Small Subunit Of Ribulose Bisphosphate Carboxylase Research Articles

The effects of illumination and trophic strategy on gene expression in Chlamydomonas reinhardtii

Microalgae are of substantial biotechnological interest due their polyphyletic nature which grants them access to a wide array of high-value metabolites. The inherent genetic diversity of microalgae combined with their metabolic plasticity when grown using different trophic and illumination strategies necessitate the establishment of a reference knowledge base. In the present study we present a detailed characterisation of the combined effects of wavelength selection and trophic strategy on the growth kinetics and gene expression profile of the model microalgae Chlamydomonas reinhardtii grown under moderate to high light intensity (400 μmolph m−2·s−1). The aim is twofold: (a) to establish a list of reliable housekeeping genes valid for quantitative comparisons across several combinations of different wavelengths and trophic strategies and (b) to enable the investigation of the response of central carbon metabolic pathways under these process conditions. White, blue and red light emitting diodes (LEDs) were used to grow pH controlled photo- and mixo-trophic cultures over a period of 136 h in batch mode. A panel of 10 candidate genes, along with biomass growth rate and pigment content were dynamically monitored across all conditions. Statistical analysis identified genes (acetyl-CoA carboxylase subunit α and photosystem I reaction centre subunit II) with less variability observed in their expression levels across the entirety of conditions evaluated compared to housekeeping genes often referred to in literature (receptor of activated protein kinase C and ribosomal protein (large subunit) 19). Further analysis of gene expression profiles revealed substantial differences in response to changes in both wavelength selection (upregulation of ribulose bisphosphate carboxylase small subunit under red phototrophic growth) and trophic strategy (upregulation of malate synthase from phototrophic to mixotrophic conditions). The systematic approach used to establish reliable reference genes presented herein enables robust comparisons of cellular responses across different conditions to better understand algal metabolism and improve process performance.

Morpho-physiological and proteomic analysis to identify and characterise the traditional rice genotypes for drought tolerance

Rice is the major staple food for one-third of the world’s population. Rice requires high amount of water as compared to other cereal crops. In this context, we screened traditional rice genotypes collected from RARS, Pattambi and Ambalavayal for drought tolerance. Based on the morpho-physiological characters, the plants which showed wilting symptoms were screened according to international standard i.e., IRRI leaf score 7.0. The genotype which showed no wilting symptom even after 90% of the plants were showing wilting was identified as tolerant and the plant which showed early wilting symptom was identified as susceptible genotype. The genotype Chuvanna Modan has taken 25 days and the genotype Parambuvattan has taken only 10 days for wilting. After 90% of plants reached IRRI score 7.0 phenotypic observations i.e., electrical conductivity, measurement of stomatal conductance and transpiration rate using Infra red gas analyser were recorded. Root traits were also measured. The identified tolerant and susceptible genotypes were further subjected to 2D gel electrophoresis to identify and characterize the differentially expressed proteins for drought tolerance. Totally four differentially expressed protein spots were identified in Chuvanna Modan. The differential protein spots were sequenced by Matrix Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry (MALDI–TOF/MS). Spot 1 and 2 were identified as ribulose bisphosphate carboxylase small subunit, spot 3 was identified as Protein tyrosine phosphatases and spot 4 was identified as Photosystem II stability/assembly factor HCF136.

Expression of Pinellia pedatisecta Lectin Gene in Transgenic Wheat Enhances Resistance to Wheat Aphids.

Wheat aphids are major pests during the seed filling stage of wheat. Plant lectins are toxic to sap-sucking pests such as wheat aphids. In this study, Pinellia pedatisecta agglutinin (ppa), a gene encoding mannose binding lectin, was cloned, and it shared 92.69% nucleotide similarity and 94% amino acid similarity with Pinellia ternata agglutinin (pta). The ppa gene, driven by the constitutive and phloem-specific ribulose bisphosphate carboxylase small subunit gene (rbcs) promoter in pBAC-rbcs-ppa expression vector, was transferred into the wheat cultivar Baofeng104 (BF104) by particle bombardment transformation. Fifty-four T0 transgenic plants were generated. The inheritance and expression of the ppa gene were confirmed by PCR and RT-PCR analysis respectively, and seven homozygous transgenic lines were obtained. An aphid bioassay on detached leaf segments revealed that seven ppa transgenic wheat lines had lower aphid growth rates and higher inhibition rates than BF104. Furthermore, two-year aphid bioassays in isolated fields showed that aphid numbers per tiller of transgenic lines were significantly decreased, compared with wild type BF104. Therefore, ppa could be a strong biotechnological candidate to produce aphid-resistant wheat.

Differential Expression of Proteins in Response to Molybdenum Deficiency in Winter Wheat Leaves Under Low-Temperature Stress

Molybdenum (Mo) is an essential micronutrient for plants. To obtain a better understanding of the molecular mechanisms of cold resistance enhanced by molybdenum application in winter wheat, we applied a proteomic approach to investigate the differential expression of proteins in response to molybdenum deficiency in winter wheat leaves under low-temperature stress. Of 13 protein spots that were identified, five spots were involved in the light reaction of photosynthesis, five were involved in the dark reaction of photosynthesis, and three were highly involved in RNA binding and protein synthesis. Before the application of cold stress, four differentially expressed proteins between the Mo deficiency (−Mo) vs. Mo application (+Mo) comparison are involved in carbon metabolism and photosynthetic electron transport. After 48 h of cold stress, nine differentially expressed proteins between the −Mo vs. +Mo comparison are involved in carbon metabolism, photosynthetic electron transport, RNA binding, and protein synthesis. Under −Mo condition, cold stress induced a more than twofold decrease in the accumulation of six differential proteins including ribulose bisphosphate carboxylase large-chain precursor, phosphoglycerate kinase, cp31BHv, chlorophyll a/b-binding protein, ribulose bisphosphate carboxylase small subunit, and ribosomal protein P1, whereas under +Mo condition cold stress only decreased the expression of RuBisCO large subunit, suggesting that Mo application might contribute to the balance or stability of these proteins especially under low-temperature stress and that Mo deficiency has greater influence on differential protein expression in winter wheat after low-temperature stress. Further investigations showed that Mo deficiency decreased the concentrations of chlorophyll a, chlorophyll b, and carotenoids; the maximum net photosynthetic rate; the apparent quantum yield; and carboxylation efficiency, even before the application of the cold stress, although the decrease rates were greater after 48 h of cold treatment, which is consistent with changes in the expressions of differential proteins in winter wheat under low-temperature stress. These findings provide some new evidence that Mo might be involved in the light and dark reaction of photosynthesis and protein synthesis.

Separation and Identification of Proteins Related to Fruits Ripening in Mulberry (<i>Morus alba</i>)

To elucidate the physiological mechanism of mulberry fruit ripening in protein level, differential proteome expression of mulberry fruits was analyzed by using 2-DE and mass spectrometry in different ripening stages, green ripe stage(G), half ripe stage(R) and pan ripe stage(P). A mulberry cultivator, “Da10” was used as experimental material. The results showed that separation of proteins with 2-DE were significantly improved by using phenol/SDS buffer for protein extraction. 441, 222, 328 protein spots were detected respectively in ripening stage G, R and P. Among them, differential expression of 31 proteins was more than 2-fold and 6 proteins were stage-specific expression. 8 differential proteins were identified by MALDI-TOF/TOF MS analysis and database search, which were photosynthesis related proteins (ribulose bisphosphate carboxylase/oxygenase activase and ribulose bisphosphate carboxylase small subunit), stress related protein (18kD winter accumulating protein), glucose metabolism related protein (cell wall invertase）and so on, suggesting that these proteins may play the specific physiological role in mulberry fruits ripening.

Development of a gene silencing DNA vector derived from a broad host range geminivirus

BackgroundGene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.ResultsThe construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV), named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS), transketolase, the sulfur allele of magnesium chelatase (ChlI), and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant.ConclusionThe unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.

Adaptive response to salt involving carbohydrate metabolism in leaves of a salt-sensitive tomato cultivar

A salt-sensitive genotype of Solanum lycopersicum cv. Volgogradskij was submitted to a 6-day treatment with high salt (100, 200 mM NaCl), allowed to recover for 6 days and then submitted to a second period of salt stress in order to study changes in carbohydrate metabolism related to salt adaptation. The ion, soluble sugar and starch contents, as well as sucrose biosynthetic and sugar mobilizing enzyme activities and transcript levels were determined during the salt stress/recovery/stress cycle. Sodium ions were found to accumulate preferentially in old leaves. Young leaves accumulated lower levels of sodium ions but maintained control levels of potassium ions. Hexoses accumulated to higher levels and starch was better maintained in young compared to old leaves during the two salt treatments. Sucrose accumulated dramatically only in old leaves during the initial salt treatment. Sugar accumulation was not related to decreases in the activities of sugar mobilizing enzymes, acid (EC 3.2.1.25) and neutral (EC 3.2.1.26) invertases, sucrose synthase (EC 2.4.1.13) and hexokinase (EC 2.7.1.1). The activity of the biosynthetic enzyme sucrose phosphate synthase (EC 2.3.1.14) was linked to changes in sucrose levels but not with transcript levels. These results point to the importance of post-transcriptional regulation. Transcriptional regulation could nevertheless be seen in the down-regulation of ribulose bisphosphate carboxylase small subunit (EC 4.1.1.39) in old compared to young leaves, but this was not related to sugar levels.

Heterologous signals allow efficient targeting of a nuclear‐encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

A Novel Approach to Functional Analysis of the Ribulose Bisphosphate Carboxylase Small Subunit Gene by Agrobacterium-Mediated Gene Silencing

A novel approach to virus-induced post-transcriptional gene silencing for studying the function of the ribulose bisphosphate carboxylase small subunit (rbcS) gene was established and optimized using potato virus X vector and Nicotiana benthamiana as experimental material. The analysis of silencing phenomena, transcriptional level, protein expression, and pigment measurement showed that the expression of the rbcS endogenous gene was inactivated by the expression of a 500-bp homologous cDNA fragment carried in the virus vector.

Effects of mutations at the two processing sites of the precursor for the small subunit of ribulose-bisphosphate carboxylase in Chlamydomonas reinhardtii.

The role of the two processing sites in the precursor of the small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSS) of Chlamydomonas reinhardtii was studied by introducing mutations at the cleavage sites for the stromal processing peptidases SPP-1 and SPP-2, which hydrolyse wild-type pSS (20.6 kDa) to an intermediate-sized product iSS (18.3 kDa) and to the mature SS (16.3 kDa), respectively. The mutations introduced into cDNA resulted in exchange of (a) two amino acids flanking processing site 1, or (b) one or (c) both amino acids flanking processing site 2. Mutation (a) prevented pSS from being processed at site 1 but not from cleavage at site 2. Mutation (c) abolished the action of SPP-2 but not SPP-1. When pSS with mutation (c) was imported into isolated chloroplasts, iSS accumulated while SS formation was abolished. However, mature SS was produced even in the absence of iSS synthesis (mutation a). Import of pSS bearing mutation (b), which only partially inhibited processing at the SPP-2 site, slowed the rate of SS formation down whereas iSS and some slightly smaller derivatives accumulated. These experiments suggested that in Chlamydomonas processing of pSS can occur in two steps, whereby the first step is facultative. The same three mutations were studied in vivo after transformation of SS-deficient C. reinhardtii T60-3 with mutated genomic DNA. Growth and photosynthesis was as in control transformants, except for the slower-growing transformants (mutation c) where no mature SS was immuno-detected. However, pSS fragments with molecular masses between those of iSS and SS were present even in the ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme.

Arabidopsis seedling growth, storage lipid mobilization, and photosynthetic gene expression are regulated by carbon:nitrogen availability.

The objective of the current work was to establish the degree to which the effects of carbon and nitrogen availability on Arabidopsis seedling growth and development are due to these nutrients acting independently or together. Growth of seedlings on low (0.1 mM) nitrogen results in a significant reduction of seedling and cotyledon size, fresh weight, chlorophyll, and anthocyanin content but a slight increase in endogenous sugars. The addition of 100 mM sucrose (Suc) to the nitrogen-depleted growth media results in a further reduction in cotyledon size and chlorophyll content and an overall increase in anthocyanins and endogenous sugars. Storage lipid breakdown is almost completely blocked in seedlings grown on low nitrogen and 100 mM Suc and is significantly inhibited when seedlings are grown on either low nitrogen or high Suc. Carbohydrate repression of photosynthetic gene expression can only be observed under low nitrogen conditions. Low (0.1 mM) nitrogen in the absence of exogenous carbohydrate results in a significant decrease in chlorophyll a/b-binding protein and ribulose bisphosphate carboxylase small subunit gene transcript levels. Thus, carbon to nitrogen ratio rather than carbohydrate status alone appears to play the predominant role in regulating various aspects of seedling growth including storage reserve mobilization and photosynthetic gene expression.

Arabidopsis Seedling Growth, Storage Lipid Mobilization, and Photosynthetic Gene Expression Are Regulated by Carbon:Nitrogen Availability

The objective of the current work was to establish the degree to which the effects of carbon and nitrogen availability on Arabidopsis seedling growth and development are due to these nutrients acting independently or together. Growth of seedlings on low (0.1 mm) nitrogen results in a significant reduction of seedling and cotyledon size, fresh weight, chlorophyll, and anthocyanin content but a slight increase in endogenous sugars. The addition of 100 mm sucrose (Suc) to the nitrogen-depleted growth media results in a further reduction in cotyledon size and chlorophyll content and an overall increase in anthocyanins and endogenous sugars. Storage lipid breakdown is almost completely blocked in seedlings grown on low nitrogen and 100 mm Suc and is significantly inhibited when seedlings are grown on either low nitrogen or high Suc. Carbohydrate repression of photosynthetic gene expression can only be observed under low nitrogen conditions. Low (0.1 mm) nitrogen in the absence of exogenous carbohydrate results in a significant decrease in chlorophyll a/b-binding proteinand ribulose bisphosphate carboxylase small subunit gene transcript levels. Thus, carbon to nitrogen ratio rather than carbohydrate status alone appears to play the predominant role in regulating various aspects of seedling growth including storage reserve mobilization and photosynthetic gene expression.

Plastid state- and light-dependent regulation of the expression of nucleus-encoded genes for chloroplast proteins in the flagellate Euglena gracilis.

Interorganellar regulatory interactions in the flagellate Euglena gracilis were shown to be more complicated than in green algae and higher plants. Euglena plastids have a much more complex influence on nuclear gene expression than was previously thought. The petJ gene for cytochrome c6 represents a group of nucleus-encoded genes for chloroplast proteins, the expression of which is influenced by the state of plastids at the transcriptional level. Moroever, the regulation of these genes might be light-dependent. In contrast, for nucleus-encoded small subunit of ribulose-bisphosphate carboxylase, chlorophyll a/b binding protein, and porphobilinogen deaminase transcript levels are unchanged in wild-type cells relative to white mutants. In these cases there is no plastid-derived signal operative during transcription. Porphobilinogen deaminase appeared to be regulated even at the post-translational level.

Characterisation of cDNAs for ribulose bisphosphate carboxylase small subunit genes in different species of Avena (Poaceae)

ABSTRACT We report the cloning and sequencing of 14 rbcS cDNAs in six species of Avena (Poaceae) with different genome and ploidy levels. The nucleotide sequences 504 bp long were aligned with the published sequences of cultivated hexaploid oat, wheat, barley, rye, rice and corn and subjected to cladistic and phenetic analyses. The parsimonious analysis generated a tree with a topology very similar to the phenogram generated by the Neighbor-Joining analysis based on the Jukes and Cantor distances. Within the monophyletic assemblage of the tribe Aveneae, consistent clades composed of rbcS clones belonging to different species are recognized. It is suggested that they correspond to orthologous genes belonging to different subfamilies, and that the “within-species˚d homogenisation may have occurred at a slow rate with respect to species evolution. In the monophyletic group of Pooideae, the topologies place barley rbcS sequences closer to wheat and rye than to oat sequences. This grouping agrees with most taxonomic and phylogenetic views.

The promoter of rbcS in a C3 plant (rice) directs organ-specific, light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression.

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding beta-glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-specific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.

Transcription of a maize cDNA in Lotus corniculatus is regulated by T-DNA methylation and transgene copy number

Lotus corniculatus plants transformed with a maize cDNA (G1L) encoding a sulphur-rich γ-zein were obtained by using two fusion genes: one with the CaMV 35S promoter, the other with the ribulose bisphosphate carboxylase small subunit (rbcS) promoter. The highest expression of G1L mRNA was found in plants transformed with G1L under the rbcS promoter. The steady state level of G1L mRNA in the leaves was generally directly correlated with the G1L copy number. However, due to a transcriptional block, no G1L mRNA was detected in some of the 35S-G1L multi-copy transformants. Analyses with methylation-sensitive restriction enzymes revealed that the T-DNA of the silenced 35S-G1L transformants was methylated. T-DNA copy number, G1L silencing activity, and the state of methylation were strictly correlated in primary transformants. A cross between two 35S-G1L transformed plants, one (S) with the T-DNA methylated and the other (NS) without, showed that: (1) the methylated state of T-DNA passed through meiosis; and (2) when T-DNA copies from the two parents were combined in the progeny, the unmethylated T-DNA copies of parent NS became methylated at different levels and G1L mRNA production was dependent on the degree of methylation.

Arabidopsis bZIP protein HY5 directly interacts with light-responsive promoters in mediating light control of gene expression.

The Arabidopsis HY5 gene has been defined genetically as a positive regulator of photomorphogenesis and recently has been shown to encode a basic leucine zipper type of transcription factor. Here, we report that HY5 is constitutively nuclear localized and is involved in light regulation of transcriptional activity of the promoters containing the G-box, a well-characterized light-responsive element (LRE). In vitro DNA binding studies suggested that HY5 can bind specifically to the G-box DNA sequences but not to any of the other LREs present in the light-responsive promoters examined. High-irradiance light activation of two synthetic promoters containing either the consensus G-box alone or the G-box combined with the GATA motif (another LRE) and the native Arabidopsis ribulose bisphosphate carboxylase small subunit gene RBCS-1A promoter, which has an essential copy of the G-box, was significantly compromised in the hy5 mutant. The hy5 mutation's effect on the high-irradiance light activation of gene expression was observed in both photosynthetic and nonphotosynthetic tissues. Furthermore, the characteristic phytochrome-mediated red light- and far-red light-reversible low-fluence induction of the G-box-containing promoters was diminished specifically in hy5 plants. These results suggest that HY5 may interact directly with the G-box in the promoters of light-inducible genes to mediate light-controlled transcriptional activity.

Differential expression of a γ-zein gene in Medicago sativa, Lotus corniculatus and Nicotiana tabacum

With the aim of increasing the content of sulphur amino acids (SAAs) in the aerial part of the plants, Nicotiana tabacum and two leguminous species, Medicago sativa and Lotus corniculatus, were transformed with a maize γ-zein gene (G1L), coding for a SAAs-rich protein. Gene fusions with two different promoters were constructed; one promoter was the constitutive cauliflower mosaic virus (CaMV) 35S and the other was the light-regulated ribulose bisphosphate carboxylase small subunit (rbcS) from a tobacco gene. The constructs were introduced by Agrobacterium-mediated gene transfer. All the transformants produced the G1L RNA (two differently sized transcripts were detected) and no relevant differences were found between the two promoters. Despite the presence of G1L message the γ-zein was found only in tobacco plants (up to 0.05% of the alcohol soluble proteins). Post-transcriptional mechanisms seemed to inhibit protein accumulation. The low level of G1L RNA in the transformed plants was probably the main factor that limited the production of γ-zein protein.

Expression and chloroplast-targeting of active phosphoenolpyruvate synthetase from Escherichia coli in Solanum tuberosum

Solanum tuberosum was transformed with a chimeric gene consisting of the constitutive 35S CaMV promoter, a potato ribulose bisphosphate carboxylase (Rubisco) small subunit transit sequence (rbcS), and the phosphoenolpyruvate synthetase gene (ppsA) from E. coli. Transgenic plants were regenerated producing (phospho enolpyruvate-) PEP-synthetase in amounts as much as 0.1% of total soluble protein. Western blot analysis indicated that most of the protein is located in the chloroplasts and that the transit sequence is cleaved off. Electron microscopy of leaves revealed that PEP-synthetase specific immunogold labeling was most pronounced over the chloroplast matrix occurring in between the thylakoid stacks. PEP-synthetase activity was detected in isolated chloroplasts of transgenic plants. Chloroplast morphology and starch production in leaves were affected.

Effects of chloroplast targeted potato virus Y coat protein on transgenic plants

Studies of several plant viruses have shown that virus coat protein (CP) may play a role in symptom development. In order to determine how and where potato virus Y (PVY)-CP may contribute to symptom development, two sets of plants were generated, one expressing PVY-CP, and one transformed with a chimeric construct consisting of PVY-CP fused to the transit peptide (TP) of pea ribulose bisphosphate carboxylase small subunit (rbcs) to target PVY-CP to the chloroplast stroma. Numerous transgenic plants developed chlorotic or white mosaic symptoms after transformation with the TP-CP construct, but not after transformation with PVY-CP alone. Both western blot analysis of stroma and thylakoid protein fractions and immunocytochemistry revealed that the imported PVY-CP was preferentially associated with thylakoid membranes in TP-CP+plants. Light and electron microscopy showed that PVY-CP disrupted chloroplast development and cell structure in TP-CP+transformants. Virus challenge of non-transformed CP+and a range of TP-CP+tobacco plants indicated that high level PVY-CP expressing TP-CP+plants were more susceptible to PVY. These observations suggest that PVY-CP action in chloroplasts may contribute to symptom development in PYV-infected plants.