Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants

In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional gene silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transgene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are viruses that suppress in all tissues of all infected leaves, whereas others are able to suppress only in the veins of new emerging leaves. This variation existed even between closely related members of the potexvirus group. Collectively, these results suggest that virus-encoded suppressors of gene silencing have distinct modes of action, are targeted against distinct components of the host gene-silencing machinery, and that there is dynamic evolution of the host and viral components associated with the gene-silencing mechanism.

Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato

The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site–leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.

Expression of alfalfa mosaic virus coat protein in tobacco mosaic virus (TMV) deficient in the production of its native coat protein supports long-distance movement of a chimeric TMV

Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in Nicotiana benthamiana, Nicotiana tabacum MD609, and Spinacia oleracea. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus.

Evolution of a quadripartite hybrid virus by interspecific exchange and recombination between replicase components of two related tripartite RNA viruses

Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) belong to the Cucumovirus genus. They have a tripartite genome consisting of single-stranded RNAs, designated 1, 2, and 3. Previous studies have shown that viable pseudorecombinants could be created in vitro by reciprocal exchanges between CMV and TAV RNA 3, but exchanges of RNAs 1 and 2 were replication deficient. When we coinoculated CMV RNAs 2 and 3 along with TAV RNAs 1 and 2 onto Nicotiana benthamiana, a hybrid quadripartite virus appeared that consisted of TAV RNA 1, CMV RNAs 2 and 3, and a distinctive chimeric RNA originating from a recombination between CMV RNA 2 and the 3′-terminal 320 nucleotides of TAV RNA 2. This hybrid arose by means of segment reassortment and RNA recombination to produce an interspecific hybrid with the TAV helicase subunit and the CMV polymerase subunit. To our knowledge, this is the first report demonstrating the evolution of a new plant or animal virus strain containing an interspecific hybrid replicase complex.

Ancient origin of pathogen recognition specificity conferred by the tomato disease resistance gene Pto

We have investigated the origin of the Pto disease resistance (R) gene that was previously identified in the wild tomato species Lycopersicon pimpinellifolium and isolated by map-based cloning. Pto encodes a serine-threonine protein kinase that specifically recognizes strains of Pseudomonas syringae pv. tomato (Pst) that express the avirulence gene avrPto. We examined an accession of the distantly related wild species Lycopersicon hirsutum var. glabratum that exhibits avrPto-specific resistance to Pst. The Pst resistance of L. hirsutum was introgressed into a susceptible Lycopersicon esculentum background to create the near-isogenic line 96T133-3. Resistance to Pst(avrPto) in 96T133-3 was inherited as a single dominant locus and cosegregated with a restriction fragment length polymorphism detected by the Pto gene. This observation suggested that a member of the Pto gene family confers Pst(avrPto) resistance in this L. hirsutum line. Here we report the cloning and characterization of four members of the Pto family from 96T133-3. One gene (LhirPto) is 97% identical to Pto and encodes a catalytically active protein kinase that elicits a hypersensitive response when coexpressed with avrPto in leaves of Nicotiana benthamiana. In common with the Pto kinase, the LhirPto protein physically interacts with AvrPto and downstream members of the Pto signaling pathway. Our studies indicate that R genes of the protein kinase class may not evolve rapidly in response to pathogen pressure and rather that their ability to recognize specific Avr proteins can be highly conserved.

Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes

Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434–438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants.

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

Horizontal gene transfer and mutation: Ngrol genes in the genome of Nicotiana glauca

Ngrol genes (NgrolB, NgrolC, NgORF13, and NgORF14) that are similar in sequence to genes in the left transferred DNA (TL-DNA) of Agrobacterium rhizogenes have been found in the genome of untransformed plants of Nicotiana glauca. It has been suggested that a bacterial infection resulted in transformation of Ngrol genes early in the evolution of the genus Nicotiana. Although the corresponding four rol genes in TL-DNA provoked hairy-root syndrome in plants, present-day N. glauca and plants transformed with Ngrol genes did not exhibit this phenotype. Sequenced complementation analysis revealed that the NgrolB gene did not induce adventitious roots because it contained two point mutations. Single-base site-directed mutagenesis at these two positions restored the capacity for root induction to the NgrolB gene. When the NgrolB, with these two base substitutions, was positioned under the control of the cauliflower mosaic virus 35S promoter (P35S), transgenic tobacco plants exhibited morphological abnormalities that were not observed in P35s-RirolB plants. In contrast, the activity of the NgrolC gene may have been conserved after an ancient infection by bacteria. Discussed is the effect of the horizontal gene transfer of the Ngrol genes and mutations in the NgrolB gene on the phenotype of ancient plants during the evolution of N. glauca.

A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana

Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind SC10-RNase in SI N. alata SC10SC10 and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia × SI N. alata SC10SC10) hybrids with reduced levels of HT-protein continued to express SC10-RNase but failed to reject SC10-pollen. Control hybrids expressing both SC10-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

Expression Studies of the Zeaxanthin Epoxidase Gene in Nicotiana plumbaginifolia1

Abscisic acid (ABA) is a plant hormone involved in the control of a wide range of physiological processes, including adaptation to environmental stress and seed development. In higher plants ABA is a breakdown product of xanthophyll carotenoids (C40) via the C15 intermediate xanthoxin. The ABA2 gene of Nicotiana plumbaginifolia encodes zeaxanthin epoxidase, which catalyzes the conversion of zeaxanthin to violaxanthin. In this study we analyzed steady-state levels of ABA2 mRNA in N. plumbaginifolia. The ABA2 mRNA accumulated in all plant organs, but transcript levels were found to be higher in aerial parts (stems and leaves) than in roots and seeds. In leaves ABA2 mRNA accumulation displayed a day/night cycle; however, the ABA2 protein level remained constant. In roots no diurnal fluctuation in mRNA levels was observed. In seeds the ABA2 mRNA level peaked around the middle of development, when ABA content has been shown to increase in many species. In conditions of drought stress, ABA levels increased in both leaves and roots. A concomitant accumulation of ABA2 mRNA was observed in roots but not in leaves. These results are discussed in relation to the role of zeaxanthin epoxidase both in the xanthophyll cycle and in the synthesis of ABA precursors.

Directional Guidance of Nicotiana alata Pollen Tubes in Vitro and on the Stigma1

Pollen tubes navigate the route from stigma to ovule with great accuracy, but the cues that guide them along this route are not known. We reproduced the environment on the stigma of Nicotiana alata by immersing pollen in stigma exudate or oil close to an interface with an aqueous medium. The growth of pollen in this culture system mimicked growth on stigmas: pollen grains hydrated and germinated, and pollen tubes grew toward the aqueous medium. The rate-limiting step in pollen germination was the movement of water through the surrounding exudate or oil. By elimination of other potential guidance cues, we conclude that the directional supply of water probably determined the axis of polarity of pollen tubes and resulted in growth toward the interface. We propose that a gradient of water in exudate is a guidance cue for pollen tubes on the stigma and that the composition of the exudate must be such that it is permeable enough for pollen hydration to occur but not so permeable that the supply of water becomes nondirectional. Pollen tube penetration of the stigma may be the most frequently occurring hydrotropic response of higher plants.

Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia1

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5′-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions

The ability of Nicotiana tabacum cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [3H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [3H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [3H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum proteins were metabolically labeled with [3H]F-OH or [3H]geranylgeraniol ([3H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by in vitro enzymatic studies. [3H]F-P and [3H]F-P-P were synthesized when exogenous [3H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor–product relationship between [3H]F-P and [3H]F-P-P. In agreement with this kinetic pattern of labeling, [32P]F-P and [32P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [γ-32P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [3H]GG-OH to [3H]geranylgeranyl monophosphate and [3H]geranylgeranyl pyrophosphate ([3H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [3H]CTP was formed when microsomes were incubated with [3H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein isoprenylation.

Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein from Pyrus communis suspension-cultured cells

Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development. We recently have shown that two AGPs, NaAGP1 (from Nicotiana alata styles) and PcAGP1 (from Pyrus communis cell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor. However, paradoxically, both AGPs were buffer soluble rather than membrane associated. We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs. This GPI anchor has the minimal core oligosaccharide structure, d-Manα(1–2)-d-Manα(1–6)-d-Manα(1–4)-d-GlcN-inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial β(1–4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid. The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D. The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways.