Isolation of a cDNA encoding a 31-kDa, pathogenesis-related 5/thaumatin-like (PR5/TL) protein abundantly expressed in apple fruit (Nalus domestica cv. Fuji)

A fruit-specific and pathogenesis-related 5/thaumatin-like (PR5/TL), 31-kDa protein was isolated by 2D-PAGE from fully-grown apples (Malus domestica cv. Fuji) and named Mdtl1 (Malus domestica thaumatin-like protein 1). Using the N-terminal sequence of the protein, the full-length cDNA encoding Mdtll was isolated. The cDNA clone (Mdtl1) consists of 944 bp with an open reading frame (ORF) of 744 bp encoding a protein of 247 amino acids. The deduced amino acid sequence of Mdtl1 shows high similarity to the sequences of PR5/TL proteins. Mdtl1 is a slightly acidic protein with a putative signal peptide and a putative N-glycosylation site, and lacks a C-terminal extension. This suggests that Mdtl1 is an apoplastic glycoprotein. Results of northern blotting indicated that expressions of Mdtl1 are developmentally regulated. Southern blot analysis showed that Mdtl1 may be present as a single copy, and there exist other genes closely related to Mdtl1 in the apple genome.     

Analysis of a Nicotiana plumbaginifolia cDNA encoding a novel small GTP-binding protein.

Small GTP-binding proteins belonging to the Ras superfamily have been found in evolutionarily divergent organisms. Here, we report the isolation and analysis of a cDNA encoding a putative small GTP-binding protein, designated Rhn1, from the plant, Nicotiana plumbaginifolia. The 21.8-kDa protein has 60% amino acid similarity with the mammalian Rab5 proteins. The Rhn1 protein is encoded by a small multigene family. Northern analysis shows the highest steady-state mRNA levels to be in roots and flowers. Furthermore, the Rhn1 protein has 80% amino acid similarity with an Arabidopsis small GTP-binding protein, designated Rha1.     

A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds

Based on the N-terminal sequence of a sunflower antifungal protein, a full length cDNA (Ha-LTP5) encoding a putative lipid transfer protein from sunflower seeds was cloned using a RT-PCR based strategy. However, the sequence of the deduced protein is not identical to that of the antifungal protein previously isolated. The nucleotide sequence presents an ORF of 116 amino acids with a putative signal peptide, thus encoding a mature protein of 90 amino acids that is basic and hydrophobic. In contrast to the pattern of expression described for most LTP-like genes from dicots, Northern blot analyses detected constitutive expression of Ha-LTP5 in seeds, but not in aerial parts of sunflower plants.     

Plant-based heterologous expression of Mal d 2, a thaumatin-like protein and allergen of apple (Malus domestica), and its characterization as an antifungal protein

Mal d 2 is a thaumatin-like protein and important allergen of apple fruits that is associated with IgE-mediated symptoms in apple allergic individuals. We obtained a full-length cDNA clone of Mal d 2 from RNA isolated from ripe apple (Malus domestica cv. Golden Delicious). The cDNA's open reading frame encodes a protein of 246 amino acid residues including a signal peptide of 24 residues and two putative glycosylation sites. The deduced amino acid sequence of the mature Mal d 2 protein results in a predicted molecular mass of 23,210.9Da and a calculated pI of 4.55. Sequence comparisons and molecular modeling place Mal d 2 among those pathogenesis-related thaumatin-like proteins that contain a conserved acidic cleft. In order to ensure the correct formation of the protein's eight conserved disulfide bridges we expressed Mal d 2 in Nicotiana benthamiana plants by the use of a tobacco mosaic viral vector. Transfected N.benthamiana plants accumulated Mal d 2 to levels of at least 2% of total soluble protein. MALDI-TOF mass spectrometric analyses of the recombinant Mal d 2 and its proteolytic fragments showed that the apple-specific leader peptide was correctly cleaved off by the host plant and that the mature recombinant protein was intact and not glycosylated. Purified recombinant Mal d 2 displayed the ability to bind IgE from apple-allergic individuals equivalent to natural Mal d 2. In addition, the recombinant thaumatin-like Mal d 2 exhibited antifungal activity against Fusarium oxysporum and Penicillium expansum, implying a function in plant defense against fungal pathogens.     

The sterol‐binding activity of PATHOGENESIS‐RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein

Pathogenesis‐related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS‐RELATED 1 (PR‐1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR‐1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR‐1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol‐auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR‐1, whereas sterol‐prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR‐1 expression are particularly well protected against oomycete pathogens.     

A novel calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings

A clone for a novel Arabidopsisthaliana calmodulin (CaM)-binding protein of 25 kDa (AtCaMBP25) has been isolated by using a radiolabelled CaM probe to screen a cDNA expression library derived from A. thaliana cell suspension cultures challenged with osmotic stress. The deduced amino acid sequence of AtCaMBP25 contains putative nuclear localization sequences and shares significant degree of similarity with hypothetical plant proteins only. Fusion of the AtCaMBP25 coding sequence to reporter genes targets the hybrid protein to the nucleus. Bacterially expressed AtCaMBP25 binds, in a calcium-dependent manner, to a canonical CaM but not to a less conserved isoform of the calcium sensor. AtCaMBP25 is encoded by a single-copy gene, whose expression is induced in Arabidopsis seedlings exposed to dehydration, low temperature or high salinity. Transgenic plants overexpressing AtCaMBP25 exhibits an increased sensitivity to both ionic (NaCl) and non-ionic (mannitol) osmotic stress during seed germination and seedling growth. By contrast, transgenic lines expressing antisense AtCaMBP25 are significantly more tolerant to mannitol and NaCl stresses than the wild type. Thus, the AtCaMBP25 gene functions as a negative effector of osmotic stress tolerance and likely participates in stress signal transduction pathways.     

Identification, cloning and characterization of a novel nuclear protein, HA95, homologous to A-kinase anchoring protein 95

Previously, we have identified and characterized nuclear AKAP95 from man which targets cyclic AMP (cAMP)-dependent protein kinase (PKA)-type II to the condensed chromatin/spindle region at mitosis. Here we report the cloning of a novel nuclear protein with an apparent molecular mass of 95 kDa that is similar to AKAP95 and is designated HA95 (homologous to AKAP95). HA95 cDNA sequence encodes a protein of 646 amino acids that shows 61% homology to the deduced amino acid sequence of AKAP95. The HA95 gene is located on chromosome 19p13.1 immediately upstream of the AKAP95 gene. Both HA95 and AKAP95 genes contain 14 exons encoding similar regions of the respective proteins, indicating a previous gene duplication event as the origin of the two tandem genes. Despite their apparent similarity, HA95 does not bind RII in vitro. HA95 contains a putative nuclear localization signal in its N-terminal domain. It is localized exclusively into the nucleus as demonstrated in cells transfected with HA95 fused to either green fluorescence protein or the c-myc epitope. In the nucleus, the HA95 protein is found as complexes directly associated with each other or indirectly associated via other nuclear proteins. In interphase, HA95 is co-localized with AKAP95, but the two proteins are not biochemically associated. At metaphase, both proteins co-localize with condensed chromosomes. The similarity in sequence and localization of HA95 and AKAP95 suggests that the two molecules constitute a novel family of nuclear proteins that may exhibit related functions.     

Style-specific and developmentally regulated accumulation of a glycosylated thaumatin/PR5-like protein in Japanese pear (Pyrus serotina Rehd.)

The stylar proteins of Japanese pear (Pyrus serotina Rehd.) were analyzed by two-dimensional gel electrophoresis, and a 32-kDa protein with an isoelectric point of 4.8 was found to be a major component in the style. The 32-kDa protein was a soluble glycoprotein which reacted with concanavalin A. The 32-kDa protein specifically accumulated in the style in a developmentally regulated manner, but was not detected in the other floral organs and leaves. An oligonucleotide representing the N-terminal amino acid sequence of the 32-kDa protein was used to amplify a cDNA fragment by polymerase chain reaction (PCR). The generated PCR product was used to screen a style cDNA library. The selected cDNA clone encoded 244 amino acid residues containing the N-terminal sequence of the 32-kDa protein. The N-terminus of the protein was preceded by putative signal peptide of 22 amino acid residues. The 32-kDa protein showed significant homology with the thaumatin/PR5-like proteins, and was named PsTL1 (Pyrus serotina thaumatin-like protein 1). The possible biological role of PsTL1 in the styles is discussed. Received: 27 November 1997 / Accepted: 19 January 1998     

Characterization of the arabidopsis formin-like protein AFH1 and its interacting protein

A partial cDNA encoding an Arabidopsis thaliana FH (Formin Homology) protein (AFH1) was used as a probe to clone a full length AFH1 cDNA. The deduced protein encoded by the cDNA contains a FH1 domain rich in proline residues and a C-terminal FH2 domain which is highly conserved amongst FH proteins. In contrast to FH proteins of other organisms, the predicted AFH1 protein also contains a putative signal peptide and a transmembrane domain suggesting its association with membrane. Cell fractionation by differential centrifugation demonstrated the presence of AFH1 in the Triton X-100 insoluble microsomal fraction. An Arabidopsis cDNA library was screened to identify proteins that interact with the C-terminal region of AFH1 using yeast two-hybrid assays, and one of the isolated cDNAs encoded a novel protein, FIP2. Experiments using recombinant proteins expressed in E. coli demonstrated that FIP2 interacted directly with AFH1. The amino acid sequence of FIP2 has partial homology to bacterial putative membrane proteins and animal A-type K+ ATPases. AFH1 may form a membrane anchored complex with FIP2, which might be involved in the organization of the actin cytoskeleton.     

Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.     

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.     

A novel pepper membrane-located receptor-like protein gene CaMRP1 is required for disease susceptibility, methyl jasmonate insensitivity and salt tolerance

Plant receptor proteins are involved in the signaling networks required for defense against pathogens. The novel pepper pathogen-induced gene CaMRP1 was isolated from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). This gene is predicted to encode a membrane-located receptor-like protein that has an N-terminal signal peptide and a C-terminal transmembrane helix. A CaMRP1-GFP fusion protein localized primarily to the plasma membrane of plant cells. Strong and early induction of CaMRP1 expression occurred following exposure of pepper plants to Xcv, Colletotricum coccodes, methyl jasmonate (MeJA) and wounding stress. Virus-induced gene silencing (VIGS) of CaMRP1 in pepper conferred enhanced basal resistance to Xcv infection, accompanied by induction of genes encoding basic PR1 (CaBPR1), defensin (CaDEF1) and SAR8.2 (CaSAR82A). In contrast, CaMRP1 overexpression (OX) in transgenic Arabidopsis plants resulted in increased disease susceptibility to Hyaloperonospora parasitica infection. Arabidopsis plants overexpressing CaMRP1 exhibited insensitivity to MeJA by causing reduced expression of MeJA-responsive genes. Overexpression also resulted in tolerance to NaCl and during salt stress, the expression of several abscisic acid-responsive genes was induced. Together, these results suggest that pepper CaMRP1 may belong to a new subfamily of membrane-located receptor-like proteins that regulate disease susceptibility, MeJA-insensitivity and salt tolerance.