Molecular studies on a complex of potyviruses infecting solanaceous crops, and some specific virus-host interactions

This thesis constitutes a comprehensive analysis of the molecular and biological characteristics of three potyviruses (genus Potyvirus, family Potyviridae) naturally occurring in cultivated and wild species of family Solanaceae: Peru tomato virus (PTV), Potato virus V (PVV) and Wild potato mosaic virus (WPMV). In addition, the studies presented in this thesis focus on the genetic variability of isolates of PTV and PVV and on the role of the Potato virus A (PVA) 6K2 protein as a host-specific determinant of virus movement and symptom induction. Determination of the complete genomic sequences of PVV, PTV and WPMV demonstrated that these viruses are typical members of the genus Potyvirus. Furthermore, comparison of the polyprotein amino acid sequences and the biological and serological characteristics of these three viruses supported their current taxonomic position as independent species of the genus Potyvirus. The nucleotide sequences of the P1 protein, coat protein and non-translated regions of European and South American PVV isolates were determined and compared. Results showed limited genetic variability among the European isolates, in contrast to the higher variability found among the South American isolates of PVV. Phylogenetic analysis defined two distinct clusters, grouping the European isolates together but placing two South American isolates to a different group; these two isolates of PVV did not induce a hypersensitive response in an Nv gene-carrying potato cultivar in contrast to the European PVV isolates. Thus, European and South American PVV isolates belong to different strain groups. In addition, great genetic variability was detected among PTV isolates. Analysis of phylogenetic relationships among PTV, PVV, WPMV and other members of the genus Potyvirus commonly found infecting solanaceous crop plants showed that PTV, PVV and WPMV are the most closely related viruses which together with Potato virus Y, Pepper mottle virus, Pepper severe mosaic virus and Pepper yellow mosaic virus constitute a group distinguishable from other potyviruses. Thus, members of this group seem to share a common ancestor. The 6K2 protein of PVA was modified by deleting various portions or by introducing six histidine residues (6xHis) into various positions of this protein. These modifications disturbed functions required for viral infection in Nicotiana tabacum. Furthermore, inoculation of the insertion constructs to N. benthamiana plants did not result in systemic infection with the exception of one plant. This plant lacked typical PVA symptoms but had virus titers similar to the plants infected with the wild type virus: a single point mutation (Gly2 ® Cys2) in the 6xHis-containing 6K2 had restored the viral movement functions. However, partial deletion of the 6xHis-tag to gain the original size of the 6K2 protein was required to restore the induction of symptoms in N. benthamiana and to enable systemic infection of N. tabacum. Taken together, these results indicate the 6K2 is a host-specific determinant for long-distance movement and exemplify that mutations that arise during viral propagation represent a mechanisms by which viruses can evolve and adapt to different hosts

Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Rysto , from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Rysto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Rysto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single-molecule real-time sequencing). Rysto was found to encode a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Rysto -dependent extreme resistance was temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops

The coevolution of plants and viruses: Resistance and pathogenicity

Virus infection may damage the plant, and plant defenses are effective against viruses; thus, it is currently assumed that plants and viruses coevolve. However, and despite huge advances in understanding the mechanisms of pathogenicity and virulence in viruses and the mechanisms of virus resistance in plants, evidence in support of this hypothesis is surprisingly scant, and refers almost only to the virus partner. Most evidence for coevolution derives from the study of highly virulent viruses in agricultural systems, in which humans manipulate host genetic structure, what determines genetic changes in the virus population. Studies have focused on virus responses to qualitative resistance, either dominant or recessive but, even within this restricted scenario, population genetic analyses of pathogenicity and resistance factors are still scarce. Analyses of quantitative resistance or tolerance, which could be relevant for plant–virus coevolution, lag far behind. A major limitation is the lack of information on systems in which the host might evolve in response to virus infection, that is, wild hosts in natural ecosystems. It is presently unknown if, or under which circumstances, viruses do exert a selection pressure on wild plants, if qualitative resistance is a major defense strategy to viruses in nature, or even if characterized genes determining qualitative resistance to viruses did indeed evolve in response to virus infection. Here, we review evidence supporting plant–virus coevolution and point to areas in need of attention to understand the role of viruses in plant ecosystem dynamics, and the factors that determine virus emergence in crops

Genetic control and biodiversity of tolerance to Verticillium albo-atrum and Verticillium dahliae in Medicago truncatula

La verticilliose est une maladie vasculaire des plantes dont les symptômes typiques sont un flétrissement des parties aériennes, des feuilles chlorosées puis séchées, et dans les cas de maladie grave la mort de la plante. Au niveau des racines on observe une coloration brune du tissu conducteur. Cette maladie est causée par un champignon du sol du genre Verticillium. Les espèces majeures V. dahliae et V. albo-atrum sont responsables de pertes importantes de rendement sur de nombreuses cultures. Le champignon entre dans la racine par des blessures ou par des fissures au niveau de sites d’émergence de racines latérales, puis il avance vers le cylindre central et envahit les vaisseaux du xylème. Sa croissance reste pendant longtemps limitée aux vaisseaux qu’il colonise en avançant vers les parties aériennes de la plante. Aux stades tardifs, le champignon sort du cylindre central et colonise les autres tissus. En Europe, V. albo-atrum constitue l’une des principales causes de maladies chez la luzerne pérenne et est à l’origine de pertes économiques très importantes. La capacité de V. albo-atrum de survivre dans le sol ainsi que sa localisation protégée dans le cylindre centrale des plantes infectées en font un pathogène difficile à combattre, la lutte génétique par sélection de variétés tolérantes apparaissant comme une approche prometteuse. Cependant, la capacité des microorganismes pathogènes de s’adapter rapidement à des nouvelles plantes hôtes est une menace bien connue de la durabilité des variétés résistantes. Au laboratoire, des travaux ultérieurs ont établi que la plante modèle des légumineuses Medicago truncatula, une espèce sauvage proche de la luzerne cultivée, peut être utilisée pour étudier les mécanismes de résistance et sensibilité vis-à-vis de V. alboatrum. Une lignée résistant et une lignée sensible ont été identifiées et l’étude de la descendance d’un croisement entre ces deux lignées a permis d’identifier un locus majeur (Quantitative trait locus, QTL) contrôlant la résistance à une souche de V. albo-atrum isolée de la Luzerne (Ben et al., 2013 ; Negahi en 6e co-auteur). Ce travail a également montré qu’il existait une grande biodiversité au sein de l’espèce M. truncatula par rapport à la réponse à cette souche de V. albo-atrum. ABSTRACT : Verticillium wilt, caused by Verticillium albo-atrum (Vaa) and Verticillium dahliae (Vd), is responsible for yield losses in many economically important crops. The capacity of pathogenic fungi to adapt to new hosts is a well-known threat to the durability of resistant crop varieties. Medicago truncatula is a good model for studying resistance and susceptibility to Verticillium wilt in legume plants. Phenotyping a population of inbred lines from a cross between resistant parent line A17 and susceptible parent F83005.5 contributed to the identification of a first QTL controlling resistance to an alfalfa strain of Vaa in M. truncatula. Then, 25 M. truncatula genotypes from a core collection and six Vaa and Vd strains were used to study the potential of non-host Verticillium strains isolated from different plant species to infect this legume plant, and the plant’s susceptibility to the pathogens. The experiment was arranged as factorial based on a randomized complete block design with three replications. The wilt symptoms caused by Vaa and Vd were scored on a disease index scale from 0 to 4, during 30 days after inoculation of ten day-old plantlets. Disease severity was quantified by the parameters Maximum Symptom Scores (MSS) and Areas Under the Disease Progress Curves (AUDPC). Highly significant differences were observed among plant genotypes and fungal strains, and their interaction was also significant. The correlation between MSS and AUDPC was 0.86 and highly significant. The most severe symptoms were caused by the alfalfa strain Vaa-V31-2 and the least severe by Vd-JR2, as shown by mean values obtained on the 25 genotypes. M. truncatula genotype TN8.3 was identified as the most susceptible genotype by mean values obtained with the 6 fungal strains, whereas F11013-3, F83005.9 and DZA45.6 were highly resistant to all strains studied. The results were used to choose parents for studying the genetics of resistance in M. truncatula to a nonalfalfa Verticillium strain. So, in the second part of this work, genotype A17 which was susceptible and genotype F83005.5 which was resistant to the potato strain Vaa-LPP0323 and recombinant inbred lines (RILs) from a cross between these genotypes were selected in order to study the genetic control of resistance to this strain of the pathogen. Our experimental design was a randomized complete blocks with 116 RILs and three replications. High genetic variability and transgressive segregation for resistance to Vaa-LPP0323 were observed among RILs. A total of four QTLs controlling resistance to Vaa-LPP0323 were detected for the parameters MSS and AUDPC. The phenotypic variance explained by each QTL (R2) was moderate, ranging from 3 to 21%. A negative sign of additive gene effects showed that favourable alleles for resistance come from the resistant parent