Patrick Berndt

Daniel Lanver

Prof. Dr Regine Kahmann
email: kahmann@mpi-marburg.mpg.de

appressorium formation and maintenance of the actin cytoskeleton in the smut fungus Ustilago maydis

Keywords: plant pathogenic fungi, actin cytoskeleton, appressoria, AGC family-kinase, virulence plant pathogenic fungi, AGC family-kinase, appressoria, actin cytoskeleton

The AGC Ser/Thr kinase Aga1 is essential for appressorium formation and maintenance of the actin cytoskeleton in the smut fungus Ustilago maydis

Introduction

Plant-pathogenic fungi need to penetrate the cuticle in order to successfully invade their host plants. This occurs by specialized infection structures, so called appressoria, which either facilitate penetration by secretion of lytic enzymes or the generation of mechanical force [START_REF] Tucker | Surface attachment and pre-penetration stage development by plant pathogenic fungi[END_REF]. After contact with the plant surface, the plant cuticle provides a variety of chemical and physical cues that can be perceived by fungi and induce differentiation and appressorium formation. These include physical stimuli like hydrophobicity, hardness and topography as well as chemical stimuli like ethylene, cutin and epicuticular waxes [START_REF] Kolattukudy | Surface signaling in pathogenesis[END_REF][START_REF] Tucker | Surface attachment and pre-penetration stage development by plant pathogenic fungi[END_REF][START_REF] Doehlemann | Different signalling pathways involving a Galpha protein, cAMP and a MAP kinase control germination of Botrytis cinerea conidia[END_REF][START_REF] Kumamoto | Molecular mechanisms of mechanosensing and their roles in fungal contact sensing[END_REF]. Appressorium formation in plant pathogenic fungi is a complex process and includes changes in cell wall composition [START_REF] Ishida | Electron microscopic observation of cell wall structure during appressorium formation in Colletotrichum lagenarium[END_REF][START_REF] Snetselaar | Infection of maize stigmas by Ustilago maydis: Light and electron microscopy[END_REF][START_REF] Wessels | Developmental regulation of fungal cell wall formation[END_REF][START_REF] Xu | Targeted Cell Wall Degradation at the Penetration Site of Cowpea Rust Basidiosporelings[END_REF] and cytoskeletal organization [START_REF] Kwon | Cytoskeletal organization in Uromyces urediospore germling apices during appressorium formation[END_REF][START_REF] Mendgen | Morphogenesis and mechanisms of penetration by plant pathogenic fungi[END_REF]. A key element in the regulation of these processes is a conserved signaling module consisting of TOR (target of rapamycin, Ser/Thr kinase), PDK (3-phosphoinositidedependent protein kinase-1) and AGC kinase [START_REF] Cybulski | TOR complex 2: a signaling pathway of its own[END_REF]. The TOR kinase is highly conserved in fungi, worms, flies and mammals [START_REF] Cybulski | TOR complex 2: a signaling pathway of its own[END_REF]. Unlike S. cerevisiae and Schizosaccharomyces pombe, which posess two TOR genes, most fungi and higher eukaryotes possess only a single TOR gene [START_REF] Wullschleger | TOR signaling in growth and metabolism[END_REF]. In S. cerevisiae and S. pombe it has been demonstrated 3 that TOR and PDK1 activate a set of AGC-kinases [START_REF] Chen | A pair of putative protein kinase genes (YPK1 and YPK2) is required for cell growth in Saccharomyces cerevisiae[END_REF][START_REF] Roelants | Pkh1 and pkh2 differentially phosphorylate and activate ypk1 and ykr2 and define protein kinase modules required for maintenance of cell wall integrity[END_REF][START_REF] Matsuo | Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases[END_REF].

AGC-kinases are a subgroup of Ser/Thr protein kinases which are most closely related to cAMP-dependent protein kinase 1 (PKA), cGMP-dependent protein kinase (PKG) and protein kinase C (PKC) [START_REF] Hanks | Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification[END_REF][START_REF] Pearce | The nuts and bolts of AGC protein kinases[END_REF]. So far, 14 AGC kinase domain structures have been determined, all showing a bilobal kinase fold first decribed for PKA [START_REF] Knighton | Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase[END_REF][START_REF] Pearce | The nuts and bolts of AGC protein kinases[END_REF]. AGC-kinases have been grouped into 21 subfamilies according to their homology outside the catalytic domain [START_REF] Pearce | The nuts and bolts of AGC protein kinases[END_REF]. Isoforms of the S6K1 (ribosomal protein S6 kinase 1) subfamily in mammalian systems are phosphorylated by TOR at their hydrophobic motif, Phe-X-X-Ar-Ser*/Thr*-Ar (where X represents any amino acid and Ar an aromatic residue), located near the C-terminus [START_REF] Toker | Cellular signaling: pivoting around PDK-1[END_REF][START_REF] Vanhaesebroeck | The PI3K-PDK1 connection: more than just a road to PKB[END_REF]. While this alone is not sufficient for activation, it stimulates phosphorylation of a Thr*-Phe-Cys-Gly-Thr-X-Glu-Tyr motif in the activation loop by PDK1 [START_REF] Hanks | Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification[END_REF][START_REF] Biondi | The PIFbinding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB[END_REF].

S. cerevisiae possesses two S6K1-kinases, Ypk1p [START_REF] Maurer | Isolation of a yeast protein kinase gene by screening with a mammalian protein kinase cDNA[END_REF] and Ypk2p [START_REF] Kubo | A novel yeast gene coding for a putative protein kinase[END_REF], while in S. pombe Gad8p is the only kinase of this type [START_REF] Matsuo | Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases[END_REF]. In S. cerevisiae phosphorylation of Ypk2p by Tor2p is required for actin organization and regulation of cell wall integrity [START_REF] Helliwell | TOR2 is part of two related signaling pathways coordinating cell growth in Saccharomyces cerevisiae[END_REF][START_REF] Kamada | Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization[END_REF] while TOR1p dependent activation of Ypk1p is required for the regulation of cell growth [START_REF] Yu | A survey of essential gene function in the yeast cell division cycle[END_REF]. In the fungal pathogen Colletotrichum trifolii, the causal agent of anthracnose in alfalfa (Medicago sativa L.), the AGC kinase lipk was identified as being induced by long-chain fatty acids. lipk mutants failed to develop appressoria and were unable to cause disease in intact host tissue [START_REF] Dickman | A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation[END_REF]. [START_REF] Dickman | A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation[END_REF] speculate that this lipk mediated pathway is necessary to induce differentiation of appressoria in response to components of the plant cuticle.

In Ustilago maydis, the causative agent of the corn smut disease, infection related development occurs after two compatible haploid sporidia fuse on the leaf surface and form the filamentous dikaryon. The older parts of filaments are septated and appear empty and only the tip cell contains cytoplasm [START_REF] Snetselaar | Sporidial fusion and infection of maize seedlings by the smut fungus Ustilago maydis[END_REF]. The tip cell eventually differentiates an appressorium leading to plant penetration and colonization [START_REF] Feldbrugge | Regulation of mating and pathogenic development in Ustilago maydis[END_REF][START_REF] Doehlemann | Establishment of compatibility in the Ustilago maydis/maize pathosystem[END_REF]Mendoza-Mendoza et al., 2009). In contrast to the dome shaped melanized appressoria of Magnaporthe oryzae or Colletotrichum spp. that penetrate by high turgor pressure (Howard et [START_REF] Bechinger | Optical measurements of invasive forces exerted by appressoria of a plant pathogenic fungus[END_REF], appressoria of U. maydis are unmelanized and are proposed to penetrate the cuticle by secretion of plant cell wall degrading enzymes [START_REF] Schirawski | Endoplasmic reticulum glucosidase II is required for pathogenicity of Ustilago maydis[END_REF]. Two plant-derived stimuli, a hydrophobic surface and hydroxy-fatty acids induce appressorium formation in U. maydis [START_REF] Mendoza-Mendoza | Physical-chemical plant-derived signals induce differentiation in Ustilago maydis[END_REF]. While perception of the hydrophobic signal is likely to involve two membrane proteins related to S. cerevisiae Sho1p and Msb2p [START_REF] Lanver | Sho1 and Msb2-related proteins regulate appressorium development in the smut fungus Ustilago maydis[END_REF], the sensor for hydroxy-fatty acids is unknown. Based on the observation in C. trifolii, that cutin monomers induce lipk expression and differentiation of appressoria [START_REF] Dickman | A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation[END_REF] we wanted to investigate, whether AGC-kinases are also involved in the perception of plant-derived hydroxy-fatty acids in U. maydis. One AGC-kinase, Ukb1, has already been characterized in U. maydis. ukb1 mutants show irregularities in bud emergence and fail to develop tumors but show no phenotype in mating and are still able to differentiate functional appressoria [START_REF] Abramovitch | The ukb1 gene encodes a putative protein kinase required for bud site selection and pathogenicity in Ustilago maydis[END_REF][START_REF] Aichinger | Identification of plant-regulated genes in Ustilago maydis by enhancer-trapping mutagenesis[END_REF], ruling out a role in perception of plant stimuli.

Here we characterize aga1, the only S6K1-type AGC-kinase of U. maydis. We demonstrate that aga1 controls growth and appressorium formation in U. maydis through its effects on the actin cytoskeleton.

Results

Aga1 is a S6K1-AGC kinase

Aga1 (GenBank XM_751538.1) was identified in the U. maydis genome by homology to the fungal S6K1 Ypk1p and Ypk2p from S. cerevisiae [START_REF] Maurer | Isolation of a yeast protein kinase gene by screening with a mammalian protein kinase cDNA[END_REF][START_REF] Chen | A pair of putative protein kinase genes (YPK1 and YPK2) is required for cell growth in Saccharomyces cerevisiae[END_REF], LIPK from C. trifolii [START_REF] Dickman | A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation[END_REF] and Gad8p from S. pombe [START_REF] Matsuo | Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases[END_REF]. The aga1 gene contains a 79 bp intron and encodes a 577 amino acid protein (http://mips.helmholtz-muenchen.de/genre/proj/ustilago/). Aga1 has all characteristics of S6K1-type AGC-kinases and shows high amino acid identity to the central catalytic domains of Gad8p (81%), Ypk1p (76%), Ypk2p (74%) and human S6K1 (51%) (Fig. S1A andS1B). In Aga1 and other family members the region following the kinase domain also shows amino acid conservation, but to a lesser extent than the kinase domain itself (Fig. S1B). The kinase domains are preceded by non-conserved N-terminal regions differing in size in the various members of this family. To become active, members of the AGC kinase family need to undergo phosphorylation at three conserved sites, the activation loop, the turn motif and the hydrophobic motif [START_REF] Matsuo | Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases[END_REF][START_REF] Pearce | The nuts and bolts of AGC protein kinases[END_REF]. In Aga1 theses sites correspond to Thr403, Ser542 and Ser562 respectively (Fig. S1C).

S. cerevisiae ypk1 mutants display reduced growth while ypk2 mutants show no apparent phenotype [START_REF] Chen | A pair of putative protein kinase genes (YPK1 and YPK2) is required for cell growth in Saccharomyces cerevisiae[END_REF]. Since aga1 deletion mutants in U. maydis also displayed a growth phenotype (see below), ypk1 was considered to be the most likely ortholog of aga1. To test this, aga1 was expressed under the control of the gal promoter in the ypk1 deletion strain Y04976. As control Y04976 and its progenitor strain Y00000 were transformed with the empty plasmid pGREG536. Immunoblot analysis showed that U. maydis Aga1 was expressed in Y04976-pAga1 (Fig. 1A) and Aga1 could complement the growth phenotype of Y04976 (Fig. 1B). This demonstrates that U. maydis aga1 can functionally replace ypk1 in S. cerevisiae.

aga1 expression is not induced by plant-derived stimuli

To see whether transcription of aga1 can be induced by fatty-acids, SG200 was exposed to an inductive hydrophobic surface in the presence or absence of 16hydroxy-hexadecanoic acid, a cutin monomer with a strong inducing effect on appressorium formation [START_REF] Mendoza-Mendoza | Physical-chemical plant-derived signals induce differentiation in Ustilago maydis[END_REF]. aga1 expression was then monitored by quantitative real-time PCR. No significant changes in aga1 expression could be detected compared to the expression after incubation on a hydrophilic glass surface (Fig. S2A). In line with this, no significant change in aga1 expression could be observed during the differentiation of appressoria after 18 hours on the leaf surface (Fig S2B). This shows that in contrast to lipk [START_REF] Dickman | A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation[END_REF], aga1 expression is not triggered by these surface cues.

aga1 has a role in growth, perception of pheromone and stress resistance

To study the function of aga1 in U. maydis, deletion mutants were generated by replacement of the open reading frame with a hygromycin resistance cassette in the solopathogenic strain SG200 as well as in the haploid strains FB1 and FB2. In addition SG200∆aga1 was complemented by introducing an aga1-mcherry-HA fusion gene under the control the aga1 promoter in single copy into the ip locus [START_REF] Loubradou | A homologue of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis[END_REF], yielding strain SG200∆aga1-C. Expression of the full length Aga1-mCherry-HA fusion protein could be verified by immunoblot analysis (Fig. S3). On solid CM-glucose medium, SG200∆aga1 colonies were significantly reduced in growth and displayed a light brown color that was not observed in SG200 colonies (Fig. 2A). The nature of this brown pigment is currently unknown. In SG200∆aga1-C colony diameter and color were comparable to SG200, which illustrates that the Aga1-mCherry-HA fusion protein is able to complement the growth phenotype of the aga1 mutant strain. Growth curves in liquid YEPSL medium revealed that the SG200∆aga1-C strain was slightly attenuated in growth compared to SG200 (Fig. 2B), suggesting that complementation was incomplete, most likely because the fusion protein had reduced activity. In the same medium, 17.8 ± 3.4% of the SG200∆aga1 formed aggregates of three or more cells, while this was observed in only 1.8 ± 0.3% of the SG200 cells. In the haploid strains we could also observe a higher frequency of aggregates in FB1∆aga1 (29 ± 2.6%) compared to only 2.3 ± 1.5% of the FB1 cells (Fig. S4A).

When spotted on charcoal containing medium, SG200 responds with formation of filaments, a process depending on autocrine pheromone stimulation [START_REF] Di Stasio | The dual specificity phosphatase Rok1 negatively regulates mating and pathogenicity in Ustilago maydis[END_REF]. In SG200∆aga1 formation of the filaments was seen (Fig. 2C) but their appearance was delayed (not shown). When compatible strains FB1∆aga1 and FB2∆aga1 were mated by co-spotting on charcoal containing media the formation of dikaryotic hyphae was again delayed and became evident only after 48 h while the respective wild-type mixtures showed dikaryotic filaments already after 24 h (Fig. S5A). To follow this up, haploid FB1 and FB1∆aga1 cells were treated with a2 pheromone and the formation of conjugation hyphae was determined after 2 and 3 hours of incubation. While 26 ± 7.8% and 41.5 ± 4.6% of FB1 cells showed conjugation hyphae after 2 h and 3 h, respectively, in FB1∆aga1 only 5 ± 1.7% and 10.5 ± 1.8% of cells, respectively, had formed conjugation hyphae at these time points (Fig. S5B). In line with this, stimulation of FB1 and FB1∆aga1 with synthetic a2 pheromone and subsequent analysis of the expression of the pheromone precursor gene mfa1 revealed a 50% decrease of mfa1 expression in FB1∆aga1 compared to FB1 (Fig S5C ), while the basal levels of mfa1 expression in DMSO treated FB1 and FB1∆aga1 cells were not significantly different (Fig S5C). Taken together, this indicates that aga1 plays a role in pheromone perception, but is not an essential component in the mating reaction.

To further characterize the phenotype of ∆aga1, we tested growth under different stress conditions i.e. high/low temperatures, osmotic as well as cell wall stress. SG200 and SG200∆aga1 were spotted on solid media (Fig. 2A andS6). Compared to SG200, SG200∆aga1 displayed slightly enhanced sensitivity towards the cell wall stressors congo red (100µM) and calcofluor (75µM) (Fig. 2A) and towards osmotic stress (Fig. S6). In addition, growth of SG200∆aga1 at 22°C and 34°C was more significantly affected than at 28°C (Fig. S6). These results show that aga1 does not only contribute to growth and pheromone perception but also affects resistance to cell wall and temperature stress.

Aga1 is essential for appressorium formation and pathogenicity

To determine whether aga1 is required for pathogenic development of U. maydis, seven-day-old maize plants were infected with SG200, SG200∆aga1 and SG200∆aga1-C. Disease symptoms were scored 12 days after infection according to severity [START_REF] Kamper | Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis[END_REF] and averages from three independent experiments are shown. The deletion of aga1 resulted in the complete loss of tumor formation (Fig. 3A). The only symptoms detectable in plants inoculated with SG200∆aga1 were small chlorotic spots in about 30% of the infected plants (Fig. 3B). In contrast, plants inoculated with SG200 showed symptoms like anthocyanine accumulation and tumor formation in about 60% of the infected plants (Fig. 3A andB). Virulence of SG200∆aga1-C was not significantly different (p>0.18) to SG200 in any of the disease categories, indicating successful complementation (Fig. 3A). Disease symptoms were also monitored 21 d after infection with SG200∆aga1 without revealing any symptoms (not shown). Compatible aga1 deletion strains FB1∆aga1 and FB2∆aga1 also failed to cause disease while a mixture of wild-type strains FB1 and FB2 induced tumors in about 70% of the infected plants (Fig. S5D).

To examine at which point pathogenic development of SG200∆aga1 is impaired, leaves inoculated with either SG200 or SG200∆aga1 were analysed by confocal microscopy after staining fungal material with WGA-AF488 and the plant cell wall with propidium iodide, respectively [START_REF] Doehlemann | Establishment of compatibility in the Ustilago maydis/maize pathosystem[END_REF]. While strong colonization of the plant tissue could be observed in plants inoculated with SG200 after three days (Fig. 3C), hardly any fungal material could be found inside plant Strong colonization of the plant tissue could be observed 10 days after inoculation with SG200, while no colonization could be detected in SG200∆aga1 infected plants (Fig 3C,lower panels). This rules out that plant colonization by aga1 mutants is simply delayed. We take this as an indication that SG200∆aga1 has a strong defect in cuticle penetration and subsequent colonization of the plant tissue.

To investigate whether the aga1 mutant forms infection structures, appressorium formation was quantified on the leaf surface and also analyzed under previously established in vitro conditions [START_REF] Mendoza-Mendoza | Physical-chemical plant-derived signals induce differentiation in Ustilago maydis[END_REF]. To facilitate quantification, strains carrying the AM1-marker gene construct (Mendoza-Mendoza et al., 2009) were generated. The AM1-marker shows GFP expression exclusively in those tip cells of filaments that differentiate an appressorium (Mendoza-Mendoza et al., 2009). Appressorium formation by U. maydis is preceded by formation of a characteristic crook-like structure (Fig. 4; [START_REF] Snetselaar | Sporidial fusion and infection of maize seedlings by the smut fungus Ustilago maydis[END_REF]. Under in vitro conditions as well as on the leaf surface SG200AM1 formed appressoria expressing the GFP-reporter gene and on the leaf surface this could be observed in 15.33 ± 2.52% of the filaments 18 h after inoculation. In strain SG200AM1∆aga1 the formation of septated filaments could be observed, but these filaments were morphologically altered, the tip cells displayed characteristic zigzag sections as well as wavy sections and a change in growth direction towards the surface could not be observed (Fig. 4A and4B). On the leaf surface SG200AM1∆aga1 cells expressed the GFP reporter in comparable frequency to SG200AM1, however, in stark contrast to SG200AM1, the mutant hyphae did not show the characteristic tip swellings and failed to develop appressoria (Fig. 4C). Consequently, SG200AM1∆aga1 hyphae could only rarely be found inside epidermal cells (Fig. 4C).

To determine whether the penetration defect of SG200∆aga1 is caused by a reduced capability of the mutant to secrete cutinolytic enzymes, esterase activity was assayed in supernatants of SG200 and SG200∆aga1 cells that were incubated under the in vitro conditions for filamentation and appressoria formation. We could not detect any significant differences in esterase activity between the two strains incubated either on hydrophilic glass, ParafilmM or ParafilmM plus 100µM 16-hydroxy hexadecanoic acid (Fig. S7). The phosphorylation sites in the activation loop, the hydrophobic-and the turn-motif are conserved in Aga1 and other S6K1-kinases (Fig. S1C). To examine the role of the phosphorylation site in the activation loop, which is supposedly a target of PDK1-like kinases, and the putative TOR phosphorylation sites in the hydrophobic and the turn motifs, respectively, we generated SG200 derivatives containing mutated alleles.

SG200aga1-GFP, harboring an aga1-GFP fusion gene in the aga1 locus, displayed full virulence when compared with SG200 (Fig. 5A), and this strain served as control.

In SG200aga1T403A-GFP, Thr403 of Aga1 was replaced by Ala and in SG200aga1S542AS562A-GFP Ser542 and Ser562 of Aga1 were replaced by Ala. In both cases the mutated genes were expressed as C-terminal GFP fusions. In growth tests on CM-glucose, SG200aga1T403A-GFP showed only a mild growth reduction compared to SG200aga1-GFP while SG200aga1S542AS562A-GFP showed reduced growth comparable to the aga1 deletion strain (Fig. S7). Under cell wall (congo red) and osmotic stress conditions SG200aga1T403A-GFP was even more sensitive than SG200aga1S542AS562A-GFP (Figs 5A and S8) while both mutants showed similar sensitivity to cell wall stress inflicted by calcofluor (Fig. 5A). This suggests that phosphorylation at Thr403 of Aga1 is important to develop resistance against cell wall and osmotic stress, while phosphorylation at Ser542 and Ser562 contributes to growth on complete medium.

To study the role of the phosphorylation sites during pathogenic development, plant infections were carried out with strains SG200, SG200aga1-GFP, SG200aga1T403A-GFP and SG200aga1S542AS562A-GFP. SG200 and SG200aga1-GFP exhibited no significant differences in virulence in any of the seven disease categories (p>0.38, Fig. 5B). None of the strains carrying point mutations was able to cause tumor formation or other disease symptoms except mild chlorosis (Fig. 5B) and all mutant strains failed to induce anthocyanine (not shown). We take this as evidence, that the conserved phosphorylation sites are essential for the function of Aga1 during pathogenesis. For S. cerevisiae it was reported that the Aga1 homolog Ypk2p is phosphorylated by Tor2p to regulate actin polarization [START_REF] Kamada | Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization[END_REF]. To examine the role of Aga1 in actin distribution in U. maydis, we visualized actin by immunofluorescence in budding cells of FB1 and FB1∆aga1. Actin in FB1 cells was predominantly detectable in patches at the bud tip in the region of active cell wall growth and could also be weakly detected throughout the cell (Fig. 6) as described previously [START_REF] Banuett | Bud morphogenesis and the actin and microtubule cytoskeletons during budding in the corn smut fungus, Ustilago maydis[END_REF]. In FB1∆aga1 the actin signal appeared hyperpolarized and could be detected only in a narrow area close to the bud tip (Fig. 6). In filaments of SG200 induced by exposure to a hydrophobic surface, actin was primarily localized cortically in the tip cell of the filament (Fig. S9A). Unfortunately, the immunofluorescence method applied here failed to detect actin in appressoria (Fig. S9A). In SG200∆aga1 filaments that displayed zigzag and wavy growth zones, i.e. those filaments that also express the AM1 marker (Fig. 4), cortical actin could be detected in the tip cell excluding the segment displaying wavy growth (Fig. S9B). We take this as indication that appressoria and the wavy segments of aga1 mutant filaments have similar properties with respect to the actin recognition by the antibody.

Aga1 is required for actin-mediated endocytosis

To investigate the role of the actin cytoskeleton during appressorium formation, we used strain SG200AM1 and induced appressorium formation in vitro on a hydrophobic surface in the presence of 1 µM Latrunculin A (LatA). LatA inhibits repolymerization of monomeric actin into filaments [START_REF] Morton | Latrunculin alters the actin-monomer subunit interface to prevent polymerization[END_REF]. The low concentration of LatA was chosen because filament formation in SG200AM1 was not impaired, while higher concentrations of 50 µM and higher lead to complete depolymerization of F-actin and hence inhibition of filament formation [START_REF] Weber | A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis[END_REF]. SG200AM1 treated with DMSO as solvent control expressed the appressorial marker in 7.9 ± 2.5% of the tip cells and of those 96 ± 3.9% had differentiated an appressorium. Cells that were treated with LatA showed expression of the AM1 marker at a rate not significantly different (p = 0.13) from control cells (13.33 ± 4%) but of these only 9.4 ± 2.8% had differentiated an appressorium (Fig. 7). This demonstrates the importance of F-actin in appressorium formation and suggests that Aga1 has a crucial role in actin organization during this differentiation step.

Since frequent formation of cell clusters was observed in FB1∆aga1 during budding growth in liquid medium (Fig. S4A) we tested whether this is caused by disturbed actin polymerization. To this end we treated FB1 cells with 1 µM LatA for 8 h. Microscopic quantification showed that 7.5 ± 0.9% of the FB1 cells treated with 11 DMSO as solvent control formed clusters, while this could be observed in 38.9 ± 1.5% of the cells treated with LatA (Fig. S4B). To reinforce that aga1 mutant strains display an impaired function of the actin cytoskeleton, we tested actin dependent endocytosis by visualizing uptake of the membrane selective dye FM4-64 [START_REF] Geli | Endocytic internalization in yeast and animal cells: similar and different[END_REF][START_REF] Yamashita | Constitutive activation of endocytosis by mutation of myoA, the myosin I gene of Aspergillus nidulans[END_REF]. 20 min after addition of FM4-64 to strain FB1 the fluorescent dye was detectable mainly in the tonoplast of FB1 cells, while in FB1∆aga1 cells fluorescence localized to the plasma membrane and the cytosol (Fig. S10). When depolymerization of actin filaments in FB1 cells was induced with LatA (50 µM) 5 min prior to addition of FM4-64, FM4-64 localized to the plasma membrane and the cytosol, resembling the localization in FB1∆aga1 cells (Fig. S10).

We take this as evidence for an impaired function of the actin cytoskeleton in aga1 deletion strains.

Discussion

In this communication we have shown, that the AGC-kinase Aga1 is an essential virulence factor of U. maydis. Besides its role in growth, mating and resistance to cell wall stress, Aga1 is essential for the formation of appressoria most likely by regulating actin polymerization.

The slow growth phenotype of U. maydis ∆aga1 cells in rich medium could indicate that Aga1 has a role in nutrient sensing or regulation of protein biosynthesis, as demonstrated for S. cerevisiae Ypk1p [START_REF] Gelperin | Loss of ypk1 function causes rapamycin sensitivity, inhibition of translation initiation and synthetic lethality in 14-3-3-deficient yeast[END_REF]. Additionally, based on the finding that aga1 deletion mutants displayed enhanced sensitivity towards calcofluor and congo red, Aga1 contributes to the maintenance of cell wall integrity.

Point mutations in the putative TOR phosphorylation sites in Aga1 phenocopied the aga1 deletion phenotype with respect to growth and response to cell wall stress, indicating that the S542 and S562 phosphorylation sites are essential for function of Aga1. Interestingly, mutating the putative PDK1 phosphorylation site only weakly affected cell growth, suggesting that phosphorylation of T403 is not needed for normal growth. However, sensitivity to cell wall stress in this mutant was enhanced and exceeded sensitivity observed in the aga1 mutant strain. It is known that S6K1 kinases need to be phosphorylated both at the hydrophobic motif and the activation loop to become fully activated and kinase activity depends specifically on phosphorylation of the threonine at the activation loop [START_REF] Matsuo | Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases[END_REF][START_REF] Pearce | The nuts and bolts of AGC protein kinases[END_REF]. Furthermore, kinase dead mutants of Ypk1p exhibit a dominant negative [START_REF] Tanoue | The requirement for the hydrophobic motif phosphorylation of Ypk1 in yeast differs depending on the downstream events, including endocytosis, cell growth, and resistance to a sphingolipid biosynthesis inhibitor, ISP-1[END_REF]. Therefore, the enhanced sensitivity of SG200Aga1T403A-GFP to cell wall stress is likely to result from a dominant negative effect of the aga1T403A allele.

In budding cells of the aga1 deletion strain actin appeared hyperpolarized indicating a defect in actin distribution. Furthermore, a defect in actin mediated endocytosis in aga1 mutants was evident after staining with the endocytotic marker FM4-64. In U. maydis constant actin-mediated endocytosis of the Pra1 receptor is essential for perception of the a2 pheromone [START_REF] Fuchs | Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis[END_REF]. Therefore, the impaired endocytosis seen in aga1 deletion strains could provide an explanation for the attenuated response to pheromone, reduced induction of mfa1 expression after pheromone stimulation and the delayed mating reaction observed. Additionally, budding ∆aga1 cells grown in liquid media showed a strong cell separation defect. Such a morphological alteration was not reported for S. pombe gad8, S. cerevisiae ypk1 and the lipk mutant of C. trifolii [START_REF] Chen | A pair of putative protein kinase genes (YPK1 and YPK2) is required for cell growth in Saccharomyces cerevisiae[END_REF][START_REF] Dickman | A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation[END_REF][START_REF] Matsuo | Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases[END_REF]. In U. maydis this phenotype could be copied by addition of low doses of LatA, strongly suggesting that an impairment of the actin cytoskeleton is responsible for this defect in aga1 mutants. In S. cerevisiae Ypk2p has been reported to regulate actin polarization [START_REF] Kamada | Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization[END_REF][START_REF] Cybulski | TOR complex 2: a signaling pathway of its own[END_REF]. Since the actin cytoskeleton also plays a decisive role in the polar transport of secretory vesicles [START_REF] Weber | A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis[END_REF][START_REF] Fuchs | Endocytosis is essential for pathogenic development in the corn smut fungus Ustilago maydis[END_REF], disturbances could affect cell wall biogenesis and lead to susceptibility towards cell wall interfering agents. Studies in S.

pombe have suggested that the actin cytoskeleton controls secretion of β-1,6-glucan and other cell wall components [START_REF] Osumi | Dynamics of cell wall formation in fission yeast, Schizosaccharomyces pombe[END_REF]. Insufficient delivery of secretory vesicles containing cell wall modifying enzymes to the sites of cell separation could also account for the cell separation defect observed in aga1 mutants. Since we could not observe differences in secreted esterase activity in SG200 and SG200∆aga1 strains the cell wall defects of aga1 mutants might result from specific defects in the secretion of cell wall components rather than a general secretion defect.

The most striking phenotype of the aga1 deletion mutant, as well as of mutants in the phosphorylation motifs, was their inability to differentiate appressoria and cause disease. This phenotype is reminiscent to the phenotype of lipk mutants in C. trifolii. However, C. trifolii lipk deletion mutants did not form morphologically aberrant structures [START_REF] Dickman | A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation[END_REF]. In filaments of U. maydis aga1 deletion strains we 13 observed a zigzag growth pattern which we consider likely to result from several crooking attempts. This is followed by a wavy growth pattern which we consider to result from repeated attempts to make appressoria, as discussed below. Following this aberrant growth zone normal filamentous growth was often resumed and these hyphae failed to penetrate the cuticle. In rare cases, penetration of the leaf surface could be observed but in these cases hyphae became arrested immediately after penetration.

This could indicate that the secretion machinery which is required for the delivery of effectors suppressing plant defense responses [START_REF] Doehlemann | Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells[END_REF] or enzymes required for fungal cell wall modification [START_REF] Wu | A fungal kinesin required for organelle motility, hyphal growth, and morphogenesis[END_REF][START_REF] El Gueddari | Developmentally regulated conversion of surface-exposed chitin to chitosan in cell walls of plant pathogenic fungi[END_REF][START_REF] Tucker | Common Genetic Pathways Regulate Organ-Specific Infection-Related Development in the Rice Blast Fungus[END_REF] is impaired in aga1 mutants. Such a wavy growth phenotype has been observed in other fungi like Neurospora crassa and Aspergillus nidulans where it could be linked to defects in either the actin or microtubule cytoskeleton or the secretory pathway [START_REF] Mcgoldrick | myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth[END_REF][START_REF] Wu | A fungal kinesin required for organelle motility, hyphal growth, and morphogenesis[END_REF][START_REF] Seiler | The genetic basis of cellular morphogenesis in the filamentous fungus Neurospora crassa[END_REF]. The observed arrest in epidermal cells in rare instances of colonization by the aga1 mutant may again result from the inability to differentiate appressoria-like structures required for crossing into neighboring cells [START_REF] Doehlemann | Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells[END_REF].

Actin is an integral part of the cytoskeleton and during appressorium formation reorganization of the actin cytoskeleton could be shown in the phytopathogenic fungi Uromyces appendiculatus, and Magnaporthe grisea [START_REF] Kwon | Cytoskeletal organization in Uromyces urediospore germling apices during appressorium formation[END_REF][START_REF] Bourett | Actin in penetration pegs of the fungal rice blast pathogen, Magnaporthe grisea[END_REF]. By immunoflourescence we have demonstrated a cortical localization of actin in filamentous cells differentiating appressoria; however, actin was undetectable in appressorial structures. This could reflect that actin within the appressorium is not accessible to the antibody or that actin is in a conformation in which it is not recognized by the antibody. Since ∆aga1 filaments showed cortical actin accumulation but did not stain with the antibody in the region of wavy growth while cortical staining was seen again in the outgrowing tip region, we consider the wavy region to result from successive attempts to make appressoria. However, the final conversion into the differentiated form is not possible in the absence of Aga1.

Since LatA at low concentration inhibited appressorium formation but not filamentous growth, we propose that the defect in the aga1 mutant to differentiate appressoria is also caused by impaired actin polymerization. The demonstration that cell wall integrity is disturbed in aga1 mutants makes it conceivable that actin dependent cell Based on the assertion that the appressorial differentiation process is initiated in aga1 mutants we suggest that Aga1 plays a prominent role during appressorium maturation.

In line with this we observed that the appressorium specific AM1 marker was expressed in the aga1 deletion strain and the percentage of cells that expressed this marker was comparable to wild-type. This leads us to conclude that the perception of the plant derived stimuli, i.e. hydrophobicity and hydroxy fatty acids, which are transduced through the pathogenesis-related MAP kinase module [START_REF] Mendoza-Mendoza | Physical-chemical plant-derived signals induce differentiation in Ustilago maydis[END_REF] occurs independently of Aga1.

This study has demonstrated a crucial role of the actin cytoskeleton in appressorium differentiation in U. maydis. At the same time it has demonstrated the current limitations that prevent a detailed analysis of the cytoskeletal dynamics in differentiating appressoria. In U. maydis a direct visualization of actin by application of labeled phalloidine is not possible while immunofluorescence allows to localize actin in budding cells [START_REF] Banuett | Bud morphogenesis and the actin and microtubule cytoskeletons during budding in the corn smut fungus, Ustilago maydis[END_REF] but fails to locate actin in appressorial structures. It is evident that these problems, which are not U. maydis specific [START_REF] Howard | Breaking and entering: host penetration by the fungal rice blast pathogen Magnaporthe grisea[END_REF][START_REF] Czymmek | Live-cell imaging of tubulin in the filamentous fungus Magnaporthe grisea treated with anti-microtubule and anti-microfilament agents[END_REF], need to be resolved to visualize how plant pathogenic fungi reorganize their cytoskeleton during appressorium differentiation and penetration into the host tissue.

Experimental procedures

Strain construction and growth conditions

The Escherichia coli strains DH5α (Bethesda Research Laboratories) and Top10 (Invitrogen) were used for cloning purposes. The S. cerevisiae strains Y00000 and Y04976 (Euroscarf, Frankfurt, Germany) were used for complementation studies. U. maydis strains were grown in liquid YEPSL (0.4% yeast extract, 0.4% peptone, 2 % sucrose) or on solid potato dextrose (PD) plates. For mating assays and filament induction PD plates containing 1% activated charcoal were used [START_REF] Holliday | Ustilago maydis[END_REF]. For growth assays CM glucose medium was used [START_REF] Holliday | Ustilago maydis[END_REF]. Stress assays were performed as described by [START_REF] Lanver | Sho1 and Msb2-related proteins regulate appressorium development in the smut fungus Ustilago maydis[END_REF]. Haploid U. maydis strains FB1 and FB2, solopathogenic strains SG200 and SG200AM1 have been described previously [START_REF] Banuett | Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis[END_REF][START_REF] Kamper | Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis[END_REF][START_REF] Mendoza-Mendoza | Physical-chemical plant-derived signals induce differentiation in Ustilago maydis[END_REF]. For gene disruptions, the PCR strategy described by Kämper (2004) and the SfiI insertion cassette system [START_REF] Brachmann | A reverse genetic approach for generating gene replacement mutants in Ustilago maydis[END_REF] were used. All gene replacement constructs were sequenced after cloning.

For the generation of aga1 deletion mutants, a 1.1 kb and 1.3 kb fragment containing the 5'-and the 3'-flanking region of the aga1 gene respectively were amplified by PCR using FB1 genomic DNA as template with the primer combinations 10123LBFW (5'-ATGCTGCTAGTCGTGATTGG-3') and 10123LBRV (5'-TTCGGCCATCTAGGCCGTCGGAGCACGAGGTGATTG-3') for the left border and 10123RBFW (5'-CACGGCCTGAGTGGCCCAGACGCACGAGAAGCATAC-3') and 10123RBRV (5'-CGTTCTCCACACGCCAATTC-3') for the right border, respectively. The PCR fragments were digested with SfiI and ligated to the hygromycin resistance cassette isolated as 2.7 kb SfiI fragment from plasmid pMF1-h [START_REF] Brachmann | A reverse genetic approach for generating gene replacement mutants in Ustilago maydis[END_REF]. The ligation product served as template for a subsequent PCR amplification of the deletion construct using the primer combination 10123LBFW1 (5'-ACCGTCGTGAGTCGTAAGTG-3') and 10123RBRV1 (5'-GTGCTTTACGTCCCGAATCC-3'). The obtained PCR product was then cloned into PCRII-TOPO (Invitrogen) to generate p∆aga1-hyg. From the plasmid the deletion construct was amplified with primers 10123LBFW1 and10123RBRV1 and transformed into SG200AM1. To create noursethricin resistant deletion mutants in SG200, FB1 and FB2, the resistance cassette was changed in p∆aga1-hyg by SfiI digestion and ligation of a 1.4 kb SfiI fragment from plasmid pMF1-n creating the plasmid p∆aga1-nat. Deletion mutants were identified by Southern-blot analysis.

To complement SG200∆aga1, a 1.2 kb fragment corresponding to the promoter of aga1 and the 1.8 kb ORF was amplified from FB1 genomic DNA using the primer combination P10123SfiIFW(5'-ATAGGCCTGAGTGGCCCAGAATGTGCTCCAAGTTGAAGCGC-3') and 10123SfiIRV

(5'-TATGGCCGCGTTGGCCGCGTCTCTCAAGCTCTCGCCTGCACC-3'), subsequently digested with SfiI and ligated into the 5.4 kb, SfiI-opened plasmid pUNG, a derivative of pONG, lacking the otef promoter region [START_REF] Lanver | Sho1 and Msb2-related proteins regulate appressorium development in the smut fungus Ustilago maydis[END_REF].

In the resulting plasmid pP aga1 :aga1-mCherryHA aga1 is fused to mcherryHA. The plasmid was digested with SspI and integrated in single copy into the ip locus of SG200∆aga1 [START_REF] Loubradou | A homologue of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis[END_REF]. To generate strain SG200otefmCherryHA, p123mCherryHA carrying the constitutive otef promoter [START_REF] Spellig | Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis[END_REF] fused to mCherryHA was constructed. Using primers oDL114 (5'-AATACCATGGTGAGCAAGGGCGAGGAGG-3') and oDL115 (5'-TATGCGGCCGCTTTAAGCGTAATCTGGAACATCGTATGGGTA-3') and pONG [START_REF] Lanver | Sho1 and Msb2-related proteins regulate appressorium development in the smut fungus Ustilago maydis[END_REF] as template a 0.7 kb product containing the mcherryHA gene was amplified and digested with NcoI and NotI. This fragment was ligated into the NcoI and NotI sites of p123 [START_REF] Aichinger | Identification of plant-regulated genes in Ustilago maydis by enhancer-trapping mutagenesis[END_REF].

p123otefmCherryHA was digested with SspI and subsequently integrated into the ip locus of SG200.

To generate the plasmids pAga1-GFP, pAga1TA-GFP and pAga1SSA-GFP two flanks were amplified from FB1 genomic DNA by PCR. One flank contains a 1 kb fragment extending from bp 707 until the end of the aga1 ORF excluding the stop codon and was amplified using primers 10123FW707

(5'-CGCTCAACATGGACGCCTTTGAAC-3') and 10123SfiIRV

(5'-TATGGCCGCGTTGGCCGCGTCTCTCAAGCTCTCGCCTGCACC-3'). The second 1.1 kb flank contains the intergenic region between aga1 and um10124 and a 787 bp part of the ORF of um10124. It was amplified using primer combination 10123ilSfiIFW

(5'-GTGGGCCTGAGTGGCCGACGCACGAGAAGCATACAGTATCC-3') and 10123ilRV (5'-TCTGGCCGATCAGTTGAGCTGTGTAG-3'). Both flanks were digested with SfiI and then ligated to a 3.7 kb SfiI fragment of pMF3-h carrying eGFP and a hygromycin resistance cassette [START_REF] Brachmann | A reverse genetic approach for generating gene replacement mutants in Ustilago maydis[END_REF]. In the second step the ligation product was cloned into PCRII-TOPO (Invitrogen) to yield plasmid pAga-GFP. From this plasmid aga-GFP was amplified using primers 10123FW707 and to create pAgaSSA-GFP. From these plasmids aga1T403A-GFP and aga1S542AS562A-GFP were amplified using primers 10123FW707 and 10123ilRV described above and transformed in SG200 generating the strains SG200aga1T403A-GFP and SG200aga1S542AS562A-GFP. All strains were verified by Southern-blot analysis and sequencing.

To complement the S. cerevisiae ypk1 mutant with U. maydis aga1, drag and drop cloning was used as described by [START_REF] Jansen | Drag&Drop cloning in yeast[END_REF]. To generate pAga1, an intronless aga1 gene was cloned from cDNA. First-strand cDNA synthesis was performed using the SuperScript III first-strand synthesis SuperMix assay (Invitrogen)

according to the provided protocol to generate cDNA on RNA isolated from SG200 cells grown in liquid YEPSL. The aga1 ORF was then amplified from cDNA by PCR using the primers 10123FWDD

(5'-GAATTCGATATCAAGCTTATCGATACCGTCGACAATGTCCTGGAAGCTTG GAAAG-3') and 10123RVDD

(5'-GCGTGACATAACTAATTACATGACTCGAGGTCGACCTAGTCTCTCAAGCTC TCGCCTGCAC3'). These primers carry the flanks necessary for the recombination into pGREG536 after transformation into S. cerevisiae [START_REF] Jansen | Drag&Drop cloning in yeast[END_REF]. S. cerevisiae strain Y04976 was then co-transformed with SalI linearized pGREG536 and the PCR product described above. Additionally, strains Y00000 and Y04976 were transformed with pGREG536 as control. Transformation, selection and screening were performed as described in [START_REF] Jansen | Drag&Drop cloning in yeast[END_REF].

Plant infections

Strains were grown in YEPSL medium to an OD 600 of 0.8 and concentrated in H 2 O to a final OD 600 of 1.0. This suspension was inoculated into seven day old corn seedlings of the variety Early Golden Bantam (Olds Seeds, Madison). Compatible haploid strains were mixed (1:1) prior to infection. Disease symptoms were evaluated according to the disease rating criteria reported by Kämper et al. (2006). Three independent experiments were carried out and the average values (for each symptom category) were expressed as percentage of the total number of infected plants.

Analysis of filaments and appressoria

The in vitro system for inducing filaments and appressoria in U. maydis was applied as described previously [START_REF] Mendoza-Mendoza | Physical-chemical plant-derived signals induce differentiation in Ustilago maydis[END_REF] with minor modifications: Briefly, SG200 and derivates were grown in YEPSL at 28°C to an OD 600 of 0.6 to 0.8.

The cells were resuspended in 2% YEPSL to an OD 600 of 0.1 and supplemented with either 100 µM (f.c.) 16-hydroxy-hexadecanoic acid (Sigma), or an appropriate amount of ethanol (1%, f.c.), the solvent for 16-hydroxy-hexadecanoic acid. To inhibit appressorium formation in SG200AM1, 1 µM of LatA (Sigma) was added before spraying the cells on ParafilmM. To quantify appressorium formation on a hydrophobic surface, SG200AM1 derivatives were sprayed with an aerosol sprayer (EcoSpray, Roth) on ParafilmM and incubated in 100% humidity at 28°C for 18 h.

The samples were stained with calcofluor to visualize fungal cells and the percentage of filaments that had developed appressoria or express the AM1-marker were determined using fluorescence microscopy. All experiments were done in three biological replicates.

To measure esterase activity, cells of the respective strains were treated as described above. After 18 h supernatants were collected by centrifugation and esterase activity was determined as described in [START_REF] Reis | Molecular and functional characterization of a secreted lipase from Botrytis cinerea[END_REF].

Cytological methods

For pheromone stimulation, strains were grown in CM-glucose to an OD 600 of 0.6. Synthetic a2 pheromone [START_REF] Szabo | Structure-function analysis of lipopeptide pheromones from the plant pathogen Ustilago maydis[END_REF] dissolved in DMSO was added to a final concentration of 2.5 µg/ml. Cells were subjected to microscopic observation after 2 h and 3 h of incubation in polypropylene tubes on a rotating wheel at 28°C.

For analysis of endocytosis in U. maydis sporidia, FB1 cells were grown at 28°C to an OD 600 of 0.6 following staining of plasma membranes by the addition of 1 µM of the endocytic marker dye FM4-64 (Molecular Probes, Invitrogen). Uptake was visualized by microscopic analysis 5 min after FM4-64 addition. In experiments where endocytosis was blocked with LatA (Sigma), LatA was added to 10 µM f.c., cells were incubated for 30 min at room temperature and then stained with FM4-64 as described above.

To determine the role of actin in cell separation, cells were adjusted to an OD 600 of 0.1, treated with 1 µM LatA in YEPSL and grown at 28°C for 8 h. DMSO treated cells served as control. To visualize mitochondria and the nucleus, cells were grown at 28°C to an OD 600 of 0.6. The cells were collected by centrifugation, resuspended in PBS (140 mM NaCl, 16 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , 3.5 mM KCl, 1 mM Na 2 -EDTA, pH 7.4), supplemented with DAPI (0.5 µg/ml, Sigma), incubated for 5 min and washed in PBS. For immunofluorescence in sporidia, we adapted the protocol established by [START_REF] Banuett | Bud morphogenesis and the actin and microtubule cytoskeletons during budding in the corn smut fungus, Ustilago maydis[END_REF] with slight modifications. For fixation of sporidia, 3% formaldehyde (f.c.) was added directly to the culture and the cells were left at 20°C

with gentle shaking for 30 min. Then the cells were washed with PBS (pH 7.0) and resuspended in PBS to an OD 600 of 8.0. A 1:10 dilution in PBS was applied to a Poly-L-lysine coated glass slide. The cells were allowed to settle for 1 min and then the slide was rinsed with PBS. After washing with PBS, incubations with 1mg/ml Novozyme in PBS for 1 min and Triton X-100 (0.1%) in PBS for 10 min followed.

After washing with PBS, cells were blocked with 5% BSA in PBS followed by incubation with α-actin Antibody (Merck, CP01) 1:500 diluted in PBS. As secondary antibody, an α-mouse Antibody coupled to AF488 (Molecular Probes, A11017) 1:500 diluted in PBS was applied. Actin structures were visualized by confocal microscopy as described above.

To detect actin by immunofluorescence in filaments and appressoria these differentiation steps were induced as described above. Then samples were briefly washed in H 2 O to eliminate Sporidia not attached to the surface and the cells were fixed with 3% formaldehyde in PBS (pH 7.0) for 30 min. All following steps were carried out as described for immunofluorescence of sporidia.

Microscopy

To stain fungal material, samples were incubated in calcofluor (Fluorescent Brightner 28, 100 µg/ml in 0.2 M Tris/HCL, pH 8; Sigma) for 30 s.

To examine fungal colonization inside the leaf tissue six days after infection, the third leaf was de-stained in ethanol, transferred to 10% KOH, incubated at 95°C overnight, washed once with PBS and incubated under vacuum in staining solution (10 µg/ml propidium iodide; 10 µg/ml WGA-AF488 (Invitrogen) in PBS, pH 7.4) according to [START_REF] Doehlemann | Establishment of compatibility in the Ustilago maydis/maize pathosystem[END_REF]. For fluorescence microscopy an Axioplan II microscope (Zeiss) with differential interference contrast optics was used. Fluorescence of GFP, mCherry and calcofluor was observed using GFP (ET470/40BP, ET495LP, ET525/50BP), TexasRed (HC562/40BP, HC593LP, HC624/40BP) and DAPI (HC375/11BP, HC409BS, HC447/60BP) filter sets (Semrock) respectively. Pictures were taken with a CoolSNAP-HQ charge-coupled device camera (Photometrics).

Image processing was done with MetaMorph software (Universal Imaging). Confocal microscopy was performed using a TCS-SP5 confocal microscope (Leica nm and detected at 580-630 nm. To visualize AF488 an excitation of 488 nm and subsequent detection at 500-540 nm was employed. Calcofluor was exited with a 405 nm laser and detected at 415-460 nm. Images were processed using LAS-AF software (Leica Microsystems).

Quantitative real-time PCR

First-strand cDNA synthesis was performed as described above. Real-time PCR was performed using the Platinum SYBR qPCR Supermix-UDG kit (Invitrogen) in the presence of 10 nM fluorescein (Biorad), 0.2 mM of each primer and the various cDNA preparations. Fifty cycles (95°C for 15 s, 60°C for 30 s, 72°C for 30 s) were run on an iCycler iQ (Biorad). Results were normalized against the expression of the peptidyl-prolyl-isomerase gene cpr1 (um03726). The experiments were carried out in three biological replicates with technical duplicates. Gene specific primers for aga1 (um10123) 10123-RT-FW (5'-TCACGCTGTTGCTCAACC-3') and 10123-RT-RV

(5'-GAGCAGCAACTTCCAGTC-3') and for cpr1 (um03726) Rt-ppi-F2 (5'-ACATCGTCAAGGCTATCG-3') and Rt-ppi-R2 (5'-AAAGAACACCGGACTTGG-3') were used. To quantify mfa1 (um02382) expression gene specific primers Rt-mfa1-FW (5'-GCTTTCGATCTTCGCTCAGAC-3') and Rt-mfa1-RV (5'-CAACAACACAGCTGGAGTAGC-3') were used. For data analysis the iCycler™ Software (Biorad) was used.

Molecular techniques

For molecular analysis standard procedures were followed [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF].

Transformation of U. maydis was performed as published previously [START_REF] Schulz | The b alleles of U. maydis, whose combinations program pathogenic development, code for polypeptides containing a homeodomain-related motif[END_REF] and DNA was isolated according to the protocol of [START_REF] Hoffman | A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli[END_REF].

For extraction of proteins from S. cerevisiae, 10 ml cells grown in SD-ura medium to an OD 600 of 1.0 were washed with 50 mM Tris-HCl (pH 7.5) and re-suspended in 100 µl ESB buffer (80 mM Tris-HCI pH 8.0, 2% SDS, 10% glycerol, 100 mM DTT, 0.01% bromophenol blue). Samples were boiled for 3 min at 95°C, glass beads were added and cells were disrupted by incubation for 3 min on a Vibrax shaker (IKA). For isolation of proteins from U. maydis, 2 ml cells were grown in YEPSL to an OD 600 of 1, washed in water and re-suspended in 100 µl Thorner buffer (8 M urea, 5% SDS, 0.1 mM EDTA, 0.01% bromophenol blue, 100 mM DTT, 100 mM Tris-HCl, pH 6.8). Samples were boiled for 10 min at 95°C, glass beads were added followed by a 5 min incubation on a Vibrax shaker (IKA). After a second boiling step (10 min at 95°C) and subsequent centrifugation, 10 µl were loaded for SDS polyacrylamide gel electrophoresis.

Transfer of proteins to PVDF membranes (Amersham Pharmacia Biotech) was conducted in a semidry blot chamber (UniEquip). Blots were probed with monoclonal anti-HA (Sigma), anti-tubulin (Merck) and monoclonal anti-actin (Sigma) antibodies.

HRP conjugated antimouse IgG (Cell Signaling) was used as secondary antibody and the ECL Plus system (Amersham Pharmacia Biotech) for detection of proteins. could be observed with respect to AM1 marker expression (p=0.97), while the difference in appressorium formation was highly significant and is indicated by asterisk (p=0.0005).

  tissue three days after infection with SG200∆aga1 (Fig 3C). Only rarely penetration attempts on epidermal cells could be detected and in such cases the fungal hyphae arrested immediately after the penetration. Around the site of the penetration attempt staining was observed, indicating plant defense responses (Fig 3C, upper right panel).

  and maintenance of the actin cytoskeleton

  necessary for appressorium maturation and that this process is regulated by Aga1.

  10123ilRV described above and transformed in SG200. In the resulting strain SG200aga1-GFP, aga1-GFP has replaced the aga1 locus. To generate plasmids carrying point mutations, pAga-GFP served as template. The QuickChange Site-Directed Mutagenesis-Kit (Stratagene) was used to introduce the mutations T403A and the double mutation S542A S562A, respectively. The mutations were

  For GFP fluorescence an excitation of 488 nm and detection at 495-530 nm was used. Propidium iodide and FM4-64 fluorescence was excited with 561

Fig. 1 .

 1 Fig. 1. U. maydis aga1 complements the ypk1 deletion phenotype in S. cerevisiae. S. cerevisiae strains Y00000 and the ypk1 deletion strain Y04976 were transformed with the indicated plasmids pGREG and pAga1 yielding the strains Y00000/pGREG, Y04976/pGREG and Y04976/pAga1. (A) The indicated strains were grown overnight in selection media (-ura) and total protein was isolated. Expression of the Aga1 fusion protein was demonstrated by Immunoblot. Proteins were detected with antibodies against the HA-epitope of HA-Aga1 (top panel) and actin (lower panel). The proteins are indicated by arrowheads on the right and the molecular weight marker is shown on the left. (B) Strains containing the indicated plasmids were grown overnight in selection media (-ura) and spotted on selection plates (-ura). Plates were incubated for two days at 22°C.

Fig. 2 .

 2 Fig. 2. The aga1 deletion mutant is impaired in growth and stress responses. (A) Serial dilutions of exponentially growing cells of SG200, SG200∆aga1 and SG200∆aga1-C were spotted on CM-glucose plates and CM-glucose plates supplemented with congo red or calcofluor. Plates were incubated for three days at 28°C. (B) Growth curve of SG200, SG200∆aga1 and SG200∆aga1-C in YEPSL. OD 600 was determined during exponential growth at the indicated time points. Growth rate of the respective strains is depicted relative to the initial OD 600 of 0.06 at time point 0. (C) The indicated strains were adjusted to an OD 600 of 1.0 in H 2 O, spotted on a PD charcoal plate and incubated for 48 h. White fuzzy colonies indicate formation of b-dependent filaments.

Fig. 3 .

 3 Fig. 3. Deletion of aga1 results in loss of pathogenicity. (A) Disease symptoms caused by SG200, SG200∆aga1 and SG200∆aga1-C were scored 12 days after infection of seven-day-old maize seedlings. Based on the severity of symptoms observed on each plant, symptoms were grouped into color-coded categories according to Kämper et al. (2006) depicted below. Three independent experiments were carried out and the average values are expressed as percentage of the total number of infected plants (n) given above each column. Tested strains are listed below each column. Significant differences to SG200 infected plants (p<0.005) are indicated by an asterisk in the respective category columns. (B) Photographs of representative leaves 12 days after infection with the indicated strains. A magnified section of the SG200∆aga1 infected leaf is depicted and a chlorotic spot is indicated by arrowhead. (C) Confocal projections of epidermal cells down to vascular bundles of representative leaves three days (upper panels) and ten days (lower panels) after infection with the indicated strains are shown. Fungal material was stained by WGA-AF488 (green). Plant material was stained by propidium iodide (red). Rare colonization attempts of the aga1 mutant are indicated by arrowheads.

Fig. 4 .

 4 Fig. 4. aga1 deletion mutants are unable to differentiate appressoria. (A) Cell suspensions of the indicated strains in 2% YEPSL were sprayed on ParafilmM with 100 µM 16-hydroxy-hexadecanoic acid dissolved in ethanol and incubated for 18 h at 28°C. After staining of fungal cells with calcofluor,

Fig. 5 .

 5 Fig. 5. The conserved phosphorylation sites T403, S542 and S562 in Aga1 are essential for function. (A) Serial dilutions of exponentially growing cells of SG200aga1-GFP, SG200aga1T403A-GFP and SG200aga1S542AS562A-GFP were spotted on CMglucose plates supplemented with congo red, calcofluor and NaCl at the indicated concentrations. Plates were incubated for two to three days at 28°C. (B) Disease symptoms caused by the indicated strains were scored 12 days after infection of seven-day-old maize seedlings as described in legend to Fig. 3A. Highly significant differences to SG200 infected plants (p<0.005) are indicated by an asterisk in the respective boxes.

Fig. 6 .

 6 Fig. 6. Visualization of actin distribution in sporidia by immunofluorescence.Exponentially growing haploid FB1 (upper panel) and FB1∆aga1 (lower panel) cells were fixed with formaldehyde, actin was stained with a monoclonal α-actin antibody that was detected by α-mouse-AlexaFluor488 conjugated antibody (AF488). The samples were analysed by confocal microscopy. Calcofluor stained cell wall appears in white while AF488 appears in green.

Fig. 7 .

 7 Fig. 7. Treatment of cells with LatA prevents appressorium formation. Cell suspensions of SG200AM1 in 2% YEPSL were sprayed on ParafilmM and incubated for 18 h either in presence of 1 µM LatA or DMSO as solvent control. (A) Following incubation, the samples were analysed by epifluorescence microscopy after staining with calcofluor (DAPI-filter set). DMSO treated SG200AM1 cells produce appressoria (indicated by asterisk) that express the AM1 marker (GFP-filter set) (upper panel). Cells treated with LatA express the appressorial marker but are reduced in their ability to form appressoria (lower panel). (B) Quantification of appressoria (white bars) and AM1 marker gene expression (grey bars) of cells grown and treated as in (A). The bars represent results of three independent experiments, evaluating at least 100 filaments per sample, n indicates the total number of cells that were analysed. Error bars represent the standard deviation. The number of cells expressing the appressorial marker is significantly higher in SG200AM1 treated with LatA (*p=0.024) while appressorium formation in SG200AM1∆aga1 is significantly lower compared to SG200AM1 treated with DMSO (**p=0.0003).

  al.,

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Molecular Microbiology

Acknowledgements

We would like to thank Gunther Doehlemann for stimulating discussions and for providing plasmid p123otefmCherry and Stefanie Reissman for critical comments on the manuscript. P.B. and D.L. were supported by fellowships through the GRK1216 of the Deutsche Forschungsgemeinschaft.


Vassilios ; Ladis

Vassilis Ladis

Helen Berdousi

Efstathios Gotsis

Antonis Kattamis

Vassilis Ladis

transfusional iron overload in patients with thalassaemia intermedia

Keywords: thalassaemia intermedia, IRON OVERLOAD, iron chelation therapy, deferasirox, chelation efficiency thalassaemia intermedia, iron overload, iron chelation therapy, deferasirox, chelation efficiency

Deferasirox administration for the treatment of non-

Introduction

Many patients diagnosed with thalassaemia intermedia never (or rarely) receive red blood cell (RBC) transfusion therapy to treat the symptoms of anaemia, however, they remain vulnerable to ongoing iron loading and associated clinical sequelae [START_REF] Taher | Thalassemia intermedia: revisited[END_REF]. The mechanism of iron loading is similar to that observed in patients with hereditary haemochromatosis, whereby enhanced erythropoiesis leads to inappropriately low hepcidin levels resulting in increased intestinal iron absorption [START_REF] Origa | Liver iron concentrations and urinary hepcidin in β-thalassemia[END_REF]. Effective iron chelation therapies have been available for many years, yet there are few studies that have evaluated iron chelation in thalassaemia intermedia-specific patient populations [START_REF] Cossu | Iron overload and desferrioxamine chelation therapy in beta-thalassemia intermedia[END_REF][START_REF] Olivieri | Reduction of tissue iron stores and normalization of serum ferritin during treatment with the oral iron chelator L1 in thalassemia intermedia[END_REF][START_REF] Pippard | Iron balance and the management of iron overload in β-thalassemia intermedia[END_REF][START_REF] Pootrakul | Clinical trial of deferiprone iron chelation therapy in betathalassaemia/haemoglobin E patients in Thailand[END_REF]. In one small-scale study of paediatric patients with thalassaemia intermedia, subcutaneous infusion of deferoxamine (Desferal ® ; DFO; Novartis Pharma AG, Basel, Switzerland) resulted in significant increases in urinary iron excretion [START_REF] Cossu | Iron overload and desferrioxamine chelation therapy in beta-thalassemia intermedia[END_REF]. However, the practical limitations and inconvenience of frequent, prolonged subcutaneous infusions of DFO has a negative impact on quality of life and compliance [START_REF] Cappellini | Overcoming the challenge of patient compliance with iron chelation therapy[END_REF][START_REF] Treadwell | Improving adherence with deferoxamine regimens for patients receiving chronic transfusion therapy[END_REF]. Deferasirox (Exjade ® ; Novartis Pharma AG, Basel, Switzerland) is a once-daily orally administered iron-chelating agent that has been investigated in patients with various transfusion-dependent anaemias [START_REF] Cappellini | Tailoring iron chelation by iron intake and serum ferritin trends: the prospective multicenter EPIC study of deferasirox in 1744 patients with various transfusion-dependent anemias[END_REF] including a pilot study of patients with thalassaemia intermedia over 12 months [START_REF] Voskaridou | Treatment with deferasirox (Exjade) effectively decreases iron burden in patients with thalassaemia intermedia: results of a pilot study[END_REF]. Here, we have evaluated deferasirox therapy for up to 24 months in a population of non-transfused patients with thalassaemia intermedia. 4 transfusion-independent although some had received <20 packed RBC transfusions in their lifetime (Table 1). Patients with impaired kidney or liver function (creatinine clearance ≤70 mL/min, serum creatinine levels greater than the upper limit of normal [ULN], alanine aminotransferase [ALT] levels >3 x ULN), or a life expectancy of <2 years were excluded.

Patients, materials and methods

Patients

Study design

Eleven patients received deferasirox. Patients initially received deferasirox doses of 10 or 20 mg/kg/day. Dose adjustments were permitted at the end of each year of treatment. Therapy was discontinued when patients reached serum ferritin levels <250 ng/mL and LIC <2 mg Fe/g dw. Patients provided written, informed consent before entering the study. The study was conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki.

MRI measurements

All patients were examined at 1.5 Tesla (Signa HDx, GE, Milwaukee, USA) using an FDA-approved multi-echo gradient sequence with electrocardiograph-triggering and breath-holding. For very high iron overload (>15 mg Fe/g dw tissue and equivalently T2* <1.7 ms) an additional single-echo, multi-breath sequence was used, covering a range of 8 echo time values between 0.9 and 3.0 ms, allowing accurate and repeatable measurements of T2* as low as 0.4 ms (equivalent to 64 mg Fe/g dw tissue). We note that the MRI protocol used is identical to that used by Wood et al. [START_REF] Wood | MRI R2 and R2* mapping accurately estimates hepatic iron concentration in transfusion-dependent thalassemia and sickle cell disease patients[END_REF], so that the equation linking 1/T2* to LIC published in the aforementioned paper could be applied to our relaxation data to obtain LIC values.

Efficacy and safety assessments

Statistical analysis

The paired Wilcoxon-test was used for testing significant differences and correlations were analysed using the Spearman's rho correlation coefficient. The analyses were conducted on the data set for all patients who completed at least 12 months of treatment. A type I error (a) of 0.05 was used to determine statistical significance in all tests.

Results

Patients

Eleven patients entered the study between June 2006 and July 2008 (Table I). One patient discontinued after 6 months of therapy (patient #6) and was excluded from the analysis. His ferritin levels after 6 months and 24 months were 4740 ng/mL and 4400 ng/mL, respectively. MRI-estimated LIC after 20 months of observation did not change from baseline (31.8 and 32 mg Fe/g dw tissue, respectively).

Reason for discontinuation was non-compliance due to both inability to cope

with chronic treatment and mild gastrointestinal disturbances. One patient completed only 12 months of deferasirox therapy, as he reached low iron levels, while the remaining nine patients continued treatment for a total of 24 months.

Deferasirox was initiated at a dose of 10 mg/kg/day in seven patients, with four of them increasing their dose to 20 mg/kg/day after 12 months. Four patients started deferasirox at the dose of 20 mg/kg/day, mainly because of significant iron burden.

Dose was escalated from 20 to 25 mg/kg/day in one patient (patient #7) and reduced to 15 mg/kg/day in another patient after achieving low serum ferritin levels (patient #9). A significant decrease in transaminase levels was noted during both the first (P=0.005) and second year of treatment (P=0.005). Mean ± SD ALT levels decreased from 50.5 ± 20.4 U/L at baseline to 29.9 ±14.0 U/L at 12 months and 20.0 ±9.5 U/L at 24 months. AST levels decreased from 42.8 ± 13.5 U/L at baseline to 30.8 ± 10.7 U/L at 12 months and 25.9 ± 7.2 U/L at 24 months.

Discussion

Significant reductions in iron burden as assessed by serum ferritin levels and LIC were achieved with deferasirox therapy over 24 months in this non-transfused population of patients with thalassaemia intermedia, expanding on other previously reported observations over 12 months [START_REF] Voskaridou | Treatment with deferasirox (Exjade) effectively decreases iron burden in patients with thalassaemia intermedia: results of a pilot study[END_REF]. The rate of improvement was faster in the first 12 months compared with the later 12 months, suggesting that iron chelation efficiency was more pronounced when iron overload was greater in these patients. Many patients completing 24 months of therapy had reached more acceptable levels of iron burden with serum ferritin levels of <500 µg/L and LIC of <3 mg Fe/g dw. Cardiac siderosis was not observed in this patient group and no evidence of cardiac iron accumulation or reduced cardiac function was seen over the course of the study. This observation confirms findings from other institutes [START_REF] Origa | No evidence of cardiac iron in 20 never or minimally transfused patients with thalassemia intermedia[END_REF]Roghi et al, 2009).

Relatively low deferasirox doses of 10-20 mg/kg/day were used in this study and were sufficient to reduce iron burden even in severely iron-overload patients, possibly since patients were receiving few or no transfusions. This compares with transfused, heavily iron-overloaded patients with β-thalassaemia [START_REF] Taher | Efficacy and safety of deferasirox doses of >30 mg/kg per d in patients with transfusion-dependent anaemia and iron overload[END_REF].

The efficacy of the treatment was not affected by potential genetic factors, which may influence iron accumulation, such as thalassaemia genotype or HFE.

Previous studies have suggested a fairly constant chelation efficiency for deferasirox; estimated to be 27% and independent of the dosage used [START_REF] Porter | Iron chelation efficiency of deferasirox (Exjade ® , ICL670) in patients with transfusional hemosiderosis[END_REF]. However, we showed a decrease in the chelation efficiency of deferasirox at lower LIC values. We should underline that the equation for determining chelation efficiency was based on a mathematical formula for estimating total-body iron load derived from studies in regularly transfused ex-thalassaemic patients. Our data may suggest a different behaviour of the chelator in patients with thalassaemia intermedia, who do not generally receive chronic transfusion therapy and therefore may develop different chelatable iron pools compared to patients with thalassaemia major. Nevertheless, these findings suggest that at low LIC values, lower deferasirox doses may be more suitable in order to increase the percentage of chelator that is excreted bound to iron. On the other hand, treatment was well tolerated in this study and did not appear to be associated with significant adverse events, even when Baseline iron burden according to LIC and serum ferritin levels in this study population were above threshold levels identified in patients with thalassaemia major to be associated with significant morbidity and mortality [START_REF] Porter | Monitoring chelation therapy to achieve optimal outcome in the treatment of thalassaemia[END_REF]. The Thalassaemia International Federation acknowledge the issue of iron overload in patients with thalassaemia intermedia and suggest initiation of iron chelation when 12 LIC exceeds 7 mg Fe/g dw [START_REF] Taher | Thalassemia intermedia: revisited[END_REF]Thalassaemia International Federation, 2008). However, effective management guidelines are still required to monitor patients with thalassaemia intermedia for iron overload and provide effective treatment to prevent iron overload-related organ damage.

In

Adult

  male and female patients with thalassaemia intermedia with variable genotypes and evidence of iron overload (serum ferritin levels of ≥500 µg/L and liver iron concentration [LIC] of ≥2 mg Fe/g dry weight [dw] estimated by quantitative magnetic resonance imaging [MRI]) were included in this single-centre investigatorinitiated trial conducted at the 'Aghia Sofia' Children's Hospital. All patients were

  Treatment efficacy was assessed according to changes in serum ferritin levels, as well as hepatic and cardiac iron levels estimated by MRI (multi-echo gradient echo sequence and T2* measurements, respectively) and left ventricular ejection fraction (LVEF, calculated from fast imaging employing steady state acquisition sequence multi-phase MR images). Safety parameters included assessment of transaminases (ALT and aspartate aminotransferase [AST]), creatinine, creatinine clearance and . Biochemical parameters were measured on a monthly basis, whereas MRI sequences were measured yearly.

F

Fig 1 .Fig 2 .

 12 Fig 1. Change from baseline in A) Serum ferritin levels and B) LIC. Mean values are represented by a single black line, individual patient data by grey lines.

  >30 mg/kg/day required to achieve therapeutic goals in regularly

  levels of low iron load were reached. The improvement in transaminase levels over the course of the study may reflect a decrease in hepatic siderosis as a result of reduced iron burden in the liver, although further analyses are required to confirm this hypothesis. The observed changes in renal function were within the expected range reported with the use of deferasirox (Cappelini et al., 2010). Creatinine and creatine clearance did not reach abnormal levels in any of the patients. Consequently, close follow up and dosage adjustments are required.

  conclusion, our results indicate that deferasirox provides effective control of iron levels in rarely or non-transfused patients with thalassaemia intermedia over a 24-month treatment period. Results need to be verified in randomized placebocontrolled trials. Ongoing, large-scale studies (NCT00873041) will serve to further characterize the utility of deferasirox therapy in this specific patient group, to define appropriate dosages and help guide the development of effective management strategies for the future.

  Change from baseline in A) Serum ferritin levels and B) LIC. Mean values are represented by a black dashed line, individual patient data by grey lines. 142x222mm (600 x 600 DPI)

British Journal of Haematology

Acknowledgements

Financial support for medical editorial assistance was provided by Novartis Pharmaceuticals. We thank Rebecca Helson for medical editorial assistance with this manuscript.

Vassilis Ladis received grant support, consulting fees and lecture fees from both Apopharma and Novartis Pharmaceuticals Antonis Kattamis reports receiving grant support, consulting fees and lecture fees from Novartis