Regulation of planar growth by the Arabidopsis AGC protein kinase UNICORN.

The spatial coordination of growth is of central importance for the regulation of plant tissue architecture. Individual layers, such as the epidermis, are clonally propagated and structurally maintained by symmetric cell divisions that are oriented along the plane of the layer. The developmental control of this process is poorly understood. The simple cellular basis and sheet-like structure of Arabidopsis integuments make them an attractive model system to address planar growth. Here we report on the characterization of the Arabidopsis UNICORN (UCN) gene. Analysis of ucn integuments reveals localized distortion of planar growth, eventually resulting in an ectopic multicellular protrusion. In addition, ucn mutants exhibit ectopic growth in filaments and petals, as well as aberrant embryogenesis. We further show that UCN encodes an active AGC VIII kinase. Genetic, biochemical, and cell biological data suggest that UCN suppresses ectopic growth in integuments by directly repressing the KANADI transcription factor ABERRANT TESTA SHAPE. Our findings indicate that UCN represents a unique plant growth regulator that maintains planar growth of integuments by repressing a developmental regulator involved in the control of early integument growth and polarity.

Melanin is not required for turgor generation but enhances cell-wall rigidity in appressoria of the corn pathogen Colletotrichum graminicola.

The ascomycete and causative agent of maize anthracnose and stem rot, Colletotrichum graminicola, differentiates melanized infection cells called appressoria that are indispensable for breaching the plant cell wall. High concentrations of osmolytes accumulate within the appressorium, and the internal turgor pressure of up to 5.4 MPa provides sufficient force to penetrate the leaf epidermis directly. In order to assess the function of melanin in C. graminicola appressoria, we identified and characterized the polyketide synthase 1 (CgPKS1) gene which displayed high similarity to fungal polyketide synthases (PKS) involved in synthesis of 1,3,6,8-tetrahydronaphthalene, the first intermediate in melanin biosynthesis. Cgpks1 albino mutants created by targeted gene disruption were unable to penetrate intact leaves and ruptured frequently but, surprisingly, were able to penetrate ultrathin polytetrafluoroethylene membranes mimicking the plant surface. Nonmelanized Cgpks1 appressoria were sensitive to externally applied cell-wall-degrading enzymes whereas melanized appressoria were not affected. Expression studies using a truncated CgPKS1 fused to green fluorescent protein revealed fluorescence in immature appressoria and in setae, which is in agreement with transcript data obtained by RNA-Seq and quantitative polymerase chain reaction. Unexpectedly, surface scans of mutant and wild-type appressoria revealed considerable differences in cell-wall morphology. Melanization of appressoria is indispensable for successful infection of intact leaves. However, cell collapse experiments and analysis of the appressorial osmolyte content by Mach-Zehnder interferometry convincingly showed that melanin is not required for solute accumulation and turgor generation, thus questioning the role of melanin as a barrier for osmolytes in appressoria of C. graminicola.

New gene models and alternative splicing in the maize pathogen Colletotrichum graminicola revealed by RNA-Seq analysis.

BACKGROUND: An annotated genomic sequence of the corn anthracnose fungus Colletotrichum graminicola has been published previously, but correct identification of gene models by means of automated gene annotation remains a challenge. RNA-Seq offers the potential for substantially improved gene annotations and for the identification of posttranscriptional RNA modifications, such as alternative splicing and RNA editing. RESULTS: Based on the nucleotide sequence information of transcripts, we identified 819 novel transcriptionally active regions (nTARs) and revised 906 incorrectly predicted gene models, including revisions of exon-intron structure, gene orientation and sequencing errors. Among the nTARs, 146 share significant similarity with proteins that have been identified in other species suggesting that they are hitherto unidentified genes in C. graminicola. Moreover, 5'- and 3'-UTR sequences of 4378 genes have been retrieved and alternatively spliced variants of 69 genes have been identified. Comparative analysis of RNA-Seq data and the genome sequence did not provide evidence for RNA editing in C. graminicola. CONCLUSIONS: We successfully employed deep sequencing RNA-Seq data in combination with an elaborate bioinformatics strategy in order to identify novel genes, incorrect gene models and mechanisms of transcript processing in the corn anthracnose fungus C. graminicola. Sequence data of the revised genome annotation including several hundreds of novel transcripts, improved gene models and candidate genes for alternative splicing have been made accessible in a comprehensive database. Our results significantly contribute to both routine laboratory experiments and large-scale genomics or transcriptomic studies in C. graminicola.

The Transient Receptor Potential (TRP) Channel Family in Colletotrichum graminicola: A Molecular and Physiological Analysis.

Calcium (Ca2+) is a universal second messenger in all higher organisms and centrally involved in the launch of responses to environmental stimuli. Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae) contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt) elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM) domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by influx of Ca2+ from the extracellular space. Cgtrpf mutants did not show pathogenicity defects in leaf infection assays. In summary, our study reveals major differences between different fungi in the contribution of TRP channels to Ca2+-mediated signal transduction.

Chloroplast proteins without cleavable transit peptides: rare exceptions or a major constituent of the chloroplast proteome?

Most chloroplast proteins (cp proteins) are nucleus-encoded, synthesized on cytosolic ribosomes as precursor proteins containing a presequence (cTP), and post-translationally imported via the Tic/Toc complex into the organelle, where the cTP is removed. Only a few unambiguous instances of cp proteins that do not require cTPs (non-canonical cp proteins) have been reported so far. However, the survey of data from large-scale proteomic studies presented here suggests that the fraction of such proteins in the total cp proteome might be as large as approximately 30%. To explore this discrepancy, we chose a representative set of 28 putative non-canonical cp proteins, and used in vitro import and Red Fluorescent Protein (RFP)-fusion assays to determine their sub-cellular destinations. Four proteins, including embryo defective 1211, glycolate oxidase 2, protein disulfide isomerase-like protein (PDII), and a putative glutathione S-transferase, could be unambiguously assigned to the chloroplast. Several others ('potential cp proteins') were found to be imported into chloroplasts in vitro, but failed to localize to the organelle when RFP was fused to their C-terminal ends. Extrapolations suggest that the fraction of cp proteins that enter the inner compartments of the organelle, although they lack a cTP, might be as large as 11.4% of the total cp proteome. Our data also support the idea that cytosolic proteins that associate with the cp outer membrane might account for false positive cp proteins obtained in earlier studies.