The fungus Fusarium fujikuroi causes “bakanae” disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.

SET domains are conserved amino acid motifs present in chromosomal proteins that function in epigenetic control of gene expression. These proteins can be divided into four classes as typified by their Drosophila members E(Z), TRX, ASH1 and SU(VAR)3-9. Homologs of all four classes have been identified in yeast and mammals, but not in plants. A BLASTP screening of the Arabidopsis genome identified 37 genes: three E(z) homologs, five trx homologs, four ash1 homologs and 15 genes similar to Su(var)3-9. Seven genes were assigned as trx-related and three as ash1-related. Only four genes have been described previously. Our classification is based on the characteristics of the SET domains, cysteine-rich regions and additional conserved domains, including a novel YGD domain. RT-PCR analysis, cDNA cloning and matching ESTs show that at least 29 of the genes are active in diverse tissues. The high number of SET domain genes, possibly involved in epigenetic control of gene activity during plant development, can partly be explained by extensive genome duplication in Arabidopsis. Additionally, the lack of introns in the coding region of eight SU(VAR)3-9 class genes indicates evolution of new genes by retrotransposition. The identification of putative nuclear localization signals and AT-hooks in many of the proteins supports an anticipated nuclear localization, which was demonstrated for selected proteins.

SU(VAR)3-9 like histone methyltransferases control heterochromatic domains in eukaryotes. In Arabidopsis, 10 SUVH genes encode SU(VAR)3-9 homologues where SUVH1, SUVH2 and SUVH4 (KRYPTONITE) represent distinct subgroups of SUVH genes. Loss of SUVH1 and SUVH4 causes weak reduction of heterochromatic histone H3K9 dimethylation, whereas in SUVH2 null plants monoand dimethyl H3K9, mono-and dimethyl H3K27, and monomethyl H4K20, the histone methylation marks of Arabidopsis heterochromatin are significantly reduced. Like animal SU(VAR)3-9 proteins SUVH2 displays strong dosage-dependent effects. Loss of function suppresses, whereas overexpression enhances, gene silencing, causes ectopic heterochromatization and significant growth defects. Furthermore, modification of transgene silencing by SUVH2 is partially transmitted to the offspring plants. This epigenetic stability correlates with heritable changes in DNA methylation. Mutational dissection of SUVH2 indicates an implication of its N-terminus and YDG domain in directing DNA methylation to target sequences, a prerequisite for consecutive histone methylation. Gene silencing by SUVH2 depends on MET1 and DDM1, but not CMT3. In Arabidopsis, SUVH2 with its histone H3K9 and H4K20 methylation activity has a central role in heterochromatic gene silencing.

Cereals including wheat and barley are of primary importance to ensure food security for the 21st century. A combination of lab- and field-based approaches has led to a considerably improved understanding of the importance of organ and particularly of whole-plant (monocarpic) senescence for wheat and barley yield and quality. A delicate balance between senescence timing, grain nutrient content, nutrient-use efficiency, and yield needs to be considered to (further) improve cereal varieties for a given environment and end use. The recent characterization of the Gpc-1 (NAM-1) genes in wheat and barley demonstrates the interdependence of these traits. Lines or varieties with functional Gpc-1 genes demonstrate earlier senescence and enhanced grain protein and micronutrient content but, depending on the environment, somewhat reduced yields. A major effort is needed to dissect regulatory networks centred on additional wheat and barley transcription factors and signalling pathways influencing the senescence process. Similarly, while important molecular details of nutrient (particularly nitrogen) remobilization from senescing organs to developing grains have been identified, important knowledge gaps remain. The genes coding for the major proteases involved in senescence-associated plastidial protein degradation are largely unknown. Membrane transport proteins involved in the different transport steps occurring between senescing organ (such as leaf mesophyll) cells and protein bodies in the endosperm of developing grains remain to be identified or further characterized. Existing data suggest that an improved understanding of all these steps will reveal additional, important targets for continued cereal improvement.

The development of non-petrochemical sources for the plastics industry continues to progress as large multinationals focus on renewable resources to replace fossil carbon. Many bacteria are known to accumulate polyoxoesters as water-insoluble granules in the cytoplasm. The thermoplastic and/or elastomeric behaviour of these biodegradable polymers holds promise for the development of various technological applications. Here, we report the synthesis and characterization of microbial polythioesters (PTEs), a novel class of biopolymers of general technological relevance. Biosynthesis of PTE homopolymers was achieved using a recombinant strain of Escherichia coli that expressed a non-natural pathway consisting of a butyrate kinase, a phosphotransbutyrylase, and a PHA synthase. Different homopolymers were produced, consisting of either 3-mercaptopropionate, 3-mercaptobutyrate, or 3-mercaptovalerate repeating units, if the respective mercaptoalkanoic acids were provided as precursor substrates to the fermentative process. The PTEs contributed up to 30% (w/w) of the cellular dry weight and were identified as hydrophobic inclusions in the cytoplasm. The chemical and stereochemical homogeneity of the purified PTEs were identified by different methods, and the estimated physical properties were compared to the oxypolyester equivalents, revealing low crystalline order and, for the poly(3-mercaptopropionate) improved thermal stability. The ability to produce PTEs through a biosynthetic route opens up new avenues in the field of biomaterials.

Plant cell vacuoles may have either storage or degradative functions. Vegetative storage proteins (VSPs) are synthesized in response to wounding and to developmental switches that affect carbon and nitrogen sinks. Here we show that VSPs are stored in a unique type of vacuole that is derived from degradative central vacuoles coincident with insertion of a new tonoplast intrinsic protein (TIP), ␦-TIP, into their membranes. This finding demonstrates a tight coupling between the presence of ␦-TIP and acquisition of a specialized storage function and indicates that TIP isoforms may determine vacuole identity.Uniquely, in contrast to yeast or mammalian cells, plant cells may contain separate vacuoles with protein storage and digestive functions (1, 2). It is not known how functionally distinct vacuoles are generated or maintained. Plant vacuole tonoplast membranes contain abundant tonoplast intrinsic proteins (TIPs) that may function as aquaporins (3), but the quantities present seem to be far in excess of what is required for water transport (4). Protein storage vacuoles (PSVs), containing seed-type storage proteins, are marked by the presence of ␣-TIP, and lytic or degradative vacuoles (LVs) are marked by the presence of ␥-TIP (1, 2). These observations indicate that a specific TIP isoform correlates with a specific vacuole function (5). A further test of this hypothesis would require demonstration that a third functionally distinct vacuole carried a different TIP isoform. Here we define a third functionally distinct vacuole in plant cells and demonstrate that it is marked specifically by ␦-TIP. MATERIALS AND METHODSAntibodies and Immunocytochemistry. Anti-␣-TIP protein antiserum (6) and VM23 antiserum to purified ␥-TIP protein from radish root (7) were generously provided by M. Chrispeels (6) and M. Maeshima (7), respectively. Synthetic peptides were synthesized and antisera to the peptides coupled to keyhole limpet hemocyanin were generated by Quality Controlled Biochemicals, Hopkinton, MA. For antibody purification, the peptides were coupled via an amino-terminal Cys residue to SulfoLink agarose (Pierce) according to the manufacturer's instructions, and peptide-specific antibodies were affinity-purified as described previously (8) for use in all procedures. Antisera to proteinase inhibitor I (Inh I) and II (Inh II) have been described previously (9-11). Fluorochrometagged secondary antibodies were purchased from Jackson ImmunoResearch. Plant tissues were fixed in either formaldehyde͞acetic acid͞ethyl alcohol or 3.7% paraformaldehyde, and paraffin-embedded sections were prepared as described (12). After removal of paraffin and rehydration, the sections were blocked as described (2). The double-label protocol to identify two different rabbit antibodies separately with immunofluorescence has been described (2). Briefly, the first primary antibody was completely covered by incubation with an excess of anti-rabbit F(abЈ) 2 fragment coupled to lissamine rhodamine before incubating with the second primary antibo...

Summary• Leaf senescence can be described as the dismantling of cellular components during a specific time interval before cell death. This has the effect of remobilizing N in the form of amino acids that can be relocalized to developing seeds. High levels of carbohydrates have previously been shown to promote the onset of the senescence process.• Carbohydrate accumulation in barley ( Hordeum vulgare ) plants was induced experimentally by steam-girdling at the leaf base, occluding the phloem, and gene regulation under these conditions was investigated using the Affymetrix Barley GeneChip array and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR).• Transcript levels of plastidial (aminopeptidases, cnd41) and vacuolar (thiol and serine) proteases clearly increase in girdled leaves. Of special interest are cnd41, a plastidial aspartyl peptidase that has been implicated in Rubisco degradation in tobacco; and cp-mIII, a highly upregulated carboxypeptidase. SAG12 , hexokinases and other senescence-specific genes are also upregulated under these conditions.• Applying a genomic approach to the innovative experimental system described here significantly enhances our knowledge of leaf proteolysis and whole-plant N recycling.

Trimethoprim-sulfamethoxazole (SXT)-resistant