Emmanuel Jacquot

Aphid transmission of cauliflower mosaic virus requires the viral PIII protein.

The open reading frame (ORF) III product (PIII) of cauliflower mosaic virus is necessary for the infection cycle but its role is poorly understood. We have used in vitro protein binding (‘far Western’) assays to demonstrate that PIII interacts with the cauliflower mosaic virus (CaMV) ORF II product (PII), a known aphid transmission factor. Aphid transmission of purified virions of the PII‐defective strain CM4‐184 was dependent upon added PII, but complementation was efficient only in the presence of PIII, demonstrating the requirement of PIII for transmission. Deletion mutagenesis mapped the interaction domains of PIII and PII to the 30 N‐terminal and 61 C‐terminal residues of PIII and PII, respectively. A model for interaction between PIII and PII is proposed on the basis of secondary structure predictions. Finally, a direct correlation between the ability of PIII and PII to interact and aphid transmissibility of the virus was demonstrated by using mutagenized PIII proteins. Taken together, these data argue strongly that PIII is a second ‘helper’ factor required for CaMV transmission by aphids.

The Helper Component Proteinase Cistron of Potato virus Y Induces Hypersensitivity and Resistance in Potato Genotypes Carrying Dominant Resistance Genes on Chromosome IV

The Nc(tbr) and Ny(tbr) genes in Solanum tuberosum determine hypersensitive reactions, characterized by necrotic reactions and restriction of the virus systemic movement, toward isolates belonging to clade C and clade O of Potato virus Y (PVY), respectively. We describe a new resistance from S. sparsipilum which possesses the same phenotype and specificity as Nc(tbr) and is controlled by a dominant gene designated Nc(spl). Nc(spl) maps on potato chromosome IV close or allelic to Ny(tbr). The helper component proteinase (HC-Pro) cistron of PVY was shown to control necrotic reactions and resistance elicitation in plants carrying Nc(spl), Nc(tbr), and Ny(tbr). However, inductions of necrosis and of resistance to the systemic virus movement in plants carrying Nc(spl) reside in different regions of the HC-Pro cistron. Also, genomic determinants outside the HC-Pro cistron are involved in the systemic movement of PVY after induction of necroses on inoculated leaves of plants carrying Ny(tbr). These results suggest that the Ny(tbr) resistance may have been involved in the recent emergence of PVY isolates with a recombination breakpoint near the junction of HC-Pro and P3 cistrons in potato crops. Therefore, this emergence could constitute one of the rare examples of resistance breakdown by a virus which was caused by recombination instead of by successive accumulation of nucleotide substitutions.

Interaction between the Open Reading Frame III Product and the Coat Protein Is Required for Transmission of Cauliflower Mosaic Virus by Aphids

ABSTRACT Transmission of cauliflower mosaic virus (CaMV) by aphids requires two viral nonstructural proteins, the open reading frame (ORF) II and ORF III products (P2 and P3). An interaction between a C-terminal domain of P2 and an N-terminal domain of P3 is essential for transmission. Purified particles of CaMV are efficiently transmitted only if aphids, previously fed a P2-containing solution, are allowed to acquire a preincubated mixture of P3 and virions in a second feed, thus suggesting a direct interaction between P3 and coat protein. Herein we demonstrate that P3 directly interacts with purified viral particles and unassembled coat protein without the need for any other factor and that P3 mediates the association of P2 with purified virus particles. The interaction domain of P3 is located in its C-terminal half, downstream of the P3-P2 interaction domain but overlapping a region which binds nucleic acids. Mutagenesis of P3 which interferes with the interaction between P3 and virions is correlated with the loss of transmission by aphids. Taken together, our results demonstrate that P3 plays a crucial role in the formation of the CaMV transmissible complex by serving as a bridge between P2 and virus particles.

Early detection of cacao swollen shoot virus using the polymerase chain reaction.

A polymerase chain reaction assay was developed which allows early detection of cacao swollen shoot virus (CSSV) in DNA extracts from cacao plantlets agroinoculated with the Togolese isolate Agou 1. The primers used were derived from badnavirus conserved sequences and nucleic acid was extracted with the Plant DNeasy extraction kit (Qiagen). CSSV genome was detectable between 6 and 20 days after inoculation. The first leaf symptoms appeared after 4 weeks and the first shoot swelling symptoms after 8 weeks.

In situ localization of cacao swollen shoot virus in agroinfected Theobroma cacao

SummaryCacao swollen shoot virus (CSSV) is a small non-enveloped bacilliform virus with a double-stranded DNA genome. A very restricted host range and difficulties in transmitting the virus, either mechanically or via its natural vector, have hindered the study of cacao swollen shoot disease. As an alternative to the particle-bombardment method previously reported, we investigated another approach to infect Theobroma cacao. A greater-than-unit length copy (1.2) of the CSSV DNA genome was cloned into the Agrobacterium binary vector pBin19 and was transferred into young plants via Agrobacterium tumefaciens. Typical leaf symptoms and stem swelling were observed seven and eleven weeks post inoculation, respectively. Viral DNA, CSSV coat protein and virions were detected in leaves with symptoms. Agroinfected plants were used to study the in situ localization of CSSV and its histopathologic effects in planta. In both leaves and petioles, virions were only seen in the cytoplasm of phloem companion cells and of a few xylem parenchyma cells. Light microscopy showed that stem swelling results from a proliferation of the xylem, phloem and cortex cells.

Fluorescently Tagged Potato virus Y: A Versatile Tool for Functional Analysis of Plant-Virus Interactions

Potato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 ± 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Désirée and NahG-Désirée and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild potato relatives. PVY N605-GFP is therefore a powerful tool for future studies of PVY-host interactions, such as functional analysis of viral and plant genes involved in viral movement.Potato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 ± 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Desiree and NahG-Desiree and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild pot...

Metagenomics Approaches Based on Virion-Associated Nucleic Acids (VANA): An Innovative Tool for Assessing Without A Priori Viral Diversity of Plants

This chapter describes an efficient approach that combines quality and yield extraction of viral nucleic acids from plants containing high levels of secondary metabolites and a sequence-independent amplification procedure for both the inventory of known plant viruses and the discovery of unknown ones. This approach turns out to be a useful tool for assessing the virome (the genome of all the viruses that inhabit a particular organism) of plants of interest. We here show that this approach enables the identification of a novel Potyvirus member within a single plant already known to be infected by two other Potyvirus species.

Potato virus Y: biodiversity, pathogenicity, epidemiology and management

Diseases caused by plant viruses can have significant and devastating impacts on many cultivated crops worldwide. The impact of disease caused by a virus depends on the virus species, strains, type of inoculum, host plant characteristics, vector pressure, climatic conditions, trade, changes in agricultural landscape and intensive production practices. Viruses affect plants by causing a large variety of symptoms such as alteration of shape, pigmentation, necrosis on different parts of the plant, thus affecting plant development. In most of the cases, these lead to a decrease in crop yield and quality. There are numerous viruses that affect potato; among them, Potato virus Y is considered to be one of the ten most important plant viruses of crops, because of its worldwide distribution and economic impact. Some PVY isolates are able to cause potato ringspot necrotic disease in infected tubers rendering them unmarketable. Understanding the genetic diversity and molecular biology of PVY is essential to understand its infectious cycle, epidemiology and developing efficient methods of control and management for the virus itself and its vector. In spite of an ever-increasing wealth of data in these topics, several major scientific challenges remain in understanding the molecular nature of the interaction between PVY, its hosts, aphid vector in different environments and the epidemiology of PVY. This and following chapters will present the context and current state of our knowledge for these different topics and attempt to provide some answers to these important questions. C. Lacomme (*) Science and Advice for Scottish Agriculture (SASA), 1 Roddinglaw Road, Edinburgh EH12 9FJ, UK e-mail: Christophe.Lacomme@sasa.gsi.gov.uk E. Jacquot UMR Biologie et Génétique des Interactions Plante-Parasite, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France

General Characteristics of Potato virus Y (PVY) and Its Impact on Potato Production: An Overview

Diseases caused by plant viruses can have significant and devastating impacts on many cultivated crops worldwide. The impact of disease caused by a virus depends on the virus species, strains, type of inoculum, host plant characteristics, vector pressure, climatic conditions, trade, changes in agricultural landscape and intensive production practices. Viruses affect plants by causing a large variety of symptoms such as alteration of shape, pigmentation, necrosis on different parts of the plant, thus affecting plant development. In most of the cases, these lead to a decrease in crop yield and quality. There are numerous viruses that affect potato; among them, Potato virus Y is considered to be one of the ten most important plant viruses of crops, because of its worldwide distribution and economic impact. Some PVY isolates are able to cause potato ringspot necrotic disease in infected tubers rendering them unmarketable. Understanding the genetic diversity and molecular biology of PVY is essential to understand its infectious cycle, epidemiology and developing efficient methods of control and management for the virus itself and its vector. In spite of an ever-increasing wealth of data in these topics, several major scientific challenges remain in understanding the molecular nature of the interaction between PVY, its hosts, aphid vector in different environments and the epidemiology of PVY. This and following chapters will present the context and current state of our knowledge for these different topics and attempt to provide some answers to these important questions.