Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene, AvrLmS-Lep2.

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene-for-gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus 'Surpass 400'. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map-based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas-LepR2, and an RlmS-line. The gene, renamed AvrLmS-Lep2, encodes a small cysteine-rich protein of unknown function with an N-terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS-Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS-Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.

Role of beneficial microbes with nitrogen and phosphorous levels on canola productivity / Papel de microbes benéficos em associação com níveis de nitrogênio e fósforo na produtividade de canola

A research was conducted to evaluate the impact of various nitrogen and phosphorus levels along with beneficial microbes to enhance canola productivity. The research was carried out at Agronomy Research Farm, The University of Agriculture Peshawar in winter 2016-2017. The experiment was conducted in randomized complete block factorial design. The study was comprised of three factors including nitrogen (60, 120 and 180 kg ha-¹), phosphorous (70, 100 and 130 kg ha-¹) and beneficial microbes (with and without BM). A control treatment with no N, P and BM was also kept for comparison. Application of beneficial microbes significantly increased pods plant, seed pod, seed filling duration, 1000 seed weight, biological yield and seed yield as compared to control plots. Nitrogen applied at the rate of 180 kg ha-¹ increased pods plant-¹, seed pod, seed filling duration, seed weight, biological yield and seed yield. Maximum pods plant-¹, seed pod, early seed filling, heavier seed weight, biological yield, seed yield, and harvest index were observed in plots treated with 130 kg.ha-¹ phosphorous. As comparison, the combine treated plots have more pods plant-¹, seeds pod-¹, seed filling duration, heaviest seeds, biological yield, seed yield and harvest index as compared to control plots. It is concluded that application of beneficial microbes with N and P at the rate of 180 kg ha-¹ and 130 kg ha-¹, respectively, increased yield and its attributes for canola.

Uma pesquisa foi realizada para avaliar o impacto de vários níveis de nitrogênio e fósforo, juntamente com micróbios benéficos, para aumentar a produtividade da canola. A pesquisa foi realizada no inverno de 2016-17 no Agronomy Research Farm, Universidade de Agricultura do Peshawar. O experimento foi conduzido por planejamento fatorial aleatorizado em blocos. O estudo focou-se em três fatores, incluindo o teor de nitrogênio, N, (60, 120 e 180 kg.ha-¹), o teor de fósforo, P, (70, 100 e 130 kg ha-¹) e a presença de micróbios benéficos (com BM e sem BM). Para fins de comparação, um tratamento controle sem N, P e BM também foi incluído no estudo. A aplicação de micróbios benéficos aumentou significativamente as vagens das plantas e de sementes, a duração do enchimento das sementes, o peso de 1000 sementes, o rendimento biológico e o rendimento de sementes em comparação com os resultados do controle. O nitrogênio aplicado na taxa de 180 kg ha-¹ aumentou as vagens por planta, vagem, duração do enchimento, peso da semente, rendimento biológico e rendimento de sementes. Vagens máximas por planta, vagem, enchimento precoce de sementes, peso maior de semente, rendimento biológico, rendimento de sementes e índice de colheita foram observados em parcelas tratadas com 130 kg.ha-¹ de fósforo. Em comparação aos blocos cultivados de controle, os blocos cultivados tratados combinados têm mais vagens por planta e sementes por vagem, maior duração do enchimento das sementes, maior número de sementes mais pesadas e maior rendimento biológico, rendimento de sementes e índice de colheita. Conclui-se que a aplicação de micróbios benéficos junto com N e P nas doses de 180 kg ha-¹ e 130 kg ha-¹, respectivamente, aumentou a produtividade e atributos de produtividade para a canola.

Rhizobium rhizophilum sp. nov., an indole acetic acid-producing bacterium isolated from rape (Brassica napus L.) rhizosphere soil.

A novel Gram-stain-negative, aerobic, rod-shaped and indole acetic acid-producing strain, designated 7209-2T, was isolated from rhizosphere of rape (Brassica napus L.) grown in the Yakeshi City, Inner Mongolia, PR China. The 16S rRNA gene sequence analysis indicated that strain 7209-2T belongs to the genus Rhizobium and is closely related to Rhizobium rosettiformans W3T, Rhizobium ipomoeae shin9-1T and Rhizobium wuzhouense W44T with sequence similarities of 98.2, 98.1 and 97.9 %, respectively. Phylogenetic analysis based on concatenated housekeeping recA and atpD gene sequences showed that strain 7209-2T formed a group together with R. wuzhouense W44T and R. rosettiformans W3T, with sequences similarities of 92.6 and 91.1 %, respectively. The genome size of strain 7209-2T was 5.25 Mb, comprising 5027 predicted genes with a DNA G+C content of 61.2 mol%. The average nucleotide identity and digital DNA-DNA hybridization comparisons among 7209-2T and reference strains for the most closely related species showed values below the accepted threshold for species discrimination. The major fatty acids of strain 7209-2T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 2 (C12 : 0 aldehyde and/or unknown 10.953) . The major polar lipids were found to consist of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and an unidentified aminophospholipid. The predominant ubiquinone was identified as quinone 10. Based on all the above results, strain 7209-2T represents a novel species of the genus Rhizobium, for which the name Rhizobium rhizophilum sp. nov. is proposed with 7209-2T (=CGMCC 1.15691T=DSM 103161T) as the type strain.

Cover crops restore declining soil properties and suppress bacterial wilt by regulating rhizosphere bacterial communities and improving soil nutrient contents.

Bacterial wilt (BW) disease causes huge economic loss. Heretofore there is no effective way to completely control BW. Here, cover crops (pea, rapeseed, and wheat) were used to restore declining soil properties and control BW. Cover crops can increase content of soil organic matter, alkali-hydrolyzable nitrogen and enzymatic activities, as well as suppress BW. Different kinds of cover crops are distinguished in recovering different soil properties. For instance, rapeseed can inhibit BW more effectively than wheat and pea, while wheat has the best effect on increasing soil organic matter, urease, and invertase. Nevertheless, pea improves catalase better than rapeseed and wheat. Moreover, relative abundance of plant-beneficial bacteria in cover crop treatments is higher than that in the control, with a negative correlation with disease index. For example, wheat has the best effect on improving the growth of plant-beneficial bacteria, followed by rapeseed. The bacteria involved in nitrogen cycling are enriched in pea treatments. However, the relative abundance of pathogen and denitrifying bacteria in cover crop treatments is lower than that in the control, with a positive correlation with disease index. The count of bacteria genes involved in nutrients cycling, antibiotics synthesis, and biodegradation of toxic compounds in cover crop treatments is higher than that in the control. Wheat includes more these genes than rapeseed and pea. Overall, cover crops can restore declining soil properties and suppress BW by increasing soil nutrients and beneficial bacteria as well as decreasing pathogen. Among all cover crops, wheat is considered as the optimal one.

Selenium as a potential fungicide could protect oilseed rape leaves from Sclerotinia sclerotiorum infection.

Sclerotinia sclerotiorum (S. sclerotiorum) is a soil-borne pathogen causing serious damage to the yield of oilseed rape. Selenium (Se) acted as a beneficial element for plants, and also proved to inhibit the growth of plant pathogens. However, whether Se could reduce S. sclerotiorum infection in oilseed rape, the related mechanism is still unclear. In this study, proper Se levels (0.1 mg/kg and 0.5 mg/kg) applied in soil decreased the lesion diameter and incidence of S. sclerotiorum in rape leaves. Se enfeebled the decrease of net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr), and maintained leaf cell structure. Se enhanced the antioxidant system of leaves, as evidenced by the maintenance of mitochondrial function, reduction of reactive oxygen species (ROS) accumulation and malondialdehyde (MDA) content, and the improvement of antioxidant enzyme activities including catalase (CAT), polyphenol oxidase (PPO) and peroxidase (POD). The upregulated defense gene expressions (CHI, ESD1, NPR1 and PDF1.2) of leaves were also observed under Se treatments. Furthermore, metabolome analysis revealed that Se promoted the metabolism of energy and amino acids in leaves infected with S. sclerotiorum. These findings inferred that Se could act as a potential eco-fungicide to protect oilseed rape leaves from S. sclerotiorum attack. The result arising from this study not only introduces an ecological method to control S. sclerotiorum, but also provides a deep insight into microelement for plant protection.

The quality of cold-pressed rapeseed oil obtained from seeds of Brassica napus L. with increased moisture content.

BACKGROUND: The basic parameter influencing the quality of cold-pressed oil is the quality of seeds used  for pressing. Adverse moisture content and storage temperature of rape seeds may affect the quality of oil obtained from them. This paper presents the effects of increased rapeseed moisture content on the quality of the oil pressed. METHODS: The material used for the tests was rapeseed (canola) cv. PR 46 W14. The moisture content of the seeds was adjusted to 10%, 12% and 20%, and the seeds were stored at room temperature for 14 days. The samples were then dried to a seed moisture equal to 7% and oil was pressed from them. Acid and peroxide values, as well as the content of water, tocopherols and phenolic acids, were determined. In addition, a sensory analysis of the oil samples was carried out, and structural changes in the association colloids in the oil were determined using a fluorescent probe. RESULTS: With the increase in seed moisture, the increase in peroxide and acid values of the analyzed oils was recorded. A slight decrease in tocopherol content and a significant increase in phenolic acid concentration, depending on the seed moisture content, was observed. Sensory analysis showed odor sensory profile changes that probably indicate microflora development. CONCLUSIONS: The rapeseed moisture content has a crucial influence on the quality of oil obtained from them. Along with an increase in seed moisture, the possibility of developing undesirable microflora grows, which results in a deterioration in the quality of the obtained oil.

Electrospun Microbial-Encapsulated Composite-Based Plasticized Seed Coat for Rhizosphere Stabilization and Sustainable Production of Canola ( Brassica napus L.).

Plant-growth-promoting bacteria show promises in crop production; nevertheless, innovation in their stable delivery is required for practical use by farmers. Herein, the composite of poly(vinyl alcohol)/poly(vinylpyrrolidone) plasticized with glycerol and loaded with the microbial consortium ( Bacillus subtilis plus Seratia marcescens) was fabricated and engineered onto canola ( Brassica napus L.) seed via electrospinning. Scanning electron microscopy showed that the biocomposite is a one-dimensional membrane, which encapsulated microbes in a multilayered nanostructure, and their interfacial behavior between microorganism and seed is beneficial for safer farming. A universal testing machine and thermogravimetric analysis demonstrated that the biocomposite holds sufficient thermomechanical properties for stable handling and practical management. A spectroscopic study resolved the living hybrid-polymer structure of the biocomposite and proved the plasticizing role of glycerol. A swelling study supports the degradation of the biocomposite in the hydrophilic environment as a result of the leaching of the plasticizer, which is important for the sustained release of microbial cells. A shelf life study supported that the biocomposite seed coat placed a threshold level of microbes [5.675 ± 0.48 log10 colony forming units (CFU)/seed] and maintained their satisfactory viability for 15 days at room temperature. An antifungal and nutrient-solubilizing study supported that the biocomposite seed coat could provide opportunities to biocontrol diseases and improve nutrient acquisition by the plant. A pot study documents the better performance of the biocomposite seed coat on seed germination, seedling growth, leaf area, plant dry biomass, and root system. A chemical and microbial study demonstrated that the biocomposite seed coat improved the effectiveness of the bioinoculant in the root-soil interface, where they survive, flourish, and increase the nutrient pool status. In particular, this study presents advances in the fabrication of the biocomposite for encapsulation, preservation, sustained release, and efficacious use of microorganisms onto seeds for precision farming.

Characterization of cadmium-resistant rhizobacteria and their promotion effects on Brassica napus growth and cadmium uptake.

Excessive cadmium (Cd) accumulation in soil can adversely affect plants, animals, microbes, and humans; therefore, novel and uncharacterized Cd-resistant plant-growth-promoting rhizobacteria (PGPR) are required to address this issue. In the paper, 13 bacteria were screened, their partial 16S rRNA sequences determined, and the isolates, respectively, clustered into Curtobacterium (7), Chryseobacterium (4), Cupriavidus (1), and Sphingomonas (1). Evaluation of PGP traits, including indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, siderophore secretion, and cyanhydric acid production, identified Cupriavidus necator GX_5, Sphingomonas sp. GX_15, and Curtobacterium sp. GX_31 as promising candidates for PGPR based on high IAA or ACC deaminase production. Additionally, root-elongation assays indicated that inoculating GX_5, _15, or _31 increased Brassica napus root length both in the presence and absence of Cd by 19.75-29.96% and 19.15-31.69%, respectively. Pot experiments indicated that inoculating B. napus with GX_5, _15, and _31 significantly increased the dry weight of above-ground tissues and root biomass by 40.97-85.55% and 18.99-103.13%, respectively. Moreover, these isolates significantly increased Cd uptake in the aerial parts and root tissue of B. napus by 7.38-11.98% and 48.09-79.73%, respectively. These results identified GX_5, _15, or _31 as excellent promoters of metal remediation by using microorganism-associated phytoremediation.

Live cell imaging of Plasmodiophora brassicae-host plant interactions based on a two-step axenic culture system.

Plasmodiophora brassicae, a parasitic protist, induces club-shaped tumor-like growth of host Brassicas roots and hypocotyls after infection. Due to its soil-borne nature and intracellular, biotrophic parasitism the infection biology and early pathogenesis remains in doubt. In this study, we have established a new protocol, based on a two-step axenic culture of P. brassicae with its host tissues, for easy and in planta observation of cellular interactions between P. brassicae and host plants: first, coculture of P. brassicae with infected canola root tissues, on growth-medium plates, enables the propagation of P. brassicae that serves as pure inoculum for pathogenicity assays, and second, the pure inoculum is subsequently used for pathogenicity tests on both canola and Arabidopsis seedlings grown on medium plates in Petri dishes. During the first axenic culture, we established a staining protocol by which the pathogen was fluorescently labeled with Nile red and calcofluor white, thus allowing in planta observation of pathogen development. In the pathogenicity assays, our results showed that axenic cultures of P. brassicae, in calli, remains fully virulent and completes its life cycle in both canola and Arabidopsis roots grown in Petri dishes. Combining visualization of fluorescent probe-labeled P. brassicae structures with fluorescent protein tagging of Arabidopsis cellular components, further revealed dynamic responses of host cells at the early stages of P. brassicae infection. Thus, established protocols for in planta detection of P. brassicae structures and the live cell imaging of P. brassicae-Arabidopsis interactions provide a novel strategy for improving our detailed knowledge of P. brassicae infection in host tissues.

Syntenic quantitative trait loci and genomic divergence for Sclerotinia resistance and flowering time in Brassica napus.

Oilseed rape (Brassica napus) is an allotetraploid with two subgenomes descended from a common ancestor. Accordingly, its genome contains syntenic regions with many duplicate genes, some of which may have retained their original functions, whereas others may have diverged. Here, we mapped quantitative trait loci (QTL) for stem rot resistance (SRR), a disease caused by the fungus Sclerotinia sclerotiorum, and flowering time (FT) in a recombinant inbred line population. The population was genotyped using B. napus 60K single nucleotide polymorphism arrays and phenotyped in six (FT) and nine (SSR) experimental conditions or environments. In total, we detected 30 SRR QTL and 22 FT QTL and show that some of the major QTL associated with these two traits were co-localized, suggesting a genetic linkage between them. Two SRR QTL on chromosome A2 and two on chromosome C2 were shown to be syntenic, suggesting the functional conservation of these regions. We used the syntenic properties of the genomic regions to exclude genes for selection candidates responsible for QTL-associated traits. For example, 152 of the 185 genes could be excluded from a syntenic A2-C2 region. These findings will help to elucidate polyploid genomics in future studies, in addition to providing useful information for B. napus breeding programs.

Responses of bacterial community to dibutyl phthalate pollution in a soil-vegetable ecosystem.

Phthalate esters (PAEs) are a type of plasticizer that has aroused great concern due to their mutagenic, teratogenic, and carcinogenic effects, wherefore dibutyl phthalate (DBP) and other PAEs have been listed as priority pollutants. In this study, the impacts of DBP on a soil-vegetable ecosystem were investigated. The results showed that DBP could accumulate within vegetable tissues, and the accumulative effect was enhanced with higher levels of DBP contamination in soils. DBP accumulation also decreased vegetable quality in various ways, including decreased soluble protein content and increased nitrate content. The diversity of bacteria in soils gradually decreased with increasing DBP concentration, while no clear association with endophytic bacteria was observed. Also, the relative abundance, structure, and composition of soil bacterial communities underwent successional change during the DBP degradation period. The variation of bulk soil bacterial community was significantly associated with DBP concentration, while changes in the rhizosphere soil bacteria community were significantly associated with the properties of both soil and vegetables. The results indicated that DBP pollution could increase the health risk from vegetables and alter the biodiversity of indigenous bacteria in soil-vegetable ecosystems, which might further alter ecosystem functions in agricultural fields.

Multi-environment QTL studies suggest a role for cysteine-rich protein kinase genes in quantitative resistance to blackleg disease in Brassica napus.

BACKGROUND: Resistance to the blackleg disease of Brassica napus (canola/oilseed rape), caused by the hemibiotrophic fungal pathogen Leptosphaeria maculans, is determined by both race-specific resistance (R) genes and quantitative resistance loci (QTL), or adult-plant resistance (APR). While the introgression of R genes into breeding material is relatively simple, QTL are often detected sporadically, making them harder to capture in breeding programs. For the effective deployment of APR in crop varieties, resistance QTL need to have a reliable influence on phenotype in multiple environments and be well defined genetically to enable marker-assisted selection (MAS). RESULTS: Doubled-haploid populations produced from the susceptible B. napus variety Topas and APR varieties AG-Castle and AV-Sapphire were analysed for resistance to blackleg in two locations over 3 and 4 years, respectively. Three stable QTL were detected in each population, with two loci appearing to be common to both APR varieties. Physical delineation of three QTL regions was sufficient to identify candidate defense-related genes, including a cluster of cysteine-rich receptor-like kinases contained within a 49 gene QTL interval on chromosome A01. Individual L. maculans isolates were used to define the physical intervals for the race-specific R genes Rlm3 and Rlm4 and to identify QTL common to both field studies and the cotyledon resistance response. CONCLUSION: Through multi-environment QTL analysis we have identified and delineated four significant and stable QTL suitable for MAS of quantitative blackleg resistance in B. napus, and identified candidate genes which potentially play a role in quantitative defense responses to L. maculans.

Leptosphaeria maculans effector AvrLm4-7 affects salicylic acid (SA) and ethylene (ET) signalling and hydrogen peroxide (H2 O2 ) accumulation in Brassica napus.

To achieve host colonization, successful pathogens need to overcome plant basal defences. For this, (hemi)biotrophic pathogens secrete effectors that interfere with a range of physiological processes of the host plant. AvrLm4-7 is one of the cloned effectors from the hemibiotrophic fungus Leptosphaeria maculans 'brassicaceae' infecting mainly oilseed rape (Brassica napus). Although its mode of action is still unknown, AvrLm4-7 is strongly involved in L. maculans virulence. Here, we investigated the effect of AvrLm4-7 on plant defence responses in a susceptible cultivar of B. napus. Using two isogenic L. maculans isolates differing in the presence of a functional AvrLm4-7 allele [absence ('a4a7') and presence ('A4A7') of the allele], the plant hormone concentrations, defence-related gene transcription and reactive oxygen species (ROS) accumulation were analysed in infected B. napus cotyledons. Various components of the plant immune system were affected. Infection with the 'A4A7' isolate caused suppression of salicylic acid- and ethylene-dependent signalling, the pathways regulating an effective defence against L. maculans infection. Furthermore, ROS accumulation was decreased in cotyledons infected with the 'A4A7' isolate. Treatment with an antioxidant agent, ascorbic acid, increased the aggressiveness of the 'a4a7' L. maculans isolate, but not that of the 'A4A7' isolate. Together, our results suggest that the increased aggressiveness of the 'A4A7' L. maculans isolate could be caused by defects in ROS-dependent defence and/or linked to suppressed SA and ET signalling. This is the first study to provide insights into the manipulation of B. napus defence responses by an effector of L. maculans.

Genome-wide transcriptomic analyses provide insights into the lifestyle transition and effector repertoire of Leptosphaeria maculans during the colonization of Brassica napus seedlings.

Molecular interaction between the causal agent of blackleg disease, Leptosphaeria maculans (Lm), and its host, Brassica napus, is largely unknown. We applied a deep RNA-sequencing approach to gain insight into the pathogenicity mechanisms of Lm and the defence response of B. napus. RNA from the infected susceptible B. napus cultivar Topas DH16516, sampled at 2-day intervals (0-8 days), was sequenced and used for gene expression profiling. Patterns of gene expression regulation in B. napus showed multifaceted defence responses evident by the differential expression of genes encoding the pattern recognition receptor CERK1 (chitin elicitor receptor kinase 1), receptor like proteins and WRKY transcription factors. The up-regulation of genes related to salicylic acid and jasmonic acid at the initial and late stages of infection, respectively, provided evidence for the biotrophic and necrotrophic life stages of Lm during the infection of B. napus cotyledons. Lm transition from biotrophy to necrotropy was also supported by the expression function of Lm necrosis and ethylene-inducing (Nep-1)-like peptide. Genes encoding polyketide synthases and non-ribosomal peptide synthetases, with potential roles in pathogenicity, were up-regulated at 6-8 days after inoculation. Among other plant defence-related genes differentially regulated in response to Lm infection were genes involved in the reinforcement of the cell wall and the production of glucosinolates. Dual RNA-sequencing allowed us to define the Lm candidate effectors expressed during the infection of B. napus. Several candidate effectors suppressed Bax-induced cell death when transiently expressed in Nicotiana benthamaina leaves.

A game of hide and seek between avirulence genes AvrLm4-7 and AvrLm3 in Leptosphaeria maculans.

Extending the durability of plant resistance genes towards fungal pathogens is a major challenge. We identified and investigated the relationship between two avirulence genes of Leptosphaeria maculans, AvrLm3 and AvrLm4-7. When an isolate possesses both genes, the Rlm3-mediated resistance of oilseed rape (Brassica napus) is not expressed due to the presence of AvrLm4-7 but virulent isolates toward Rlm7 recover the AvrLm3 phenotype. Combining genetic and genomic approaches (genetic mapping, RNA-seq, BAC (bacterial artificial chromosome) clone sequencing and de novo assembly) we cloned AvrLm3, a telomeric avirulence gene of L. maculans. AvrLm3 is located in a gap of the L. maculans reference genome assembly, is surrounded by repeated elements, encodes for a small secreted cysteine-rich protein and is highly expressed at early infection stages. Complementation and silencing assays validated the masking effect of AvrLm4-7 on AvrLm3 recognition by Rlm3 and we showed that the presence of AvrLm4-7 does not impede AvrLm3 expression in planta. Y2H assays suggest the absence of physical interaction between the two avirulence proteins. This unusual interaction is the basis for field experiments aiming to evaluate strategies that increase Rlm7 durability.

Crystal structure of the effector AvrLm4-7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins.

The avirulence gene AvrLm4-7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4-7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4-7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well-conserved among AvrLm4-7 homologs. Loss of recognition of AvrLm4-7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well-conserved C-terminal motif or close to the glycine involved in Rlm4-mediated recognition, resulting in the loss of Rlm7-mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4-7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4-7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.

Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2 using an intraspecific comparative genomics approach.

Five avirulence genes from Leptosphaeria maculans, the causal agent of blackleg of canola (Brassica napus), have been identified previously through map-based cloning. In this study, a comparative genomic approach was used to clone the previously mapped AvrLm2. Given the lack of a presence-absence gene polymorphism coincident with the AvrLm2 phenotype, 36 L. maculans isolates were resequenced and analysed for single-nucleotide polymorphisms (SNPs) in predicted small secreted protein-encoding genes present within the map interval. Three SNPs coincident with the AvrLm2 phenotype were identified within LmCys1, previously identified as a putative effector-coding gene. Complementation of a virulent isolate with LmCys1, as the candidate AvrLm2 allele, restored the avirulent phenotype on Rlm2-containing B. napus lines. AvrLm2 encodes a small cysteine-rich protein with low similarity to other proteins in the public databases. Unlike other avirulence genes, AvrLm2 resides in a small GC island within an AT-rich isochore of the genome, and was never found to be deleted completely in virulent isolates.

Homoeologous duplicated regions are involved in quantitative resistance of Brassica napus to stem canker.

BACKGROUND: Several major crop species are current or ancient polyploids. To better describe the genetic factors controlling traits of agronomic interest (QTL), it is necessary to understand the structural and functional organisation of these QTL regions in relation to genome duplication. We investigated quantitative resistance to the fungal disease stem canker in Brassica napus, a highly duplicated amphidiploid species, to assess the proportion of resistance QTL located at duplicated positions. RESULTS: Genome-wide association analysis on a panel of 116 oilseed rape varieties genotyped with 3228 SNP indicated that 321 markers, corresponding to 64 genomic regions, are associated with resistance to stem canker. These genomic regions are relatively equally distributed on the A (53%) and C (47%) genomes of B. napus. Overall, 44% of these regions (28/64) are duplicated homoeologous regions. They are located in duplications of six (E, J, R, T, U and W) of the 24 ancestral blocks that constitute the B. napus genome. Overall, these six ancestral blocks have 34 duplicated copies in the B.napus genome. Almost all of the duplicated copies (82% of the 34 regions) harboured resistance associated markers for stem canker resistance, which suggests structural and functional conservation of genetic factors involved in this trait in B. napus. CONCLUSIONS: Our study provides information on the involvement of duplicated loci in the control of stem canker resistance in B. napus. Further investigation of the similarity/divergence in sequence and gene content of these duplicated regions will provide insight into the conservation and allelic diversity of the underlying genes.

Metabolomic shifts in Brassica napus lines with enhanced BnPLC2 expression impact their response to low temperature stress and plant pathogens.

Phosphatidylinositol-specific phospholipase C2 (PLC2) is a signaling enzyme with hydrolytic activity against membrane-bound phosphoinositides. It catalyzes the cleavage of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P 2) into two initial second messengers, myo-inositol-1,4,5-trisphosphate (InsP 3) and diacylglycerol (DAG). The former, as well as its fully phosphorylated derivative, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP 6), play a major role in calcium signaling events within the cell, while DAG may be used in the regeneration of phospholipids or as a precursor for phosphatidic acid (PA) biosynthesis, an important signaling molecule involved in both biotic and abiotic types of stress tolerance. Overexpression of the gene for Brassica napus phospholipase C2 (BnPLC2) in Brassica napus has been shown to enhance drought tolerance, modulate multiple genes involved in different processes and favorably affect hormonal levels in different tissues. We, therefore, undertook the current study with a view to examining, at the metabolome level, its effect on both abiotic (low temperature) and biotic (stem white rot disease) types of stress in canola. Thus, while transgenic plants exhibited a significant rise in maltose levels and a concomitant elevation in some unsaturated free fatty acids (FFAs), glycerol, and glycerol 3-phosphate under subzero temperatures, they accumulated high levels of raffinose, stachyose and other sugars as well as some flavonoids under acclimatization conditions. Collectively, overexpression of BnPLC2 appears to have triggered different metabolite patterns consistent with its abiotic and, to a limited extent, biotic stress tolerance phenotypes.

Use of Plackett-Burman design for rapid screening of nitrogen and carbon sources for the production of lipase in solid state fermentation by Yarrowia lipolytica from mustard oil cake (Brassica napus)

Mustard oil cake (Brassica napus), the residue obtained after extraction of mustard oil from mustard oil seeds, was investigated for the production of lipase under solid state fermentation (SSF) using the marine yeast Yarrowia lipolytica NCIM 3589. Process parameters such as incubation time, biomass concentration, initial moisture content, carbon source concentration and nitrogen source concentration of the medium were optimized. Screening of ten nitrogen and five carbon sources has been accomplished with the help of Plackett-Burman design. The highest lipase activity of 57.89 units per gram of dry fermented substrate (U/gds) was observed with the substrate of mustard oil cake in four days of fermentation.