Carol E. Jenner

High-Resolution Temporal Profiling of Transcripts during Arabidopsis Leaf Senescence Reveals a Distinct Chronology of Processes and Regulation

This work presents a high-resolution time-course analysis of gene expression during development of a leaf from expansion through senescence. Enrichment in ontologies, sequence motifs, and transcription factor families within genes showing altered expression over time identified both metabolic pathways and potential regulators active at different stages of leaf development and senescence. 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.

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.

The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the brassica resistance gene TuRB01.

The viral component of Turnip mosaic virus (TuMV) determining virulence to the Brassica napus TuRB01 dominant resistance allele has been identified. Sequence comparisons of an infectious cDNA clone of the UK 1 isolate of TuMV (avirulent on TuRB01) and a spontaneous mutant capable of infecting plants possessing TuRB01 suggested that a single nucleotide change in the cylindrical inclusion (CI) protein coding region (gene) of the virus was responsible for the altered phenotype. A second spontaneous mutation involved a different change in the CI gene. The construction of chimeric genomes and subsequent inoculations to plant lines segregating for TuRB01 confirmed the involvement of the CI gene in this interaction. Site-directed mutagenesis of the viral coat protein (CP) gene at the ninth nucleotide was carried out to investigate its interaction with TuRB01. The identity of this nucleotide in the CP gene did not affect the outcome of the viral infection. Both mutations identified in the CI gene caused amino acid changes in the C terminal third of the protein, outside any of the conserved sequences reported to be associated with helicase or cell-to-cell transport activities. This is the first example of a potyvirus CI gene acting as a determinant for a genotype-specific resistance interaction.

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.

Expression of avrPphB, an Avirulence Gene from Pseudomonas syringae pv. phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean

Protein production encoded by the avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola was examined. Incorporation of [35S]-labeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa product. The 28-kDa native peptide was isolated from E. coli following over-expression of avrPphB and found not to elicit the hypersensitive response (HR) after infiltration into bean leaves. Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P. syringae pv. phaseolicola; immunoreactive peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucrose. Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPphB protein, revealed the accumulation of peptides corresponding to the smaller cleavage products, in both E. coli and P. syringae pv. phaseolicola. Biochemical localization experiments showed that all AvrPphB peptides were cytoplasmic in P. syringae pv. phaseolicola. No AvrPphB peptides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to wild type were observed in a hrpY- strain, although it also failed to cause a confluent HR. Growth of P. syringae pv. phaseolicola under inducing conditions removed the requirement for rifampicin-sensitive mRNA synthesis by bacteria to allow HR development (the induction time) in bean and lettuce leaves. Constitutive expression of hrpL reduced but did not remove the induction time. Expression of the hrp gene cluster of P. syringae pv. phaseolicola from plasmid pPPY430 in E. coli enabled phenotypic expression of avrPphE (also carried by pPPY430) and avrPphB (if over-expressed from pPPY3031). Despite constitutive expression of the hrp and avr genes in E. coli, a protein synthesis dependent induction time was still required for development of the HR in bean genotypes with matching resistance genes. The significance of processing for the function of AvrPphB peptides and the delivery of elicitors of the HR are discussed.

The phylogeny of Turnip mosaic virus ; comparisons of 38 genomic sequences reveal a Eurasian origin and a recent emergence in east Asia

The genomes of a representative world‐wide collection of 32 Turnip mosaic virus (TuMV) isolates were sequenced and these, together with six previously reported sequences, were analysed. At least one‐fifth of the sequences were recombinant. In phylogenetic analyses, using genomic sequences of Japanese yam mosaic virus as an outgroup, the TuMV sequences that did not show clear recombination formed a monophyletic group with four well‐supported lineages. These groupings correlated with differences in pathogenicity and provenance; the sister group to all others was of Eurasian B‐strain isolates from nonbrassicas, and probably represents the ancestral TuMV population, and the most recently ‘emerged’ branch of the population was probably that of the BR‐strain isolates found only in east Asia. Eight isolates, all from east Asia, were clear recombinants, probably the progeny of recent recombination events, whereas a similar number, from other parts of the world, were seemingly older recombinants. This difference indicates that the presence of clear recombinants in a subpopulation may be a molecular signature of a recent ‘emergence’.

Characterisation of resistance to turnip mosaic virus in oilseed rape (Brassica napus) and genetic mapping of TuRB01

Abstract Turnip mosaic virus (TuMV) is the major virus infecting Brassica crops. A dominant gene, TuRB01, that confers extreme resistance to some isolates of TuMV on Brassica napus (oilseed rape), has been mapped genetically. The mapping employed a set of doubled-haploid lines extracted from a population used previously to develop a reference RFLP map of the B. napus genome. The positioning of TuRB01 on linkage group N6 of the B. napus A–genome indicated that the gene probably originated from Brassica rapa. Resistance phenotypes were confirmed by indirect plate-trapped antigen ELISA using a monoclonal antibody raised against TuMV. The specificity of TuRB01 was determined using a wide range of TuMV isolates, including representatives of the European and American/Taiwanese pathotyping systems. Some isolates of TuMV that did not normally infect B. napus plants possessing TuRB01 produced mutant viruses able to overcome the action of the resistance gene. TuRB01 is the first gene for host resistance to TuMV to be mapped in a Brassica crop. A second locus, TuRB02, that appeared to control the degree of susceptibility to the TuMV isolate CHN 1 in a quantitative manner, was identified on the C-genome linkage group N14. The mapping of other complementary genes and the selective combining of such genes, using marker-assisted breeding, will make durable resistance to TuMV a realisable breeding objective.

Different classes of resistance to turnip mosaic virus in Brassica rapa

Pathotype-specific and broad-spectrum resistance to turnip mosaic virus (TuMV) have been identified in the diploid A genome brassica species Brassica rapa. The pathotype-specific resistance is effective against pathotype 1 isolates of TuMV, which are the most common in Europe. It is almost identical in its specificity to that of a mapped resistance gene (TuRB01) present in the A genome of the amphidiploid species Brassica napus. A mutant of a pathotype 1 isolate of TuMV (UK 1M) that is able to overcome TuRB01 also overcame the B. rapa resistance. This, combined with the fact that a single-nucleotide mutation in the cylindrical inclusion gene of TuMV that has been shown to induce a change from avirulence to virulence against TuRB01, had an identical effect on the B. rapa resistance, suggest that the two resistances are conditioned by the same gene. A second source of resistance in B. rapa prevented systemic spread of all TuMV isolates tested. A third source of resistance that appears to provide immunity to, or severely restrict replication of most isolates of TuMV has been characterised. This resistance source also prevented systemic spread of all TuMV isolates tested. Prior to this study, no resistance to pathotype 4 or pathotype 12 isolates of TuMV had ever been identified. For each of these three resistance sources, plant lines that are not segregating for some of the resistance phenotypes and that are presumably homozygous for the genes controlling these phenotypes have been generated. Strategies for further characterising and deploying these resistances in different Brassica species are described.

Strains of Turnip mosaic potyvirus as defined by the molecular analysis of the coat protein gene of the virus.

Turnip mosaic virus (TuMV) is a member of the potyvirus genus with a wide host range and highly variable in its biological characteristics. Analysis of the CP gene sequences from databases, combined with the experimental analysis of the CP gene of further isolates, using data derived from sequence or restriction analysis, has allowed the genetic classification of 60 TuMV isolates or sequences. Two main genetic clusters MB (mostly Brassica isolates) and MR (mostly Radish isolates) were found, together with several apparently independent lineages. Isolates in the latter could be grouped as Intermediate between Brassica and Radish clusters (IBR) or outside Brassica and Radish clusters (OBR), according to their genetic distance to the main clusters. The genetic diversity of TuMV isolates deposited in the databases was increased with the sequences of the CP gene of seven selected isolates, mainly belonging to IBR or OBR groups. There was a correlation between the MR genetic cluster and JPN 1 serotype.

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.