Parental origin of the allotetraploid tobacco Nicotiana benthamiana

Nicotiana section Suaveolentes is an almost all‐Australian clade of allopolyploid tobacco species including the important plant model Nicotiana benthamiana. The homology relationships of this clade and its formation are not completely understood. To address this gap, we assessed phylogenies of all individual genes of N. benthamiana and the well studied N. tabacum (section Nicotiana) and their homologues in six diploid Nicotiana species. We generated sets of 44 424 and 65 457 phylogenetic trees of N. benthamiana and N. tabacum genes, respectively, each collectively called a phylome. Members of Nicotiana sections Noctiflorae and Sylvestres were represented as the species closest to N. benthamiana in most of the gene trees. Analyzing the gene trees of the phylome we: (i) dated the hybridization event giving rise to N. benthamiana to 4–5 MyA, and (ii) separated the subgenomes. We assigned 1.42 Gbp of the genome sequence to section Noctiflorae and 0.97 Gbp to section Sylvestres based on phylome analysis. In contrast, read mapping of the donor species did not succeed in separating the subgenomes of N. benthamiana. We show that the maternal progenitor of N. benthamiana was a member of section Noctiflorae, and confirm a member of section Sylvestres as paternal subgenome donor. We also demonstrate that the advanced stage of long‐term genome diploidization in N. benthamiana is reflected in its subgenome organization. Taken together, our results underscore the usefulness of phylome analysis for subgenome characterization in hybrid species.     

Ultrastructure of Necrotic Lesions in Nicotiana glutinosa Leaves Locally Infected with a Variant of Cucumber mosaic virus

CMV(Y/GM2)tr is a variant of Cucumber mosaic virus strain Y [CMV(Y)] which infects Nicotiana species, including N. glutinosa, to induce necrotic local lesions (NLLs) in inoculated leaves, although all other CMV strains including CMV(Y) systemically infect Nicotiana species. To investigate the morphological features of this unique host response in N. glutinosa leaves infected with CMV(Y/GM2)tr, the ultrastructure of cells surrounding completely collapsed NLLs in virus‐inoculated N. glutinosa leaves was compared with that of normal cells of mock‐inoculated N. glutinosa leaves. The changes, which have been reported in other several virus–host plant systems showing the hypersensitive response (HR), were frequently observed in cells surrounding the NLLs. Furthermore, clumping of the nuclear matrix within the nuclei, which is a feature of programmed cell death, also occurred in these cells. These results indicated that the HR‐like host response occurred at the fine structural level in the cells of N. glutinosa plants infected with CMV(Y/GM2)tr.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

Heterologous virus-induced gene silencing as a promising approach in plant functional genomics

VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death.     

Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component

Virus‐induced gene silencing (VIGS) is currently recognized as a powerful reverse genetics tool for application in functional genomics. DNA1, a satellite‐like and single‐stranded DNA molecule associated with begomoviruses (Family Geminiviridae), has been shown to replicate autonomously but requires the helper virus for its dissemination. We developed a VIGS vector based on the DNA1 component of tobacco curly shoot virus (TbCSV), a monopartite begomovirus, by inserting a multiple cloning site between the replication‐associated protein open reading frame and the A‐rich region for subsequent insertion of DNA fragments of genes targeted for silencing. When a host gene (sulphur, Su) or transgene (green fluorescent protein, GFP) was inserted into the modified DNA1 vector and co‐agroinoculated with TbCSV, efficient silencing of the cognate gene was observed in Nicotiana benthamiana plants. More interestingly, we demonstrated that this modified DNA1 could effectively suppress GFP in transgenic N. benthamiana or endogenous Su in tobacco plants when co‐agroinoculated with tomato yellow leaf curl China virus (TYLCCNV), another monopartite begomovirus that does not induce any viral symptoms. A gene‐silencing system in Nicotiana spp., Solanum lycopersicum and Petunia hybrida plants was then established using TYLCCNV and the modified DNA1 vector. The system can be used to silence genes involved in meristem and flower development. The modified DNA1 vector was used to silence the AtTOM homologous genes (NbTOM1 and NbTOM3) in N. benthamiana. Silencing of NbTOM1 or NbTOM3 can reduce tobamovirus multiplication to a lower level, and silencing of both genes simultaneously can completely inhibit tobamovirus multiplication. Previous studies have reported that DNA1 is associated with both monopartite and bipartite begomoviruses, as well as curtoviruses. This vector system can therefore be applied for the study, analysis and discovery of gene function in a variety of important crop plants.     

A geminivirus-induced gene silencing system for gene function validation in cassava

We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacumsulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow–white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2endogenous gene, sharing 89% homology with CYP79D1endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.     

NbLRK1, a lectin-like receptor kinase protein of <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>, interacts with <Emphasis Type="Italic">Phytophthora infestans</Emphasis> INF1 elicitin and mediates INF1-induced cell death

Phytophthora infestans INF1 elicitin causes the hypersensitive response (HR) in Nicotiana benthamiana (Kamoun et al. in Plant Cell 10:1413–1425, 1998). To identify N. benthamiana proteins that interact with INF1, we carried out a yeast two-hybrid screen. This screen resulted in the isolation of a gene NbLRK1 coding for a novel lectin-like receptor kinase. NbLRK1 interacted with INF1 through its VIb kinase subdomain. Purified INF1 and NbLRK1 proteins also interacted in vitro. INF1 treatment of N. benthamiana leaves induced autophosphorylation of NbLRK1. Most importantly, virus-induced gene silencing (VIGS) of NbLRK1 delayed INF1-mediated HR in N. benthamiana. These data suggest that NbLRK1 is a component of the N. benthamiana protein complex that recognizes INF1 elicitor and transduces the HR signal.     

Translationally controlled tumour protein (TCTP) from tomato and Nicotiana benthamiana is necessary for successful infection by a potyvirus

Translationally controlled tumour protein (TCTP) is a ubiquitously distributed protein in eukaryotes, involved in the regulation of several processes, including cell cycle progression, cell growth, stress protection, apoptosis and maintenance of genomic integrity. Its expression is induced during the early stages of tomato (Solanum lycopersicum) infection by the potyvirus Pepper yellow mosaic virus (PepYMV, a close relative of Potato virus Y). Tomato TCTP is a protein of 168 amino acids, which contains all the conserved domains of the TCTP family. To study the effects of TCTP silencing in PepYMV infection, Nicotiana benthamiana plants were silenced by virus‐induced gene silencing (VIGS) and transgenic tomato plants silenced for TCTP were obtained. In the early stages of infection, both tomato and N. benthamiana silenced plants accumulated less virus than control plants. Transgenic tomato plants showed a drastic reduction in symptoms and no viral accumulation at 14 days post‐inoculation. Subcellular localization of TCTP was determined in healthy and systemically infected N. benthamiana leaves. TCTP was observed in both the nuclei and cytoplasm of non‐infected cells, but only in the cytoplasm of infected cells. Our results indicate that TCTP is a growth regulator necessary for successful PepYMV infection and that its localization is altered by the virus, probably to favour the establishment of virus infection. A network with putative interactions that may occur between TCTP and Arabidopsis thaliana proteins was built. This network brings together experimental data of interactions that occur in other eukaryotes and helps us to discuss the possibilities of TCTP involvement in viral infection.     

Qualitative and quantitative immunofluorescence studies of chloroplast ferredoxin

Antibodies were raised in rabbits against 2Fe–2S ferredoxin from N. tabacum L. The antibodies showed partial cross-reactivity in the double diffusion test with ferredoxins from Spinacia oleracea L., Petunia inflata Fries., P. axillaris Lam., Phaseolus vulgaris L., Chlamydomonas remhardii Dang. A complete cross-reaction was observed with ferredoxins from five other Nicotiana species, thus with this test it was impossible to discriminate between these ferredoxins. Therefore the following test was performed. Heterologous ferredoxin (i.e., ferredoxin other than from N. tabacum) was coupled covalently to Sepharose beads. Rabbit anti-N. tabacum-serum was then pre-incubated with this ferredoxin which resulted in complete abolition of cross-reactivity with free heterologous ferredoxin. However, the serum retained antibody activity against specific antigenic determinants of N. tabacum ferredoxin. When this serum was tested against ferredoxin purified from the hybrid: N. tabacum ()xN. glutinosa () it gave a positive reaction. The relative content of maternal N. tabacum ferredoxin in the hybrid was estimated by using a fluorescent derivative of this specific antibody and estimating the cross-reactivity compared with that of artificial mixtures of pure N. tabacum and N. glutinosa ferredoxins. The hybrid contained 50% of maternal ferredoxin. This technique was also applied to ferredoxins of other species of Nicotiana and to the ferredoxin from the hybrid N. clevelandii ()xN. glutinosa (). We conclude that it provides a good test system for the study of the expression of chloroplast ferredoxin in Nicotiana hybrids in general.Abbreviations PBS phosphate buffered saline - FITC fluorescein isothiocyanate - S.E.M. standard error of means     

Chilli leaf curl virus-based vector for phloem-specific silencing of endogenous genes and overexpression of foreign genes

Geminiviruses are the largest and most devastating group of plant viruses which contain ssDNA as a genetic material. Geminivirus-derived virus-induced gene silencing (VIGS) vectors have emerged as an efficient and simple tool to study functional genomics in various plants. However, previously developed VIGS vectors have certain limitations, owing to their inability to be used in tissue-specific functional study. In the present study, we developed a Chilli leaf curl virus (ChiLCV)-based VIGS vector for its tissue-specific utilization by replacing the coat protein gene (open reading frame (ORF) AV1) with the gene of interest for phytoene desaturase (PDS) of Nicotiana benthamiana. Functional validation of ChiLCV-based VIGS in N. benthamiana resulted in systemic silencing of PDS exclusively in the phloem region of inoculated plants. Furthermore, expression of enhanced green fluorescence protein (EGFP) using the same ChiLCV vector was verified in the phloem region of the inoculated plants. Our results also suggested that, during the early phase of infection, ChiLCV was associated with the phloem region, but at later stage of pathogenesis, it can spread into the adjoining non-vascular tissues. Taken together, the newly developed ChiLCV-based vector provides an efficient and versatile tool, which can be exploited to unveil the unknown functions of several phloem-specific genes.

     

Nicotiana species as surrogate host for studying the pathogenicity of Acidovorax citrulli,the causal agent of bacterial fruit blotch of cucurbits

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management.     

Efficient and high-throughput pseudorecombinant-chimeric Cucumber mosaic virus-based VIGS in maize

Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

A pseudorecombinant-chimeric Cucumber mosaic virus-based virus-induced gene silencing system rapidly and efficiently triggers persistent gene silencing in maize.     

Probable mechanical transmission of a virus-like agent from rose rosette disease-infected multiflora rose to Nicotiana species

As part of efforts to identify the causal agent of the rose rosette disease (RRD) of multiflora rose (Rosa multiflora Thunb.), root tip extracts from both symptomatic and nonsymptomatic roses were used to mechanically inoculate leaves of Nicotiana glutinosa. Pale green spots were observed along the margins of the major leaf veins only on leaves inoculated with extracts prepared from symptomatic rose plants. Light microscopy revealed abnormal development of the palisade and spongy mesophyll cells in the symptomatic tissue, although no virus‐like particles (VLPs) were observed by electron microscopy. However, VLPs were observed in cells from tissue adjacent to the leaf veins and bordered by the pale green spots. Inoculation of N. benthamiana with extracts from symptomatic N. glutinosa initially did not result in visible symptoms on N. benthamiana inoculated leaves. However, approximately 4 wk post inoculation, splitting of leaf tissue across and along major leaf veins in expanding leaves occurred. In later stages of leaf expansion some leaves split in regions not associated with veins. Light microscopy of thick sections revealed separation between palisade cells and groups of small dead cells in the mesophyll tissue of expanding systemically infected leaf blades. Electron microscopy revealed crystalline arrays in the cytoplasm of mesophyll cells. No abnormal cellular changes were observed in plants inoculated with extracts prepared from nonsymptomatic rose plants.     

Genomic factors responsible for abnormal morphology of pollen tubes in the interspecific cross Nicotiana tabacum ×N. rustica

Nicotiana tabacum shows unilateral pollen-pistil incongruity with N. rustica. If N. tabacum is pollinated with N. rustica, growth of the pollen tube is arrested in the middle of the style, and abundant callose deposition, tube swelling and tube winding are observed. An attempt was made to clarify the genomic factors responsible for this pollen-pistil incongruity. N. tabacum was pollinated with N. paniculata or N. undulata, progenitors of amphidiploid N. rustica. When pollinated with N. undulata, growth of the pollen tube was arrested in the middle of the style and showed abnormal morphology similar to that with N. rustica, but when pollinated with N. paniculata the pollen tube reached near the base of the style and was almost normal in appearance. These observations suggest that the factors responsible for the pollen tube abnormality of N. rustica are derived from the N. undulata genome.We also used N. sylvestris, N. glutinosa and N. otophora as pistilar parents and N. rustica or its progenitors as pollen parents to examine the genomic factors of the pistilate parents. The pollen tube features of these three species in the pistils of N. sylvestris were similar to those in the pistil of N. tabacum. Received: 25 October 1999 / Revision accepted: 2 February 2000     

A Nicotiana Gametosomatic Hybrid and its Progenies

A Nicotiana gametosomatic hybrid between N. tabacum and N. glutinosawas studied. It was shown to have a stable somatic chromosomecomplement of 2n=5x=60. Karyotypes and measures of DNA contentshow that there have been no major changes in the parental chromosomenumbers or morphology accompanying hybridization. Forty-eightchromosomes are derived from N. tabacum (2n=4x=48); 12 fromN. glutinosa (2n=2x=24). Homologous pairs of N. tabacum chromosomesusually pair normally at meiosis. Trivalents incorporating aglutinosa chromosome do occur but usually these remain as univalents. The fertility of the hybrid is low but some products of self-pollinationwere obtained. These carry the complete N. tabacum genome witha few glutinosa chromosomes, some of which form supplementarybivalents at meiosis. All derivatives studied were mixoploidbut progressive selfing reduces the extent of abnormality ofmitotic divisions. The study of morphological and developmentaltraits indicates that the addition of even a few chromosomesof N. glutinosa to the N. tabacum complement can modify thetabacum phenotype substantially. There is considerable variationamong the derivatives and scope for fixing desirable qualitiesthrough selection. The presence of only a haploid set of glutinosachromosomes in the original hybrid makes the return to a desirablegenotype more efficient than that achieved through more conventionalbreeding methods. Key words: Nicotiana, gametosomatic hybrid, selfed progeny, cytology     

Sesquiterpenoid stress compounds from Nicotiana species

Solavetivone, 3-hydroxysolavetivone, solanascone, phytuberin and phytuberol were identified as stress compounds in leaves of Nicotiana tabacum cv Samsun NN. N. sylvestris, which is the maternal progenitor of N. tabacum, produced all the above compounds except 3-hydroxysolavetivone. In the F₁, hybrid of N. tabacum and N. glutinosa, all the stress compounds produced by N. tabacum and N. glutinosa, respectively, were accumulated.     

Stable Plastid Transformation in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.     

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement

Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2‐YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2‐YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2‐YFP fluorescence were affected in fibrillarin‐silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.