only two conserved regions, one of which is presumed to function as the nuclear localization signal, whereas the COOH-terminal domain is highly conserved, the frequency of identical amino acids in all four XPAC proteins being 50%, and the four cysteine residues predicted to form a zinc-finger motif, and three other cysteine residues are all conserved. These results strongly suggest that the COOH-terminal domain containing a zinc-finger motif plays an important role in the function of these proteins.     

Structural characterization of the 60-kDa bermuda grass pollen isoallergens, a covalent flavoprotein

Our studies suggest a tripartite structure for the 60-kDa allergen of Bermuda grass pollen (BG60) including a short N-terminal segment, a FAD-binding domain, and a C-terminal domain. The lower molecular weight isoallergens lack the N-terminal segment. The higher protease susceptibility and the lower melting temperature of approximately 20 degrees C of the lower molecular weight isoforms suggest that the N-terminal segment is essential for a compact structure. Database screening reveals that the protease-digested peptide sequences (approximately 180 residues in total) share 40% identity with the plant berberine bridge enzymes. In particular, a 24-residue peptide sequence displays high similarity to a conserved FAD-binding motif. The spectroscopic and SDS-PAGE analyses suggest that the cofactor FAD is covalently linked to the central domain. Therefore, we conclude that BG60 is identified as the first flavinylated allergen.