<Emphasis Type="Italic">VpUR9</Emphasis>, a novel RING-type ubiquitin ligase gene from <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>, is involved in powdery mildew response in transgenic <Emphasis Type="Italic">V. vinifera</Emphasis> plants

Ubiquitination plays important roles in disease resistance in plants. We report the identification and functional characterization of the RING-type ubiquitin ligase gene VpUR9 from Chinese wild Vitis pseudoreticulata accession Baihe-35-1. VpUR9, encodes 164 amino acids and possesses a RING conserved motif. It is homologously cloned from the cDNA library of the high powdery mildew (Erysiphe necator [Schw.] Burr) resistant V. pseudoreticulata accession Baihe-35-1 inoculated with E. necator. The gene is induced in response to powdery mildew and salicylic acid. VpUR9 fused with FLAG-tag controlled by 35S promoter was transformed into 15 regenerated V. vinifera L. cv. Red Globe lines via Agrobacterium tumefaciens-mediated transformation. Twelve of these lines were confirmed by Western blot of FLAG-tag. As a result, the powdery mildew-resistance of Red Globe transformed with VpUR9 was repressed. Furthermore, the expression of some disease-resistant related genes (NPR1, PR1, PR10 and PAL) of the transgenic Red Globe declined compared with wild type grapes when inoculated with powdery mildew or salicylic acid. When treated with jasmonic acid methyl ester, its PR1 gene expression decreased, while the expressions of NPR1, PR10 and PAL all increased, contrasting with the wild type grape.     

Characterisation of the <Emphasis Type="Italic">Vitis vinifera</Emphasis>PR10 multigene family

Background  

Genes belonging to the pathogenesis related 10 (PR10) group have been studied in several plant species, where they form multigene families. Until now, such an analysis has not been performed in Vitis vinifera, although three different PR10 genes were found to be expressed under pathogen attack or abiotic stress, and during somatic embryogenesis induction. We used the complete genome sequence for characterising the whole V. vinifera PR10 gene family. The expression of candidate genes was studied in various non-treated tissues and following somatic embryogenesis induction by the auxin 2,4-D.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) with a novel stilbene synthase gene from Chinese wild <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>

A novel stilbene synthase gene (STS), cloned from Chinese wild Vitis pseudoreticulata (W. T. Wang) and responsible for synthesis of the phytoalexin resveratrol in grapevine, was successfully transferred into V. vinifera L. cv. Thompson Seedless via Agrobacterium tumefaciens-mediated transformation. Using transformation procedures developed in the present study, 72% GFP-positive germinated embryos were produced with about 38% of transformed embryos regenerated into normal plantlets. Integration of the STS gene into the transgenic plants was verified by PCR and Southern blot analysis. Expression of the STS gene was detected by high performance liquid chromatography (HPLC), which showed that the resveratrol concentration in the transgenic plants was 5.5 times higher than that in non-transformed control plants. Chaohong Fan and Ni Pu contributed equally to this work.     

Enhancement of crystallinity of cellulose produced by <Emphasis Type="Italic">Escherichia coli</Emphasis> through heterologous expression of <Emphasis Type="Italic">bcsD</Emphasis> gene from <Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>

Objectives

To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001.

Results

The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD.

Conclusion

The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

     

Molecular cloning and characterization of cold-responsive gene <Emphasis Type="Italic">Cbrci35</Emphasis> from <Emphasis Type="Italic">Capsella bursa-pastoris</Emphasis>

A new rare cold-inducible (RCI) gene designated Cbrci35 was cloned from Capsella bursa-pastoris, an edible wild herb, using the rapid amplification of cDNA ends (RACE) method. The full-length cDNA of Cbrci35 (Database Accession No.: AY566573) was 1300 bp and contained a 978 bp ORF encoding a precursor of 326 amino acid residues with a 23 amino acids signal peptide. The predicted Cbrci35 protein contained a peroxidase active site and proximal heme-ligand signatures, an RGD cell attachment sequence motif and two leucine zipper pattern motifs. Bioinformatics analysis revealed that Cbrci35 has a high level of similarity with RCI genes from Arabidopsis thaliana and peroxidases genes from other plants. RT-PCR analysis revealed that Cbrci35 expressed only in root. A cold acclimation assay showed that Cbrci35 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. But expression was not induced exposed to dehydration, salt stress or abscisic acid, indicating that it might be subjected specifically to cold regulation. These results indicate that Cbrci35 is an analogue of RCI genes and may participate in cold-response or increasing the freezing tolerance of plants.     

Expression Profiles of Pathogenesis-Related Gene, <Emphasis Type="Italic">TaLr35PR1</Emphasis>, as it Relate to <Emphasis Type="Italic">Lr35</Emphasis>-Mediated Adult Plant Leaf Rust Resistance

Plants have developed sophisticated mechanisms to combat pathogen infection. One of the acquired modes in response to pathogen attack is the production of the pathogenesis-related (PR) proteins. Our earlier studies reported that TaLr35PR1, a PR1 gene encoding a protein with conserved serine carboxypeptidase (SCP) domain, has been cloned from wheat near-isogenic line TcLr35. However, the involvement of TaLr35PR1 in wheat growth and Lr35-mediated adult resistance to Puccinia triticina remains unclear. Here, we showed that TaLr35PR1 was strongly induced by P. triticina in wheat plant containing Lr35 (TcLr35), in which the expression level of TaLr35PR1 significantly increased and reached the maximum at 12 hpi. The accumulations of TaLr35PR1 increased stably and showed significant peak challenged by P. triticina at different growth and development periods of TcLr35 wheat while it maintained similar level and changed little in mock inoculated. Western blotting was conducted to confirm that TaLr35PR1 protein was increasingly accumulated in the TcLr35 adult plants after P. triticina inoculation and maintained at a similar level from 120 to 168 h post-inoculation. Similar to the expression patterns of TaLr35PR1 at RNA levels, the accumulations of TaLr35PR1 protein were weak in the seedling stage and then increased to the peak and kept constant levels at the mature stage which is consistent with the expression feature of Lr35 gene as an adult plant resistance gene. All these findings suggest that TaLr35PR1 is involved in wheat growth and Lr35-mediated adult wheat defense response to leaf rust pathogen attack.     

Characteristic expression of rice pathogenesis-related proteins in rice leaves during interactions with <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Pathogenesis-related (PR) proteins play an important role in the disease resistance response. To better understand the function of rice PR proteins, we examined the expressions of ten PR proteins in rice leaves at different developmental stages with or without the interaction between rice and Xanthomonas oryzae pv. oryzae (Xoo). The results showed that most of the PR proteins were expressed in rice leaves in normal growth conditions, suggesting that they play a role in rice growth. Six out of ten PR proteins (PR1, PR2, PR3, PR4b, PR8, and PR-pha) showed enhanced expression in Xa21-mediated resistance responses at late stages after inoculation with Xoo. The remaining four PR proteins (PR5, PR6, PR15, and PR16) did not show changes in expression in the resistance response. The expressions of PR proteins in the resistance reaction were further compared with those in the susceptible reaction and a mock treatment. Interestingly, several of the PR proteins were expressed at the highest levels in the susceptible reaction and at the lowest levels in the mock treatment. Among the other four PR proteins, PR5 and PR16 showed changes in the abundance only in the susceptible response, while PR6 and PR15 showed no detectable difference in expression. These data provide fundamental knowledge about the expression of PR proteins in the interaction between rice and Xoo.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">pathogenesis related</Emphasis> protein 6 from <Emphasis Type="Italic">Panax ginseng</Emphasis>

Panax ginseng Meyer is one of the important medicinal plants in the world, particularly in Asian countries. Ginseng encounters many stress exposure during its long cultivation period. However, the molecular mechanism of stress resistance is still poorly understood in spite of its importance. In this study, pathogenesis-related protein 6 (PR6), also called proteinase inhibitor (PI), was isolated from ginseng embryogenic callus, named PgPR6. The small size of PR6, containing an open reading frame of 219 bp encoding 72 amino acids, the typical characteristic of PR6 protein, shares the highest sequence similarity to PR6 of Theobroma cacao (69% identity). Sequence and structural analysis indicated that PgPR6 belongs to class Kunitz-type PI family. This is the first report pertaining to the identification of PR6 gene from the P. ginseng genome. The high-level expression of PgPR6 was observed in root as revealed by quantitative real-time PCR. The temporal expression analysis demonstrated that PgPR6 expression was highly up-regulated by signaling molecules, heavy metals, mechanical wounding, chilling, salt, sucrose, and mannitol stress, indicating that PgPR6 may play an important role in the molecular defense response of ginseng to a various range of environmental stresses.     

Expression of CoQ10-producing <Emphasis Type="Italic">ddsA</Emphasis> transgene by efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Panicum meyerianum</Emphasis>

Panicum meyerianum Nees is a wild relative of Panicum maximum Jacq. (guinea grass), which is an important warm-season forage grass and biomass crop. We investigated the conditions that maximized the transformation efficiency of P. meyerianum by Agrobacterium infection by monitoring the expression of the β-glucuronidase (GUS) gene. The highest activities of GUS in calli were achieved by the co-cultivation of plants with Agrobacterium at 28°C for 6 days. We transferred the ddsA gene, which encodes decaprenyl diphosphate synthase and is required for coenzyme Q10 (CoQ10) synthesis, into P. meyerianum by using our optimized co-cultivation procedure for transformation. We confirmed by PCR and DNA gel blot hybridization that all hygromycin-resistant plants retained stable insertion of the hpt and ddsA genes. We also demonstrated strong expression of S14:DdsA protein in the leaves of transgenic P. meyerianum. Furthermore, we showed that transgenic P. meyerianum produced CoQ10 at levels 11–20 times higher than that of non-transformants. By comparison, the CoQ9 level in transgenic plants was dramatically reduced. This is the first report of efficient Agrobacterium-mediated transfer of a foreign gene into the warm-season grass P. meyerianum.     

Isolation and Expression Analysis of <Emphasis Type="Italic">PeDWF1</Emphasis> in <Emphasis Type="Italic">Phyllostachys edulis</Emphasis>

Brassinosteroids (BRs) are steroidal hormones that play crucial roles in various processes of plant growth and development. DWF1 encodes a delta(24)-sterol reductase that participates in one of the early stage in the brassinosteroids’ biosynthetic pathway: the conversion of 24-methylenecholesterol to campesterol. Here we report the isolation and expression of one DWF1 homologous gene, PeDWF1, in moso bamboo (Phyllostachys edulis (Carrière) J. Houz.). Sequence analysis revealed that the open reading frame of PeDWF1 was 1686-bp encoding a protein composed of 561 amino acid residues with a calculated molecular weight of 65.1 kD and a theoretic isoelectric point of 8.32. Phylogenetic analysis indicated that PeDWF1 was very close to the cell elongation protein Dwarf1 in rice (Oryza sativa). Furthermore, transient expression of a PeDWF1::GFP fusion protein showed that PeDWF1 was an integral membrane protein most probably associated with the endoplasmic reticulum similar to Dwarf1. Tissue specific expression analysis showed that PeDWF1 was constitutively expressed in moso bamboo with the highest level in shoots and the lowest level in mature leaves. In the early growing stage of shoots, the expression level of PeDWF1 had a rising trend with the increasing height of shoots. These results indicated that PeDWF1 might be involved in the regulation of shoot development by participating in BRs biosynthesis. Moreover, PeDWF1 was heterologously expressed in Escherichia coli and the recombinant protein was about 65 kD, which facilitated further study on the gene function of PeDWF1 in bamboo.     

Characterization of directly transformed weedy <Emphasis Type="Italic">Brassica rapa</Emphasis> and introgressed <Emphasis Type="Italic">B. rapa</Emphasis> with Bt <Emphasis Type="Italic">cry1Ac</Emphasis> and <Emphasis Type="Italic">gfp</Emphasis> genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.     

Proteomic approach: Identification of <Emphasis Type="Italic">Medicago truncatula</Emphasis> proteins induced in roots after infection with the pathogenic oomycete <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>

The legume root rot disease caused by the oomycete pathogen Aphanomyces euteiches is one major yield reducing factor in legume crop production. A comparative proteomic approach was carried out in order to identify proteins of the model legume Medicago truncatula which are regulated after an infection with A. euteiches. Several proteins were identified by two dimensional gel electrophoresis to be differentially expressed after pathogen challenge. Densitometric evaluation of expression values showed different regulation during the time-course analysed. Proteins regulated during the infection were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Among the differentially expressed proteins, two encoded putative cell wall proteins and two were designated as small heat shock proteins. Furthermore, an isoform of the chalcone-O-methyltransferase was found to be increased in infected roots. The majority of induced proteins belonged to the family of class 10 of pathogenesis related proteins (PR10). Previously, various PR10-like proteins have been shown to be regulated by general stress or abscisic acid (ABA). Therefore, these proteins were further investigated concerning their regulation in response to drought stress and exogenous ABA-application. Complex regulation patterns were identified: three of the A. euteiches-induced PR10-like proteins were also induced by exogenous ABA- but none of them is induced after drought stress. In contrast, three of these proteins are down-regulated by drought stress. Hence, the strong expression of different PR10-family members and their regulation profiles indicates that this set of proteins plays a major role during root adaptations to various stress conditions.