The APSES transcription factor Vst1 is a key regulator of development in microsclerotium- and resting mycelium-producing Verticillium species.

Plant pathogens of the genus Verticillium pose a threat to many important crops worldwide. They are soil-borne fungi which invade the plant systemically, causing wilt symptoms. We functionally characterized the APSES family transcription factor Vst1 in two Verticillium species, V. dahliae and V. nonalfalfae, which produce microsclerotia and melanized hyphae as resistant structures, respectively. We found that, in V. dahliae Δvst1 strains, microsclerotium biogenesis stalled after an initial swelling of hyphal cells and cultures were never pigmented. In V. nonalfalfae Δvst1, melanized hyphae were also absent. These results suggest that Vst1 controls melanin biosynthesis independent of its role in morphogenesis. The absence of vst1 also had a great impact on sporulation in both species, affecting the generation of the characteristic verticillate conidiophore structure and sporulation rates in liquid medium. In contrast with these key roles in development, Vst1 activity was dispensable for virulence. We performed a microarray analysis comparing global transcription patterns of wild-type and Δvst1 in V. dahliae. G-protein/cyclic adenosine monophosphate (G-protein/cAMP) signalling and mitogen-activated protein kinase (MAPK) cascades are known to regulate fungal morphogenesis and virulence. The microarray analysis revealed a negative interaction of Vst1 with G-protein/cAMP signalling and a positive interaction with MAPK signalling. This analysis also identified Rho signalling as a potential regulator of morphogenesis in V. dahliae, positively interacting with Vst1. Furthermore, it exposed the association of secondary metabolism and development in this species, identifying Vst1 as a potential co-regulator of both processes. Characterization of the putative Vst1 targets identified in this study will aid in the dissection of specific aspects of development.

Hip Replacement Surgery in 14-Year-Old Girl with Factor V Deficiency: Haemostatic Treatment and Thromboprophylaxis.

Factor V (FV) is a pivotal coagulation factor present in plasma and platelets. It plays an essential role in secondary haemostasis acting as a cofactor in the prothrombinase complex, catalysing the conversion of prothrombin to thrombin. There is little evidence on the management of mayor orthopaedic surgery in paediatric or adolescents subjects with this coagulopathy and almost no information about thromboprophylaxis in these situations. We report a case of a hip replacement in a 14-year-old girl with moderate FV deficiency (0.07 IU mL-1). As haemostatic replacement, inactivated fresh frozen plasma (FFP) was transfused at doses of 600 mL (15 mL kg-1, 45 kg weight) 2 hours before surgery and then sequential FFP infusions of 250 mL (7 mL kg-1) every 12 hours for 7 days. Plasma factor VIII, von Willebrand factor antigen, and von Willebrand ristocetin cofactor were monitored to avoid supranormal levels. Since the patient was sexually mature (Marshall and Tanner stage 5) with the hormone replacement therapy, she was immobilized and the surgery was considered as a high thrombotic risk. Thus, low molecular weight heparin was administered at doses of intermediate risk (Enoxaparin 20 mg daily, by weight) after finishing the daily infusion of plasma: 24 hours and during the 7 days after intervention. No tranexamic acid was used. No haemorrhagic or thrombotic adverse event was described.

Conserved and Distinct Functions of the "Stunted" (StuA)-Homolog Ust1 During Cell Differentiation in the Corn Smut Fungus Ustilago maydis.

Ustilago maydis, causal agent of corn smut, can proliferate saprobically in a yeast form but its infectious filamentous form is an obligate parasite. Previously, we showed that Ust1, the first APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) transcription factor functionally characterized in the phylum Basidiomycota, controlled morphogenesis and virulence in this species. Here, we further analyzed Ust1 function using multiple experimental approaches and determined that i) Ust1 activity was able to partially reverse stuA− conidiophore defects in Aspergillus nidulans; ii) in U. maydis, normal development and virulence were strongly dependent on precise induction or repression of Ust1 activity; iii) consistent with its role as a transcription factor regulating multiple processes, Ust1 accumulated in the nucleus at various stages of the life cycle; iv) however, it was undetectable at specific stages of pathogenic growth, indicating that Ust1 repression is part of normal development in planta; v) StuA response elements upstream of the ust1 open reading frame exhibited affinity for U. maydis DNA-binding proteins; vi) however, loss of regulated ust1 transcription had minor phenotypic effects; and vii) Ust1 was subject to post-translational phosphorylation but is not a target of cAMP signaling. Thus, the broad functional conservation between Ust1 and Ascomycota APSES proteins does not extend to the mechanisms regulating their activity.

Isolation of UmRrm75, a gene involved in dimorphism and virulence of Ustilago maydis.

Ustilago maydis displays dimorphic growth, alternating between a saprophytic haploid yeast form and a filamentous dikaryon, generated by mating of haploid cells and which is an obligate parasite. Induction of the dimorphic transition of haploid strains in vitro by change in ambient pH has been used to understand the mechanisms governing this differentiation process. In this study we used suppression subtractive hybridization to generate a cDNA library of U. maydis genes up-regulated in the filamentous form induced in vitro at acid pH. Expression analysis using quantitative RT-PCR showed that the induction of two unigenes identified in this library coincided with the establishment of filamentous growth in the acid pH medium. This expression pattern suggested that they were specifically associated to hyphal development rather than merely acid pH-induced genes. One of these genes, UmRrm75, encodes a protein containing three RNA recognition motifs and glycine-rich repeats and was selected for further study. The UmRrm75 gene contains 4 introns, and produces a splicing variant by a 3'-alternative splicing site within the third exon. Mutants deleted for UmRrm75 showed a slower growth rate than wild type strains in liquid and solid media, and their colonies showed a donut-like morphology on solid medium. Interestingly, although ΔUmRrm75 strains were not affected in filamentous growth induced by acid pH and oleic acid, they exhibited reduced mating, post-mating filamentous growth and virulence. Our data suggest that UmRrm75 is probably involved in cell growth, morphogenesis, and pathogenicity in U. maydis.

One step construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi.

Increasing availability of genomic data and sophistication of analytical methodology in fungi has elevated the need for functional genomics tools in these organisms. Previously we reported a method called DelsGate for rapid preparation of deletion constructs for protoplast-mediated fungal transformation systems, which is based on Gateway® technology. However, over the past several years Agrobacteriumtumefaciens-mediated transformation (ATMT) has become the preferred genetic transformation method for an increasing number of fungi. Therefore, we developed a method for One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), to rapidly create deletion constructs for ATMT systems. The OSCAR methodology involves PCR amplification of the upstream and downstream flanks of the gene of interest, using gene specific primers each with a 5' extension containing one of four different attB recombination sites, modified from the Invitrogen MultiSite Gateway® system. Amplified gene flanks are then mixed with specifically designed marker and binary vectors and treated with BP clonase, generating the deletion construct in a single cloning step. The entire process of deletion construct preparation can be accomplished in just 2days. Using OSCAR we generated eight targeted deletion constructs and used two of them to generate deletion mutants in Verticillium dahliae by ATMT. In summary, OSCAR methodology combines PCR and Gateway® technology to rapidly and robustly generate precise deletion constructs for fungal ATMT and homologous gene replacement.

DelsGate: a robust and rapid method for gene deletion.

Gene deletion is one of the most powerful tools to study gene function. In the genomics era there is great demand for fast, simple high-throughput methods for gene deletion to study the roles of the large numbers of genes that are being identified. Here we present an approach that speeds up the process of generation of deletion mutants by greatly simplifying the production of gene deletion constructs. With this purpose we have developed a method, which we named DelsGate (Deletion via Gateway), that combines PCR and Gateway cloning technology together with the use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. DelsGate consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for the transformation of Ascomycetes and the Basidiomycete fungus Ustilago maydis. The PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. We describe here the entire process, from the generation of the deletion construct with DelsGate to the analysis of the fungal transformants to test for gene replacement, with the Basidiomycete fungus Ustilago maydis. Application of DelsGate to other fungal species is also underway. Additionally, we describe how this basic approach can be adapted to other genetic manipulations with minor changes. We specifically describe its application to create unmarked deletions in Ralstonia solanacearum, a Gram-negative phytopathogenic bacterium.

Regulation of Ustilago maydis dimorphism, sporulation, and pathogenic development by a transcription factor with a highly conserved APSES domain.

In Ustilago maydis, the causal agent of corn smut, the morphological transition from yeast to filamentous growth is inextricably linked to pathogenicity; budding haploid cells are saprobic and, upon mating of compatible strains, the fungus converts to dikaryotic filamentous growth and obligate parasitism. The filamentous dikaryon proliferates in the host plant, inducing tumor formation and undergoing additional morphological changes that eventually result in the production of melanized diploid teliospores. In an attempt to identify new trans-acting factors that regulate morphogenesis in U. maydis, we searched for the presence of common binding sequences in the promoter region of a set of 37 genes downregulated in the filamentous form. Putative cis-acting regulatory sequences fitting the consensus binding site for the Aspergillus nidulans transcription factor StuA were identified in 13 of these genes. StuA is a member of the APSES transcription factors which contain a highly conserved DNA-binding domain with a basic helix-loop-helix (bHLH)-like structure. This class of proteins comprises critical regulators of developmental processes in ascomycete fungi such as dimorphic growth, mating, and sporulation but has not been studied in any fungus of the phylum Basidiomycota. A search for StuA orthologs in the U. maydis genome identified a single closely related protein that we designated Ust1. Deletion of ust1 in budding haploid wild-type and solopathogenic strains led to filamentous growth and abolished mating, gall induction, and, consequently, in planta teliosporogenesis. Furthermore, cultures of ust1 null mutants produced abundant thick-walled, highly pigmented cells resembling teliospores which are normally produced only in planta. We showed that ssp1, a gene highly induced in teliospores produced in the host, is also abundantly expressed in cultures of ust1 null mutants containing these pigmented cells. Our results are consistent with a major role for ust1 in regulating dimorphism, virulence, and the sporulation program in U. maydis.