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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.

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.

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.

The extracellular matrix (ECM) of animals and plants cells plays important roles in viral diseases. While in animal cells, extracellular matrix components can be exploited by viruses for recognition, attachment and entry, the plant cell wall (CW) acts as a physical barrier to viral entry and adds a higher level of difficulty to intercellular movement of viruses. Interestingly, both in plant and animal systems, ECM can be strongly remodeled during virus infection, and the understanding of remodeling mechanisms and molecular players offers new perspectives for therapeutic intervention. This review focuses on the different roles played by the ECM in plant and animal hosts during virus infection with special emphasis on the similarities and differences. Possible biotechnological applications aimed at improving viral resistance are discussed.

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.

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.

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.