Increased vegetative development and sturdiness of storekeeper-transgenic tobacco

The STOREKEEPER (STK) family of DNA-binding proteins work as transcription factors and the ectopic expression of two stk-like genes from Arabidopsis thaliana, stk01 (At1g61730) and stk03 (At4g00238), in tobacco increased the number of vegetative internodes and promoted plant and leaf size, stem diameter and sturdiness. The development of these plants started with rosette formation while pronounced shoot elongation and flowering was delayed. Moreover, when the STK01 and STK03 proteins were fused to the Herpes Simplex Virus VP16 transcriptional activation domain and expressed in tobacco the vigorous storekeeper-phenotype did not appear indicating that transgenic STK-like proteins in part worked as repressors of tobacco reproductive development. Furthermore, Yeast Two-Hybrid screenings proved that STK01 and STK03 can form homodimers and heterodimers with further members of the STKlike family. Therefore, we assume that interactions between transgenic Arabidopsis STKs and resident tobacco STKs could have contributed to the observed developmental changes in transgenic tobacco. Our findings open up promising applications for overexpression of stk-like genes in crops that benefit from increased sturdiness and vegetative organ development, such as tobacco in molecular farming approaches, biomass-based energy crops and medicinal plants that produce bioactive compounds in leaves

Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis

Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens

The Arabidopsis protein phosphatase PP2C38 negatively regulates the central immune kinase BIK1

Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component

Relocalization of nuclear ALY proteins in the cytoplasm by the Tomato bushy stunt virus P19 pathogenicity protein

The P19 protein of tomato bushy stunt virus (TBSV) is a multifunctional pathogenicity determinant involved in suppression of posttranscriptional gene silencing, virus movement, and symptom induction. Here, we report that P19 interacts with the conserved RNA-binding domain of an as yet uncharacterized family of plant ALY proteins that, in animals, are involved in export of RNAs from the nucleus and transcriptional coactivation. We show that the four ALY proteins encoded by the Arabidopsis genome and two ALY proteins from Nicotiana benthamiana are localized to the nucleus. Moreover, and in contrast to animal ALY, all but one of the proteins are also in the nucleolus, with distinct subnuclear localizations. Infection of plants by TBSV or expression of P19 from Agrobacterium results in relocation of three of the six ALY proteins from the nucleus to the cytoplasm demonstrating specific targeting of the ALY proteins by P19. The differential effects on subcellular localization indicate that, in plants, the various ALY proteins may have different functions. Interaction with and relocalization of ALY is prevented by mutation of P19 at residues previously shown to be important for P19 function in plants. Down-regulation of expression of two N. benthamiana ALY genes by virus-induced gene silencing did not interfere with posttranscriptional gene silencing. Targeting of ALY proteins during TBSV infection may therefore be related to functions of P19 in addition to its silencing suppression activityThis work was supported by the Scottish Executive Environment and Rural Affairs Department and by The Royal Society (ESEP grant no. 12822 to J.F.U. and S.A.M.)Peer reviewe

Relocalization of Nuclear ALY Proteins to the Cytoplasm by the Tomato Bushy Stunt Virus P19 Pathogenicity Protein

The P19 protein of tomato bushy stunt virus (TBSV) is a multifunctional pathogenicity determinant involved in suppression of posttranscriptional gene silencing, virus movement, and symptom induction. Here, we report that P19 interacts with the conserved RNA-binding domain of an as yet uncharacterized family of plant ALY proteins that, in animals, are involved in export of RNAs from the nucleus and transcriptional coactivation. We show that the four ALY proteins encoded by the Arabidopsis genome and two ALY proteins from Nicotiana benthamiana are localized to the nucleus. Moreover, and in contrast to animal ALY, all but one of the proteins are also in the nucleolus, with distinct subnuclear localizations. Infection of plants by TBSV or expression of P19 from Agrobacterium results in relocation of three of the six ALY proteins from the nucleus to the cytoplasm demonstrating specific targeting of the ALY proteins by P19. The differential effects on subcellular localization indicate that, in plants, the various ALY proteins may have different functions. Interaction with and relocalization of ALY is prevented by mutation of P19 at residues previously shown to be important for P19 function in plants. Down-regulation of expression of two N. benthamiana ALY genes by virus-induced gene silencing did not interfere with posttranscriptional gene silencing. Targeting of ALY proteins during TBSV infection may therefore be related to functions of P19 in addition to its silencing suppression activity

Increased vegetative development and sturdiness of storekeeper-transgenic tobacco

The STOREKEEPER (STK) family of DNA-binding proteins work as transcription factors and the ectopic expression of two stk-like genes from Arabidopsis thaliana, stk01 (At1g61730) and stk03 (At4g00238), in tobacco increased the number of vegetative internodes and promoted plant and leaf size, stem diameter and sturdiness. The development of these plants started with rosette formation while pronounced shoot elongation and flowering was delayed. Moreover, when the STK01 and STK03 proteins were fused to the Herpes Simplex Virus VP16 transcriptional activation domain and expressed in tobacco the vigorous storekeeper-phenotype did not appear indicating that transgenic STK-like proteins in part worked as repressors of tobacco reproductive development. Furthermore, Yeast Two-Hybrid screenings proved that STK01 and STK03 can form homodimers and heterodimers with further members of the STKlike family. Therefore, we assume that interactions between transgenic Arabidopsis STKs and resident tobacco STKs could have contributed to the observed developmental changes in transgenic tobacco. Our findings open up promising applications for overexpression of stk-like genes in crops that benefit from increased sturdiness and vegetative organ development, such as tobacco in molecular farming approaches, biomass-based energy crops and medicinal plants that produce bioactive compounds in leaves

MIDGET Unravels Functions of the Arabidopsis Topoisomerase VI Complex in DNA Endoreduplication, Chromatin Condensation, and Transcriptional Silencing[W]

The plant homologs of the archaeal DNA topoisomerase VI complex are required for the progression of endoreduplication cycles. Here, we describe the identification of MIDGET (MID) as a novel component of topoisomerase VI. We show that mid mutants show the same phenotype as rhl1, rhl2, and top6B mutants and that MID protein physically interacts with RHL1. The phenotypic analysis revealed new phenotypes, indicating that topoisomerase VI is involved in chromatin organization and transcriptional silencing. In addition, genetic evidence is provided suggesting that the ATR-dependent DNA damage repair checkpoint is activated in mid mutants, and CYCB1;1 is ectopically activated. Finally, we demonstrate that overexpression of CYCB1;2 can rescue the endoreduplication defects in mid mutants, suggesting that in mid mutants, a specific checkpoint is activated preventing further progression of endoreduplication cycles