Disease symptoms and schematic presentation of effector cocktail use in different maize organs and tissues infected by <i>U. maydis</i>.

<p>A) Confocal microscopy of a <i>U. maydis</i> strain expressing cytosolic mRFP (yellow arrows) during intracellular growth in epidermal maize cells expressing PIN-YFP as a plasma membrane marker (white arrows). B) <i>U. maydis</i> tumor on field-grown maize plant (picture kindly provided by S. Krombach). C–F depict schematically the different tissues infected by <i>U. maydis</i> (the width of the interaction zone between hyphae and host plasma membrane is not drawn to scale): C) epidermal cell of an infected maize seedling (light green); D) epidermal cell of an infected mature leaf (yellow); E) epidermal cell of infected tassel (orange; F) epidermal cell (light green) and mesophyll cells (dark green) of infected seedling. Core effectors, organ-specific effectors, and cell type–specific effectors with either apoplastic or cytoplasmic function inside plant cells are indicated with different symbols.</p

Strategies for successful host invasion.

<p>Plant-colonizing microbes employ effectors fulfilling various functions during the host invasion, which are visualized symbolically in this cartoon. Different modes of action (self-binding and self-modifying, activating or inhibiting activities) of effectors described in the text may be applied to serve the listed strategies (text on grey oval background).</p

In vivo insertion pool sequencing identifies virulence factors in a complex fungal–host interaction

<div><p>Large-scale insertional mutagenesis screens can be powerful genome-wide tools if they are streamlined with efficient downstream analysis, which is a serious bottleneck in complex biological systems. A major impediment to the success of next-generation sequencing (NGS)-based screens for virulence factors is that the genetic material of pathogens is often underrepresented within the eukaryotic host, making detection extremely challenging. We therefore established insertion Pool-Sequencing (iPool-Seq) on maize infected with the biotrophic fungus <i>U</i>. <i>maydis</i>. iPool-Seq features tagmentation, unique molecular barcodes, and affinity purification of pathogen insertion mutant DNA from in vivo-infected tissues. In a proof of concept using iPool-Seq, we identified 28 virulence factors, including 23 that were previously uncharacterized, from an initial pool of 195 candidate effector mutants. Because of its sensitivity and quantitative nature, iPool-Seq can be applied to any insertional mutagenesis library and is especially suitable for genetically complex setups like pooled infections of eukaryotic hosts.</p></div

iPool-Seq library preparation workflow features tagmentation and UMIs.

<p>(<b>a</b>) Library preparation was carried out for the input mutant collection and for the output after infection. For the output, we harvested infected areas of the second and third maize leaves and isolated gDNA. (<b>b</b>) Extracted gDNA was fragmented with Tn5 Transposase loaded with custom adapters containing an SBS (green), 12-bp UMI, and Tn5 hyperactive MEs (blue). Genome–hpt resistance cassette junctions were PCR-amplified with biotinylated primers directed against UPSs (magenta) and adapter-specific primers directed at the SBS. (<b>c</b>) Biotinylated PCR products were streptavidin-affinity–purified and Illumina-compatible P5 (purple; NGS1) and P7 (purple; NGS2) ends were introduced by nested PCR. Final products were subjected to Illumina PE sequencing on a MiSeq platform. gDNA, genomic DNA; hpt, hygromycin phosphotransferase; iPool-Seq, insertion Pool-Sequencing ME, mosaic end; PE, paired-end; ROI, region of interest; SBS, sequencing primer binding site; UMI, unique molecular identifier; UPS, unique primer binding site.</p

Identification of a conserved phosphorylation motif within RNA metabolic phosphoproteins

<p><b>Copyright information:</b></p><p>Taken from "Phosphoproteomics reveals extensive phosphorylation of Arabidopsis proteins involved in RNA metabolism"</p><p>Nucleic Acids Research 2006;34(11):3267-3278.</p><p>Published online 17 Jul 2006</p><p>PMCID:PMC1904105.</p><p>© 2006 The Author(s)</p> The phosphorylated Ser residue is indicated in boldface. Identity and homology of amino acids are marked by black and gray shades, respectively

SR protein-specific kinase 4 phosphorylates splicing factor RSp31 () Purification of SR protein-specific kinase 4 (SRPK4) and RSp31 from (left panel)

<p><b>Copyright information:</b></p><p>Taken from "Phosphoproteomics reveals extensive phosphorylation of Arabidopsis proteins involved in RNA metabolism"</p><p>Nucleic Acids Research 2006;34(11):3267-3278.</p><p>Published online 17 Jul 2006</p><p>PMCID:PMC1904105.</p><p>© 2006 The Author(s)</p> Autoradiogram of SRPK4 and RSp31 incubated together in a kinase buffer-containing radioactive ATP (right panel). As controls, SRPK4 and RSp31 were separately incubated in the same buffer. The faint signal in the SRPK4 lane represents low autophosphorylation activity of the kinase. GST alone was not phosphorylated at all by SRPK4 (data not shown). () Mass spectra of the RpLpPVYR peptide. The upper panel shows an MS3 spectrum of the peptide of the phosphorylated protein. The lower panel shows an MS3 spectrum of the peptide found in the analysis (). p indicates a phospho-Ser residue; indicates a phospho-Ser residue that has lost its phosphate group

Conservation of phosphorylation sites in homologous proteins from other species and phosphorylation of DExD/H box RNA helicases

<p><b>Copyright information:</b></p><p>Taken from "Phosphoproteomics reveals extensive phosphorylation of Arabidopsis proteins involved in RNA metabolism"</p><p>Nucleic Acids Research 2006;34(11):3267-3278.</p><p>Published online 17 Jul 2006</p><p>PMCID:PMC1904105.</p><p>© 2006 The Author(s)</p> () Alignment of the region surrounding the Arabidopsis SF1-like protein phosphorylation site and related sequences from rice (Os, ), potato (St, ), human (Hs, ) and (Ce) homologs. The two corresponding Ser residues of HsSF1 are phosphorylated as well (,). () Alignment of the phosphosite in At4g17720 and corresponding regions of homologs from the dicot species Arabidopsis (At, ), (Lj), (Mt), tomato (Le, ), and the monocots maize (Zm, ), barley (Hv, ), and rice (Os, ). () Positioning of the phosphorylation sites in the DEAD box RNA helicase RH26. The phosphorylation sites are both conserved in AtRH25, but not in AtRH31. The gray arrow (at RSZ33) represents a pSer also identified in non-phosphorylated form. () Phosphorylation of the DEAH box RNA helicase encoded by At1g32490. One of the two pSer residues are positionally conserved in the human homolog DBP2 and the fission yeast homolog Cdc28/Prp8. DEAD/H, DEAD/H-like helicase domain; HELICc, helicase superfamily c-terminal domain; HA2, Helicase associated domain (PFAM accession no. PF04408), DUF1605, domain of unknown function (PFAM accession no. PF07717). DUF1605 is always found in association with HA2 in helicases