The transcriptional response of apple alcohol acyltransferase (MdAAT2) to salicylic acid and ethylene is mediated through two apple MYB TFs in transgenic tobacco.

Volatile esters are major factors affecting the aroma of apple fruits, and alcohol acyltransferases (AATs) are key enzymes involved in the last steps of ester biosynthesis. The expression of apple AAT (MdAAT2) is known to be induced by salicylic acid (SA) or ethylene in apple fruits, although the mechanism of its transcriptional regulation remains elusive. In this study, we reveal that two apple transcription factors (TFs), MdMYB1 and MdMYB6, are involved in MdAAT2 promoter response to SA and ethylene in transgenic tobacco. According to electrophoretic mobility shift assays, MdMYB1 or MdMYB6 can directly bind in vitro to MYB binding sites in the MdAAT2 promoter. In vivo, overexpression of the two MYB TFs can greatly enhance MdAAT2 promoter activity, as demonstrated by dual luciferase reporter assays in transgenic tobacco. In contrast to the promoter of MdMYB1 or MdMYB6, the MdAAT2 promoter cannot be induced by SA or ethephon (ETH) in transgenic tobacco, even in stigmas in which the MdAAT2 promoter can be highly induced under normal conditions. However, the induced MYB TFs can dramatically enhance MdAAT2 promoter activity under SA or ETH treatment. We conclude that MdMYB1 and MdMYB6 function in MdAAT2 responses to SA and ethylene in transgenic tobacco, suggesting that a similar regulation mechanism may exist in apple.

A multifunctional protein encoded by turkey herpesvirus suppresses RNA silencing in Nicotiana benthamiana.

Many plant and animal viruses counteract RNA silencing-mediated defense by encoding diverse RNA silencing suppressors. We characterized HVT063, a multifunctional protein encoded by turkey herpesvirus (HVT), as a silencing suppressor in coinfiltration assays with green fluorescent protein transgenic Nicotiana benthamiana line 16c. Our results indicated that HVT063 could strongly suppress both local and systemic RNA silencing induced by either sense RNA or double-stranded RNA (dsRNA). HVT063 could reverse local silencing, but not systemic silencing, in newly emerging leaves. The local silencing suppression activity of HVT063 was also verified using the heterologous vector PVX. Further, single alanine substitution of arginine or lysine residues of the HVT063 protein showed that each selected single amino acid contributed to the suppression activity of HVT063 and region 1 (residues 138 to 141) was more important, because three of four single amino acid mutations in this region could abolish the silencing suppressor activity of HVT063. Moreover, HVT063 seemed to induce a cell death phenotype in the infiltrated leaf region, and the HVT063 dilutions could decrease the silencing suppressor activity and alleviate the cell death phenotype. Collectively, these results suggest that HVT063 functions as a viral suppressor of RNA silencing that targets a downstream step of the dsRNA formation in the RNA silencing process. Positively charged amino acids in HVT063, such as arginine and lysine, might contribute to the suppressor activity by boosting the interaction between HVT063 and RNA, since HVT063 has been demonstrated to be an RNA binding protein.

A novel late embryogenesis abundant like protein associated with chilling stress in Nicotiana tabacum cv. bright yellow-2 cell suspension culture.

Low temperature is one of the major abiotic stresses limiting the productivity and geographical distribution of many important crops. To identify proteins associated with chilling stress in Nicotiana tabacum cv. bright yellow-2 (BY-2) cell suspension culture, we utilized a proteomic approach with two-dimensional electrophoresis to compare proteins from samples of treated with or without chilling treatment at 4 °C. One protein specifically more abundant in chilling treated sample was identified and designated as NtLEA7-3. Rapid amplification of cDNA ends gave rise to a full-length NtLEA7-3 cDNA with a complete open reading frame of 1267 bp, encoding a 322 amino acid polypeptide. Homology search and sequence multi-alignment demonstrated that the deduced NtLEA7-3 protein sequence shared a high identity with LEA-like proteins from other plants. Subcellular localization analysis indicated that the NtLEA7-3 was localized exclusively in the nucleus. When the gene was overexpressed in bright yellow-2 cells, the transgenic bright yellow-2 cells show more resistant to chilling stress than the wild-type cells. In addition, transgenic Arabidopsis plants overexpressing the NtLEA7-3 are much more resistant to cold, drought, and salt stresses. Interestingly, the expression of NtLEA7-3 in tobacco was not tissue-specific and induced by chilling, drought and salt stresses. All of these, taken together, suggest that NtLEA7-3 is worthwhile to elucidate the contribution of the proteins to the tolerance mechanism to chilling stress, and can be considered as a potential target for crop genetic improvement in the future.

NtKTI1, a Kunitz trypsin inhibitor with antifungal activity from Nicotiana tabacum, plays an important role in tobacco's defense response.

A cDNA library from tobacco inoculated with Rhizoctonia solani was constructed, and several cDNA fragments were identified by differential hybridization screening. One cDNA clone that was dramatically repressed, NtKTI1, was confirmed as a member of the Kunitz plant proteinase inhibitor family. RT-PCR analysis revealed that NtKTI1 was constitutively expressed throughout the whole plant and preferentially expressed in the roots and stems. Furthermore, RT-PCR analysis showed that NtKTI1 expression was repressed after R. solani inoculation, mechanical wounding and salicylic acid treatment, but was unaffected by methyl jasmonate, abscisic acid and NaCl treatment. In vitro assays showed that NtKTI1 exerted prominent antifungal activity towards R. solani and moderate antifungal activity against Rhizopus nigricans and Phytophthora parasitica var. nicotianae. Bioassays of transgenic tobacco demonstrated that overexpression of NtKTI1 enhanced significantly the resistance of tobacco against R. solani, and the antisense lines exhibited higher susceptibility than control lines towards the phytopathogen. Taken together, these studies suggest that NtKTI1 may be a functional Kunitz trypsin inhibitor with antifungal activity against several important phytopathogens in the tobacco defense response.

GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5.

* Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. * A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH-type zinc finger protein, was isolated from a salt-induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two-hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis-related protein 5), which interacted with GhZFP1, were isolated. * GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. * Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

Tissue-specific expression of the PNZIP promoter is mediated by combinatorial interaction of different cis-elements and a novel transcriptional factor.

Recent studies demonstrated that PNZIP and its homologs encode a special cyclase and play an important role in chlorophyll biosynthesis in higher plants. To investigate the molecular mechanism governing the PNZIP gene, the PNZIP promoter was isolated and analyzed. Deletion analysis indicated that G-box is an important element in the regulation of the reporter gene expression. Further mutation assay demonstrated that G-box and GATACT elements are necessary and sufficient for the high and tissue-specific expression of the GUS gene. Using yeast one-hybrid screening, we have isolated a novel tobacco bZIP protein, NtbZIP, which can specifically recognize the G-box of the PNZIP promoter. The NtbZIP protein shares a limited amino acid homology to Arabidopsis ABI5 and AtAREB1 and very low homology to other bZIP proteins. Northern blot analysis showed that the NtbZIP gene is not induced by exogenous ABA and is expressed in different tobacco organs. Cotransformation assays showed that the NtbZIP protein could activate the transcription of the GUS gene driven by the PNZIP promoter. Transgenic tobaccos analysis demonstrated that constitutively expressing antisense NtbZIP gene resulted in a lower NTZIP synthesis and reduced chlorophyll levels. We suggest that NTZIP is a target gene of NtbZIP, which is involved in the regulation of chlorophyll biosynthesis.

Cotton GhDREB1 increases plant tolerance to low temperature and is negatively regulated by gibberellic acid.

The transcription factors C-repeat binding factors/dehydration-responsive element binding proteins (CBFs/DREBs) control the expression of many stress-inducible genes in Arabidopsis. A cDNA clone, designated GhDREB1, was isolated from cotton (Gossypium hirsutum) by cDNA library screening. Northern blot analysis indicated that mRNA accumulation of GhDREB1 was induced by low temperatures and salt stress, but was not induced by abscisic acid (ABA) or drought stress in cotton seedlings. Transgenic tobacco (Nicotiana tabacum) plants overexpressing GhDREB1 displayed stronger chilling tolerance than wild-type plants. Their leaf chlorophyll fluorescence, net photosynthetic rate and proline concentrations were higher than those of control plants during low-temperature treatment. However, under normal growth conditions, the transgenic tobacco plants exhibited retarded growth and delayed flowering. Interestingly, GhDREB1 transcripts in cotton seedlings were negatively regulated by gibberellic acid (GA(3)) treatment. Analysis of the promoter of the GhDREB1 gene revealed the presence of one low-temperature and four gibberellin-responsive elements. Green fluorescent protein (GFP) signal intensity or beta-glucuronidase (GUS) activity driven by the GhDREB1 promoter was clearly enhanced by low temperature but repressed by GA(3). These results suggest that GhDREB1 functions as a transcription factor and plays an important role in improving cold tolerance, and also affects plant growth and development via GA(3).

Expression and characterization of Helicobacter pylori heat-shock protein A (HspA) protein in transgenic tobacco (Nicotiana tabacum) plants.

Helicobacter pylori infection is prevalent worldwide, especially in developing countries, and is associated with several upper-gastrointestinal-tract diseases. Vaccination is the most effective method to prevent and cure H. pylori infection. By using transgenic plants, plant organs could serve as factories to produce antigens of biotechnological interest. HspA (heat-shock protein A) is an effective antigen and one common to all strains of H. pylori. In the present study, the PCR technique was employed to amplify the gene fragment of the HspA from H. pylori chromosomal DNA. The pGEM-T vector was used for the insertion of the gene fragment of the HspA, and the vector pBI121 was used to construct the plant expression vector. After transformation, the regenerated tobacco plants were identified by PCR and by Northern- and Western-blot analyses. The results verified the integration of this gene into the genome of tobacco (Nicotiana tabacum) and the expression of this gene in transgenic tobacco. Mucosal immunization of mice with transgenic tobacco extracts containing the HspA protein elicited anti-HspA serum antibody that specifically bound to the purified bacterial HspA protein. The present study, using transgenic tobacco plants, provides useful data for the production of an edible plant vaccine.

Colonization by the arbuscular mycorrhizal fungus Glomus versiforme induces a defense response against the root-knot nematode Meloidogyne incognita in the grapevine (Vitis amurensis Rupr.), which includes transcriptional activation of the class III chitinase gene VCH3.

Inoculation of the grapevine (Vitis amurensis Rupr.) with the arbuscular mycorrhizal (AM) fungus Glomus versiforme significantly increased resistance against the root-knot nematode (RKN) Meloidogyne incognita. Studies using relative quantitative reverse transcription-PCR (RQRT-PCR) analysis of grapevine root inoculation with the AM fungus revealed an up-regulation of VCH3 transcripts. This increase was greater than that observed following infection with RKN. However, inoculation of the mycorrhizal grapevine roots with RKN was able to enhance VCH3 transcript expression further. Moreover, the increase in VCH3 transcripts appeared to result in a higher level of resistance against subsequent RKN infection. Constitutive expression of VCH3 cDNA in transgenic tobacco under the control of the cauliflower mosaic virus 35S promoter also conferred resistance against RKN, but had no significant effect on the growth of the AM fungus. We analyzed beta-glucuronidase (GUS) activity directed by a 1,216 bp VCH3 promoter in transgenic tobacco following inoculation with both the AM fungus and RKN. GUS activity was negligible in the root tissues before inoculation, and was more effectively induced after inoculation with the AM fungus than with RKN. Moreover, GUS staining in the mycorrhizal transgenic tobacco roots was enhanced by subsequent RKN infection, and was found ubiquitously throughout the whole root tissue. Together, these results suggest that AM fungus induced a defense response against RKN in the mycorrhizal grapevine roots, which appeared to involve transcriptional control of VCH3 expression throughout the whole root tissue.

Isolation and characterization of a chitinase gene VCH3 promoter from grapevine (Vitis amurensis).

The 1216-bp 5' upstream region of the gene encoding a class III chitinase VCH3 was isolated from grapevine (Vitis amurensis Rupr.) by adaptor-PCR (GenBank accession number AF441123), and the transcriptional start site of the VCH3 gene was identified by primer extension, which corresponds to the second A in the DNA sequence 5'-ATCAAGCAC-3'. Sequence analysis revealed that the VCH3 promoter sequence contains CAAT and TATA motifs that are located at the -122 and -29 nucleotide upstream of the transcriptional start site, respectively. Both motifs are characteristic of the eukaryotic gene promoter. In addition, within the VCH3 promoter, we found two inverse salicylic acid (SA)-responsive cis-acting motifs (TGACG) at -1181 bp and -293 bp upstream of the transcriptional start site, respectively. To characterize the VCH3 promoter, the VCH3 promoter-GUS chimera was constructed and transferred to Nicotiana tobacum cv. NC89 by Agrobacterium tumefaciens-mediated leaf disc transformation. Fluorometric and histochemical analysis of GUS activity in the transgenic tobacco root and leaf treated with SA showed that the VCH3 promoter was induced by SA, which indicates that it may have potential use in genetic engineering.

TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants.

Nuclear matrix attachment regions (MARs) are thought to influence the expression of the flanking genes. TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro. In this study, we investigated the effect of TM2 on transgene expression under the control of three different promoters in stably transformed rice calli and plants. The presence of TM2 flanking the transgene increased the expression of constructs based on the constitutive CaMV 35S and maize ubiquitin gene promoters in both resistant calli and transformed plants. The GUS expression directed by the photosynthetic-tissue-specific PNZIP promoter was also increased in photosynthetic tissues of transformants. However, TM2 did not change the gene expression pattern controlled by the PNZIP promoter. The effect of TM2 in transgenic plants was stronger than that in transgenic calli based on all three promoters. Our results indicate that TM2, as a novel strong MAR, can be used to increase the transgene expression levels in the whole plant or in particular tissues of monocotyledons.

The cotton GhNHX1 gene encoding a novel putative tonoplast Na(+)/H(+) antiporter plays an important role in salt stress.

A cDNA clone was isolated from cotton (Gossypium hirsutum) cDNA library and characterized with regard to its sequence, regulation in response to salt stress and functions in yeast mutants and transgenic tobacco plants. The clone, designated as GhNHX1, contains 2485 nucleotides with an open reading frame of 1629 nucleotides, and the deduced amino acid sequence showed high identities with other plant vacuolar-type Na(+)/H(+) antiporters. Northern blot analysis indicated that the mRNA accumulation of GhNHX1 was strongly induced by salt stress and abscisic acid in cotton seedlings. The expression of GhNHX1 in yeast Na(+)/H(+) antiporter mutant showed function complementation. The transgenic tobacco plants overexpressing GhNHX1 also had higher salt tolerance than the wild-type plants. The salt-induced mRNA level of GhNHX1 was 3 and 7 times higher in the salt-tolerant cotton cultivar ZM3 than those in the salt-sensitive cotton cultivars ZMS17 and ZMS12, respectively. Together, these results suggest that the products of the novel gene, GhNHX1, function as a tonoplast Na(+)/H(+) antiporter and play an important role in salt tolerance of cotton.

NTZIP antisense plants show reduced chlorophyll levels.

We have isolated and characterized a new photosynthetic tissue-specific gene NTZIP (Nicotiana tabacum leucine zipper) from tobacco (N. tabacum). Its deduced amino acid sequence has two highly conserved regions, leucine zipper and [EX(n)DEXRH](2) motifs, which are related to the gene's biochemical functions. NTZIP was expressed in leaves and stems, but was not detected in roots or flowers, suggesting that its physiological functions might be associated with photosynthesis. Northern blot analysis showed that NTZIP mRNA accumulation was induced by light signals, increased greatly under low temperatures and was repressed by strong light illumination. Furthermore, a number of homologs of NTZIP were isolated from cucumber (Cucumis sativus), rape (Brassica napus), clover (Trifolium repens), willow (Salix babylonica), rosebush (Rusa dovurica), wheat (Triticum aestivum) and spinach (Spinacia oleracea), proving the ubiquitous existence of the NTZIP-like genes in higher plants. Transgenic tobaccos constitutively expressing antisense RNA to NTZIP displayed chlorosis and a lack of ability to turn green even under normal growth conditions. The chlorophyll deficiency was further confirmed by chlorophyll content determination and gas exchange analysis. Based on these observations, we propose that NTZIP may be involved in chlorophyll biosynthesis, and might define a novel family of evolutionarily conserved proteins with its homologs in other plant species.