Characterization of the swede midge, Contarinia nasturtii, first instar larval salivary gland transcriptome.

Proteins in saliva of gall-forming insect larvae govern insect-host plant interactions. Contarinia nasturtii, the swede midge, is a pest of brassicaceous vegetables (cabbage, cauliflower, broccoli) and canola. We examined the salivary gland (SG) transcriptome of first instar larvae reared on Brassica napus and catalogued genes encoding secreted proteins that may contribute to the initial stages of larval establishment, the synthesis of plant growth hormones, extra-oral digestion and evasion of host defenses. A significant portion of the secreted proteins with unknown functions were unique to C. nasturtii and were often members of larger gene families organized in genomic clusters with conservation patterns suggesting that they are undergoing selection.

Genetic variation and structural diversity in major seed proteins among and within Camelina species.

MAIN CONCLUSION: Genetic variation in seed protein composition, seed protein gene expression and predictions of seed protein physiochemical properties were documented in C. sativa and other Camelina species. Seed protein diversity was examined in six Camelina species (C. hispida, C. laxa, C. microcarpa, C. neglecta, C. rumelica and C. sativa). Differences were observed in seed protein electrophoretic profiles, total seed protein content and amino acid composition between the species. Genes encoding major seed proteins (cruciferins, napins, oleosins and vicilins) were catalogued for C. sativa and RNA-Seq analysis established the expression patterns of these and other genes in developing seed from anthesis through to maturation. Examination of 187 C. sativa accessions revealed limited variation in seed protein electrophoretic profiles, though sufficient to group the majority into classes based on high MW protein profiles corresponding to the cruciferin region. C. sativa possessed four distinct types of cruciferins, named CsCRA, CsCRB, CsCRC and CsCRD, which corresponded to orthologues in Arabidopsis thaliana with members of each type encoded by homeologous genes on the three C. sativa sub-genomes. Total protein content and amino acid composition varied only slightly; however, RNA-Seq analysis revealed that CsCRA and CsCRB genes contributed > 95% of the cruciferin transcripts in most lines, whereas CsCRC genes were the most highly expressed cruciferin genes in others, including the type cultivar DH55. This was confirmed by proteomics analyses. Cruciferin is the most abundant seed protein and contributes the most to functionality. Modelling of the C. sativa cruciferins indicated that each type possesses different physiochemical attributes that were predicted to impart unique functional properties. As such, opportunities exist to create C. sativa cultivars with seed protein profiles tailored to specific technical applications.

Engineering a feedback inhibition-insensitive plant dihydrodipicolinate synthase to increase lysine content in Camelina sativa seeds.

Camelina sativa (camelina) is emerging as an alternative oilseed crop due to its short growing cycle, low input requirements, adaptability to less favorable growing environments and a seed oil profile suitable for biofuel and industrial applications. Camelina meal and oil are also registered for use in animal and fish feeds; however, like meals derived from most cereals and oilseeds, it is deficient in certain essential amino acids, such as lysine. In higher plants, the reaction catalyzed by dihydrodipicolinate synthase (DHDPS) is the first committed step in the biosynthesis of lysine and is subject to regulation by lysine through feedback inhibition. Here, we report enhancement of lysine content in C. sativa seed via expression of a feedback inhibition-insensitive form of DHDPS from Corynebacterium glutamicums (CgDHDPS). Two genes encoding C. sativa DHDPS were identified and the endogenous enzyme is partially insensitive to lysine inhibition. Site-directed mutagenesis was used to examine the impact of alterations, alone and in combination, present in lysine-desensitized DHDPS isoforms from Arabidopsis thaliana DHDPS (W53R), Nicotiana tabacum (N80I) and Zea mays (E84K) on C. sativa DHDPS lysine sensitivity. When introduced alone, each of the alterations decreased sensitivity to lysine; however, enzyme specific activity was also affected. There was evidence of molecular or structural interplay between residues within the C. sativa DHDPS allosteric site as coupling of the W53R mutation with the N80V mutation decreased lysine sensitivity of the latter, but not to the level with the W53R mutation alone. Furthermore, the activity and lysine sensitivity of the triple mutant (W53R/N80V/E84T) was similar to the W53R mutation alone or the C. glutamicum DHDPS. The most active and most lysine-insensitive C. sativa DHDPS variant (W53R) was not inhibited by free lysine up to 1 mM, comparable to the C. glutamicums enzyme. Seed lysine content increased 13.6 -22.6% in CgDHDPS transgenic lines and 7.6-13.2% in the mCsDHDPS lines. The high lysine-accumulating lines from this work may be used to produce superior quality animal feed with improved essential amino acid profile.

Horizontally transmitted parasitoid killing factor shapes insect defense to parasitoids.

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor (pkf), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts.

Silencing of an ABC transporter, but not a cadherin, decreases the susceptibility of Colorado potato beetle larvae to Bacillus thuringiensis ssp. tenebrionis Cry3Aa toxin.

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), is a major pest of potato plants worldwide and is notorious for its ability to develop resistance to insecticides. Cry3 toxins synthesized by Bacillus thuringiensis ssp. tenebrionis have been used successfully to manage this pest. Resistance to Cry toxins is a concerning problem for many insect pests; therefore, it is important to determine the mechanisms by which insects acquire resistance to these toxins. Cadherin-like and ABC transporter proteins have been implicated in the mode of action of Cry toxins as mutations in these genes render lepidopterans resistant to them; however, clear consensus does not exist on whether these proteins also play a role in Cry3 toxin activity and/or development of resistance in coleopterans. In the current study, we identified the L. decemlineata orthologues of the cadherin (LdCAD) and the ABCB transporter (LdABCB1) that have been implicated in the mode of action of Cry toxins in other coleopterans. Suppression of LdABCB1 via RNA interference reduced toxin-related larval mortality, whereas partial silencing of LdCAD did not. Our results suggest that the ABCB is involved in the mode of action of Cry3Aa toxins; however, no evidence was found to support the role of cadherin as a receptor of Cry3Aa in L. decemlineata.

De Novo Whole-Genome Assembly of the Swede Midge (Contarinia nasturtii), a Specialist of Brassicaceae, Using Linked-Read Sequencing.

The swede midge, Contarinia nasturtii, is a cecidomyiid fly that feeds specifically on plants within the Brassicaceae. Plants in this family employ a glucosinolate-myrosinase defense system, which can be highly toxic to nonspecialist feeders. Feeding by C. nasturtii larvae induces gall formation, which can cause substantial yield losses thus making it a significant agricultural pest. A lack of genomic resources, in particular a reference genome, has limited deciphering the mechanisms underlying glucosinolate tolerance in C. nasturtii, which is of particular importance for managing this species. Here, we present an annotated, scaffolded reference genome of C. nasturtii using linked-read sequencing from a single individual and explore systems involved in glucosinolate detoxification. The C. nasturtii genome is similar in size and annotation completeness to that of the Hessian fly, Mayetiola destructor, but has greater contiguity. Several genes encoding enzymes involved in glucosinolate detoxification in other insect pests, including myrosinases, sulfatases, and glutathione S-transferases, were found, suggesting that C. nasturtii has developed similar strategies for feeding on Brassicaceae. The C. nasturtii genome will, therefore, be integral to continued research on plant-insect interactions in this system and contribute to effective pest management strategies.

Gene expression patterns in shoots of Camelina sativa with enhanced salinity tolerance provided by plant growth promoting bacteria producing 1-aminocyclopropane-1-carboxylate deaminase or expression of the corresponding acdS gene.

Growth of plants in soil inoculated with plant growth promoting bacteria (PGPB) producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase or expression of the corresponding acdS gene in transgenic lines reduces the decline in shoot length, shoot weight and photosynthetic capacity triggered by salt stress in Camelina sativa. Reducing the levels of ethylene attenuated the salt stress response as inferred from decreases in the expression of genes involved in development, senescence, chlorosis and leaf abscission that are highly induced by salt to levels that may otherwise have a negative effect on plant growth and productivity. Growing plants in soil treated with Pseudomonas migulae 8R6 negatively affected ethylene signaling, auxin and JA biosynthesis and signalling, but had a positive effect on the regulation of genes involved in GA signaling. In plants expressing acdS, the expression of the genes involved in auxin signalling was positively affected, while the expression of genes involved in cytokinin degradation and ethylene biosynthesis were negatively affected. Moreover, fine-tuning of ABA signaling appears to result from the application of ACC deaminase in response to salt treatment. Moderate expression of acdS under the control of the root specific rolD promoter or growing plants in soil treated with P. migulae 8R6 were more effective in reducing the expression of the genes involved in ethylene production and/or signaling than expression of acdS under the more active Cauliflower Mosaic Virus 35S promoter.

Characterization of calcium signaling proteins from the fat body of the Colorado Potato Beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae): Implications for diapause and lipid metabolism.

Calcium (Ca2+) regulates many cellular and physiological processes from development to reproduction. Ca2+ is also an important factor in the metabolism of lipids, the primary energy source used during insect starvation and diapause. Ca2+ signaling proteins bind to Ca2+ and maintain intracellular Ca2+ levels. However, knowledge about Ca2+ signaling proteins is mostly restricted to the model Drosophila melanogaster and the response of Ca2+ signaling genes to starvation or diapause is not known. In this study, we identified three Ca2+ signaling proteins; the primary Ca2+ binding protein Calmodulin (LdCaM), phosphatase Calcineurin B (LdCaNB), and the senescence marker protein Regucalcin (LdRgN), from the fat body of the Colorado Potato Beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). This insect is a major pest of potato worldwide and overwinters under hibernation diapause as adults while utilizing lipids as the primary energy source. Putative EF-hand domains involved in Ca2+ binding were present in LdCaM, LdCaNB, but absent in LdRgN. LdCaM and LdCaNB were expressed in multiple tissues, while LdRgN was primarily expressed in the fat body. LdCaM was constitutively-expressed throughout larval development and at the adult stage. LdCaNB was primarily expressed in feeding larvae, and LdRgN in both feeding larvae and adults at comparable levels; however, both genes were down-regulated by molting. A response to starvation was observed only for LdRgN. Transcript abundance analysis in the entire body in relation to diapause revealed differential regulation with a general suppression during diapause, and higher mRNA levels in favor of females at post-diapause for LdCaM, and in favor of males at non-diapause for LdCaNB. Fat body-specific transcript abundance was not different between non-diapause and post-diapause for LdCaNB, but both LdCaM and LdRgN were down-regulated in males and both sexes, respectively by post-diapause. Silencing LdCaNB or LdRgN in larvae led to decreased fat content, indicating their involvement in lipid accumulation, while RNAi of LdCaM led to lethality.

Two calcium-binding chaperones from the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) involved in diapause.

Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin-type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N-globular domain, a P-arm domain, and a highly charged C-terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause-related expression analysis in the whole body revealed an upregulation of both genes by post-diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause-related expression pattern in the entire body for both genes. Fat body-specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post-diapause. This study suggests that calcium-binding chaperones play similar and possibly gender-specific roles during diapause.

A look into Colorado potato beetle lipid metabolism through the lens of lipid storage droplet proteins.

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) inflicts serious damage to potato plants by feeding ravenously on their leaves. Adult L.decemlineata have a photoperiod-induced dormancy response, also known as diapause, which allows them to survive severe winter conditions by digging into soil. Most insects that undergo diapause accumulate abundant lipid reserves prior to diapause and utilize most of them during the diapause. This process is likely to be governed by the interplay of lipid storage droplet proteins (LSDs), also known as perilipins, with the help of other proteins. Here, genes encoding L. decemlineata LSD1 and LSD2 were identified. Both were expressed primarily in the fat body with LdLSD1 and LdLSD2 being primarily expressed in adult and larval stages, respectively. LdLSD1 was up-regulated in starving larvae, while LdLSD2 was primarily expressed in feeding larvae. The expression pattern of LdLSD1 in adults during feeding, diapause and post-diapause contrasted to the total body fat levels, while the expression pattern of LdLSD2 was positively correlated with total body fat levels. RNA interference (RNAi) of LdLSD2 in larvae suggested a core role for LSD2 in the protection/assembly of storage lipids as this treatment reduced overall lipid droplet volume. These data shed light on the functions of these proteins in L. decemlineata and their roles in both diapause and during starvation.

Subunit composition affects formation and stabilization of o/w emulsions by 11S seed storage protein cruciferin.

The 11S globulin cruciferin is the major storage protein in Brassicaceae/Cruciferae seeds and exists as a hexamer in its natural configuration. Arabidopsis thaliana cruciferin is composed of CRUA, CRUB and CRUC subunits. Wild type (WT) cruciferin and cruciferins composed only of identical CRUA, CRUB and CRUC subunits were examined for their ability to form and stabilize oil-in-water (o/w) emulsions. All proteins (0.9% at pH 7.4 and 2.0), except CRUC, formed stable canola oil or triolein emulsions with a dispersed phase volume fraction of 22-23%. A fine emulsion was formed by CRUB at pH 7.4 with droplet sizes of 6.8 and 8.6 µm for canola oil and triolein, respectively. The presence of 0.5 M NaCl reduced the level of adsorbed protein and protein load at the interface at pH 7.4, and resulted in emulsions that were less stable. Emulsions of CRUA and CRUB (pH 7.4, zero ionic strength, canola oil or triolein) had higher stability than emulsions with WT cruciferin up to 15 days after formation. CRUC formed a stable emulsion only at pH 2.0. The low solubility, low surface hydrophobicity and compact structure of the CRUC protein may contribute to its inferior emulsifying properties at neutral pH; however, acidic pH-induced dissociation of the hexameric assembly improved these properties. The abundance and exposure of hydrophobic residues in the hypervariable regions, extended loop regions, and solvent exposed surfaces of cruciferin are critical factors affecting o/w interface stabilization.

Receptor-Like Kinases BAK1 and SOBIR1 Are Required for Necrotizing Activity of a Novel Group of Sclerotinia sclerotiorum Necrosis-Inducing Effectors.

Sclerotinia sclerotiorum is a characteristic necrotrophic plant pathogen and is dependent on the induction of host cell death for nutrient acquisition. To identify necrosis-inducing effectors, the genome of S. sclerotiorum was scanned for genes encoding small, secreted, cysteine-rich proteins. These potential effectors were tested for their ability to induce necrosis in Nicotiana benthamiana via Agrobacterium-mediated expression and for cellular localization in host cells. Six novel proteins were discovered, of which all but one required a signal peptide for export to the apoplast for necrotizing activity. Virus-induced gene silencing revealed that the five necrosis-inducing effectors with a requirement for secretion also required the plant co-receptor-like kinases Brassinosteroid Insensitive 1-Associated Receptor Kinase 1 (BAK1) and Suppressor of BAK1-Interacting Receptor-like Kinase 1 (SOBIR1) for the induction of necrosis. S. sclerotiorum necrosis-inducing effector 2 (SsNE2) represented a new class of necrosis-inducing proteins as orthologs were identified in several other phytopathogenic fungi that were also capable of inducing necrosis. Substitution of conserved cysteine residues with alanine reduced, but did not abolish, the necrotizing activity of SsNE2 and full-length protein was required for function as peptides spanning the entire protein were unable to induce necrosis. These results illustrate the importance of necrosis-inducing effectors for S. sclerotiorum virulence and the role of host extracellular receptor(s) in effector-triggered susceptibility to this pathogen.

Examining population structure of a bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae), outbreak in western North America: Implications for gene flow and dispersal.

The bertha armyworm (BAW), Mamestra configurata, is a significant pest of canola (Brassica napus L. and B. rapa L.) in western North America that undergoes cyclical outbreaks every 6-8 years. During peak outbreaks millions of dollars are spent on insecticidal control and, even with control efforts, subsequent damage can result in losses worth millions of dollars. Despite the importance of this pest insect, information is lacking on the dispersal ability of BAW and the genetic variation of populations from across its geographic range which may underlie potential differences in their susceptibility to insecticides or pathogens. Here, we examined the genetic diversity of BAW populations during an outbreak across its geographic range in western North America. First, mitochondrial cytochrome oxidase 1 (CO1) barcode sequences were used to confirm species identification of insects captured in a network of pheromone traps across the range, followed by haplotype analyses. We then sequenced the BAW genome and used double-digest restriction site associated DNA sequencing, mapped to the genome, to identify 1000s of single nucleotide polymorphisms (SNP) markers. CO1 haplotype analysis identified 9 haplotypes distributed across 28 sample locations and three laboratory-reared colonies. Analysis of genotypic data from both the CO1 and SNP markers revealed little population structure across BAW's vast range. The CO1 haplotype pattern showed a star-like phylogeny which is often associated with species whose population abundance and range has recently expanded and combined with pheromone trap data, indicates the outbreak may have originated from a single focal point in central Saskatchewan. The relatively recent introduction of canola and rapid expansion of the canola growing region across western North America, combined with the cyclical outbreaks of BAW caused by precipitous population crashes, has likely selected for a genetically homogenous BAW population adapted to this crop.

Proteomics analysis of Trichoplusia ni midgut epithelial cell brush border membrane vesicles.

The insect midgut epithelium is composed of columnar, goblet, and regenerative cells. Columnar epithelial cells are the most abundant and have membrane protrusions that form the brush border membrane (BBM) on their apical side. These increase surface area available for the transport of nutrients, but also provide opportunities for interaction with xenobiotics such as pathogens, toxins and host plant allelochemicals. Recent improvements in proteomic and bioinformatics tools provided an opportunity to determine the proteome of the T. ni BBM in unprecedented detail. This study reports the identification of proteins from BBM vesicles (BBMVs) using single dimension polyacrylamide gel electrophoresis coupled with multi-dimensional protein identification technology. More than 3000 proteins were associated with the BBMV, of which 697 were predicted to possess either a signal peptide, at least one transmembrane domain or a GPI-anchor signal. Of these, bioinformatics analysis and manual curation predicted that 185 may be associated with the BBMV or epithelial cell plasma membrane. These are discussed with respect to their predicted functions, namely digestion, nutrient uptake, cell signaling, development, cell-cell interactions, and other functions. We believe this to be the most detailed proteomic analysis of the lepidopteran midgut epithelium membrane to date, which will provide information to better understand the biochemical, physiological and pathological processes taking place in the larval midgut.

A high-quality chromosome-level genome assembly of a generalist herbivore, Trichoplusia ni.

The cabbage looper, Trichoplusia ni, is a globally distributed highly polyphagous herbivore and an important agricultural pest. T. ni has evolved resistance to various chemical insecticides, and is one of the only two insect species that have evolved resistance to the biopesticide Bacillus thuringiensis (Bt) in agricultural systems and has been selected for resistance to baculovirus infections. We report a 333-Mb high-quality T. ni genome assembly, which has N50 lengths of scaffolds and contigs of 4.6 Mb and 140 Kb, respectively, and contains 14,384 protein-coding genes. High-density genetic maps were constructed to anchor 305 Mb (91.7%) of the assembly to 31 chromosomes. Comparative genomic analysis of T. ni with Bombyx mori showed enrichment of tandemly duplicated genes in T. ni in families involved in detoxification and digestion, consistent with the broad host range of T. ni. High levels of genome synteny were found between T. ni and other sequenced lepidopterans. However, genome synteny analysis of T. ni and the T. ni derived cell line High Five (Hi5) indicated extensive genome rearrangements in the cell line. These results provided the first genomic evidence revealing the high instability of chromosomes in lepidopteran cell lines known from karyotypic observations. The high-quality T. ni genome sequence provides a valuable resource for research in a broad range of areas including fundamental insect biology, insect-plant interactions and co-evolution, mechanisms and evolution of insect resistance to chemical and biological pesticides, and technology development for insect pest management.

Changes in gene expression in Camelina sativa roots and vegetative tissues in response to salinity stress.

The response of Camelina sativa to salt stress was examined. Salt reduced shoot, but not root length. Root and shoot weight were affected by salt, as was photosynthetic capacity. Salt did not alter micro-element concentration in shoots, but increased macro-element (Ca and Mg) levels. Gene expression patterns in shoots indicated that salt stress may have led to shuttling of Na+ from the cytoplasm to the tonoplast and to an increase in K+ and Ca+2 import into the cytoplasm. In roots, gene expression patterns indicated that Na+ was exported from the cytoplasm by the SOS pathway and that K+ was imported in response to salt. Genes involved in chelation and storage were up-regulated in shoots, while metal detoxification appeared to involve various export mechanisms in roots. In shoots, genes involved in secondary metabolism leading to lignin, anthocyanin and wax production were up-regulated. Partial genome partitioning was observed in roots and shoots based on the expression of homeologous genes from the three C. sativa sub-genomes. Sub-genome I and II were involved in the response to salinity stress to about the same degree, while about 10% more differentially-expressed genes were associated with sub-genome III.

Changes in the Sclerotinia sclerotiorum transcriptome during infection of Brassica napus.

BACKGROUND: Sclerotinia sclerotiorum causes stem rot in Brassica napus, which leads to lodging and severe yield losses. Although recent studies have explored significant progress in the characterization of individual S. sclerotiorum pathogenicity factors, a gap exists in profiling gene expression throughout the course of S. sclerotiorum infection on a host plant. In this study, RNA-Seq analysis was performed with focus on the events occurring through the early (1 h) to the middle (48 h) stages of infection. RESULTS: Transcript analysis revealed the temporal pattern and amplitude of the deployment of genes associated with aspects of pathogenicity or virulence during the course of S. sclerotiorum infection on Brassica napus. These genes were categorized into eight functional groups: hydrolytic enzymes, secondary metabolites, detoxification, signaling, development, secreted effectors, oxalic acid and reactive oxygen species production. The induction patterns of nearly all of these genes agreed with their predicted functions. Principal component analysis delineated gene expression patterns that signified transitions between pathogenic phases, namely host penetration, ramification and necrotic stages, and provided evidence for the occurrence of a brief biotrophic phase soon after host penetration. CONCLUSIONS: The current observations support the notion that S. sclerotiorum deploys an array of factors and complex strategies to facilitate host colonization and mitigate host defenses. This investigation provides a broad overview of the sequential expression of virulence/pathogenicity-associated genes during infection of B. napus by S. sclerotiorum and provides information for further characterization of genes involved in the S. sclerotiorum-host plant interactions.

The Sclerotinia sclerotiorum Slt2 mitogen-activated protein kinase ortholog, SMK3, is required for infection initiation but not lesion expansion.

Mitogen-activated protein kinases (MAPKs) play a central role in transferring signals and regulating gene expression in response to extracellular stimuli. An ortholog of the Saccharomyces cerevisiae cell wall integrity MAPK was identified in the phytopathogenic fungus Sclerotinia sclerotiorum. Disruption of the S. sclerotiorum Smk3 gene severely reduced virulence on intact host plant leaves but not on leaves stripped of cuticle wax. This was attributed to alterations in hyphal apical dominance leading to the inability to aggregate and form infection cushions. The mutation also caused loss of the ability to produce sclerotia, increased aerial hyphae formation, and altered hyphal hydrophobicity and cell wall integrity. Mutants had slower radial expansion rates on solid media but more tolerance to elevated temperatures. Loss of the SMK3 cell wall integrity MAPK appears to have impaired the ability of S. sclerotiorum to sense its surrounding environment, leading to misregulation of a variety of functions. Many of the phenotypes were similar to those observed in S. sclerotiorum adenylate cyclase and SMK1 MAPK mutants, suggesting that these signaling pathways co-regulate aspects of fungal growth, physiology, and pathogenicity.

The developmental transcriptome atlas of the biofuel crop Camelina sativa.

Camelina sativa is currently being embraced as a viable industrial bio-platform crop due to a number of desirable agronomic attributes and the unique fatty acid profile of the seed oil that has applications for food, feed and biofuel. The recent completion of the reference genome sequence of C. sativa identified a young hexaploid genome. To complement this work, we have generated a genome-wide developmental transcriptome map by RNA sequencing of 12 different tissues covering major developmental stages during the life cycle of C. sativa. We have generated a digital atlas of this comprehensive transcriptome resource that enables interactive visualization of expression data through a searchable database of electronic fluorescent pictographs (eFP browser). An analysis of this dataset supported expression of 88% of the annotated genes in C. sativa and provided a global overview of the complex architecture of temporal and spatial gene expression patterns active during development. Conventional differential gene expression analysis combined with weighted gene expression network analysis uncovered similarities as well as differences in gene expression patterns between different tissues and identified tissue-specific genes and network modules. A high-quality census of transcription factors, analysis of alternative splicing and tissue-specific genome dominance provided insight into the transcriptional dynamics and sub-genome interplay among the well-preserved triplicated repertoire of homeologous loci. The comprehensive transcriptome atlas in combination with the reference genome sequence provides a powerful resource for genomics research which can be leveraged to identify functional associations between genes and understand the regulatory networks underlying developmental processes.

The eukaryotic protein kinase superfamily of the necrotrophic fungal plant pathogen, Sclerotinia sclerotiorum.

Protein kinases have been implicated in the regulation of many processes that guide pathogen development throughout the course of infection. A survey of the Sclerotinia sclerotiorum genome for genes encoding proteins containing the highly conserved eukaryotic protein kinase (ePK) domain, the largest protein kinase superfamily, revealed 92 S. sclerotiorum ePKs. This review examines the composition of the S. sclerotiorum ePKs based on conserved motifs within the ePK domain family, and relates this to orthologues found in other filamentous fungi and yeasts. The ePKs are also discussed in terms of their proposed role(s) in aspects of host pathogenesis, including the coordination of mycelial growth/development and deployment of pathogenicity determinants in response to environmental stimuli, nutrients and stress.