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Context Class III acyl-CoA-binding proteins such as those from dicots (Arabidopsis and grapevine) play a role in defence against biotrophic pathogens. The overexpression of the monocot Oryza sativa (rice) OsACBP5 in Arabidopsis and rice has been demonstrated to enhance broad-spectrum disease resistance against selected phytopathogens in OsACBP5-overexpressing (OsACBP5-OE) lines. Aims We aimed to develop transgenic rapid-cycling Brassica napus (B. napus-RC) and canola cv. Westar OsACBP5-OEs using tissue culture-based Agrobacterium-mediated transformation and to evaluate transgenic plants for resistance against Alternaria blight, blackleg and Sclerotinia rot diseases. Methods Transgenic B. napus-RC and cv. Westar OsACBP5-OEs were generated through Agrobacterium-mediated transformation using Agrobacterium strain LBA4404 harbouring a plasmid with the rice complementary DNA encoding OsACBP5 driven by the cauliflower mosaic virus 35S promoter. Alternaria blight and blackleg pathogen assays were based on infecting young cotyledons, while detached leaf assay was used to test the tolerance of B. napus plants toward Sclerotinia sclerotiorum. Key results Average transformation efficiencies of 24.2% and 29.1% were obtained for B. napus-RC and B. napus cv. Westar cotyledons respectively. OsACBP5-OE plants exhibited resistance 5 days after inoculation with Alternaria brassicae, 12 days after inoculation with Leptosphaeria maculans, and 24 h after inoculation with S. sclerotiorum. Conclusions Transformation of B. napus-RC was shown herein to be an effective trait testing platform for canola. This study also provides an insight into the usefulness of OsACBP5 in enhancing resistance to necrotrophic phytopathogens. Implications OsACBP5 can be overexpressed in other crops to generate pathogen-resistant varieties.

Continuous passaging in vitro can lead to the accumulation of changes in DNA sequence that potentially affect the properties of microbes, making them different from the original isolates. The identification of such genetic alterations is rare in fungi. A set of insertional mutants in the plant pathogenic fungus Leptosphaeria maculans, all derived from the same transformation experiment, had independent Agrobacterium T-DNA insertions and reduced pathogenicity on canola (Brassica napus). None of the insertions co-segregated in progeny from crosses with the reduction in pathogenicity. Genome sequences of three strains were analysed, and a mutation identified in a gene (ptf1, for pathogenicity-associated transcription factor 1) encoding a putative Zn2(II)Cys6 transcription factor. Homologs are found in other ascomycetes, and are required for pathogenicity by Fusarium graminearum, Fusarium oxysporum and Magnaporthe oryzae. The mutation in the L. maculans ptf1 gene co-segregates in progeny from crosses with the reduction in pathogenicity, a strain with an independent mutant allele isolated using CRISPR-Cas9 editing has reduced pathogenicity, and addition of wild type copies of the gene restores pathogenicity. Thus, this work defines a base pair substitution that occurred during in vitro passaging of a fungus that contributed to an attenuation of pathogenicity.

Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named “miscanthus sinensis mosaic virus” (MsiMV), isolated from Miscanthus sinensis (silver grass) held in a post entry quarantine facility following its initial import into Western Australia, Australia. The MsiMV genome encompasses 9604 nucleotides (nt) encoding a 3071 amino acids (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to sorghum mosaic virus (SrMV) with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.

Leptosphaeria maculans is an ascomycetous fungus that causes the disease blackleg on Brassica napus (canola). In spite of the importance of the disease worldwide, the mechanisms of disease development are poorly understood. Secondary metabolites, which are one of the common virulence factors of pathogenic fungi, have not been extensively explored from this fungus. An RNA-seq dataset was examined to find genes responsible for secondary metabolite synthesis by this fungus during infection. One polyketide synthase gene, pks5, was found to be upregulated during the early biotrophic stage of development. In addition to pks5, six other genes adjacent to the pks5 gene, including one encoding a Zn(II)2Cys6 transcription factor abscisic acid-like 7 gene (abl7), were also upregulated during that time. A striking feature of the L. maculans genome is that it contains large AT-rich regions that are gene-poor and large GC-rich regions that are gene rich. This set of seven co-regulated genes is embedded within and separated by two such AT-rich regions. Three of the genes in the cluster have similarities to those known to be involved in the synthesis of abscisic acid (ABA) in other fungi. When L. maculans is grown in axenic culture the genes in this cluster are not expressed and ABA is not produced. Overexpressing abl7, encoding the putative transcription factor, resulted in the transcription of the six adjacent genes in axenic culture and in the production of ABA, as detected by liquid chromatography quadrupole-time-of-flight mass spectrometry analysis. Mutation of two genes of the cluster using CRISPR/Cas9 did not affect pathogenicity on canola cotyledons. The characterization of the ABA gene cluster has led to the discovery of the co-regulation of genes within an AT-rich region by a transcription factor, and the first report of the plant hormone abscisic acid being produced by L. maculans.

Gene duplication contributes to evolutionary potential, yet many duplications in a genome arise from the activity of “selfish” genetic elements such as transposable elements. Fungi have a number of mechanisms by which they limit the expansion of transposons, including Repeat Induced Point mutation (RIP). RIP has been best characterized in the Sordariomycete Neurospora crassa, wherein duplicated DNA regions are recognized after cell fusion, but before nuclear fusion during the sexual cycle, and then mutated. While “signatures” of RIP appear in the genome sequences of many fungi, the species most distant from N. crassa in which the process has been experimentally demonstrated to occur is the Dothideomycete Leptosphaeria maculans. In the current study, we show that similar to N. crassa, nonlinked duplications can trigger RIP; however, the frequency of the generated RIP mutations is extremely low in L maculans (< 0.1%) and requires a large duplication to initiate RIP, and that multiple premeiotic mitoses are involved in the RIP process. However, a single sexual cycle leads to the generation of progeny with unique haplotypes, despite progeny pairs being generated from mitosis. We hypothesize that these different haplotypes may be the result of the deamination process occurring post karyogamy, leading to unique mutations within each of the progeny pairs. These findings indicate that the RIP process, while common to many fungi, differs between fungi and that this impacts on the fate of duplicated DNA.

Sirodesmin, the major secondary metabolite produced by the plant pathogenic fungus Leptosphaeria maculans in vitro, has been linked to disease on Brassica species since the 1970s, and yet its role has remained ambiguous. Re-examination of gene expression data revealed that all previously described genes and two newly identified genes within the sir gene cluster in the genome are down-regulated during the crucial early establishment stages of blackleg disease on Brassica napus. To test if this is a strategy employed by the fungus to avoid damage to and then detection by the host plant during the L. maculans asymptomatic biotrophic phase, sirodesmin was produced constitutively by overexpressing the sirZ gene encoding the transcription factor that coordinates the regulation of the other genes in the sir cluster. The sirZ over-expression strains had a major reduction in pathogenicity. Mutation of the over-expression construct restored pathogenicity. However, mutation of two genes, sirP and sirG, required for specific steps in the sirodesmin biosynthesis pathway, in the sirZ over-expression background resulted in strains that were unable to synthesize sirodesmin, yet were still non-pathogenic. Elucidating the basis for this pathogenicity defect or finding ways to overexpress sirZ during disease may provide new strategies for the control of blackleg disease.

Continuous passaging in vitro can lead to the accumulation of changes in DNA sequence that potentially affect the properties of microbes, making them different from the original isolates. The identification of such genetic alterations is rare in fungi. A set of insertional mutants in the plant pathogenic fungus Leptosphaeria maculans, all derived from the same transformation experiment, had independent Agrobacterium T-DNA insertions and reduced pathogenicity on canola (Brassica napus). None of the insertions co-segregated in progeny from crosses with the reduction in pathogenicity. Genome sequences of three strains were analysed, and a mutation identified in a gene (ptf1, for pathogenicity-associated transcription factor 1) encoding a putative Zn

As part of a special edition for MDPI on plant virology in Australia, this review provides a brief high-level overview on the evolution of diagnostic techniques used in Australian government Post-Entry Quarantine (PEQ) facilities for testing imported plants for viruses. A comprehensive range of traditional and modern diagnostic approaches have historically been employed in PEQ facilities using bioassays, serological, and molecular techniques. Whilst these techniques have been effective, they are time consuming, resource intensive and expensive. The review highlights the importance of ensuring the best available science and diagnostic developments are constantly tested, evaluated, and implemented by regulators to ensure primary producers have rapid and safe access to new genetics to remain productive, sustainable and competitive.

Gene duplication contributes to evolutionary potential, yet many duplications in a genome arise from the activity of “selfish” genetic elements such as transposable elements. Fungi have a number of mechanisms by which they limit the expansion of transposons, including Repeat Induced Point mutation (RIP). RIP has been best characterized in the Sordariomycete Neurospora crassa, wherein duplicated DNA regions are recognized after cell fusion, but before nuclear fusion during the sexual cycle, and then mutated. While “signatures” of RIP appear in the genome sequences of many fungi, the species most distant from N. crassa in which the process has been experimentally demonstrated to occur is the Dothideomycete Leptosphaeria maculans. In the current study, we show that similar to N. crassa, nonlinked duplications can trigger RIP; however, the frequency of the generated RIP mutations is extremely low in L maculans (< 0.1%) and requires a large duplication to initiate RIP, and that multiple premeiotic mitoses are involved in the RIP process. However, a single sexual cycle leads to the generation of progeny with unique haplotypes, despite progeny pairs being generated from mitosis. We hypothesize that these different haplotypes may be the result of the deamination process occurring post karyogamy, leading to unique mutations within each of the progeny pairs. These findings indicate that the RIP process, while common to many fungi, differs between fungi and that this impacts on the fate of duplicated DNA.

The Southern corn leaf blight (SCLB) epidemic of 1970 devastated fields of T-cytoplasm corn planted in monoculture throughout the eastern United States. The epidemic was driven by race T, a previously unseen race of Cochliobolus heterostrophus. A second fungus, Phyllosticta zeae-maydis, with the same biological specificity, appeared coincidentally. Race T produces T-toxin, while Phyllosticta zeae-maydis produces PM-toxin, both host-selective polyketide toxins necessary for supervirulence. The present abundance of genome sequences offers an opportunity to tackle the evolutionary origins of T- and PM- toxin biosynthetic genes, previously thought unique to these species. Using the C. heterostrophus genes as probes, we identified orthologs in six additional Dothideomycete and three Eurotiomycete species. In stark contrast to the genetically fragmented race T Tox1 locus that encodes these genes, all newly found Tox1-like genes in other species reside at a single collinear locus. This compact arrangement, phylogenetic analyses, comparisons of Tox1 protein tree topology to a species tree, and Tox1 gene characteristics suggest that the locus is ancient and that some species, including C. heterostrophus, gained Tox1 by horizontal gene transfer. C. heterostrophus and Phyllosticta zeae-maydis did not exchange Tox1 DNA at the time of the SCLB epidemic, but how they acquired Tox1 remains uncertain. The presence of additional genes in Tox1-like clusters of other species, although not in C. heterostrophus and Phyllosticta zeae-maydis, suggests that the metabolites produced differ from T- and PM-toxin.

The implementation of Agrobacterium tumefaciens as a transformation tool revolutionized approaches to discover and understand gene functions in a large number of fungal species. A. tumefaciens mediated transformation (AtMT) is one of the most transformative technologies for research on fungi developed in the last 20 years, a development arguably only surpassed by the impact of genomics. AtMT has been widely applied in forward genetics, whereby generation of strain libraries using random T-DNA insertional mutagenesis, combined with phenotypic screening, has enabled the genetic basis of many processes to be elucidated. Alternatively, AtMT has been fundamental for reverse genetics, where mutant isolates are generated with targeted gene deletions or disruptions, enabling gene functional roles to be determined. When combined with concomitant advances in genomics, both forward and reverse approaches using AtMT have enabled complex fungal phenotypes to be dissected at the molecular and genetic level. Additionally, in several cases AtMT has paved the way for the development of new species to act as models for specific areas of fungal biology, particularly in plant pathogenic ascomycetes and in a number of basidiomycete species. Despite its impact, the implementation of AtMT has been uneven in the fungi. This review provides insight into the dynamics of expansion of new research tools into a large research community and across multiple organisms. As such, AtMT in the fungi, beyond the demonstrated and continuing power for gene discovery and as a facile transformation tool, provides a model to understand how other technologies that are just being pioneered, e.g. CRISPR/Cas, may play roles in fungi and other eukaryotic species.

There was an error published in Development 130 , [4515-4525][1]. In Fig. 6, owing to an error in figure assembly during preparation of the proofs, the incorrect image (a duplication of panel D) was displayed in panel G. The corrected figure appears below. We apologise to the authors and readers

The fungus Leptosphaeria maculans causes blackleg of Brassica species. Here, we report the mapping and subsequent cloning of an avirulence gene from L. maculans. This gene, termed AvrLmJ1, confers avirulence towards all three Brassica juncea cultivars tested. Analysis of RNA-seq data showed that AvrLmJ1 is housed in a region of the L. maculans genome which contains only one gene that is highly expressed in planta. The closest genes are 57 and 33 kb away and, like other avirulence genes of L. maculans, AvrLmJ1 is located within an AT-rich, gene-poor region of the genome. The encoded protein is 141 amino acids, has a predicted signal peptide and is cysteine rich. Two virulent isolates contain a premature stop codon in AvrLmJ1. Complementation of an isolate that forms cotyledonary lesions on B. juncea with the wild-type allele of AvrLmJ1 confers avirulence towards all three B. juncea cultivars tested, suggesting that the gene may confer species-specific avirulence activity.

Phomenoic acid, a long chain aliphatic carboxylic acid is a major metabolite produced by Leptosphaeria maculans, a fungal pathogen of Brassica napus (canola). This fungus has 15 predicted polyketide synthases (PKS) and seven of them have the appropriate domains for the biosynthesis of phomenoic acid. The most highly expressed PKS gene after 7 days growth in 10% V8 juice, PKS2, was silenced and the resultant mutant produced very low levels of phomenoic acid, indicating that this PKS is involved in phomenoic acid biosynthesis. This gene is part of a co-regulated cluster of genes. Reduced expression of an adjacent gene encoding the transcriptional regulator C6TF, led to reduced expression of genes for PKS2, P450, a cytochrome P450 monoxygenase, YogA, an alcohol dehydrogenase/quinone reductase, RTA1, a lipid transport exporter superfamily member and MFS, a Major Facilitator Superfamily transporter, as well as a marked reduction in phomenoic acid production. Phomenoic acid is toxic towards another canola pathogen Leptosphaeria biglobosa 'canadensis', but not towards L. maculans and only moderately toxic towards the wheat pathogen Stagonospora nodorum. This molecule is detected in infected stems and stubble of B. napus, but biosynthesis of it does not appear to be essential for pathogenicity of L. maculans. Phomenoic acid may play a role in allowing L. maculans to outcompete other fungi in its environmental niche.

Plant pathogenic fungi in the Ascomycota phylum are amongst some of the most destructive agents of plant disease. They use a variety of strategies to gain entry into the plant tissue with some biotrophs causing little damage early on, whilst other necrotrophs cause plant cell death very early in the interaction. Regardless of the strategy, the early stages of the interaction between these fungi and their plant hosts set the stage for either disease or non-disease outcome. The fungal molecules involved in this process are being identified functionally and through transcriptomic approaches in which uncharacterized secreted proteins are often highly upregulated. This chapter reviews the current state of our understanding about the molecules involved in the early stages of plant infection.

Leptosphaeria maculans, a pathogen of Brassica napus, is unable to invade most wild-type accessions of Arabidopsis thaliana, although several mutants are susceptible. The infection pathway of L. maculans via a non-invasive inoculation method on A. thaliana lms1 (undefined), pmr4-1 (defective in callose deposition), and pen1-1 and pen2-1 (defective in non-host responses to several pathogens) mutants is described. On wild types Col-0 and Ler-0, hyphae are generally arrested at stomatal apertures. A T-DNA insertional mutant of L. maculans (A22) that penetrates stomatal apertures of Col-0 and Ler-0 five to seven times more often than the wild-type isolate is described. The higher penetration frequency of isolate A22 is associated with an increased hypersensitive response, which includes callose deposition. Complementation analysis showed that the phenotype of this isolate is due to T-DNA insertion in an intronless gene denoted as ipa (increased penetration on Arabidopsis). This gene is predicted to encode a protein of 702 amino acids with best matches to hypothetical proteins in other filamentous ascomycetes. The ipa gene is expressed in the wild-type isolate at low levels in culture and during infection of A. thaliana and B. napus.

We performed a structure–function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop–loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family.

Mice lacking the epidermal growth factor receptor (EGFR) develop epithelial defects and a neurodegenerative disease and die within the first month of birth. By employing a conditional knock-in approach using the human EGFR cDNA mice humanised for EGFR (hEGFRKI/KI)were generated. Homozygous hEGFRKI/KI mice are viable and live up to six months. However, these mice are growth retarded and show skin and hair defects similar to Egfr-/- mutants. Interestingly, the neurodegeneration is fully rescued in hEGFRKI/KI mice, however, they develop a severe heart hypertrophy with semilunar valve abnormalities. Moreover, hEGFRKI/KI mice display accelerated chondrocyte and osteoblast differentiation, a phenotype that is also present in Egfr-/- mice and has not been previously described. The severity of the phenotypes correlates with the expression levels of the hEGFRKI allele, which is not efficiently expressed in epithelial and bone cells, but is expressed at similar and even higher levels as the endogenous Egfr in brain and heart. These results demonstrate that mice humanised for EGFR display tissue-specific hypomorphic phenotypes and describe a novel function for EGFR in bone development.

Homologs of barley Mlo are found in syntenic positions in all three genomes of hexaploid bread wheat, Triticum aestivum, and in rice, Oryza sativa. Candidate wheat orthologs, designated TaMlo-A1, TaMlo-B1, and TaMlo-D1, encode three distinct but highly related proteins that are 88% identical to barley MLO and appear to originate from the three diploid ancestral genomes of wheat. TaMlo-B1 and the rice ortholog, OsMlo2, are able to complement powdery mildew-resistant barley mlo mutants at the single-cell level. Overexpression of TaMlo-B1 or barley Mlo leads to super-susceptibility to the appropriate powdery mildew formae speciales in both wild-type barley and wheat. Surprisingly, overexpression of either Mlo or TaMlo-B1 also mediates enhanced fungal development to tested inappropriate formae speciales. These results underline a regulatory role for MLO and its wheat and rice orthologs in a basal defense mechanism that can interfere with forma specialis resistance to powdery mildews.

In plants, defence against specific isolates of a pathogen can be triggered by the presence of a corresponding race-specific resistance gene1, whereas resistance of a more broad-spectrum nature can result from recessive, presumably loss-of-regulatory-function, mutations2. An example of the latter are mlo mutations in barley, which have been successful in agriculture for the control of powdery mildew fungus (Blumeria graminis f. sp. hordei; Bgh)3. MLO protein resides in the plasma membrane, has seven transmembrane domains, and is the prototype of a sequence-diversified family unique to plants4,5, reminiscent of the seven-transmembrane receptors in fungi and animals5. In animals, these are known as G-protein-coupled receptors and exist in three main families, lacking sequence similarity, that are thought to be an example of molecular convergence6. MLO seems to function independently of heterotrimeric G proteins. We have identified a domain in MLO that mediates a Ca2+-dependent interaction with calmodulin in vitro. Loss of calmodulin binding halves the ability of MLO to negatively regulate defence against powdery mildew in vivo. We propose a sensor role for MLO in the modulation of defence reactions.

The barley Mla locus encodes 28 characterized resistance specificities to the biotrophic fungal pathogen barley powdery mildew. We describe a single-cell transient expression assay using entire cosmid DNAs to pinpoint Mla1 within the complex 240-kb Mla locus. The MLA1 cDNA encodes a 108-kD protein containing an N-terminal coiled-coil structure, a central nucleotide binding domain, and a C-terminal leucine-rich repeat region; it also contains a second short open reading frame at the 5' end that has a possible regulatory function. Although most Mla-encoded resistance specificities require Rar1 for their function, we used the single-cell expression system to demonstrate that Mla1 triggers full resistance in the presence of the severely defective rar1-2 mutant allele. Wheat contains an ortholog of barley Mla, designated TaMla, that is tightly linked to (0.7 centimorgan) but distinct from a tested resistance specificity at the complex Pm3 locus to wheat powdery mildew. Thus, the most polymorphic powdery mildew resistance loci in barley and wheat may have evolved in parallel at two closely linked homeoloci. Barley Mla1 expressed in wheat using the single-cell transformation system failed to trigger a response to any of the wheat powdery mildew Avr genes tested, indicating that AvrMla1 is not genetically fixed in wheat mildew strains.

The fungus Leptosphaeria maculans causes blackleg of Brassica species. Here, we report the mapping and subsequent cloning of an avirulence gene from L. maculans. This gene, termed AvrLmJ1, confers avirulence towards all three Brassica juncea cultivars tested. Analysis of RNA‐seq data showed that AvrLmJ1 is housed in a region of the L. maculans genome which contains only one gene that is highly expressed in planta. The closest genes are 57 and 33 kb away and, like other avirulence genes of L. maculans, AvrLmJ1 is located within an AT‐rich, gene‐poor region of the genome. The encoded protein is 141 amino acids, has a predicted signal peptide and is cysteine rich. Two virulent isolates contain a premature stop codon in AvrLmJ1. Complementation of an isolate that forms cotyledonary lesions on B. juncea with the wild‐type allele of AvrLmJ1 confers avirulence towards all three B. juncea cultivars tested, suggesting that the gene may confer species‐specific avirulence activity.

The dimeric transcription factor AP-1 (activator protein-1) is composed of members of the Fos, Jun, and ATF family of proteins (Angel and Karin 1991). AP-1 activity is induced by a vast number of stimuli, including growth factors, cytokines, and UV irradiation, and couples cell surface signals to changes in cellular phenotype by regulating the expression of target genes (Angel and Karin 1991; Karin et al. 1997). Members of the AP-1 complex are critically involved in a multitude of cellular processes such as proliferation, differentiation, cell death, and oncogenic transformation. It has been hypothesized that the ability to regulate such a large number of biological processes is achieved by the formation of various functional dimers with distinct biological properties. Fos proteins (c-Fos, FosB, Fra-1, Fra-2) form stable dimers with Jun proteins (c-Jun, JunB, JunD), whereas Jun proteins can form homodimers and heterodimers with Fos and ATF proteins (Angel and Karin 1991; Karin et al. 1997). c-Fos (encoded by Fos) and Fra-1 (Fos-related antigen-1, encoded by the Fos-like-1 gene, Fosl1) show high homology in their leucine zipper and DNA-binding domains. Consistent with this sequence conservation, DNA binding specificity of Fra-1/c-Jun heterodimers is indistinguishable from c-Fos/c-Jun heterodimers (Cohen et al. 1989). Outside these domains, the proteins show little similarity. Most importantly, Fra-1 lacks transactivation domains and fails to activate transcription when fused to the DNA-binding domain of Gal4. In contrast, c-Fos is a potent activator of transcription (Cohen et al. 1989). Interestingly, Fosl1 is a transcriptional target of c-Fos, and induction of Fosl1 upon growth factor stimulation is delayed compared with Fos (Cohen and Curran 1988; Bergers et al. 1995; Schreiber et al. 1997; Matsuo et al. 2000). This led to the hypothesis that Fra-1 might counteract the function of c-Fos by lowering the transactivation potential of AP-1 complexes (Yoshioka et al. 1995). Fosl1 and Fos show different yet partially overlapping expression patterns in mice. Fosl1 is expressed in extraembryonic tissues during mouse development and in brain, skin, and testes of adult mice (Schreiber et al. 1997, 2000). Fos is expressed in the placenta and in several organs of the embryo, including fetal liver, heart, brain, and developing bones. In adult mice, Fos is readily detectable in most tissues, with highest levels found in brain and bone (Smeyne et al. 1993). Gain-of-function experiments have shown that overexpression of Fra-1 leads to increased bone formation and osteosclerosis (Jochum et al. 2000), whereas overexpression of c-Fos results in osteo- and chondrosarcomas (Ruther et al. 1987; Wang et al. 1992). Targeted disruption of the Fosl1 gene leads to early embryonic lethality due to impaired vascularization of the placenta (Schreiber et al. 2000). In contrast, lack of c-Fos results in pleiotropic defects, most importantly the bone disease osteopetrosis, due to the lack of bone-resorbing osteoclasts (Johnson et al. 1992; Wang et al. 1992; Grigoriadis et al. 1994). Furthermore, c-Fos-deficient photoreceptor neurons in the retina are resistant to light-induced apoptosis (Hafezi et al. 1997; Wenzel et al. 2000). These results suggest that c-Fos and Fra-1 have distinct functions in mice. On the other hand, Fosl1 is a transcriptional target of c-Fos in osteoclast progenitors, and overexpression of Fra-1 by viral gene transfer in vitro or in transgenic mice was able to rescue c-Fos-dependent osteoclast differentiation (Matsuo et al. 2000). To test whether Fra-1 can take over c-Fos functions under physiological conditions, knock-in mice were generated in which Fos is deleted and replaced by Fosl1. Interestingly, Fosl1 can fully complement for the lack of c-Fos in bone development and substitutes for the pro-apoptotic function of c-Fos in photoreceptor neurons. Rescue of bone cell differentiation, but not photoreceptor apoptosis, is gene dosage dependent. Furthermore, Fra-1 fails to rescue the expression of c-Fos target genes in fibroblasts. These data suggest that transactivation by c-Fos might be dispensable in vivo and that stage- and cell-specific modulation of expression determines the specific functions of these genes.

Osteoclasts are bone-resorbing cells derived from haematopoietic precursors of the monocyte-macrophage lineage. Mice lacking Fos (encoding c-Fos) develop osteopetrosis due to an early differentiation block in the osteoclast lineage. c-Fos is a component of the dimeric transcription factor activator protein-1 (Ap-1), which is composed mainly of Fos (c-Fos, FosB, Fra-1 and Fra-2) and Jun proteins (c-Jun, JunB and JunD). Unlike Fra-1 (encoded by Fosl1), c-Fos contains transactivation domains required for oncogenesis and cellular transformation. The mechanism by which c-Fos exerts its specific function in osteoclast differentiation is not understood. Here we show by retroviral-gene transfer that all four Fos proteins, but not the Jun proteins, rescue the differentiation block in vitro. Structure-function analysis demonstrated that the major carboxy-terminal transactivation domains of c-Fos and FosB are dispensable and that Fra-1 (which lacks transactivation domains) has the highest rescue activity. Moreover, a transgene expressing Fra-1 rescues the osteopetrosis of c-Fos-mutant mice in vivo. The osteoclast differentiation factor Rankl (also known as TRANCE, ODF and OPGL; refs 8-11) induces transcription of Fosl1 in a c-Fos-dependent manner, thereby establishing a link between Rank signalling and the expression of Ap-1 proteins in osteoclast differentiation.

Liquid chromatography-mass spectrometry (LC-MS or HPLC-MS) is an extremely sensitive analytical technique that enables the detection of metabolites with a vast range of chemistries and molecular masses. Extracts from any biological starting material are first fractionated chromatographically on a stationary phase suitable for the retention of the target molecules. The eluent is then transferred directly to the ionization source for MS detection. There is a vast range of chromatographic separation methods and MS configurations. This chapter describes a method for the detection of a broad range of metabolites using reversed phase (C18) LC-MS as well as a method for the isolation of targeted metabolites of interest.

Liquid chromatography-mass spectrometry (LC-MS or HPLC-MS) is an extremely sensitive analytical technique that enables the detection of metabolites with a vast range of chemistries and molecular masses. Extracts from any biological starting material are first fractionated chromatographically on a stationary phase suitable for the retention of the target molecules. The eluent is then transferred directly to the ionization source for MS detection. There is a vast range of chromatographic separation methods and MS configurations. This chapter describes a method for the detection of a broad range of metabolites using reversed phase (C18) LC-MS as well as a method for the isolation of targeted metabolites of interest.

Several cDNA clones encoding the human glutamate receptor subunit GluR3 flip and flop isoforms, were isolated from human hippocampus and fetal brain libraries. DNA sequence analysis revealed overlapping clones permitting the reconstruction of full-length GluR3-flip and GluR3-flop cDNAs. The GluR3 cDNAs demonstrated an 94.1–94.7% nucleotide (nt) identity with the corresponding rat cDNAs. The nt sequence of the GluR3 cDNAs would encode 894 amino acid proteins that have a 99.4% identity with the rat GluR3 isoforms. The human GluR3 cDNAs predict an additional 6 amino acid in the N-terminal signal peptide as compared to the rat GluR3.

Background Sirodesmin PL is a secondary metabolite toxin made by the ascomycetous plant pathogen, Leptosphaeria maculans. The sirodesmin biosynthetic genes are clustered in the genome. The key genes are a non-ribosomal peptide synthetase, sirP, and a pathway-specific transcription factor, sirZ. Little is known about regulation of sirodesmin production. Results Genes involved in regulation of sirodesmin PL in L. maculans have been identified. Two hundred random insertional T-DNA mutants were screened with an antibacterial assay for ones producing low levels of sirodesmin PL. Three such mutants were isolated and each transcribed sirZ at very low levels. One of the affected genes had high sequence similarity to Aspergillus fumigatus cpcA, which regulates the cross-pathway control system in response to amino acid availability. This gene was silenced in L. maculans and the resultant mutant characterised. When amino acid starvation was artificially-induced by addition of 3-aminotriazole for 5 h, transcript levels of sirP and sirZ did not change in the wild type. In contrast, levels of sirP and sirZ transcripts increased in the silenced cpcA mutant. After prolonged amino acid starvation the silenced cpcA mutant produced much higher amounts of sirodesmin PL than the wild type. Conclusions Production of sirodesmin PL in L. maculans is regulated by the cross pathway control gene, cpcA, either directly or indirectly via the pathway-specific transcription factor, sirZ.

SUMMARY Sirodesmin PL is a non-host-selective phytotoxin produced by Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). Previous studies have shown that sirodesmin PL biosynthesis involves a cluster of 18 co-regulated genes and that disruption of the two-module non-ribosomal peptide synthetase gene (sirP) in this cluster prevents the production of sirodesmin PL. Loss of sirodesmin PL did not affect the growth or fertility of the sirP mutant in vitro, but this mutant had less antibacterial and antifungal activity than the wild-type. When the sirP mutant was inoculated on to cotyledons of B. napus, it caused similar-sized lesions on cotyledons as the wild-type isolate, but subsequently caused fewer lesions and was half as effective as the wild-type in colonizing stems, as shown by quantitative PCR analyses. However, no significant difference was observed in size of lesions when either wild-type or mutant isolates were injected directly into the stem. The expression of two cluster genes, sirP and an ABC transporter, sirA, was studied in planta. Fungal isolates containing fusions of the green fluorescent protein gene with the promoters of these genes fluoresced after 10 days post-inoculation (dpi). Transcripts of sirP and sirA were detected after 11 dpi in cotyledons by reverse transcriptase PCR, and expression of both genes increased dramatically in stem tissue. This expression pattern was consistent with the distribution of sirodesmin PL in planta as revealed by mass spectrometry experiments.

Maintaining cell wall integrity is essential for fungal growth and development. We describe two mutants with altered expression of a gene, LmIFRD, from the ascomycete Leptosphaeria maculans. Truncation of the LmIFRD transcript in a T-DNA insertional mutant led to slower germination, less sporulation and loss-of-pathogenicity towards Brassica napus, whereas silencing of the LmIFRD transcript led to increased germination, sporulation and earlier infection. The increased tolerance to cell wall lysing enzymes and cell wall-disrupting compounds of the T-DNA mutant contrasts with decreased tolerance of the silenced mutant and suggests altered cell wall integrity and accessibility to 1,3-linked glucan and chitin. Lectin binding experiments and monosaccharide analysis revealed altered polysaccharide content and structure within the cell wall of the LmIFRD mutants, notably increased 1,3-linked galactose and chitin within the cell wall of the T-DNA mutant. This is the first analysis of monosaccharide linkage composition of cell walls of spores and mycelia for any dothideomycete.

Sirodesmin PL is a secondary metabolite toxin produced by Leptosphaeria maculans, a fungus that causes blackleg disease of oilseed rape (Brassica napus). Biosynthesis of this epipolythiodioxopiperazine (ETP) toxin involves a cluster of 18 co-regulated genes. Putative ETP gene clusters have been found in 14 taxa discontinuously distributed amongst ascomycetes. Movement of entire clusters by horizontal gene transfer is the most parsimonious hypothesis to explain this discontinuous distribution. A mutant of L. maculans that does not produce sirodesmin PL forms similar sized lesions on cotyledons of B. napus to those formed by the wild type. However, it is half as effective in colonising stems, as indicated by reduced lesion size and reduced fungal biomass. This implicates sirodesmin PL as a virulence factor in B. napus stems. Expression patterns of two of the cluster genes is consistent with the distribution of sirodesmin PL in planta, as revealed by mass spectrometry experiments. The sirodesmin-deficient mutant has less antibacterial and antifungal activity than the wild type, suggesting that sirodesmin PL may play a role in protecting L.maculans against competition from other micro-organisms.

Agrobacterium tumefaciens-mediated random mutagenesis was used to generate insertional mutants of the fungus Leptosphaeria maculans. Of 91 transformants screened, only one (A3) produced lesions of reduced size on cotyledons of canola (Brassica napus). Genes flanking the T-DNA insertion had the best matches to an alcohol dehydrogenase class 4 (ADH4)-like gene (Adh4L) and a 3-ketoacyl-CoA thiolase gene (Thiol) and were expressed in mutant A3 in vitro and in planta at significantly higher levels than in the wild type. This is the first report of a T-DNA insertion in fungi causing increased gene expression. Transformants of the wild-type isolate expressing both Adh4L and Thiol under the control of a heterologous promoter had similar pathogenicity to mutant A3. Ectopic expression of only thiolase resulted in loss of pathogenicity, suggesting that thiolase overexpression was primarily responsible for the reduced pathogenicity of the A3 isolate. The thiolase gene encoded a functional protein, as shown by assays in which a nontoxic substrate (2, 4 dichlorophenoxybutyric acid) was converted to a toxic product. The use of a translational fusion with a reporter gene showed thiolase expressed in organelles that are most likely peroxisomes.

International audience; The endoplasmic reticulum (ER) of eukaryotic cells serves as a checkpoint tightly monitoring protein integrity and channeling malformed proteins into different rescue and degradation routes. The degradation of several ER lumenal and membrane-localized proteins is mediated by ER-associated protein degradation (ERAD) in yeast (Saccharomyces cerevisiae) and mammalian cells. To date, evidence for the existence of ERAD-like mechanisms in plants is indirect and based on heterologous or artificial substrate proteins. Here, we show that an allelic series of single amino acid substitution mutants of the plant-specific barley (Hordeum vulgare) seven-transmembrane domain mildew resistance o (MLO) protein generates substrates for a postinsertional quality control process in plant, yeast, and human cells, suggesting conservation of the underlying mechanism across kingdoms. Specific stabilization of mutant MLO proteins in yeast strains carrying defined defects in protein quality control demonstrates that MLO degradation is mediated by HRD pathway-dependent ERAD. In plants, individual aberrant MLO proteins exhibit markedly reduced half-lives, are polyubiquitinated, and can be stabilized through inhibition of proteasome activity. This and a dependence on homologs of the AAA ATPase CDC48/p97 to eliminate the aberrant variants strongly suggest that MLO proteins are endogenous substrates of an ERAD-related plant quality control mechanism

Compared to viruses, bacteria and nematodes, filamentous fungi are the major causes of plant disease worldwide and are responsible for significant yield losses in crops. Disease control is mainly by deployment of resistant plant cultivars and by management strategies including crop rotations, disposal of inocula such as disease-infested stubble, and use of fungicides. In contrast to appreciable knowledge of plant defense mechanisms, there is little understanding at the molecular level of how fungal pathogens cause disease. Primary recognition between plants and pathogens is often mediated by single complementary resistance genes in the host and avirulence genes in the pathogen. Avirulence genes from several fungi have been cloned and in some cases also appear to play a role in fitness of the pathogen, often acting as disease determinants (for review see ref. 1).

Homologues of barley Mlo encode the only family of seven-transmembrane (TM) proteins in plants. Their topology, subcellular localization, and sequence diversification are reminiscent of those of G-protein coupled receptors (GPCRs) from animals and fungi. We present a computational analysis of MLO family members based on 31 full-size and 3 partial sequences, which originate from several monocot species, the dicot Arabidopsis thaliana, and the moss Ceratodon purpureus. This enabled us to date the origin of the Mlo gene family back at least to the early stages of land plant evolution. The genomic organization of the corresponding genes supports a monophyletic origin of the Mlo gene family. Phylo-genetic analysis revealed five clades, of which three contain both monocot and dicot members, while two indicate class-specific diversification. Analysis of the ratio of nonsynonymous-to-synonymous changes in coding sequences provided evidence for functional constraint on the evolution of the DNA sequences and purifying selection, which appears to be reduced in the first extracellular loop of 12 closely related orthologues. The 31 full-size sequences were examined for potential domain-specific intramolecular coevolution. This revealed evidence for concerted evolution of all three cytoplasmic domains with each other and the C-terminal cytoplasmic tail, suggesting interplay of all intracellular domains for MLO function.

Bone formation by osteoblasts is essential for skeletal growth and remodeling. Fra-1 is a c-Fos-related protein belonging to the AP-1 family of transcription factors. Here we show that transgenic mice overexpressing Fra-1 in various organs develop a progressive increase in bone mass leading to osteosclerosis of the entire skeleton, which is due to a cell-autonomous increase in the number of mature osteoblasts. Moreover, osteoblast differentiation, but not proliferation, was enhanced and osteoclastogenesis was also elevated in vitro. These data indicate that, unlike c-Fos, which causes osteosarcomas, Fra-1 specifically enhances bone formation, which may be exploited to stimulate bone formation in pathological conditions.

Plectin is a widely expressed protein that is very large in size and that has all the attributes of a multifunctional crosslinking and organizing element of the cytoskeleton. It displays a multidomain structure, versatile binding activities, and subcellular localizations that enable it to strengthen cells against mechanical stress forces. Moreover, hereditary gene defects in plectin cause epidermolysis bullosa simplex (EBS)-MD, a severe skin blistering disease with muscular dystrophy. Here we report the analysis of the exon–intron organization of the rat plectin gene and the identification of several different isoforms on the transcriptional level. We show that of 35 coding exons identified, 4 serve as alternative first exons splicing into the same successive exon 2, which is the first of 7 exons encoding a highly conserved actin-binding domain. RNase protection mapping of transcripts containing 3 of the identified 4 alternate first exons revealed their coexpression in rat glioma C6 cells and in a series of different rat tissues that we examined. Significant variations in expression levels of first exons indicated the possibility of tissue-specific promoter usage. In addition, plectin splice variants lacking exon 31 (>3 kb), which encodes the entire rod domain of the molecule, were identified in a variety of rat tissues. This study provides first insights into a complex plectin gene regulatory machinery with similarities to that of dystrophin.

Background Banks of mutants with random insertions of T-DNA from Agrobacterium tumefaciens are often used in forward genetics approaches to identify phenotypes of interest. Upon identification of mutants of interest, the flanking sequences of the inserted T-DNA must be identified so that the mutated gene can be characterised. However, for many fungi, this task is not trivial as widely used PCR-based methods such as thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) are not successful. Findings Next-generation Illumina sequencing was used to locate T-DNA insertion sites in four mutants of Leptosphaeria maculans, a fungal plant pathogen. Sequence reads of up to 150 bp and coverage ranging from 6 to 24 times, were sufficient for identification of insertion sites in all mutants. All T-DNA border sequences were truncated to different extents. Additionally, next-generation sequencing revealed chromosomal rearrangements associated with the insertion in one of the mutants. Conclusions Next-generation sequencing is a cost-effective and rapid method of identifying sites of T-DNA insertions, and associated genomic rearrangements in Leptosphaeria maculans and potentially in other fungal species.