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Plasmodiophora brassicae causes clubroot on cruciferous plants and causes worldwide huge economical losses on important Brassica crops. P. brassicae infection produces large root galls, the clubroots, which can also affect the upper plant parts by reduced water and nutrient uptake and redirection of assimilates from leaves to roots. P. brassicae is an obligate biotrophic protist in the plasmodiophorids within the eukaryote supergroup of Rhizaria and is unrelated to other known plant pathogens. Plasmodiophorids can be parasites of plants and oomycetes. The recently released genome of P. brassicae is only the third in the poorly studied Rhizaria and the first plant pathogenic genome of this eukaryotic group. The P. brassicae genome was estimated to be 25.5 Mb in size and predicted to contain 9730 gene models. A transcriptome of P. brassicae and Spongospora subterranea, the potato scab pathogen was also presented. Consequently, for the first time large scale data for a eukaryotic plant pathogen group outside the fungi and oomycetes are now available. This review highlights selected characteristics of the P. brassicae genome including molecular events shown or predicted to take place in each phase of its life-cycle, such as manipulation of: 1) host primary metabolism, 2) plant hormone homeostasis, and 3) plant defense. Further, future directions and challenges in the P. brassicae and plasmodiophorid genomic research are discussed.

Highlight A host-induced gene-silencing strategy for controlling potato late blight is presented, a plant disease that conventionally requires regular application of fungicides at high rates., Phytophthora infestans is an oomycete that causes severe damage to potato, and is well known for its ability to evolve rapidly in order to overcome resistant potato varieties. An RNA silencing strategy was evaluated here to clarify if small interfering RNA homologous to selected genes in P. infestans could be targeted from the plant host to reduce the magnitude of the infection. As a proof-of-concept, a hairpin RNA (hp-RNA) construct using the GFP marker gene was designed and introduced in potato. At 72 hpi, a 55-fold reduction of the signal intensity of a corresponding GFP expressing P. infestans strain on leaf samples of transgenic plants, compared with wild-type potato, was detected. This suggests that an RNA interference construct in the potato host could be processed and target a transcript of the pathogen. Three genes important in the infection process of P. infestans, PiGPB1, PiCESA2, and PiPEC, together with PiGAPDH taking part in basic cell maintenance were subsequently tested using an analogous transgenic strategy. Out of these gene candidates, the hp-PiGPB1 targeting the G protein β-subunit (PiGPB1) important for pathogenicity resulted in most restricted disease progress. Further, Illumina sequencing of inoculated transgenic potato leaves revealed sRNAs of 24/25 nt size homologous to the PiGPB1 gene in the transgenic plants indicating post-transcriptional silencing of the target gene. The work demonstrates that a host-induced gene-silencing approach is functional against P. infestans but is highly dependent on target gene for a successful outcome. This finding broadens the arsenal of control strategies to this important plant disease.

Spot blotch, caused by Bipolaris sorokiniana, has become an increasingly important disease on barley in Sweden. In order to enhance our knowledge of this disease, eight Swedish B. sorokiniana isolates were assessed for their virulence pathotype on 30 barley genotypes including eight lines of mutant origin, designated B. sorokiniana tolerant (bst). A leaf screen assay revealed the presence of virulence groups 1 and 7 in Sweden. Several mutant genotypes demonstrated enhanced resistance responses (P = 0.0001), of which bstl:3 had the lowest leaf scoring value to the most virulent isolate, Swe2 (virulence group 7). To evaluate the host root responses, two contrasting isolates Swe2 and Swe4 (virulence group 7 and 1) were used to inoculate a set of eight differential lines, including four promising mutant lines from the leaf screen. Mutant bstl:6 was significantly less susceptible (P = 0.001) compared with the other barley genotypes tested, independent of the two isolates used. Mutants bst1, bst1:3 and bstl:4, which displayed less or no spots, are also considered promising genotypes for further breeding attempts.

In order to study the genetic variation and phylogenetic relationship in Elymus repens, amplified fragment length polymorphism (AFLP) were used, together with sequence data for the nuclear gene phytochrome B, phyB, and the chloroplast ribosomal protein encoding gene rps4. A total of 83 collected E. repens samples, 3 E. repens reference samples and 18 related species accessions were analysed and compared with 13 GenBank sequences. AMOVA analysis revealed a moderate genetic differentiation between the populations of E. repens in the three Swedish provinces investigated, while no differentiation was observed due to landscape type. A moderate genetic differentiation was also found when samples from different fields in one province were compared to samples from a selected field. A common female origin was found in E. repens and seven other Elymus species, Pseudoroegneria spicata, Thinopyrum intermedium, T. junceum, Hordeum bogdanii and H. stenostachys. The latter two both harbour the H genome. Taken together, the data suggest that the Swedish E. repens population is slightly heterogeneous and comprises multiple origins of genome donors; a nuclear genome with contributions from Pseudoroegneria (St), Hordeum (H), Thinopyrum (E) and Y with an unknown donor together with a maternal genome donated from Pseudoroegneria.

Sweetpotato is an important food crop in the world as well as in Kenya. Various fungal, and viral diseases are major constraints in its production and are currently threatening the sweetpotato production in sub-Saharan Africa. Genetic engineering offers significant potential for the crop’s genetic improvement. However, this is limited by the low efi¬Âciency and strong genotype dependency in tissue culture. This study aimed to establish an efficient somatic embryogenesis and plant regeneration system using shoot apical meristem explants of sweetpotato. Three sweetpotato cultivars that are widely grown in Kenya; KSP36, Kemb36 and Mweu mutheke along with an exotic model cultivar Jewel were evaluated. The maximum somatic embryogenic induction, at 96.72%, was obtained from explants cultured on Linsmaier and Skoog salts and vitamins medium supplemented with 0.5 mg/l dichlorophenoxyacetic acid and 0.2 mg/l zeatin riboside. The highest number of shoot induction (33) was observed after transfer of embryonic callus to embryo maturation medium supplemented with 2 mg/l abscisic acid. Significant differences were observed between cultivars for somatic embryogenesis and plant regeneration. Jewel showed the best response, while Mweu mutheke was the least responsive under the culture conditions tested in this study. Regenerated plants were successfully rooted and grown to maturity after hardening in soil in the greenhouse. Such a robust, successful and efi¬Âcient system possesses the potential to become an important tool for crop improvement and functional studies of genes in sweetpotato.

Verticillium wilt of oilseed rape (Brassica napus) is caused primarily by Verticillium longisporum and has become a serious problem in northern Europe. In order to evaluate whether V. longisporum and V. dahliae differ in their interaction with oilseed rape, phenotypical and molecular assessments were made. Oilseed rape plants for fungal assessments were inoculated with V. longisporum and V. dahliae via root-dipping and samples were taken from roots, stems, leaves, flowers, pods and seeds during plant development. The infection by V. longisporum was found to start mainly in lateral roots and root-hairs, followed by colonization of the xylem vessels and extensive spread in stems and leaves, whereas V. dahliae infected the main roots and remained in the region below the cotyledon node of the plants. Re-isolation studies, together with PCR analysis of samples taken from early growth stages through to fully ripe plants, showed that the onset of flowering was a critical phase for V. longisporum to colonize plants. No seeds infected with V. longisporum were found. Mycelial growth from V. dahliae but not V. longisporum was significantly reduced on media containing tissue from a low glucosinolate B. napus genotype compared with growth on media containing tissue from a high glucosinolate cultivar. The results of this study suggest that V. longisporum favours B. napus as host and that the transition from the vegetative to the generative phase is of importance for the spread of the fungus in oilseed rape plants.

Three different genes were sequenced from isolates of five plant-pathogenic Verticillium species, Verticillium albo-atrum, Verticillium dahliae, Verticillium longisporum, Verticillium nigrescens, and Verticillium tricorpus. The sequences covered parts of the mitochondrial cytochrome b gene ( cob), the mitochondrial small subunit rRNA gene ( rns) and the nuclear ITS2 region. When the sequences were combined, the five species clustered in five monophyletic groups, with V. nigrescens distantly related to the other species while V. tricorpus displayed a somewhat closer relationship to the three remaining species. V. albo-atrum, V. dahliae and V. longisporum were found to be very similar to each other, with V. albo-atrum and V. longisporum displaying the closest relationship. The species affiliation of V. longisporum is discussed.

The phylogenetic relationships of 17 Bacillus strains isolated from plants and soil were determined from partial sequences of genes encoding 16S rRNA, gyraseA (gyrA) and the cheA histidine kinase. Five strains were closely related to Bacillus subtilis subsp. subtilis, three strains were more closely related to B. subtilis subsp. spizizeni and two strains were identified as B. mojavensis. The remaining seven strains formed a cluster closely related to, but distinct from, Bacillus amyloliquefaciens. Some of these strains formed red-pigmented colonies. The abilities of selected strains to survive in the rhizosphere and to colonize plants were studied using oilseed rape (Brassica napus), barley (Hordeum vulgare) and thale cress (Arabidopsis thaliana) as model plants. It was shown by following the titre of bacteria in seedlings and by scanning electron microscopy that survival of Bacillus cells on the roots of seedlings during the first week after treatment of seeds with spore suspensions was crucial for colonization of the rhizosphere and for biocontrol activity. The group of strains related to B. amyloliquefaciens were generally better adapted to colonization of the rhizosphere of plants than other members of the B. subtilis group and could be considered a distinct ecotype of B. amyloliquefaciens. Bacteria in this taxon could be recognized on the basis of amplification of a PCR product with primers directed to the tetB(L) locus but no product with primers directed to the alpha-amylase gene of B.amyloliquefaciens sensu stricto.

Out of 168 Arabidopsis accessions screened with isolates of Leptosphaeria maculans, one (An-1) showed clear disease symptoms. In order to identify additional components involved in containment of L. maculans in Arabidopsis, a screen for L. maculans-susceptible (lms) mutants was performed. Eleven lms mutants were isolated, which displayed differential susceptibility responses to L. maculans. lms1 was crossed with Columbia (Col-0) and Ws-0, and mapping data for both populations showed the highest linkage to a region on chromosome 2. Reduced levels of PR-1 and PDF1.2 expression were found in lms1 compared to wild-type plants 48 h after pathogen inoculation. In contrast, the lms1 mutant displayed upregulation of either marker gene upon chemical treatment, possibly as an effect of an altered ethylene (ET) response. To assess the contribution of different defence pathways, genotypes implicated in salicylic acid (SA) signalling plants expressing the bacterial salicylate hydroxylase (nahG) gene, non-expressor of PR1 (npr1)-1 and phytoalexin-deficient (pad4-1), jasmonic acid (JA) signalling (coronatine insensitive (coi)1-16, enhanced disease susceptibility (eds)8-1 and jasmonic acid resistant (jar)1-1) and ET signalling (eds4-1, ethylene insensitive (ein)2, ein3-1 and ethylene resistant (etr)1-1) were screened. All the genotypes screened were as resistant as wild-type plants, demonstrating the dispensability of the pathways in L. maculans resistance. When mutants implicated in cell death responses were assayed, responsive to antagonist 1 (ran1)-1 exhibited a weak susceptible phenotype, whereas accelerated cell death (acd)1-20 showed a rapid lesion development. Camalexin is only partially responsible for L. maculans containment in Arabidopsis, as pad3-1 and enhanced susceptibility to Alternaria (esa)1 clearly showed a susceptible response while wild-type levels of camalexin were present in An-1 and lms1. The data presented point to the existence of multiple defence mechanisms controlling the containment of L. maculans in Arabidopsis.

Using a polymerase chain reaction-based strategy, we have isolated a cDNA sequence from Brassica nigra, denoted Lm1, which significantly improves blackleg resistance when expressed in transgenic oilseed rape, B. napus. Lm1 was shown to map to locations on linkage groups 3 and 8 in the Brassica B-genome previously linked to both cotyledon, adult leaf, and stem resistance. B. napus plants transformed with Lm1 under the control of a constitutive promoter exhibited broad spectrum resistance to all L. maculans isolates tested, but enhanced resistance to Alternaria brassicae and Verticillium longisporum was not observed. A transcript corresponding to the cDNA size was induced in B. nigra 12 h after fungal challenge from a level of weak constitutive expression previous to inoculation. The Lm1 sequence bears no resemblance to previously characterized plant resistance genes but has two predicted transmembrane motifs. Several sequences with high homology to Lm1 were found in the databases. Lm1 appears to be a member of a larger group of related genes present in a variety of plant species. Most of them have unknown functions, but homology between Lm1 and the nodule inception gene of Lotus japonicus suggests an intriguing link between defense-related and symbiotic pathways.

Plasmodiophora brassicae causes clubroot, a major disease of Brassica oil and vegetable crops worldwide. P. brassicae is a Plasmodiophorid, obligate biotrophic protist in the eukaryotic kingdom of Rhizaria. Here we present the 25.5 Mb genome draft of P. brassicae, developmental stage-specific transcriptomes and a transcriptome of Spongospora subterranea, the Plasmodiophorid causing powdery scab on potato. Like other biotrophic pathogens both Plasmodiophorids are reduced in metabolic pathways. Phytohormones contribute to the gall phenotypes of infected roots. We report a protein (PbGH3) that can modify auxin and jasmonic acid. Plasmodiophorids contain chitin in cell walls of the resilient resting spores. If recognized, chitin can trigger defense responses in plants. Interestingly, chitin-related enzymes of Plasmodiophorids built specific families and the carbohydrate/chitin binding (CBM18) domain is enriched in the Plasmodiophorid secretome. Plasmodiophorids chitin synthases belong to two families, which were present before the split of the eukaryotic Stramenopiles/Alveolates/Rhizaria/Plantae and Metazoa/Fungi/Amoebozoa megagroups, suggesting chitin synthesis to be an ancient feature of eukaryotes. This exemplifies the importance of genomic data from unexplored eukaryotic groups, such as the Plasmodiophorids, to decipher evolutionary relationships and gene diversification of early eukaryotes.

Stem canker (blackleg) caused by Leptosphaeria maculans is a widespread disease of Brassica napus. In contrast, most Arabidopsis thaliana accessions are highly resistant. Hence, novel material derived from symmetric and asymmetric somatic hybrids between B. napus and A. thaliana was utilised in a screen for L. maculans resistance. Initially, both cotyledon and adult-leaf resistance traits were transferred from A. thaliana to B. napus. In later generations the two traits segregated and cotyledon resistance was lost. The adult-leaf resistance was investigated with respect to genome localisation and protein expression. Analyses of remaining A. thaliana DNA in resistant plants showed co-segregation between adult-leaf resistance and chromosome-3 molecular markers. Resistant offspring from asymmetric hybrid plants that contained fragments of chromosome 3 were studied in more detail. Two regions at positions 9.8-10.4 Mbp and 18-19.5 Mbp, where several defence-related genes are located, were identified. A proteomic approach was taken to further investigate genes involved in the defence interaction. Forty eight hours after inoculation with L. maculans, only a few proteins, such as glycolate oxidase, were identified as differentially expressed in the resistant line compared to B. napus, despite the presence of additional A. thaliana chromosomes. The plant materials described in the present study constitute a new genetic source of L. maculans resistance and are currently being incorporated into B. napus breeding programmes.

Somatic hybrids between Brassica napus (oilseed rape) and its wild relative Sinapis arvensis (Xinjiang wild mustard) from northwestern China were produced by fusing mesophyll protoplasts. Fifty-four plants were identified as symmetric hybrids and four as asymmetric hybrids by random amplified polymorphic DNA analysis and nuclear DNA content. The morphology of investigated 58 hybrid plants resembled characters from both parental species. Highly fertile hybrids were recovered where the fertility was associated with the choice of B. napus genotype. Enhanced disease resistance to Leptosphaeria maculans was found in S. arvensis and in the hybrid offspring. This plant material has great potentials not only for use as a bridge for the introduction of a number of valuable traits from the wild species to Brassica crops but also for breeding new varieties with improved blackleg resistance.

Leptosphaeria maculans isolates have been assigned to one of two groups, A or B, on the basis of differences in their characteristics. Group A can further be divided into pathogenicity groups (PG) 2, 3 and 4 and group B into PG1. To determine if isolates belonging to the aggressive canker forming group A are present in Sweden, physiological and genetic characterisation of 120 isolates collected in the year 2000 were performed. Thirty-seven isolates were classified as belonging to pathogenicity group PG3 and 63 isolates as PG4, based on a cotyledon assay. Twenty isolates did not cause any symptoms at all, and were classified as PG1. When comparing two geographical regions, Skane and Ostergotland, equal numbers of PG3 and PG4 isolates were found. By analysing the isolates by PCR, the collection was further classified into 100 group A isolates and 20 group B isolates. A corresponding classification of the isolates was observed when the ability to produce pigments in Czapek Dox broth was examined. The results showed a clear predominance of group A. This was also the case for the isolate collection from 2001. In a detailed survey of disease development in a L. maculans infected winter oilseed rape field in southern Sweden (Skane), basal stem canker was not observed until early June Although the disease index value increased from 8.4 in June to 18.0 in July, few severely damaged plants were observed before harvest in mid-July, despite infection with group A isolates.

Morphological characters and genetic relationships were studied among 36 Verticillium isolates derived from different host species. Conidial length varied from 3.5 μm to 12 μm and the DNA content ranged from 0.028 pg to 0.078 pg with a bias towards large conidia and higher DNA content among the Swedish and German isolates from Brassica napus. Ability to produce nitrate nonutilising ( nit ) mutants was also assayed. Of all 36 isolates tested, eight V. dahliae isolates from non- Brassica hosts and two from B. napus, V. nigrescens from flax and V. albo-atrum from tomato generated nit mutants. To facilitate disease diagnosis, a species-specific PCR primer pair was developed, aiming towards a PCR-based analysis to confirm Verticillium wilt infection in Brassica plants. To identify useful DNA templates for the PCR primer design, random genomic clones from isolates VD12 and VD17 were used as probes in RFLP analysis. The RFLP results were also utilized in phenetic analysis and data from 29 isolates revealed three major groups. The first group consisted of seven non- Brassica V. dahliae isolates and the V. nigrescens isolate. The second group contained the two V. albo-atrum isolates and the V. dahliae isolate from red clover. Finally, the third group contained all the 18 isolates of Verticillium from B. napus. In the third group, two subgroups were identified, separating the three French isolates from the German and Swedish isolates. The dendrogram indicated that the isolates in the latter subgroup were closely related to each other. Based on the above mentioned criteria, we confirm that the isolates of Verticillium from B. napus in Sweden and Germany should be classified as V. longisporum.

Summary The fungus Verticillium longisporum is a soil-borne plant pathogen of increasing economic importance, and information on plant responses to it is limited. To identify the genes and components involved in the early stages of infection, transcripts in roots of V. longisporum-challenged Arabidopsis Col-0 and the susceptible NON-RACE SPECIFIC DISEASE RESISTANCE 1 (ndr1-1) mutant were compared using ATH1 gene chips. The analysis revealed altered transcript levels of several terpene biosynthesis genes, including the monoterpene synthase TPS23/27. When transgenic 35S:TPS23/27 and TPS23/27-amiRNA plants were monitored the over-expresser line showed enhanced fungal colonization whereas the silenced genotype was indistinguishable from Col-0. Transcript analysis of terpene biosynthesis genes suggested that only the TPS23/27 pathway is affected in the two transgenic genotypes. To confirm changes in monoterpene production, emitted volatiles were determined using solid-phase microextraction and gas chromatography–mass spectrometry. Levels of all identified TPS23/27 monoterpene products were significantly altered in the transgenic plants. A stimulatory effect on conidial germination and hyphal growth of V. longisporum was also seen in co-cultivation with 35S:TPS23/27 plants and upon exposure to 1,8-cineole, the main product of TPS23/27. Methyl jasmonate treatments of myc2-1 and myc2-2 mutants and analysis of TPS23/27:uidA in the myc2-2 background suggested a dependence on jasmonic acid mediated by the transcription factor MYC2. Taken together, our results show that TPS23/27-produced monoterpenes stimulate germination and subsequent invasion of V. longisporum in Arabidopsis roots.

Eight isolates of Verticillium longisporum and seven of Verticillium dahliae were examined for their pathogenicity to Arabidopsis thaliana. Two-week-old seedlings of two A. thaliana ecotypes were inoculated with conidial suspensions (1.6 × 10 4 conidia/ml) and maintained in sterile conditions on microtitre plates for 25 days. The seedlings were assessed daily for flowering time, height at flowering and mortality. From this data the proportion of plants flowering was calculated and the susceptibility of each ecotype to individual isolate was quantified. The A. thaliana ecotypes were inoculated by several isolates of V. dahliae and V. longisporum with the disease symptoms observed being similar to those characteristic of Verticillium-infected Brassica napus. Responses to Verticillium isolates varied and for both ecotypes significant differences (P < 0.05) were recorded in days to flowering between the V. longisporum isolates VD6 and VD12 and the V. dahliae isolate INRA617 and all other isolates. There were no other significant effects correlated with isolate origin or Verticillium species.

The role of the glucosinolate-myrosinase system in resistance to Leptosphaeria maculans was studied by monitoring changes in glucosinolate profiles in leaf tissue surrounding the site of inoculation. Susceptible Brassica napus cv. Hanna, resistant B. nigra and near isogenic lines derived from interspecific hybrids between the two species were compared. Expression of myrosinase binding protein and presence of genetic markers were also assayed. No correlation between degree of resistance and amount of sinigrin or other aliphatic glucosinolates was found. However, in time course experiments the glucosinolate profile of the L. maculans inoculated plants differed significantly from the water-inoculated control plants in the amount of 4-methoxy-glucobrassicin observed. Five to eight days post-inoculation an increased level of 4-methoxy glucobrassicin, ranging from 30% to 47% on average, was found in the inoculated plants, whilst controls varied between 7.6% and 9.2%. This increase was seen both in susceptible and resistant material. Other changes observed could mainly be assigned as effects of wounding. Although inoculation with L. maculans elicited changes in the leaf indolyl glucosinolate profiles, in the material studied, these changes could not be correlated to resistance against the fungus.

The variation obtained in storage fatty acids induced by the procedures of tissue culture and transformation with Agrobacterium tumefaciens was investigated and compared in rapeseed, Brassica napus, cv. Hanna. An increased variation in the fatty acid profiles was noted after tissue culture and transformation compared with plants derived directly from seeds. In the second generation of rapeseed transformants, T2, the content of oleic acid ranged from 39–72%, 12–31% for linoleic acid and 7–16% for linolenic acid. This could be compared with the oleic acid content in the T2 generation of tissue culture-derived plants which ranged between 47–76% and in seed-derived material where oleic acid ranged between 55–69%.In the T3 generation the ranges in transgenic seeds were decreased but still larger than in the seed derived plants. The range in transgenic plants was 49–64% for oleic acid, 20–28% for linoleic acid and 9–18% for linolenic acid. The most extreme individuals, both highest and lowest in the common fatty acids, were found in the group of transformed plants independent of generation. The total lipid content was also affected by the two treatments and seeds with the lowest and highest lipid content were both found among the transformed plants. In conclusion, care should be taken to use proper controls when performing transformation experiments in order to distinguish variation in the fatty acid profiles induced by the transformation procedure and tissue culture treatments from the changes due to transgenic expression.

Offspring from asymmetric hybrids between Brassica napus and the three B-genome species Brassica nigra, Brassica juncea and Brassica carinata were analysed for the presence of B-genome markers and resistance to the fungus Leptosphaeria maculans, the causal agent of blackleg disease. Twenty five plants from each species combination were analysed in the first backcross (BC1) generation, 30 plants in BC2 and 60 plants in BC3. The plants were analysed by 46 RFLP markers detecting 85 loci dispersed throughout the B. nigra genome. The plants with additional B. carinata DNA had a decrease in the presence of RFLP markers ranging from 59% in BC1 to 36% in BC2 and down to 11% in BC3. Similar results were obtained in the lines with additional DNA from B. juncea where the 60% presence of RFLP markers in BC1 was reduced to 33% in BC2 and to 10% in BC3. However presence of the markers were significantly lower in the B. nigra-derived material where BC1 had 46%, BC2 25% and BC3 8%. Since at least two loci could be detected on each end of the eight linkage groups of the B genome, the degree of symmetry was estimated. After one back-cross between 0.5 and 1.25% intact chromosomes were retained, whereas in BC2 this frequency was 0.21% for all three B-genome donor species. The maintenance of half-chromosomes ranged from 2.63% to 5.38% in BC1 and between 0.73% and 1.15% in BC2. No chromosome arms were found in any of the BC3 plants. In total, four co-segregating markers for cotyledon and adult-leaf resistance to L. maculans were found which detected six loci located on linkage groups 2, 5 and 8. When the results from the three donor species were compared, one triplicate region in the B genome had preserved the resistance loci in all three species.

Offspring of somatic hybrids between the zero-erucic acid rapeseed cv Hanna and Lesquerella fendleri were analysed regarding their fatty acid profiles. In the first back-cross generation one plant was found that produced a seed containing up to 16.5% erucic acid and 15% eicosaenoic acid (Line 1), as well as a seed having 4.3% ricinoleic acid (Line 2). This was interpreted as due to a contribution of elongase and hydroxylase genes from the L. fendleri genome since these two fatty acids are not produced in the recipient rapeseed cultivar Hanna. Crosses between Line 1 and cv Hanna resulted in the production of seeds with 35% erucic acid (F2). Furthermore, crosses between the F2 plants and the rapeseed cultivar Gulle, producing 35% erucic acid in the seeds, resulted in F3 seeds with 48% erucic acid. The highest amount of erucic acid, 61.5%, was found in the F6 generation after crossing Line 1 with a high erucic acid rapeseed line, HEAR, followed by self-fertilisation for two generations. When performing Southern-blot analysis on the F6 plants, seven of the nine analysed plants hybridised with the L. fendleri species-specific repetitive probe. The presence of the hydroxylase gene was also observed in the F6 generation of Line 1 according to Southern-blot analysis. Hybridisation with a hydroxylase probe was seen although no hydroxy fatty acids could be detected in any of the F6 plants. In parallel, Line 2 was crossed with HEAR cv Gulle and self fertilised. No hydroxy fatty acids were detected in the F2 generation of Line 2 and no specific hybridisation patterns could be found in the Southern-blot analysis.

In our recent paper, we identified a TIR encoding gene, which is required for resistance against a broad range of necrotrophic fungi. Here we present this finding in a broader perspective and discuss the unique features of this gene which might explain its role as a general regulator of resistance responses against a class of pathogens that have previously not been associated to the classical resistance (R) gene type of defense.

To evaluate UV-irradiation as an alternative method for chromosome fragmentation in the production of asymmetric somatic hybrids, UV-irradiated protoplasts of Arabidopsis thaliana were fused with protoplasts of Brassica napus . The A. thaliana protoplasts were irradiated with doses between 780 and 28 080 J/m 2 ( λ =254 nm) and hybrid plants were obtained from experiments using doses of 780, 2340 and 4680 J/m 2 . In addition, a control fusion experiment without irradiation was performed. In total, 312 shoots were regenerated and of those 58 plants were analysed for presence of A. thaliana DNA, using 13 mapped A. thaliana RFLP markers (two to four per chromosome). Increased UV-irradiation dose resulted in a higher frequency of asymmetric hybrids and a higher dose was also associated with the generation of hybrids with a higher degree of asymmetry. RFLP data further indicated that irradiation primarily resulted in loss of chromosome fragments rather than whole chromosomes from A. thaliana . Fertility was studied in 119 hybrid plants by selfing as well as pollination by B. napus . The fertility after selfing increased with increasing UV-irradiation dose, while the results of back-crossing were not affected by the irradiation.

Summary Pathogenesis-related protein 2 (PR2) is known to play a major role in plant defense and general stress responses. Resistance against the fungal pathogen Leptosphaeria maculans in Arabidopsis requires abscisic acid (ABA), which promotes the deposition of callose, a β-1,3-glucan polymer. Here, we examined the role of PR2 in callose deposition in relation to ABA treatment and challenge with L. maculans and Pseudomonas syringae. Characterization of PR2-overexpressing plants and the knockout line indicated that PR2 negatively affects callose deposition. Recombinant PR2 purified from Pichia pastoris showed callose-degrading activity, and a considerable reduction in the callose-degrading activity was observed in the leaf extract of the PR2 knockout line compared with the wild-type. ABA pretreatment before challenge with L. maculans concomitantly repressed PR2 and enhanced callose accumulation. Likewise, overexpression of an ABA biosynthesis gene NCED3 resulted in reduced PR2 expression and increased callose deposition. We propose that ABA promotes callose deposition through the transcriptional repression of PR2 in Arabidopsis challenged by L. maculans and P. syringae. Callose by itself is likely to act antagonistically on salicylic acid (SA) defense signaling, suggesting that PR2 may function as a modulator of callose- and SA-dependent defense responses.

Somatic hybrids were produced between hypocotyl protoplasts of Brassica napus (rapeseed) and mesophyll protoplasts of B. tournefortii using protoplast fusion. Fusion products were selected by flow cytometry and cell sorting. Twenty-three plants regenerated from individual calli originating from 4 fusion experiments were successfully transferred to the greenhouse. Nineteen of these were characterized as hybrids according to analysis of morphological characteristics, isoenzyme and nuclear RFLP patterns. Analysis of nuclear DNA content revealed a variation in ploidy level among the hybrids, even though a large number (53%) had a DNA content corresponding to the sum of the parental nuclear DNA. Most of the hybrids were intermediate in morphology between B. napus and B. tournefortii. The flowers displayed a range of variation: from normal flowers to abnormal flowers with reduced number of anthers and petals. Fertility investigation showed that both fertile and sterile hybrids were obtained. Five were self fertile, while 10 were male sterile and 4 were completely sterile. Phoma lingam resistance was transferred from B. tournefortii to the hybrid plants. Out of the 16 hybrids examined 12 showed resistance to P. lingam. Chloroplast DNA (cpDNA) patterns of the hybrids were identified by hybridizing a cpDNA probe to total genomic DNA. Of 18 hybrids analysed, 11 had B. tournefortii and 7 B. napus cpDNA pattern. No recombinant cpDNA banding pattern was found. In contrast to the cpDNA, mitochondrial DNA (mtDNA) rearrangements were detected in the hybrids. Out of 18 analysed hybrids, 7 showed evidence of mitochondrial recombination, while 11 had mtDNA identical to either of the parental types.

Two isolates of Phoma lingam , the fungal toxin sirodesmin PL, and salicylic acid were used to induce stress in plantlets of Brassica napus and B. nigra , which are susceptible and resistant to the fungal pathogen, respectively. B. nigra showed a very rapid response to antisera raised against PR-2, PR-Q and PR-S in tobacco. In contrast, in B. napus stressed by the fungus, sirodesmin PL or salicylic acid, the production of the three PR-proteins was delayed or absent.

Background The fungal pathogen Setosphaeria turcica causes turcicum or northern leaf blight disease on maize, sorghum and related grasses. A prevalent foliar disease found worldwide where the two host crops, maize and sorghum are grown. The aim of the present study was to find genes controlling the host defense response to this devastating plant pathogen. A cDNA-AFLP approach was taken to identify candidate sequences, which functions were further validated via virus induced gene silencing (VIGS), and real-time PCR analysis. Phylogenetic analysis was performed to address evolutionary events. Results cDNA-AFLP analysis was run on susceptible and resistant sorghum and maize genotypes to identify resistance-related sequences. One CC-NB-LRR encoding gene GRMZM2G005347 was found among the up-regulated maize transcripts after fungal challenge. The new plant resistance gene was designated as St referring to S. turcica. Genome sequence comparison revealed that the CC-NB-LRR encoding St genes are located on chromosome 2 in maize, and on chromosome 5 in sorghum. The six St sorghum genes reside in three pairs in one locus. When the sorghum St genes were silenced via VIGS, the resistance was clearly compromised, an observation that was supported by real-time PCR. Database searches and phylogenetic analysis suggest that the St genes have a common ancestor present before the grass subfamily split 50-70 million years ago. Today, 6 genes are present in sorghum, 9 in rice and foxtail millet, respectively, 3 in maize and 4 in Brachypodium distachyon. The St gene homologs have all highly conserved sequences, and commonly reside as gene pairs in the grass genomes. Conclusions Resistance genes to S. turcica, with a CC-NB-LRR protein domain architecture, have been found in maize and sorghum. VIGS analysis revealed their importance in the surveillance to S. turcica in sorghum. The St genes are highly conserved in sorghum, rice, foxtail millet, maize and Brachypodium, suggesting an essential evolutionary function.

SUMMARY The Bipolaris sorokiniana tolerant 1 (bst1) barley mutant is derived from fast neutron-irradiated seeds of wild-type Bowman(Rph3). The induced mutation was genetically localized to a position on chromosome 5HL distal to the centromere using amplified fragment length polymorphism markers. In addition, the defence responses and related gene expression in the bst1 mutant after fungal challenge were compared with those occurring in wild-type plants. Hydrogen peroxide generation, determined by 3,3-diaminobenzidine staining, revealed a clearly reduced level of bst1, compared with the wild-type, during the entire experimental time: 8-120 h post-inoculation (hpi). At 48 hpi, the wild-type samples displayed twice as much fungal mass and three times greater H(2)O(2) production than bst1. At the same time, staining of B. sorokiniana showed less fungal growth in the spontaneous lesions of bst1 compared with the wild-type. Monitoring of defence-related genes at 48 hpi demonstrated strong expression of PR-1a, PR-2, PR-5 and PR-10 in bst1. A gene coding for a unique oxidoreductase enzyme, designated as HCP1, was expressed at much higher levels in inoculated leaves of the bst1 mutant than in those of the wild-type plant. Taken together, the results suggest that the defence to B. sorokiniana largely relies on salicylic acid-responsive pathogenesis-related (PR) genes, as well as selected reactive oxygen species and unknown HCP1-associated factors.

Plants have evolved different defense components to counteract pathogen attacks. The resistance locus resistance to Leptosphaeria maculans 1 (RLM1) is a key factor for Arabidopsis thaliana resistance to L. maculans. The present work aimed to reveal downstream defense responses regulated by RLM1. Quantitative assessment of fungal colonization in the host was carried out using quantitative polymerase chain reaction (qPCR) and GUS expression analyses, to further characterize RLM1 resistance and the role of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) in disease development. Additional assessments of A. thaliana mutants were performed to expand our understanding of this pathosystem. Resistance responses such as lignification and the formation of vascular plugs were found to occur in an RLM1-dependent manner, in contrast to the RLM1-independent increase in reactive oxygen species at the stomata and hydathodes. Analyses of mutants defective in hormone signaling in the camalexin-free rlm1(Ler)pad3 background revealed a significant influence of JA and ET on symptom development and pathogen colonization. The overall results indicate that the defense responses of primary importance induced by RLM1 are all associated with physical barriers, and that responses of secondary importance involve complex cross-talk among SA, JA and ET. Our observations further suggest that ET positively affects fungal colonization.

Out of 400 reciprocal crosses made between Brassica napus and Arabidopsis thaliana, 200 maternal B. napus flowers produced 31 seeds and 10 seeds developed when A. thaliana was used as the female parent. Of the latter, three seeds successfully germinated but did not develop into mature plants. In contrast, plants from all 31 seeds generated from B. napus were established. DNA from these 31 plants was analysed by 18 probes mapped to the A. thaliana genome, two selectable marker genes and one A. thaliana species-specific repetitive probe. It was found that one plant had identical restriction fragments to A. thaliana with two of the 21 probes used. The gene flow within Brassicaceae is discussed in light of this result.

Protoplast fusions between Brassica napus and Orychophragmus violaceus for transfer of valuable traits to oilseed rape resulted in 257 somatic hybrid plants. Hybridity was confirmed by morphological, cytological and molecular means. Symmetric fusions gave rise to 131 plants. Fifty eight of these plants had an intermediate morphology and contained nuclear DNA corresponding to the sum of the parental species. All 131 plants were sterile with no pollen grains observed upon flowering. Another 126 plants were derived from asymmetric fusions in which protoplasts of the donor parent O. violaceus were irradiated by 100 or 200-Gy X-rays prior to fusion. Morphologically these plants showed a larger variation compared to the plants regenerated from symmetric fusion experiments. In contrast to plants obtained from symmetric fusions, fertile hybrids were recovered among regenerants from the asymmetric fusions. Twenty four of these plants released viable pollen grains and 14 of the determined 17 plants set seeds after either selfing or backcrossing to B. napus. Fourteen male-sterile plants were identified with female fertility. This observed male sterility most-likely originated from alloplasmic recombination and would be of great potential for the development of a new cytoplasmic male sterility system. The fatty acid composition of the fertile hybrids and their progenies showed a biased distribution towards the B. napus parent, which has a high erucic acid-content type. However, increased levels of palmitic and linoleic acids compared to B. napus were found in subsequent generations, as well as a reduced level of erucic acid.