Genetic Variation of Turnip Yellows Virus in Arable and Vegetable Brassica Crops, Perennial Wild Brassicas, and Aphid Vectors Collected from the Plants.

Turnip yellows virus (TuYV; Polerovirus, Solemoviridae) infects and causes yield losses in a range of economically important crop species, particularly the Brassicaceae. It is persistently transmitted by several aphid species and is difficult to control. Although the incidence and genetic diversity of TuYV has been extensively investigated in recent years, little is known about how the diversity within host plants relates to that in its vectors. Arable oilseed rape (Brassica napus) and vegetable brassica plants (Brassica oleracea), wild cabbage (B. oleracea), and aphids present on these plants were sampled in the field in three regions of the United Kingdom. High levels of TuYV (82 to 97%) were detected in plants in all three regions following enzyme-linked immunosorbent assays. TuYV was detected by reverse transcription polymerase chain reaction in Brevicoryne brassicae aphids collected from plants, and TuYV sequences were obtained. Two TuYV open reading frames, ORF0 and ORF3, were partially sequenced from 15 plants, and from one aphid collected from each plant. Comparative analyses between TuYV sequences from host plants and B. brassicae collected from respective plants revealed differences between some ORF0 sequences, which possibly indicated that at least two of the aphids might not have been carrying the same TuYV isolates as those present in their host plants. Maximum likelihood phylogenetic analyses including published, the new TuYV sequences described above, 101 previously unpublished sequences of TuYV from oilseed rape in the United Kingdom, and 13 also previously unpublished sequences of TuYV from oilseed rape in Europe and China revealed three distinct major clades for ORF0 and one for ORF3, with some distinct subclades. Some clustering was related to geographic origin. Explanations for TuYV sequence differences between plants and the aphids present on respective plants and implications for the epidemiology and control of TuYV are discussed.

Identification and QTL mapping of resistance to Turnip yellows virus (TuYV) in oilseed rape, Brassica napus.

KEY MESSAGE: Partially dominant resistance to Turnip yellows virus associated with one major QTL was identified in the natural allotetraploid oilseed rape cultivar Yudal. Turnip yellows virus (TuYV) is transmitted by the peach-potato aphid (Myzus persicae) and causes severe yield losses in commercial oilseed rape crops (Brassica napus). There is currently only one genetic resource for resistance to TuYV available in brassica, which was identified in the re-synthesised B. napus line 'R54'. In our study, 27 mostly homozygous B. napus accessions, either doubled-haploid (DH) or inbred lines, representing a diverse subset of the B. napus genepool, were screened for TuYV resistance/susceptibility. Partial resistance to TuYV was identified in the Korean spring oilseed rape, B. napus variety Yudal, whilst the dwarf French winter oilseed rape line Darmor-bzh was susceptible. QTL mapping using the established Darmor-bzh × Yudal DH mapping population (DYDH) revealed one major QTL explaining 36% and 18% of the phenotypic variation in two independent experiments. A DYDH line was crossed to Yudal, and reciprocal backcross (BC1) populations from the F1 with either the susceptible or resistant parent revealed the dominant inheritance of the TuYV resistance. The QTL on ChrA04 was verified in the segregating BC1 population. A second minor QTL on ChrC05 was identified in one of the two DYDH experiments, and it was not observed in the BC1 population. The TuYV resistance QTL in 'R54' is within the QTL interval on Chr A04 of Yudal; however, the markers co-segregating with the 'R54' resistance are not conserved in Yudal, suggesting an independent origin of the TuYV resistances. This is the first report of the QTL mapping of TuYV resistance in natural B. napus.

Viral Strain-Specific Differential Alterations in Arabidopsis Developmental Patterns.

Turnip mosaic virus (TuMV) infections affect many Arabidopsis developmental traits. This paper analyzes, at different levels, the development-related differential alterations induced by different strains of TuMV, represented by isolates UK 1 and JPN 1. The genomic sequence of JPN 1 TuMV isolate revealed highest divergence in the P1 and P3 viral cistrons, upon comparison with the UK 1 sequence. Infectious viral chimeras covering the whole viral genome uncovered the P3 cistron as a major viral determinant of development alterations, excluding the involvement of the PIPO open reading frame. However, constitutive transgenic expression of P3 in Arabidopsis did not induce developmental alterations nor modulate the strong effects induced by the transgenic RNA silencing suppressor HC-Pro from either strain. This highlights the importance of studying viral determinants within the context of actual viral infections. Transcriptomic and interactomic analyses at different stages of plant development revealed large differences in the number of genes affected by the different infections at medium infection times but no significant differences at very early times. Biological functions affected by UK 1 (the most severe strain) included mainly stress response and transport. Most cellular components affected cell-wall transport or metabolism. Hubs in the interactome were affected upon infection.

Multiple copies of eukaryotic translation initiation factors in Brassica rapa facilitate redundancy, enabling diversification through variation in splicing and broad-spectrum virus resistance.

Recessive strain-specific resistance to a number of plant viruses in the Potyvirus genus has been found to be based on mutations in the eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E. We identified three copies of eIF(iso)4E in a number of Brassica rapa lines. Here we report broad-spectrum resistance to the potyvirus Turnip mosaic virus (TuMV) due to a natural mechanism based on the mis-splicing of the eIF(iso)4E allele in some TuMV-resistant B. rapa var. pekinensis lines. Of the splice variants, the most common results in a stop codon in intron 1 and a much truncated, non-functional protein. The existence of multiple copies has enabled redundancy in the host plant's translational machinery, resulting in diversification and emergence of the resistance. Deployment of the resistance is complicated by the presence of multiple copies of the gene. Our data suggest that in the B. rapa subspecies trilocularis, TuMV appears to be able to use copies of eIF(iso)4E at two loci. Transformation of different copies of eIF(iso)4E from a resistant B. rapa line into an eIF(iso)4E knockout line of Arabidopsis thaliana proved misleading because it showed that, when expressed ectopically, TuMV could use multiple copies which was not the case in the resistant B. rapa line. The inability of TuMV to access multiple copies of eIF(iso)4E in B. rapa and the broad spectrum of the resistance suggest it may be durable.

High-resolution temporal profiling of transcripts during Arabidopsis leaf senescence reveals a distinct chronology of processes and regulation.

Leaf senescence is an essential developmental process that impacts dramatically on crop yields and involves altered regulation of thousands of genes and many metabolic and signaling pathways, resulting in major changes in the leaf. The regulation of senescence is complex, and although senescence regulatory genes have been characterized, there is little information on how these function in the global control of the process. We used microarray analysis to obtain a high-resolution time-course profile of gene expression during development of a single leaf over a 3-week period to senescence. A complex experimental design approach and a combination of methods were used to extract high-quality replicated data and to identify differentially expressed genes. The multiple time points enable the use of highly informative clustering to reveal distinct time points at which signaling and metabolic pathways change. Analysis of motif enrichment, as well as comparison of transcription factor (TF) families showing altered expression over the time course, identify clear groups of TFs active at different stages of leaf development and senescence. These data enable connection of metabolic processes, signaling pathways, and specific TF activity, which will underpin the development of network models to elucidate the process of senescence.

Turnip mosaic virus (TuMV) is able to use alleles of both eIF4E and eIF(iso)4E from multiple loci of the diploid Brassica rapa.

Three copies of eIF4E and three copies of eIF(iso)4E have been identified and sequenced from a Turnip mosaic virus (TuMV)-susceptible, inbred, diploid Brassica rapa line, R-o-18. One of the copies of eIF4E lacked exons 2 and 3 and appeared to be a pseudogene. The two other copies of eIF4E and two of the three copies of eIF(iso)4E were isolated from a bacterial artificial chromosome library of R-o-18. Using an Arabidopsis line (Col-0::dSpm) with a transposon knock-out of the eIF(iso)4E gene which resulted in a change from complete susceptibility to complete resistance to TuMV, complementation experiments were carried out with the two versions of eIF4E and the two versions of eIF(iso)4E. When transformed into Col-0::dSpm, all four Brassica transgenes complemented the Arabidopsis eIF(iso)4E knock-out, conferring susceptibility to both mechanical and aphid challenge with TuMV. One of the copies of eIF4E did not appear to support viral replication as successfully as the other copy of eIF4E or the two copies of eIF(iso)4E. The results show that TuMV can use both eIF4E and eIF(iso)4E from B. rapa for replication and, for the first time, that a virus can use eIF4E and eIF(iso)4E from multiple loci of a single host plant.

Comparisons of the genetic structure of populations of Turnip mosaic virus in West and East Eurasia.

The genetic structure of populations of Turnip mosaic virus in Eurasia was assessed by making host range and gene sequence comparisons of 142 isolates. Most isolates collected in West Eurasia infected Brassica plants whereas those from East Eurasia infected both Brassica and Raphanus plants. Analyses of recombination sites (RSs) in five regions of the genome (one third of the full sequence) showed that the protein 1 (P1 gene) had recombined more frequently than the other gene regions in both subpopulations, but that the RSs were located in different parts of the genomes of the subpopulations. Estimates of nucleotide diversity showed that the West Eurasian subpopulation was more diverse than the East Eurasian subpopulation, but the Asian-BR group of the genes from the latter subpopulation had a greater nonsynonymous/synonymous substitution ratio, especially in the P1, viral genome-linked protein (VPg) and nuclear inclusion a proteinase (NIa-Pro) genes. These subpopulations seem to have evolved independently from the ancestral European population, and their genetic structure probably reflects founder effects.

The dual role of the potyvirus P3 protein of Turnip mosaic virus as a symptom and avirulence determinant in brassicas.

Two isolates of the potyvirus Turnip mosaic virus (TuMV), UK 1 and CDN 1, differ both in their general symptoms on the susceptible propagation host Brassica juncea and in their ability to infect B. napus lines possessing a variety of dominant resistance genes. The isolate CDN 1 produces a more extreme mosaic in infected brassica leaves than UK 1 and is able to overcome the resistance genes TuRB01, TuRB04, and TuRB05. The resistance gene TuRB03, in the B. napus line 22S, is effective against CDN 1 but not UK 1. The nucleic acid sequences of the UK 1 and CDN 1 isolates were 90% identical. The C-terminal half of the P3 protein was identified as being responsible for the differences in symptoms in B. juncea. A single amino acid in the P3 protein was found to be the avirulence determinant for TuRB03. Previous work already has identified the P3 as an avirulence determinant for TuRB04. Our results increase the understanding of the basis of plant-virus recognition, show the importance of the potyviral P3 gene as a symptom determinant, and provide a role in planta for the poorly understood P3 protein in a normal infection cycle.

Mutations in Turnip mosaic virus P3 and cylindrical inclusion proteins are separately required to overcome two Brassica napus resistance genes.

The Brassica napus differential line 165 is resistant to infection by Turnip mosaic virus (TuMV) isolates belonging to pathotypes 1 and 3. Nucleotide sequences of resistance-breaking mutants of pathotype 1 (UK 1), pathotype 3 (CHN 12), and wild-type isolates have been determined. When the mutations identified were introduced into an infectious clone of UK 1, a single mutation in the viral P3 protein induced a hypersensitive (necrotic) response in inoculated leaves of line 165 plants. Full systemic nonnecrotic infection was only possible when another mutation (in the cylindrical inclusion protein) was introduced. Tests on segregating populations derived from line 165 indicated that the two viral genes were pathogenicity determinants for two different resistance genes in line 165. One gene responsible for an extreme form of resistance (no symptoms seen) was epistatic to a second responsible for the hypersensitive reaction. These results help to explain the relative stability of the resistance in line 165 and to further define the genetic basis of the TuMV pathotyping system.

A fitness cost for Turnip mosaic virus to overcome host resistance.

The relative fitness of the Turnip mosaic virus (TuMV) isolate UK 1 was compared with that of two other wildtype isolates CZE 1 and CDN 1. The isolates CZE 1 and CDN 1 are able to overcome the effect of the resistance gene TuRB01 and at least three other resistance sources that are effective against UK 1. Comparisons were also made between the fitness of UK 1 and a recombinant virus with a single nucleotide change (v35Tunos +5570 A>G) conferring the ability to overcome TuRB01 resistance. Co-inoculation experiments were carried out where pairs of isolates were serially passaged over 5 months in a plant line possessing no known resistance genes in order to examine the relative fitness of the isolates. In each case, UK 1 dominated the mixture in the susceptible host background. It out-competed CZE 1 and v35Tunos +5570 A>G within four passages, and CDN 1 after one passage. The greater fitness of UK 1 suggests that there may be a fitness cost to TuMV overcoming resistance genes of brassica crops. This may shed some light on the frequency of naturally occurring isolates, in that pathotype 1 isolates are found much more frequently than isolates of other pathotypes. Implications for the deployment of TuRB01 are discussed.

Turnip mosaic virus and the quest for durable resistance.

UNLABELLED: Summary Taxonomy: Turnip mosaic virus (TuMV) is a member of the genus Potyvirus (type species Potato virus Y) in the family Potyviridae. To date, TuMV is the only potyvirus known to infect brassicas. There are potyvirus isolates that appear serologically similar to TuMV when tested with polyclonal antisera that do not readily infect brassicas (Lesemann and Vetten, 1985). Physical properties: Virions are approximately 720 x 15-20 nm flexuous rods (Fig. 1) and are composed of 95% coat protein (CP) and 5% RNA. Hosts: TuMV has been isolated from a wide range of crop and weed plant species. It is known to infect at least 318 species in over 43 dicot families, including Cruciferae, Compositae, Chenopodiaceae, Leguminosae and Caryophyllaceae and is also known to infect monocots. It has the broadest known host range in terms of plant genera and families of any potyvirus. TRANSMISSION: Aphid transmitted in the non-persistent manner, by at least 89 species, including Myzus persicae and Brevicoryne brassicae. Useful website: http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/57010072.htm.