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Cassava (Manihot esculenta Crantz) is a predominant food security crop in several developing countries. Its storage roots, rich in carbohydrate, are deficient in essential micronutrients, including provitamin A carotenoids.Increasing carotenoid content in cassava storage roots is important to reduce the incidence of vitamin A deficiency, a public health problem in sub-Saharan Africa. However, cassava improvement advances slowly, mainly due to limited information on the molecular factors influencing 13-carotene accumulation in cassava.To address this problem, we performed comparative transcriptomic and untargeted metabolic analyses of roots and leaves of eleven African cassava landraces ranging from white to deep yellow colour, to uncover regulators of carotenoid biosynthesis and accumulation with conserved function in yellow cassava roots.Sequence analysis confirmed the presence of a mutation, known to influence 13-carotene content, in PSY transcripts of deep yellow but not of pale yellow genotypes. We identified genes and metabolites with expression and accumulation levels significantly associated with 13-carotene content. Particularly an increased activity of the abscisic acid catabolism pathway together with a reduced amount of L-carnitine, may be related to the carot-enoid pathway flux, higher in yellow than in white storage roots. In fact, NCED_3.1 was specifically expressed at a lower level in all yellow genotypes suggesting that it could be a potential target for increasing carotenoid accumulation in cassava.These results expand the knowledge on metabolite compositions and molecular mechanisms influencing carotenoid biosynthesis and accumulation in cassava and provide novel information for biotechnological ap-plications and genetic improvement of cassava with high nutritional values.

Auxin-mediated activation of secondary growth and subsequent KNOX/BEL expression coincide with active storage metabolism in xylem parenchyma cells of the cassava tuberous root., Cassava storage roots are among the most important root crops worldwide, and represent one of the most consumed staple foods in sub-Saharan Africa. The vegetatively propagated tropical shrub can form many starchy tuberous roots from its stem. These storage roots are formed through the activation of secondary root growth processes. However, the underlying genetic regulation of storage root development is largely unknown. Here we report distinct structural and transcriptional changes occurring during the early phases of storage root development. A pronounced increase in auxin-related transcripts and the transcriptional activation of secondary growth factors, as well as a decrease in gibberellin-related transcripts were observed during the early stages of secondary root growth. This was accompanied by increased cell wall biosynthesis, most notably increased during the initial xylem expansion within the root vasculature. Starch storage metabolism was activated only after the formation of the vascular cambium. The formation of non-lignified xylem parenchyma cells and the activation of starch storage metabolism coincided with increased expression of the KNOX/BEL genes KNAT1, PENNYWISE, and POUND-FOOLISH, indicating their importance for proper xylem parenchyma function.

Despite the importance of storage root (SR) organs for cassava and the other root crops yield, their developmental origin is poorly understood. Here we use multiple approaches to shed light on the initial stages of root development demonstrating that SR and fibrous roots (FR) follow different rhizogenic processes. Transcriptome analysis carried out on roots collected before, during, and after root bulking, highlighted early and specific activation of a number of functions essential for root swelling and identified root-specific genes able to effectively discriminate emerging FR and SR. Starch and sugars start to accumulate at higher rate in SR before they swell but only after parenchyma tissue has been produced. Finally, using non-destructive computed tomography measurements, we show that SR (but not FR) contain, since their emergence from the stem, an inner channel structure in continuity with the stem secondary xylem, indicating that SR derive from a distinct rhizogenic process compared with FR., Plant, Cell & Environment, 45 (6), ISSN:0140-7791, ISSN:1365-3040

Cassava is an important food security crop in tropical regions of the world. Cassava improvement by breeding is limited by its delayed and poor production of flowers, such that cassava flowering under field conditions indirectly lengthens the breeding cycle. By studying genotype and environment interaction under two Nigerian field conditions (Ubiaja and Ibadan) and three controlled temperature conditions (22°C/18°C, 28/24°C and 34/30°C (day/night)), we found that while early flowering genotypes flowered at similar times and rates under all growing conditions (unfavorable and favorable field and controlled-temperature environments), late flowering genotypes were environmentally sensitive such that they were substantially delayed in unfavorable environments. On the basis of nodes-to-flower, flowering of late genotypes approached the flowering time of early flowering genotypes under relatively cool Ubiaja field conditions and in growth chambers at 22°C, whereas warmer temperatures elicited a delaying effect. Analysis of transcriptomes from leaves of field and controlled-temperature environments revealed that conditions which promote early flowering in cassava have low expression of the flowering repressor geneTEMPRANILLO 1(TEM1), before and after flowering. Expression data of field plants showed that the balance between flower stimulatory and inhibitory signaling appeared to correlate with flowering time across the environments and genotypes.

The prevalence of vitamin A deficiency in sub-Saharan Africa necessitates effective approaches to improve provitamin A content of major staple crops. Cassava holds much promise for food security in sub-Saharan Africa, but a negative correlation between &beta, carotene, a provitamin A carotenoid, and dry matter content has been reported, which poses a challenge to cassava biofortification by conventional breeding. To identify suitable material for genetic transformation in tissue culture with the overall aim to increase &beta, carotene and maintain starch content as well as better understand carotenoid composition, root and leaf tissues from thirteen field-grown cassava landraces were analyzed for agronomic traits, carotenoid, chlorophyll, and starch content. The expression of five genes related to carotenoid biosynthesis were determined in selected landraces. Analysis revealed a weak negative correlation between starch and &beta, carotene content, whereas there was a strong positive correlation between root yield and many carotenoids including &beta, carotene. Carotenoid synthesis genes were expressed in both white and yellow cassava roots, but phytoene synthase 2 (PSY2), lycopene-&epsilon, cyclase (LCY&epsilon, ), and &beta, carotenoid hydroxylase (CHY&beta, ) expression were generally higher in yellow roots. This study identified lines with reasonably high content of starch and &beta, carotene that could be candidates for biofortification by further breeding or plant biotechnological means.

Caulimoviridae is a family of non-enveloped reverse-transcribing plant viruses with non-covalently closed circular dsDNA genomes of 7.1–9.8 kbp in the order Ortervirales. They infect a wide range of monocots and dicots. Some viruses cause economically important diseases of tropical and subtropical crops. Transmission occurs through insect vectors (aphids, mealybugs, leafhoppers, lace bugs) and grafting. Activation of infectious endogenous viral elements occurs in Musa balbisiana, Petunia hybrida and Nicotiana edwardsonii. However, most endogenous caulimovirids are not infectious. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Caulimoviridae, which is available at ictv.global/report/caulimoviridae.

Cassava is an important staple crop in sub‐Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenol pyruvate carboxylase activity support a C3 mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin–Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse‐grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement., The Plant Journal, 102 (6), ISSN:0960-7412, ISSN:1365-313X

Plants defend themselves from pathogenic virus infections by deploying a complex defense system. Fundamental in the plant antiviral immune system is the RNA silencing (RS) pathway, which consists in a multilayered set of enzymatic components. The RS machinery targets the double-stranded RNA, generating 21–24 nucleotides (nt) small interfering RNAs (siRNAs) that guide the cleavage of viral RNA in a sequence-specific manner. RS signals are then amplified and transmitted cell-to-cell and systemically, in a process that is provided with self-regulatory feedback mechanisms. All the steps of the RS pathway were revealed to be targeted by viral counterattack strategies that allow pathogens to escape host defenses and establish a successful infection. Viral suppressors of RNA silencing (VSRs) are virus-encoded proteins that interfere with RS by blocking or destabilizing the host antiviral functions. This chapter reviews the current knowledge on VSRs mechanisms of action, and describes the latest discoveries in this fascinating evolutionary arms race between plants and viruses.

Dioecy (distinct male and female individuals) combined with scarce to non-flowering are common features of cultivated yam (Dioscorea spp.). However, the molecular mechanisms underlying flowering and sex determination in Dioscorea are unknown. We conducted SuperSAGE transcriptome profiling of male, female and monoecious individuals to identify flowering and sex-related genes in white Guinea yam (D. rotundata). SuperSAGE analysis generated a total of 20,236 unique tags, of which 13,901 were represented by a minimum of 10 tags. Of these, 88 tags were significantly differentially expressed in male, female and monoecious plants. Of the 88 differentially expressed SuperSAGE tags, 18 corresponded to genes previously implicated in flower development and sex determination in multiple plant species. We validated the SuperSAGE data with quantitative real-time PCR (qRT-PCR)-based analysis of the expression of four candidate genes. Our findings suggest that mechanisms of flowering and sex determination are likely conserved in Dioscorea. We further investigated the flowering patterns of 1938 D. rotundata accessions representing diverse geographical origins over two years, revealing that over 85% of the accessions are either male or non-flowering, and that less than 15% are female, while monoecious plants are rare. Intensity of flowering appeared to be a function of sex, with male plants flowering more abundantly than female ones. Candidate genes identified in this study can be targeted with the aim to induce regular flowering in poor to non-flowering cultivars. Findings of the study provide important inputs for further studies aiming to overcome the challenge of flowering in yams and to improve the efficiency of yam breeding.

Cassava is an important staple crop in sub-Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in the greenhouse in Erlangen, Germany and the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of Rubisco:phosphoenolpyruvate carboxylase activity support a C3 mode of photosynthesis. The high rate of photosynthesis is likely attributed to high activities of enzymes in the Calvin-Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This was especially so in greenhouse-grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhance sink capacity in the high light and low nitrogen environment in the field. Overall, the results indicate that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.

RNA silencing plays a critical role in plant resistance against viruses. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that interfere with the cellular silencing machinery through various mechanisms not always well understood. We examined the role of Mungbean yellow mosaic virus (MYMV) AC4 and showed that it is essential for infectivity but not for virus replication. It acts as a determinant of pathogenicity and counteracts virus induced gene silencing by strongly suppressing the systemic phase of silencing whereas it does not interfere with local production of siRNA. We demonstrate the ability of AC4 to bind native 21–25 nt siRNAs in vitro by electrophoretic mobility shift assay. While most of the known VSRs have cytoplasmic localization, we observed that despite its hydrophilic nature and the absence of trans-membrane domain, MYMV AC4 specifically accumulates to the plasma membrane (PM). We show that AC4 binds to PM via S-palmitoylation, a process of post-translational modification regulating membrane–protein interactions, not known for plant viral protein before. When localized to the PM, AC4 strongly suppresses systemic silencing whereas its delocalization impairs VSR activity of the protein. We also show that AC4 interacts with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), a positive regulator of the cell-to-cell movement of RNAi. The absolute requirement of PM localization for direct silencing suppression activity of AC4 is novel and intriguing. We discuss a possible model of action: palmitoylated AC4 anchors to the PM by means of palmitate to acquire the optimal conformation to bind siRNAs, hinder their systemic movement and hence suppress the spread of the PTGS signal in the plant., Author summary Plants have developed small RNA (siRNA)-mediated post-transcriptional gene silencing as a defense mechanism against viruses. In response, plant viruses encode viral suppressors of RNA silencing (VSRs) that can interfere with various steps of the silencing pathway. Mungbean yellow mosaic virus (MYMV) is a plant bipartite geminivirus responsible for a devastating disease in some areas of tropics and sub-tropics where its natural host, Vigna mungo, is a staple food crop. We discovered that the MYMV-encoded AC4 protein is a determinant of pathogenicity, binds native 21–25 nt siRNAs in vitro, and counteracts virus induced gene silencing by strongly suppressing the systemic phase of silencing but not the local production of siRNA. MYMV AC4 undergoes palmitoylation, a post-translational modification never reported before for plant viral proteins that mediates specific localization of the protein to the plasma membrane (PM). Interestingly, palmitoylation and in turns PM localization is indispensable for direct VSR function of AC4. AC4 also binds the PM-located BAM1, a trigger of cell-to-cell spread of RNAi. Taken together our results suggest that AC4 has synergic mechanisms of action, based on the specific PM localization, to prevent spreading of antiviral RNAi silencing in not yet infected cells.

Appropriately regulated gene expression requires a suitable promoter. A number of promoters have been isolated and shown to be functional in plants, but only a few of them activate transcription of transgenes at high levels constitutively. We report here the cloning and characterization of a novel, constitutively expressed promoter isolated from Cestrum yellow leaf curling virus (CmYLCV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family. The CmYLCV promoter is highly active in callus, meristems and vegetative and reproductive tissues in Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Zea mays and Oryza sativa. Furthermore, the level of expression is comparable to, or higher than, that from the CaMV 35S, the 'super-promoter' or the maize ubiquitin 1 promoters, three frequently used promoters in agricultural biotechnology. The heritable, strong and constitutive activity in both monocotyledonous and dicotyledonous plants, combined with the extremely narrow CmYLCV host range, makes the CmYLCV promoter an attractive tool for regulating transgene expression in a wide variety of plant species.

International audience; Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae, and fungi (1, 2). This broad distribution suggests the ancient origin(s) of these viruses, possibly [...]

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) mA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes. © 2014 American Society of Plant Biologists. All rights reserved.

Mulberry badnavirus 1 (MBV1) has been characterized as the aetiological agent of a disease observed on a mulberry tree in Lebanon (accession L34). A small RNA next-generation sequencing library was prepared and analysed from L34 extract, and these data together with genome walking experiments have been used to obtain the full-length virus sequence. Uniquely among badnaviruses, the MBV1 sequence encodes a single ORF containing all the conserved pararetrovirus motifs. Two genome sizes (6 kb and 7 kb) were found to be encapsidated in infected plants, the shortest of which shares 98.95 % sequence identity with the full L34 genome. In the less-than-full-length deleted genome, the translational frame for the replication domains was conserved, but the particle morphology, observed under electron microscopy, was somehow altered. Southern blot hybridization confirmed the coexistence of the two genomic forms in the original L34 accession, as well as the absence of cointegration in the plant genome. Both long and deleted genomes were cloned and proved to be infectious in mulberry. Differently from other similar nuclear-replicating viruses or viroids, the characterization of the MBV1-derived small RNAs showed a reduced amount of the 24-mer class size.

The persistent challenge of insufficient food, unbalanced nutrition, and deteriorating natural resources in the most vulnerable nations, characterized by fast population growth, calls for utilization of innovative technologies to curb constraints of crop production. Enhancing genetic gain by using a multipronged approach that combines conventional and genomic technologies for the development of stress-tolerant varieties with high yield and nutritional quality is necessary. The advent of nextgeneration sequencing (NGS) technologies holds the potential to dramatically impact the crop improvement process. NGS enables whole-genome sequencing (WGS) and re-sequencing, transcriptome sequencing, metagenomics, as well as high-throughput genotyping, which can be applied for genome selection (GS). It can also be applied to diversity analysis, genetic and epigenetic characterization of germplasm and pathogen detection, identification, and elimination. High-throughput phenotyping, integrated data management, and decision support tools form the necessary support‐ ing environment for effective utilization of genome sequence information. It is important that these opportunities for mainstreaming innovative breeding strategies, enabled by cutting-edge “Omics” technologies, are seized in Africa; however, several constraints must be addressed before the benefit of NGS can be fully realized. African breeding programs must have access to high-throughput genotyping facilities, capacity in the application of genome selection and marker-assisted breeding must be built and supported by capacity in genomic analysis and bioinformatics. This chapter demonstrates how interventions with NGS-enabled innovative strategies can be applied to increase genetic gain with insights from the Consortium of International © 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Agricultural Research (CGIAR) in general and the International Institute of Tropical Agriculture (IITA) in particular.

Plant viruses are obligate parasites that exploit host components for replication and spread inside the host. Transport of the viral genome is enabled by movement proteins (MPs) targeting the cell periphery to mediate passage throughout plasmodesmata (PD). Pectin methylesterase (PME) is one of the critical host factors facilitating MPs in PD gating, and a direct interaction of PME with Tobacco mosaic virus (TMV) MP is required for viral movement and in turn for virus viability. PME is a critical enzyme for host development and defence, acting via complex mechanisms involving multigenic and tissue specific isoforms and endogenous inhibitors. This composite activity of PME suggests that level and timing of protein accumulation, with respect to virus inoculation and MP expression, can be critical for the functional outcome of the PME-MP interaction and in turn for the success of a viral infection. Based on this notion, we tested different experimental conditions to evaluate the beneficial effect of the downregulation of PME gene expression on the development of TMV-induced disease and on plant protection. We used virus induced gene silencing technology (VIGS) to downregulate PME gene expression, which resulted in a 30–45 % reduction of TMV symptom severity and, correspondingly, to a 60 % reduction of TMV RNA accumulation in systemic leaves. VIGS proved to be a rapid and effective technology for PME gene silencing in functional assays and for plant defence from viral infection. Our findings indicate that N. benthamiana plants with hindered expression of PME survive a TMV infection, which kills non-silenced plants within a week.

Plant virus genomes cross the barrier of the host cell wall and move to neighboring cells either in the form of nucleoprotein complex or encapsidated into virions. Virus transport is facilitated by virus-encoded movement proteins (MP), which are different from one another in number, size, sequence, and in the strategy used to overcome the size exclusion limit (SEL) of plasmodesmata (PD). 1 A group of them forms tubules inside the lumen of highly modified PDs upon removal of the desmotubule. To date the molecular mechanism(s) and the host factors involved in the assembly of MP tubules as well as the mechanistic aspects of virus particle transport throughout them remain substantially unknown. In a recent study, we showed that Cauliflower mosaic virus (CaMV) MP traffics in the endocytic pathway with the help of three tyrosine-sorting signals, which are not required to target MP to the plasma membrane but are essential for tubule formation. 2 This evidence unravels a previously unknown connection between the plant endosomal system and tubule-mediated virus movement that is here supported by demonstration of hindrance of tubule assembly upon Brefeldin A (BFA) treatment. We discuss the implications of our data on the mechanisms of viral transport through tubules and draw parallels with plant mechanisms of polarized growth.

Using the yeast two-hybrid system, we show that the ORF III product of cauliflower mosaic virus (pIII) interacts through its C-terminus with the viral coat protein. The last five amino acids of pIII were essential for the interaction and virus infectivity. Deletion of the last three amino acids or the mutation F129A decreased the strength of the interaction by 90%. We further show that pIII is closely associated with virus particles found in the inclusion bodies of infected plants but not in viral particles released from the inclusion bodies by urea treatment.

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubule-guided encapsidated particles with the help of movement proteins (MPs). To explore how and why MPs specialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virus genomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNA genome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement, and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viral genome entry into the RNA-trafficking PD pathway.

Cestrum yellow leaf curling virus (CmYLCV) has been characterized as the aetiological agent of the Cestrum parqui mosaic disease. The virus genome was cloned and the clone was proven to be infectious to C. parqui. The presence of typical viroplasms in virus-infected plant tissue and the information obtained from the complete genomic sequence confirmed CmYLCV as a member of the Caulimoviridae family. All characteristic domains conserved in plant pararetroviruses were found in CmYLCV. Its genome is 8253 bp long and contains seven open reading frames (ORFs). Phylogenetic analysis of the relationships with other members of the Caulimoviridae revealed that CmYLCV is closely related to the Soybean chlorotic mottle virus (SbCMV)-like genus and particularly to SbCMV. However, in contrast to the other members of this genus, the primer-binding site is located in the intercistronic region following ORF Ib rather than within this ORF, and an ORF corresponding to ORF VII is missing.

We have cloned and evaluated two versions of a novel, strong and constitutive promoter from Cestrum yellow leaf-curling virus (CmYLCV) called CmpC (short- 346bp) and CmpS (longer- 400bp), which can be used for regulating transgene expression in a wide variety of plant species. CmYLCV belongs to the Caulimoviridae family and was first reported in Cestrum parqui from the Solanaceae by Ragozzino (1974). Recently, CmYLCV was cloned and seven open reading frames were identified in the genomic sequence (Hohn et al., 2001). To evaluate the utility of the CmYLCV promoter to drive expression of heterologous genes in plants, two versions of the full-length transcript promoter were cloned in front of the GUS, CAT and FP reporter genes and tested in transient assays in Nicotiana plumbaginifolia, Orichophragmus violaceus and Oriza sativa protoplasts as well as in stably transformed Zea mays and Lycopersicon esculentum. The transient expression experiments show that, depending on the plant system used, the expression level of CmpC and CmpS promoter fragments are higher than the expression level of the widely used 35S promoter from Cauliflower Mosaic Virus (Hohn et al., 2001) and that the longer promoter fragment is the weaker one. Expression analysis of CmpC and CmpS promoter fragments in stably transformed maize (Figurel) have shown that both fragments are on average ten times greater than the strong constitutive Ubi 1 promoter from Z. mays (Christensen et al., 1992) and that the expression levels in tomato (Figure 2) are comparable with the strong, constitutive SMAS promoter (Ni et al., 1994). Moreover, the spatial expression analysis has shown that both promoters express in various tissue types, except pollen in both maize and tomato and that both promoter fragments retain their high expression levels through at least two generations. We limited our analysis to the single copy events only (Ingham et al., 2001) since it is a widely accepted idea that the copy number of a transgene affects the expression level in transgenic plants.

Cauliflower mosaic virus (CaMV) open reading frame III (ORF III) codes for a virion-associated protein (Vap), which is one of two viral proteins essential for aphid transmission. However, unlike the aphid transmission factor encoded by CaMV ORF II, Vap is also essential for systemic infection, suggesting that it is a multifunctional protein. To elucidate the additional function or functions of Vap, we tested the replication of noninfectious ORF III-defective mutants in transfected turnip protoplasts. PCR and Western blot analyses revealed that CaMV replication had occurred with an efficiency similar to that of wild-type virus and without leading to reversions. Electron microscopic examination revealed that an ORF III frameshift mutant formed normally structured virions. These results demonstrate that Vap is dispensable for replication in single cells and is not essential for virion morphogenesis. Analysis of inoculated turnip leaves showed that the ORF III frameshift mutant does not cause any detectable local infection. These results are strongly indicative of a role for Vap in virus movement.

All plant pararetroviruses belong to theCaulimoviridaefamily. This family contains six genera of viruses with different biological, serological, and molecular characteristics. Although some important mechanisms of viral replication and host infection are understood, much remains to be discovered about the many functions of the viral proteins. The focus of this study, the virion-associated protein (VAP), is conserved among all members of the group and contains a coiled-coil structure that has been shown to assemble as a tetramer in the case of cauliflower mosaic virus. We have used the yeast two-hybrid system to characterize self-association of the VAPs of four distinct plant pararetroviruses, each belonging to a different genus ofCaulimoviridae. Chemical cross-linking confirmed that VAPs assemble into tetramers. Tetramerization is thus a common property of these proteins in plant pararetroviruses. The possible implications of this conserved feature for VAP function are discussed.

Eight turnip mosaic potyvirus (TuMV) isolates from the Campania region of Italy were characterized. Experiments based on host range and symptomatology indicated that the isolates were biologically different. In addition, the isolates, with the exception of ITA1 and ITA3, were distinguished from each other by using a combination of monoclonal antibodies recognizing the coat protein. Single-strand conformation polymorphism (SSCP) analysis of the coat protein gene revealed that each isolate produced a specific SSCP profile, except for isolates ITA1 and ITA3. This study indicates that (i) even in a small geographical region, there is a great deal of variation in TuMV isolates; (ii) the use of a set of four differential hosts does not always specify the same pathotype in different environments; (iii) the TuMV isolates with the same pathotype on Brassica napus test lines can still differ in host range, symptoms, serology, and SSCP; and (iv) there was perfect correlation between the panel of antibodies and SSCP in differentiating among the isolates; ITA1 and ITA3 were indistinguishable by either assay.