Verticillium dahliae. Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V. dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V. dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta.The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus  Verticillium spp. such as V. dahliae. Developmental processes and the production of secondary metabolites are coordinated by fungal velvet domain proteins. The four V. dahliae velvet domain proteins form the heterodimers Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 during vegetative growth. Vel1, Vel2 and Vel3 coordinate the formation of the microsclerotia resting structures and the production of the metabolite melanin, which is deposited in microsclerotia. The survival of these resting structures in the soil is essential for Verticillium spp. during the monocyclic disease. Microsclerotia germinate in the presence of a suitable host, the resulting hyphae grow to and colonize the root. We discovered that this important process of initial colonization requires the presence of Vel1. Once inside the plant, the fungus distributes in the vascular system by conidia formation, which depends on Vel1 and Vel3. Vel1 is a promising target to control the fungal disease, because it is required for the induction of disease symptoms.Verticillium wilt is a destructive disease of many economic important crop plants induced by fungal plant pathogenic  Verticillium dahliae, which infects over 200 hosts in temperate and subtropical regions and becomes a world-wide threat [V. dahliae remains in the soil for years without a suitable host as melanized resting structures, called microsclerotia [V. dahliae uses regions where the endodermis has not been fully developed, for instance the tip or sites of lateral root formation, for penetration when reaching the root [V. dahliae starts conidiation inside the vascular tissue and uses the sap stream to distribute within the plant [V. dahliae forms microsclerotia in senescent plants, that reach the soil with the fading plant and remain there until a new host is present [Many plants are destroyed by wilt causing plant pathogenic fungi such as the ascomycete e threat –3. V. daclerotia . Microscclerotia ,5. V. dathe root ,2. The fthe root . V. dahlhe plant . Germinahe plant ,2,8,9, r present ,7,9.Aspergillus nidulans. A strain with a point mutation in the veA gene resulted in a truncated VeA protein and produced more conidia but fewer fruiting bodies [A. nidulans velvet family includes four members: VeA, velvet like B (VelB), velvet like C (VelC) and viability of spores A (VosA) [Neurospora crassa VE-1 functions light-independently. VE-1 promotes hyphal growth, protoperithecia formation and secondary metabolism, but reduces asexual conidiation [Botrytis cinerea. The corresponding vel1 gene is required for virulence, but not for the production of virulence-related secondary metabolites as botcinic acid and botrydial [A. nidulans or other ascomycetes [A. nidulans VelC functions as positive regulator of sexual development and controls genes involved in asexual development. In several fungi velC deletion leads to increased conidia formation, its role in asexual development might therefore be conserved [A. nidulans VosA promotes spore viability, trehalose biosynthesis and control of secondary metabolite genes [Fungal development, resting structure formation and secondary metabolite production are connected to the velvet family of regulatory proteins . The firg bodies . VeA coog bodies –15. The A (VosA) ,16–18. VA (VosA) ,19,20. Iidiation ,22. Lighotrydial . Similaromycetes ,24. A. nonserved . A. nidute genes .A. nidulans VelB, VeA and the secondary metabolite methyltransferase LaeA [Velvet proteins comprise a velvet domain with proline residues in the middle of the motif but without sequence similarity to other known domains . The firase LaeA . Foldingase LaeA . Velvet ase LaeA ,18,27,28A. nidulans VosA has a transcription activation domain [n domain  and bindn domain . SclB han domain . Velvet n domain . Moreoven domain .Velvet proteins, which are conserved in ascomycetes and basidiomycetes , have toV. dahliae plant vascular pathogens. The goal was to address whether any of the velvet protein encoding genes is a potential target to combat the fungus outside or inside the plant. We found a prominent role in virulence for the Vel1 encoding gene, which exhibits a novel virulence-related function already at the early phase of plant root colonization. V. dahliae velvet proteins are important for microsclerotia formation and secondary metabolite production, as for instance the melanin precursor scytalone. They are also needed for fungal distribution by conidia in planta. Vel1 is exposed as central factor for all steps of V. dahliae development and disease induction with a novel role in root colonization.This study aimed to reveal the yet elusive functions of the velvet proteins in the life cycle of V. dahliae JR2 homolog encoding proteins with the velvet DNA binding and dimerization domain were identified by BLAST search and compared to corresponding genes from other ascomycetes . The architecture of the velvet proteins is largely conserved between V. dahliae and A. nidulans. Alignment of V. dahliae Vel2 with the A. nidulans VelB velvet domain [V. dahliae and Colletotrichum graminicola counterparts. Only the primary amino acid sequence of V. dahliae Vos1 is more like the corresponding protein of the phytopathogenic Magnaporthe oryzae as another representative of the Sordariomycetes  corresponds to the A. nidulans gene encoding the VeA regulator. VdVEL1 includes three exons and two introns and results in a 2612 bp transcript with a deduced protein of 552 aa, which is similar in size to AnVeA (573 aa) [VdVel1 as in AnVeA at the C-terminus. Vel1 carries a PEST domain between amino acids 162–183  resulted initially only in a deduced rather small 237 aa protein. Sequencing of the fungal cDNA revised the annotation with a changed start codon and an additional intron. The protein sequence of this improved annotation was further verified by protein pull-downs followed by mass spectrometry  . Besides 162–183 , suggest3 (AnVeA . The othtrometry . The corr AnVosA .V. dahliae VEL2 (VDAG_JR2_Chr3g06150a) and VEL3 (VDAG_JR2_Chr6g00630a) genes are homologs to A. nidulans genes encoding VelB and VelC, respectively  in comparison to AnVelB (369 aa), but vice versa, a smaller 437 aa VdVel3 protein compared to AnVelC (524aa). Vel2/VelB carry a similar C-terminal velvet domain, which is interrupted by an intrinsically disordered domain (IDD) in both proteins not interfering with the folding of the velvet domain in the X-ray structure of VelB [The velvet domains of the remaining proteins, Vel2 and Vel3, are located in the C-terminal part. The ely Figs  and S3.  of VelB . In contV. dahliae forms melanized microsclerotia for long-term survival in the soil, which can germinate even after decades in the presence of a host [V. dahliae. Deletion strains of the four V. dahliae velvet genes as well as selected double deletion strains were constructed by replacing their open reading frames with a resistance cassette using homologous recombination  and on minimal medium . Microsclerotia formation of n strain .VEL2 strain after illumination resembled the ΔVEL1 or the ΔVEL1/ΔVEL2 strain with impaired microsclerotia formation (VEL2 strain could produce reduced numbers of microsclerotia compared to wild type when incubated in darkness on simulated xylem medium (SXM), but not on minimal medium (CDM) (The phenotype of a Δormation . Howeverum (CDM) .V. dahliae forms microsclerotia and aerial hyphae in response to light and dark cycles on SXM, resulting in a characteristic ring-like structure. The ring-like structure was visible in the ΔVEL2 strain but in combination with only an initial melanization in the center and the first rings  than wild type in light , which allows conidiation as well as resting structure formation. Extracts were applied to liquid chromatography/mass spectrometry (LC/MS) combined with photodiode array detection analysis.Velvet domain protein complexes coordinate fungal resting structure and secondary metabolite formation, including toxic compounds for pathogenic interactions or signaling molecules for communication with other microorganisms ,35–37. SVEL1 strain compared to wild type , which were reduced, and at least four (VI-IX), which were increased in the Δype Figs  and S9A.ype Figs . Overexp-XI Figs  and S15.-XI Figs . A simil3 strain . The VELype Figs  and 3D. ype Figs .VOS1 or LAE1, which represents a major epigenetic secondary metabolite regulator interacting with A. nidulans velvet proteins [V. dahliae microsclerotia formation or secondary metabolism during the analyzed conditions  culture, a condition in which the Vel1 protein is present in the cell. The relative expression of CMR1 is decreased to less than 20% of the wild type level when VEL1 is deleted . Strains with functional analyzed . All fouanalyzed .V. dahliae strain with histone H2B-RFP labeled nuclei revealed a predominantly nuclear localization of Vel1, Vel3 and Vos1. The majority of Vel2 localized to the nucleus, but smaller subpopulations were found in the cytoplasm , Vel2 (42 proteins) and Vos1 (23 proteins), whereas Vos1 was the only significant interactor found for Vel3. Interactions for Vel1-Vel2 and Vel2-Vos1 heterodimers were observed  and microsclerotia formation (solid SXM medium) after three and six days of growth during illumination  suffered from strong or heavy symptoms induced by the fungal pathogen . ΔVOS1 aVEL1 strain resulted in almost 80% plants, which are either healthy (more than 50%) or show only weak symptoms and hardly any plants with heavy symptoms. This effect could be restored in a strain where the VEL1 gene was reintroduced. Plant treatment with double deletion strains as ΔVEL1/ΔVEL2 or ΔVEL1/ΔVEL3 resulted in reduced disease severity compared to wild type infected plants with approximately 30% of plants displaying strong or heavy symptoms. This suggests that the VEL1 gene plays a significant role in the V. dahliae caused disease in tomato.In contrast, infection of tomato plants with the ΔVEL2, ΔVEL3 or ΔVOS1 strain infections. Whereas 97% of wild type treated plants showed strong discolorations of the hypocotyl only approximately 60% of plants inoculated with VEL1 deletion strains had changes in hypocotyl color, which mostly were less severe.As additional indicator of disease or plant immune response, hypocotyl cross sections of the plants were investigated for discoloration caused by fungal infection . In the V. dahliae spore formation. The infection rate between wild type and the ΔVEL1 strain was compared to evaluate whether there are already differences in the potential to infect plants after inoculation with the same number of conidia. The amount of plants without disease symptoms relative to wild type was determined , which counterpart is phosphorylated in the N. crassa MAP kinase signaling pathway and important for fungal communication [V. dahliae.nication . It remaA. niger VeA is involved in spore production and dispersion [V. dahliae Vel1 together with Vel3 promote fungal conidiation, which facilitates colonization of the upper parts of host plants. A V. dahliae ΔVEL1 strain is impaired in colonization and entry into host roots. The application of wounded tomato plants allowed to circumvent this initial infection step, because a ΔVEL1 strain could enter and grow within the host. Symptom induction in plants treated with this strain was decreased in comparison to wild type, presumably due to the reduced potential of conidia production and propagation within the plant vascular system. Lack of Vel3 reduces conidiation less than the lack of Vel1. Spore numbers produced by the ΔVEL3 strain seem to be sufficient to effectively colonize plants, because symptom induction in tomatoes was comparable to wild type when this strain was used for inoculation.Among the Aspergilli, spersion . In addiVEL1 and VEL2 deletion strains are hindered in microsclerotia production and the ΔVEL3 strain increases melanization compared to wild type. Another metabolite controlled by Vel1 and Vel2 is an intermediate in 6-methoxymellein production, which might harm plant cells similar to terrein of A. terrreus [The fungus remains in the plant after successful infection until the host dies and forms microsclerotia as resting structures. Formation of microsclerotia means a readjustment of developmental programs. Vel1 and Vel2 are positive regulators of microsclerotia formation, whereas Vel3 is a negative regulator. Except of its promoting role in conidiation, Vel3 acts antagonistically towards Vel1 and Vel2. Velvet genes regulate secondary metabolite expression in different fungi ,24,49,64terrreus . The exaV. dahliae velvet proteins, except Vos1, which exhibits yet unknown cellular functions, contribute to these developmental processes and the linked secondary metabolite production, but are not as omnipresent as Vel1. The broad involvement of velvet domain proteins in the fungal life cycle leads to pleiotropic effects. This complex integration of velvet proteins makes especially Vel1 an interesting target to combat the growing threat of Verticillium wilt in our crops.In summary, we showed that Vel1 of a vascular plant pathogen is a key factor for early plant colonization. Vel1 controls fungal development including asexual spore and microsclerotia formation linked to an appropriate secondary metabolism and symptom induction and disease. Other V. dahliae, Agrobacterium tumefaciens and Escherichia coli strains used are listed in http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind) with default settings. The sequences of other fungi mentioned in this study were obtained from the Ensembl Fungi database [Annotations were used according to the Ensembl Fungi database . Sequencdatabase . MultiplE. coli and A. tumefaciens cells were cultivated in lysogeny broth medium [h medium  supplemeh medium . Media wGFP at the 3’ end and integrated endogenously. In addition, the GFP-fused genes were expressed ectopically under the strong A. nidulans gpdA promoter for localization studies. Further details can be found in the supporting information  and ground in liquid nitrogen. Genomic DNA was extracted using phenol. Southern hybridization was carried out using the Amersham AlkPhos Direct Labelling and CDP-Star Detection reagents . Further details can be found in previous publications ,58,59.H2A gene.RNA was extracted using the following protocol. Fungal strains were grown in triplicates in PDM for extraction of RNA. Approximately 0.5 ml ground mycelium was mixed with 1.4 ml TRIzol (Invitrogen) for 5 min. After adding 250 μl chloroform the solution was mixed again for 10 min. Centrifugation for 30 min at 4°C led to the formation of two phases. The upper phase containing the RNA was transferred into a fresh reaction tube and mixed with 400 μl isopropanol and 400 μl high-salt buffer . After inverting the reaction tube several times and centrifugation for 15 min at 4°C, the supernatant was discarded. The RNA was washed with 250 μl 70% ethanol, centrifuged again for 10 min at 4°C and the supernatant was removed. Afterwards the RNA was dried at RT and dissolved in 100 μl RNase-free water at 65°C. Alternatively, RNA was extracted from fungal mycelium using the Direct-zol RNA MiniPrep Kit (Zymo Research). cDNA synthesis was carried out with the QuantiTect Reverse Transcription Kit (Qiagen) using 0.8 μg of RNA. The absence of genomic DNA in the cDNA was tested with the primers SZ19 and SZ20, binding in the VEL1 the primers AO11 and AO12 were used. The annotation of VEL2 was confirmed by VelB-F1 and VelB-R1. VEL3 annotation was confirmed by using the primers AO3 and AO13. The VOS1 annotation was verified by using Vos1orf-E1 and Vos1orf-E2.Annotations from the Ensembl Fungi database  were verVEL1, VEL2 and VEL3 could be confirmed. The correct annotation of VOS1 starts already 528 base pairs upstream from the start codon predicted by Ensembl Fungi as confirmed by amplification and sequencing of cDNA as well as analysis of the peptide sequence obtained by LC/MS analysis  and EIF2B (primers SZ11 and SZ12) by qRT PCR using the primers SB40 and SB41 as described before [The expression levels of the transcription factor Cmr1 encoding gene were quantified relative to the reference genes histone d before  using Med before . Signifi4 freshly harvested spores of each strain. Plates were incubated for 10 to 14 days upside down and examined by binocular and light microscopy for microsclerotia formation. Plates were incubated either in constant light or constant darkness at 25°C or during long day conditions  in a growth chamber.Phenotypical analysis was conducted on SXM and CDM as described before  using 5x4 freshly harvested spores were inoculated in triplicates on 30 ml CDM plates and incubated at 25°C during constant illumination. After 10 days, aerial mycelium was removed, and pictures were taken from the top view. Pictures were converted to 8-bit grey scale. The brightness factor of the colony centres was measured using the ROI Manager of the ImageJ software [The melanization of fungal colonies was quantified as described before . 5x104 fsoftware . The brisoftware . Signifi5 freshly harvested spores were inoculated in 50 ml SXM and incubated at 25°C under constant agitation for seven days. Each strain was inoculated in triplicates. After seven days, the spores were filtered through Miracloth filters  and centrifuged. Spore sediments were dissolved in equal amounts of sterile water. By using the Coulter Z2 Particle Counter (Beckman Coulter), the conidiospore concentration was determined. The conidia formation was quantified relative to the wild type. Each experiment was performed with three technical replicates (n = 1). Bars represent the mean values of all experiments and error bars correspond to standard deviations. Statistical significances were calculated by t-test with the SISA online tool [For quantification of conidia production, 2x10ine tool . SignifiGFP. To visualize the nucleus, the constructs were additionally transformed with a Histone H2B-RFP overexpressing strain. 1x104 freshly harvested fungal spores were inoculated in 300 μl PDM in 15 μ-Slide 8 Well microscopy chambers (ibidi) and incubated at 25°C for 16h either in light or darkness. Subcellular localization of the proteins was examined by fluorescence microscopy (100x/1.4 oil objective). Exposure time for GFP was 500 ms and for RFP 800 ms.The velvet proteins were tested for their localization by gene overexpression and C-terminal fusion with Arabidopsis thaliana Col-0 was used for root infection studies with the indicated V. dahliae strains as described previously [5 spores/ml for 35 min. The plants were further incubated on water agarose for five days in the climate chamber. For preparation of fluorescent microscopy, the roots were stained with 0.0025% (v/v) propidium iodide and 0.005% (v/v) silwet for 5 min in darkness. Experiments were conducted with two independent transformants of ΔVEL1, expressing free GFP (VGB443 and VGB444).eviously ,58. Seedeviously , modifieeviously ) overnigSolanum lycopersicum seedlings  were used for infection experiments. Roots were slightly wounded by rubbing and infected by root dipping into a spore suspension of 1x107 spores/ml for 40 min under constant agitation at ~30 rpm. Per strain 15 plants were used. In addition, 3 ml of the spore suspension (concentration 1x107 spores/ml) was added to the soil of each pot. Plants were incubated for 21 days under long day conditions . Further details on plant infection and evaluation of disease symptoms can be found in previous publications [ications ,57.VEL1 deletion strain was verified by PCR using the primers AO24 and AO25, which bind in the 5’ or 3’ flanking region of the gene, respectively.Presence of fungal material of the wild type or the 6 freshly harvested spores. The plates were incubated at 25°C in light or darkness for two weeks. Secondary metabolite extraction was modified [2O  and 5 ml ethyl acetate  and incubated overnight during constant agitation. The samples were centrifuged for 10 min at 2500 rpm at 4°C. The upper phase was transferred into a vial and vaporized. The metabolites were dissolved in acetonitrile and H2O  (1:1), centrifuged at 8°C, 13000 rpm, 15 min and transferred into LC/MS vials. The samples were analysed with a Q Exactive Focus Orbitrap mass spectrometer coupled with an UltiMate 3000 high-performance liquid chromatography (HPLC) and a CAD-3000 detector (Thermo Fisher Scientific) as descried before [Per strain, two plates of 30 ml CDM with glucose as carbon source were inoculated with 1x10modified . The homd before . The meaFor the quantification of scytalone the corresponding peaks of the extracted ion chromatograms (EICs) at m/z = 193.0506 ± 5 ppm were detected and peak areas were determined with the PPD algorithm with a signal to noise ratio of 5 in FreeStyle 1.6.75.20 (Thermo Fisher Scientific). Each experiment was performed with one replicate (n = 1). Bars represent the mean values of all replicates and error bars correspond to standard deviations. Statistical significances were calculated by t-test with the SISA online tool . SignifiProteins were extracted as described  using gr7 freshly harvested spores were inoculated in 500 ml PDM and grown for 72 h during constant agitation in light. Proteins of VGB39, VGB45, VGB447, VGB450, VGB451 and VGB453 were extracted. The buffer was supplemented with 2 mM dithiothreitol, 1 mM phenylmethylsulfonylfluorid and 10 μl/ml cOmplete Protease Inhibitor Cocktail . VGB45 and VGB39 were mixed before protein extraction  due to the higher amount of free GFP in VGB45 in comparison to the above mentioned velvet strains, which was observed with Western experiments using a GFP-specific antibody. Prior to protein loading, the beads were washed with buffer in the column. For each sample, 12 mg of protein were applied to a GFP-trap pull-down with 10 μl GFP-TrapA beads (Chromotek) in a polyprep column (Bio-Rad). The final volume was adjusted to the same amount with extraction buffer. Beads were incubated for 2 h at 4°C rotating. Afterwards, the beads were washed twice with W300 buffer . To elute proteins, 150 μl glycine pH 2.5 were pipetted into the column and mixed with 15 μl 1 M Tris pH 10.4. The elution step was repeated twice leading to elution fractions E1-E3. Eluted protein was applied to chloroform-methanol extraction [5x10traction  and digetraction ,91. Furttraction  partner Fungal strains were grown in liquid PDM, liquid SXM or on SXM plates covered with a nylon membrane . Mycelium was harvested and ground in liquid nitrogen at the indicated time points. After extraction of proteins, western analysis was conducted as previously described ,88. For S1 FigAspergillus fumigatus Af293, Aspergillus nidulans FGSC A4, Botrytis cinerea BcDW1, Colletotrichum graminicola M1.001, Magnaporthe oryzae M68 and Neurospora crassa OR74A similar to Vel1 of V. dahliae JR2 were aligned by MegAlignPro (DNASTAR) using ClustalW multiple sequence alignment. The velvet domain predicted by InterProScan for the Vel1 protein (VDAG_JR2_Chr7g04890a) is depicted in dark blue according to IPR037525. The predicted NLS is shown in grey and the PEST motif in purple.Deduced proteins of (TIF)Click here for additional data file.S2 FigA. fumigatus Af293, A. nidulans FGSC A4, B. cinerea BcDW1, C. graminicola M1.001, M. oryzae M68 and N. crassa OR74A similar to Vel2 of V. dahliae JR2 were aligned by MegAlignPro (DNASTAR) using ClustalW multiple sequence alignment. The velvet domain predicted by InterProScan according to IPR037525 for the Vel2 protein (VDAG_JR2_Chr3g06150a) is depicted in dark blue. Homologies to other ascomycetes indicate that all Vel2-like velvet domains are interrupted by an intrinsically disordered domain (IDD), which is indicated by an orange color. The predicted NLS is shown in grey.Deduced proteins of (TIF)Click here for additional data file.S3 FigA. fumigatus Af293, A. nidulans FGSC A4, B. cinerea BcDW1, C. graminicola M1.001, M. oryzae M68 and N. crassa OR74A similar to Vel3 of V. dahliae JR2 were aligned by MegAlignPro (DNASTAR) using ClustalW multiple sequence alignment. The velvet domain predicted by InterProScan for the Vel3 protein (VDAG_JR2_Chr6g00630a) is depicted in dark blue. The domain is shown according to IPR037525. In grey, the predicted NLS is shown. The PEST motif is indicated in purple.Deduced proteins of (TIF)Click here for additional data file.S4 FigA. fumigatus Af293, A. nidulans FGSC A4, B. cinerea BcDW1, C. graminicola M1.001, M. oryzae M68 and N. crassa OR74A similar to Vos1 of V. dahliae JR2 were aligned by MegAlignPro (DNASTAR) using ClustalW multiple sequence alignment. The sequence used for the alignment is an improved version of the VDAG_JR2_Chr3g12090a prediction, which derived from cDNA sequencing and mass spectrometry verification of the protein. The Vos1 velvet domain predicted by InterProScan is depicted in dark blue according to IPR037525.Deduced proteins of (TIF)Click here for additional data file.S5 FigA. fumigatus Af293, A. nidulans FGSC A4, B. cinera BcDW1, C. graminicola M1.001, M. oryzae M68 and N. crassa OR74A similar to V. dahliae Vel1, Vel2, Vel3 or Vos1, respectively, were aligned by MegAlignPro (DNASTAR) using ClustalW multiple sequence alignment. Similarities of V. dahliae velvet proteins in different fungi are shown in sequence identity matrices and phylogenetic trees (A) Vel1, (B) Vel2, (C) Vel3 and (D) Vos1. (E) Amino acid sequence predicted by Ensembl Fungi is within the box, amino acids deduced from cDNA sequencing and missing in the prediction are outside of the box. The sequence highlighted in green was covered with peptides identified with LC/MS and MaxQuant 1.6.0.16 analysis. Black parts of the sequence were not covered.Deduced proteins of (TIF)Click here for additional data file.S6 FigVEL1 deletion strain (ΔVEL1) with hygromycin resistance cassette (HYG), two independent transformants of the VEL2 deletion strain (ΔVEL2) with nourseothricin resistance cassette (NAT), the complementation strains of VEL1 and VEL2  with nourseothricin resistance cassette (NAT) or hygromycin resistance cassette (HYG), respectively and two independent transformants of VEL1 and VEL2 (ΔVEL1/ΔVEL2) or VEL3 (ΔVEL3/ΔVEL1) double deletion strains. gDNA was restricted with XhoI and used for Southern hybridization with the 3’ region labeled as a probe for verification of the VEL1 deletion or complementation strains. gDNA was restricted with BglII for verification of the VEL2 deletion and complementation strains and the 3’ region was used as probe. (A) Restriction sites of XhoI in the wild type, deletion and complementation strains and binding site of the 3’ probe (marked in blue). For each strain the expected fragment size is depicted. (B) Southern hybridization of the VEL1 deletion and complementation strains with wild type control. For the wild type, a fragment of 3.5 kb was obtained. Both tested VEL1 deletion strains resulted in a fragment with a size of 5 kb. The complementation strain shows a fragment with a size of 5.7 kb. (C) Southern hybridization of the VEL1 single, VEL1/VEL2 as well as VEL3/VEL1 double deletion strains and wild type control. The wild type exhibits a fragment with a size of 3.5 kb. All strains containing the deletion cassette of VEL1 result in a fragment with a size of 5 kb. (D) Restriction sites of BglII in the wild type, deletion and complementation strains. The binding site of the 3’region, which was used as probe, is marked in blue. The expected fragment size for each strain is shown. (E) Southern hybridization of the VEL2 deletion and the complementation strains with wild type control. The wild type exhibits a fragment with the size of 3.3 kb. The tested deletion strains show fragments with a size of 3 kb. The complementation strain results in a fragment with a size of approximately 6.6 kb. (F) Southern hybridization of the wild type, VEL2 deletion and VEL1 and VEL2 double deletion strains restricted with BglII and incubated with the 3’ probe. The wild type exhibits a fragment size of 3.3 kb. Strains containing the VEL2 deletion cassette show a fragment with a size of 3 kb.Genomic DNA (gDNA) was extracted from the wild type (WT), two independent transformants of the (TIF)Click here for additional data file.S7 FigVEL3 deletion strain (ΔVEL3) with nourseothricin resistance cassette (NAT), two independent transformants of the VOS1 deletion strain (ΔVOS1) with hygromycin or nourseothricin resistance cassette , the VEL3 complementation strain (Comp. VEL3) with hygromycin resistance cassette (HYG) and two independent transformants of the VEL3 and VEL1 (ΔVEL3/ΔVEL1) as well as a single transformant of VOS1 and VEL3 (ΔVOS1/ΔVEL3) double deletion strains was extracted and used for Southern hybridization. For verification of the VEL3 deletion and complementation strains, the restriction enzyme XhoI was used and the 5’ region as a probe. For restriction of gDNA from the VOS1 deletion strains, BglI was used and the 3’region severed as probe. (A) Restriction sites of XhoI in the wild type, deletion and complementation strains. The binding site of the 5’ region which was used as probe is marked in blue. (B) Southern hybridization of the VEL3 deletion and the complementation strains with wild type control. The wild type results in a fragment with a size of approximately 2.7 kb. The two tested deletion transformants exhibit fragments with a size of 6.4 kb. The complementation shows a fragment with a size of 2.7 kb. (C) Southern hybridization of the VEL3 deletion and VEL3 and VEL1 as well as VOS1 and VEL3 double deletion strains with wild type control. The wild type results in a fragment of 2.7 kb. Strains containing the VEL3 deletion cassette exhibit a fragment with a size of 6.4 kb. (D) Restriction sites of BglI in the wild type and deletion strains. The 3’ flanking region was used as probe (marked by a blue line). (E) Southern hybridization of VOS1 deletion strains with hygromycin resistance cassette (HYG) with wild type control. The wild type exhibits a fragment with a size of 4.8 kb. The two tested VOS1 deletion transformants show fragments with a size of 1.8 kb. (F) Southern hybridization of the VOS1 deletion with hygromycin resistance cassette (HYG), double deletion strains with VEL3 and wild type control. The wild type results in a fragment with a size of 4.8 kb. VOS1 deletion strains show fragments with a size of 1.8 kb. (G) Southern hybridization of the VOS1 deletion strains with nourseothricin resistance cassette with wild type control. The labeled fragment of the wild type has a size of 4.8 kb. The two tested deletion transformants exhibit a fragment with a size of 1.8 kb.Genomic DNA (gDNA) of the wild type (WT), two independent transformants of the (TIF)Click here for additional data file.S8 Fig4 spores of indicated V. dahliae strains on either simulated xylem medium (SXM) or Czapek-Dox-Medium (CDM) and subsequent incubation for 10 days at 25°C in constant light or darkness. Single colonies on SXM are shown from the back and colonies on CDM are shown from the top of the plate. Cross sections were made through the middle of the colony. (A) Growth and development of the VOS1 deletion (ΔVOS1) and the wild type (WT) strains are similar. Single colonies (left column) and colony cross sections (right column) are shown. (B) A VEL3 and VOS1 double deletion strain (ΔVEL3/ΔVOS1) resembles the phenotype of the VEL3 (ΔVEL3) single deletion strain.Images give an overview of fungal colonies after spotting 5x10(TIF)Click here for additional data file.S9 Fig(A) LC/MS combined with photodiode array detection (PDA) analysis of secondary metabolites extracted from the V. dahliae wild type and deletion strains of VEL1 and VEL2 in darkness and light. Secondary metabolites were extracted from two-week-old fungal mycelium grown on Czapek-Dox-Medium (CDM) supplemented with glucose. Chromatograms of the wild type and both deletion strains exhibit the same presence and absence of secondary metabolites in darkness or light. (B) Quantification of CMR1 gene expression in the wild type and the VEL1 deletion strain. Mean values of three independent experiments with standard deviations relative to the wild type are depicted. Normalization was conducted to the reference genes H2A and EIF2B. Significant differences were calculated by t-test and indicate: *:p<0.05.(TIF)Click here for additional data file.S10 Fig(A) and (B) Single ion monitoring (SIM) for substance I with m/z 193.0493 and a mass tolerance of 5.00 ppm in negative ion mode. Depicted are the wild type (WT), VEL1 deletion strain (ΔVEL1) and complementation (Comp. VEL1) (A) as well as the wild type (WT), VEL2 deletion strain (ΔVEL2) and complementation (Comp. VEL2) (B). (C) MS2 spectrum of substance I.(TIF)Click here for additional data file.S11 Fig(A) and (B) Single ion monitoring (SIM) for substance II with m/z 235.0603 and a mass tolerance of 5.00 ppm in negative ion mode. Depicted are the wild type (WT), VEL1 deletion strain (ΔVEL1) and complementation (Comp. VEL1) (A) as well as the wild type (WT), VEL2 deletion strain (ΔVEL2) and complementation (Comp. VEL2) (B). (C) MS2 spectrum of substance II.(TIF)Click here for additional data file.S12 Fig(A) and (B) Single ion monitoring (SIM) for substance III with m/z 193.0499 and a mass tolerance of 5.00 ppm in positive ion mode. Depicted are the wild type (WT), VEL1 deletion strain (ΔVEL1) and complementation (Comp. VEL1) (A) as well as the wild type (WT), VEL2 deletion strain (ΔVEL2) and complementation (Comp. VEL2) (B). (C) MS2 spectrum of substance III.(TIF)Click here for additional data file.S13 Fig(A) and (B) Single ion monitoring (SIM) for substance IV with m/z 577.3732 and a mass tolerance of 5.00 ppm in positive ion mode. Depicted are the wild type (WT), VEL1 deletion strain (ΔVEL1) and complementation (Comp. VEL1) (A) as well as the wild type (WT), VEL2 deletion strain (ΔVEL2) and complementation (Comp. VEL2) (B). (C) MS2 spectrum of substance IV.(TIF)Click here for additional data file.S14 Fig(A) and (B) Single ion monitoring (SIM) for substance V with m/z 591.3887 and a mass tolerance of 5.00 ppm in positive ion mode. Depicted are the wild type (WT), VEL1 deletion strain (ΔVEL1) and complementation (Comp. VEL1) (A) as well as the wild type (WT), VEL2 deletion strain (ΔVEL2) and complementation (Comp. VEL2) (B). (C) MS2 spectrum of substance V.(TIF)Click here for additional data file.S15 Fig(A) Single ion monitoring (SIM) for substance X with m/z 331.0606 and a mass tolerance of 5.00 ppm in negative ion mode. Depicted are the wild type (WT) and the overexpression of VEL1 (OE-VEL1). (B) MS2 spectrum of substance X. (C) Single ion monitoring (SIM) for substance XI with m/z 317.0811 and a mass tolerance of 5.00 ppm in negative ion mode. Depicted are the wild type (WT) and the overexpression of VEL1 (OE-VEL1). (D) MS2 spectrum of substance XI.(TIF)Click here for additional data file.S16 Fig(A) LC/MS combined with photodiode array detection (PDA) analysis of secondary metabolites. Secondary metabolites were extracted from two-week-old fungal mycelium grown on Czapek-Dox-Medium (CDM) supplemented with glucose. Depicted are the wild type (WT), VEL3 deletion strain (ΔVEL3) and a VEL3 complementation strain (Comp. VEL3). All three chromatograms look similar, but increased relative absorbance was observed for the substances I-V in the deletion strain. (B) Selected single ion monitoring (SIM) for the seven substances with increased relative abundance in the VEL3 deletion strain in comparison to wild type and complementation . Substance I: m/z 193.0493, mass tolerance 5.00 ppm; substance II: m/z 235.0603, mass tolerance 5.00 ppm; substance III: m/z 193.0499, mass tolerance 5.00 ppm; substance IV: m/z 577.3722, mass tolerance 5.00 ppm; substance V: m/z 591.3887, mass tolerance 5.00 ppm; substance X: m/z 331.0606, mass tolerance 5.00 ppm; substance XI: m/z 317.0811, mass tolerance 5.00 ppm. (C) Quantification of scytalone production relative to the wild type in the VEL3 deletion (ΔVEL3) strain and the overexpression strain of VEL1 (OE-VEL1). In the left part an example of the scytalone peak is depicted for the wild type, the VEL3 deletion and the VEL1 overexpression strain. In the right part the size of the scytalone peak of at least two independent replicates was quantified relative to the wild type. Significant differences were calculated by t-test and indicate: **:p<0.01; ***:p<0.001.(TIF)Click here for additional data file.S17 Fig4 spores were spotted on plates  and incubated for 10 days at 25°C. Single colonies on SXM are shown from the back, colonies on CDM are shown from the top of the plate. Cross sections were made through the middle of the colony. (A) Lae1 is dispensable for microsclerotia formation. The LAE1 deletion strain has a similar phenotype as the wild type when grown on SXM or CDM. (B) Velvet proteins fused C-terminally to GFP under the control of their native promotor show no phenotypic alterations compared to the wild type. (C) Overexpression strains of velvet proteins fused to GFP and transformed with a strain with histone H2B-RFP labeled nuclei have a wild type-like appearance. (D) Overexpression strains of VEL1 and VEL2 fused to GFP and transformed into the deletion strain of VEL2 or VEL1, respectively, have a phenotype similar to the deletion strain. (E) Phenotype of a wild type ectopically expressing high amounts of free GFP and VEL1 deletion strain expressing the same construct.From the indicated strains 5x10(TIF)Click here for additional data file.S18 FigVOS1 and LAE1 deletion strains resemble the wild type.LC/MS combined with photodiode array detection (PDA) analysis of secondary metabolites. Secondary metabolites were extracted from two-week-old fungal mycelium grown on Czapek-Dox-Medium (CDM) supplemented with glucose. Chromatograms of (TIF)Click here for additional data file.S19 Fig(A) Single ion monitoring (SIM) for substance VI with m/z 263.1284 and a mass tolerance of 5.00 ppm in negative ion mode. Depicted are the wild type (WT), VEL1 deletion strain (ΔVEL1) and complementation (Comp. VEL1). (B) MS2 spectrum of substance VI. (C) Single ion monitoring (SIM) for substance VII with m/z 351.1264 and a mass tolerance of 5.00 ppm in negative ion mode. Depicted are the wild type (WT), VEL1 deletion strain (ΔVEL1) and complementation (Comp. VEL1). (D) MS2 spectrum of substance VII.(TIF)Click here for additional data file.S20 Fig(A) Single ion monitoring (SIM) for substance VIII with m/z 233.1544 and a mass tolerance of 5.00 ppm in positive ion mode. Depicted are the wild type (WT), VEL1 deletion strain (ΔVEL1) and complementation (Comp. VEL1). (B) MS2 spectrum of substance VIII. (C) Single ion monitoring (SIM) for substance IX with m/z 249.1483 and a mass tolerance of 5.00 ppm in positive ion mode. Depicted are the wild type (WT), VEL1 deletion strain (ΔVEL1) and complementation (Comp. VEL1). (D) MS2 spectrum of substance IX.(TIF)Click here for additional data file.S21 Fig4 freshly harvested spores were incubated in PDM for 16h at 25°C in light or darkness. Differential interference contrast (DIC), green fluorescent filter view (GFP), red fluorescent filter view (RFP) and a merge of GFP and RFP channels are shown. Scale bar = 10 μm. (A) Velvet protein subpopulations are primarily localized to the nucleus in light as well as in darkness with additional small Vel2 subpopulations in the cytoplasm. Nuclei are visualized by histone H2B-RFP. (B) High levels of Vel1 or Vel2 can enter the nucleus independently of each other in light or darkness in ΔVEL1 and ΔVEL2 strains, respectively. (C) Controls for subcellular localization studies in V. dahliae. The wild type (WT) was used as negative control, a strain with overexpressed GFP (WT/OE-GFP) as positive control and a strain in which histone H2B-RFP is overexpressed (WT/HISTONE-RFP) as positive control.Subcellular localization analysis of fusions of Vel1, Vel2, Vel3 and Vos1 with GFP by fluorescence microscopy. 1x10(TIF)Click here for additional data file.S22 Fig6 freshly harvested spores were inoculated for analysis of protein abundances in liquid potato dextrose medium (PDM) for filamentous growth, in liquid pectin-rich simulated xylem medium (SXM) for conidia formation and on SXM plates covered with a nylon membrane for microsclerotia development. The fungus was grown for three and six days at 25°C in light or darkness. Western hybridization with a GFP antibody was performed with crude extracts of the indicated strains . Presence (+) or absence (-) of full-length protein during growth in light or darkness during different developmental conditions  after three and six days. Vel2 is stable during filamentous growth, destabilized during conidiation in light and initially more stable in light than in darkness during microsclerotia formation.1x10(TIF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.S11 Table(PDF)Click here for additional data file.S12 Table(PDF)Click here for additional data file.


ARID1A-mutated cancers are currently under development, including EZH2. A synthetic lethal relationship between ARID1A and EZH2 has been revealed in several tumor entities. Although genomic alterations of ARID1A have been described in various cancers, no study has examined correlations between ARID1A gene mutation and protein expression with clinicopathologic parameters and prognosis, particularly in liver fluke-related cholangiocarcinoma (Ov-CCA). Here, we investigated the clinical significance of ARID1A mutations and protein expression in CCA tissues and determined whether there is a correlation with EZH2 protein expression.ARID1A is a member of the SWI/SNF chromatin remodeling complex. It functions as a tumor suppressor and several therapeutic targets in ARID1A were analyzed using the ICGC sequencing data in 489 of Ov and non Ov-CCA and assessed prognostic values.We evaluated ARID1A and EZH2 immunoreactivity using immunohistochemistry in 98 Ov-CCA with a wide range of clinicopathological features. Somatic mutations of ARID1A mutations . We observed that 12 of 13 tumors (92%) with loss of ARID1A expression had truncating mutations. There were nine of 13 tumors (69%) with loss of ARID1A expression and 25 of 41 tumors (61%) with low ARID1A expression exhibited distant metastasis . ARID1A was predominantly mutated in Ov-CCA compared to non Ov-CCA . There were 36 of 72 (50%) and 52 of 79 (66%) tumors with ARID1A mutation showed tumor stage IV and T3/T4, respectively. The significant mutual exclusivity and co-occurrence between ARID1A and TP53/KRAS mutations were not found in ICGC cohort. In addition, high EZH2 expression, a potential synthetic lethal target in ARID1A-mutated tumors, was detected in 49 of 98 Ov-CCA (50%). Importantly, neither ARID1A expression nor ARID1A mutations correlated with EZH2 expression in this cohort.While detecting a loss or reduction of ARID1A expression in 54 cases (55%) in Ov-CCA, ARID1A expression was associated with ARID1A inactivation, by somatic mutation or by loss of expression, frequently occurs in Ov-CCA. Reduction of ARID1A expression and/or somatic mutation was shown to be associated with CCA progression. These findings suggest that ARID1A may serve as a prognostic biomarker, and thus may be a promising therapeutic target for CCA.We found that  Opisthorchis viverri-associated CCA (Ov-CCA) has been associated with infestation of Ov. The highest incidence rates of Ov-CCA are in South-East Asia, where endemic areas of liver flukes occur (Cholangiocarcinoma (CCA) is the second most common liver cancer that develops along the epithelial bile duct, accounting for 10% to 20% of primary liver cancer . The inces occur , especiaes occur . In contes occur .Clinical trials evaluating targeted therapies in unselected CCA populations have shown minimal benefits . The ASCARID1A were detected in 7% to 36% of ICC  encodes a nuclear protein involved in chromatin remodeling. Inactivating mutations in ARID1A have been identified in a wide variety of malignancies   . EZH2 isortunity . Clinicac cancer . Thus, AARID1A have been described in CCA, no study has determined whether there are correlations between ARID1A gene mutation and protein expression with clinicopathologic parameters and prognosis, particularly in Ov-CCA. In the present study, we analyzed sequencing data from the International Cancer Genome Consortium (ICGC) of 489 tumors and performed immunohistochemical staining for ARID1A in 98 Ov-CCA which were sequenced in ICGC cohort. We evaluated whether ARID1A expression and mutational status could be a prognostic biomarker for Ov-CCA. To study if ARID1A could be a surrogate biomarker for EZH2 inhibitors in CCA, we evaluated EZH2 protein expression in Ov-CCA. The correlation between alterations of ARID1A and EZH2 expression in matched CCA tissues was evaluated for the first time in this study.Although genomic alterations of Ninety-eight paraffin embedded human CCA tissues and clinical data were obtained from Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand. All patients signed consent forms. The study was approved by the Ethic Committee for Human Research, Khon Kaen University (HE611195). The primary tumor at the time of resection was staged according to the 7th AJCC.For mutational analysis, a total of 489 mutational data of bile duct tumors were obtained from the ICGC data portal .36-6300, Invitrogen, CA, USA). IHC was performed as previously described . The primary antibodies include rabbit polyclonal anti-ARID1A  and rabbit anti-EZH2 antibody  and was scored by multiplying of the intensity and frequency of DAB-staining results . The intp-values were calculated using Benjamini–Hochberg correction. The survival analysis was determined using Kaplan–Meier estimate with log-rank test. Statistical significance was considered at p <  0.05.The statistical analysis was carried out using SPSS software (version 19.0). The association between mutational data, protein expression profile and the clinicopathological features of CCA patients were performed using Chi-squared or Fisher’s exact test. Adjusted p = 0.028, adjusted p-value = 0.168), suggesting that ARID1A may play a role in CCA progression. Of note, ARID1A expression was associated with ARID1A mutations . Most of ARID1A mutations were truncating , including nonsense , frameshift insertion/deletions . Interestingly, ARID1A was predominantly  mutated in Ov-CCA compared to non Ov-CCA . As shown in ARID1A mutations tended to associate with CCA staging , liver fluke related-CCA , and T factor . Of note, there were 36 of 72 (50%) tumors with ARID1A gene mutation showed tumor stage IV  of ARID1A wildtype tumors were non Ov-CCA .Even though the landscape of  489 CCA  and eval 489 CCA , ARID1A stage IV  and 52 oTP53 and KRAS mutations in CCA, we performed mutual exclusivity and co-occurrence analysis of ARID1A and TP53/KRAS mutations. The significant mutual exclusivity and co-occurrence between ARID1A and TP53/KRAS mutations were not found in the ICGC cohort , Ov-CCA  and non Ov-CCA  (Given the high frequency of C cohort . There w–1.727]) –2F.ARID1A mutant tumors were T3 or T4 and predominantly ICC  , but it was not statistically significant (p = 0.843).In non Ov-CCA, we found that 63% (30/48) of 49, 82%) . The assn Ov-CCA . There wARID1A mutated cancers are in development, including EZH2 inhibitors. EZH2 inhibition in ARID1A mutated tumors acts in a synthetically lethal manner to inhibit cancer progression, revealing a therapeutic opportunity. Since the response to EZH2 inhibitors correlates with EZH2 overexpression, we then investigated protein expression of EZH2 by IHC staining in tumor cells of Ov-CCA  with loss of ARID1A expression and 25 of 41 CCA (61%) with low ARID1A expression exhibited distant metastasis. Similar correlation between ARID1A expression with invasion was reported in ICC (Regarding the high frequency of d in ICC , suggestd in ICC . Reducedd in ICC . Similard in ICC .ARID1A mutation or protein loss to survival in several carcinomas (ARID1A mutation and protein expression were not significantly associated with overall survival of both Ov-CCA and non Ov-CCA. To date, overall survival affected by mutation in ARID1A has been shown in ICC (ARID1A mutation and protein expression was not determined in that studies. In contrast to ICC, there was no correlation between loss of ARID1A expression and overall survival in ECC (Previous studies have revealed the relevance of rcinomas , althougn in ICC . Low expn in ICC . These fl in ECC . We specl in ECC . In contl in ECC  and may ARID1A mutations was previously reported in ovarian and uterine endometrioid carcinoma (ARID1A mutation showed a loss of ARID1A expression (ARID1A gene. Of note, we observed half of CCA with ARID1A truncating mutations showed positive protein staining. Our findings are similar to those reported in ovarian clear-cell and endometrioid carcinomas (ARID1A mutation occurred in clones of cells within the tumor, resulting in a heterogeneous staining pattern of ARID1A expression. Moreover, the presence of ARID1A immunoreactivity in tumors positive for ARID1A mutation may indicate that haploinsufficiency is pathogenic, as has been reported in mice (Loss of ARID1A protein correlated with the presence of arcinoma . All mutpression . In thisrcinomas . It is l in mice .ARID1A mutation showed tumor stage IV and T3/T4, respectively. Moreover, ARID1A was predominantly mutated in Ov-CCA. These data suggest that ARID1A mutation may involve in CCA progression and the different etiology may be one of the underlying factors that drives CCA heterogeneity. ARID1A and KRAS mutations. However, mutually exclusivity between mutations of ARID1A, TP53 and KRAS was not found in this study.Interestingly, we found that there was 50% and 66% of CCA with ARID1A mutations have focused largely on using synthetic-lethal approaches. ARID1A-mutated cells. EZH2 was overexpressed in many solid cancers, suggesting the promise of therapeutic potential of EZH2 inhibitors for cancers (in vitro study showed that ARID1A-depletion did neither increase EZH2 protein or trimethylated H3K27 levels (ARID1A-mutated and wildtype CCA cells.Novel ways of treating patients with  cancers . The res cancers . Neither7 levels . These fARID1A mutation may involve in CCA progression and predominantly in CCA tumors with high tumor stage. Importantly, ARID1A protein expression was also correlated with ARID1A mutation, suggesting that loss of ARID1A immunoreactivity might be used as a surrogate marker to detect ARID1A mutations in tissues. To expand the therapeutic portfolio for CCA patients, EZH2 expression was investigated in Ov-CCA. Neither ARID1A mutation nor protein expression correlated with EZH2 expression. Further studies are necessary to determine the role of ARID1A-deficiency in response to EZH2 inhibitor in CCA.In conclusion, this is the first investigation that showed the correlation between mutations and expressions of ARID1A within the same Ov-CCA cohort. Based on ARID1A protein expression and mutational analysis, we found that ARID1A inactivation, by somatic mutation or by loss of expression, frequently occurs in Ov-CCA. CCA with distant metastasis had lower ARID1A expression than those without distant metastasis. 10.7717/peerj.10464/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.10464/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10464/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10464/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.10464/supp-5Supplemental Information 5Wildtype denotes tumors with ARID1A wildtype, Missense denotes tumors with ARID1A missense mutation, Truncation denotes tumors with truncating ARID1A mutation.Click here for additional data file.10.7717/peerj.10464/supp-6Supplemental Information 6ARID1A mutations and protein expression level of ARID1A and EAH2.Raw data of clinicopathological information, Click here for additional data file.


The transcription factor GhMML4_D12 interacts directly with a diverged WD40 protein (GhWDR), similar to but different from the MYB–bHLH–WD40 complex, in regulation of trichome development in cotton. In planta, a vital regulatory complex, MYB–basic helix–loop–helix (bHLH)–WD40 (MBW), is involved in trichome development and synthesis of anthocyanin and proanthocyanin in Arabidopsis. Usually, WD40 proteins provide a scaffold for protein–protein interaction between MYB and bHLH proteins. Members of subgroup 9 of the R2R3 MYB transcription factors, which includes MYBMIXTA-Like (MML) genes important for plant cell differentiation, are unable to interact with bHLH. In this study, we report that a cotton (Gossypium hirsutum) seed trichome or lint fiber-related GhMML factor, GhMML4_D12, interacts with a diverged WD40 protein (GhWDR) in a process similar to but different from that of the MBW ternary complex involved in Arabidopsis trichome development. Amino acids 250–267 of GhMML4_D12 and the first and third WD40 repeat domains of GhWDR determine their interaction. GhWDR could rescue Arabidopsis ttg1 to its wild type, confirming its orthologous function in trichome development. Our findings shed more light towards understanding the key role of the MML and WD40 families in plants and in the improvement of cotton fiber production. MIXTA genes, are important for the specification and regulation of plant cellular differentiation , was found to control the development of conical cell shape in the petal epidermis , responsible for fuzz fiber production (GhMML4 on chromosome D12 (GhMML4_D12), responsible for development of lint fiber (Li3), the largest source of natural textile in the world (2NSM) to adenine (A) in the Xuzhou142 fuzzless–lintless mutant (XZ142FLM) has resulted in the production of a TAA stop codon and early termination of GhMML4_D12 translation  derived from XZ142FLM  assay, a new kind of WD40 repeat protein (GhWDR) was identified as an interacting partner of GhMML4_D12 from the naked seed mutant nXZ142FLM . We founGossypium hirsutum acc. Texas Marker-1 (TM-1), the fuzzless or naked seed mutant n2NSM, and the fuzzless–lintless mutant XZ142FLM were used to investigate ﬁber development. All plants were grown at the Dangtu Breeding Station (DBS/NAU) with normal ﬁeld practices, and in the glasshouses of Nanjing Agriculture University. Arabidopsis seeds were sterilized with 20% bleach, plated on Murashige and Skoog medium , chilled at 4 °C for 3 d, and transferred to a growth room with a 16 h (22–24 °C)/8 h (19 °C) light/dark photoperiod. Nicotiana benthamiana was grown under a 16 h (28 °C)/8 h (22 °C) light/dark photoperiod.GhUBI1 (EU604080), as a standard control according to a previously described method (JXB online). The PCR was performed using SYBR-Green Real-time PCR Master Mix according to the manufacturer’s instructions , and the relative quantity of the target gene expression level was determined. The mean value and standard deviation of three biological replicates were calculated.Total RNA was isolated from cotton tissues and purified using a Qiagen RNeasy kit (Qiagen) according to the manufacturer’s instructions. Expression profiling of genes in cotton tissues was carried out by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using a cotton polyubiquitin gene, m, GhWDR, N-terminal-truncated domain (GhWDR-N), WD40 repeat domain (GhWDR-R), and C-terminal-truncated domain (GhWDR-C) without a stop codon were inserted into the binary vector pBINGFP4  leaf epidermal cells by A. tumefaciens infiltration  of GhWDR, GhWDR-N, GhWDR-R, and GhWDR-C were cloned into pGBKT7 vectors  and transferred into yeast strain AH109 using the high-efficiency lithium acetate transformation procedure. Yeast transformants were streaked on selective medium lacking tryptophan and adenine added to assay transcriptional activity. β-Galactosidase activity was also assayed by colony-lift ﬁlter assay using 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside  as the substrate.Phylogenetic analysis was conducted using two methods: neighbor-joining (NJ) and maximum likelihood (ML). NJ trees were constructed using MEGA 5.1 .A Y2H assay was performed using the Matchmaker GAL4 Two-Hybrid System following the manufacturer’s protocol (Clontech). Full-length cDNA of GhMML4_D12 was fused to the GAL4-DNA-binding domain of the bait vector pGBKT7 and transformed into yeast strain Y2H. A cDNA library from cotton ovules and fibers (−3 to ~25 d post-anthesis (DPA)) was constructed by transforming yeast strain AH109 with ds-cDNA and the pGADT7-Rec vector according to the manufacturer’s instructions. The library host strain was mated with bait strain Y2H, and the mating mixture was then spread onto SD/−Trp/−Leu/−His medium and incubated at 30 °C for 3~4 d. Positive clones were isolated and retransformed into bait strains to test their interaction using pGBKT7-p53/pGADT7 as the positive control and pGBKT7-Lamin c/pGADT7 as the negative control.600=1.2–1.5). Then 1 ml of yeast strain was centrifuged and resuspended in distilled water to the final concentration (OD600=0.4–0.6). The mating mixture was diluted in multiples of 10 and 100, and then spread onto SD/−Trp/−Leu/−His medium with different concentrations of AbA and incubated at 30 °C for 3~4 d.For the Y2H assay, all the GhMML4_D12 CDSs and their domain derivatives were cloned into pGBKT7, and the full-length or domain deletion forms of GhWDR were cloned into pGADT7. Primers used for the vector construction were presented in m, and GhWDR were fused with the N- or C-terminal fragment of yellow fluorescent protein (nYFP or cYFP) to generate GhWDR-nYFP, cYFP-GhMML4_D12, and cYFP-GhMML4_D12m plasmids. Primers used for plasmid construction are presented in pSPYNE and pSPYCE plasmids have been reported previously  to a final concentration of OD600≈0.6–0.8. Equal volumes and concentrations of different combinations of Agrobacterium strains were co-infiltrated into N. benthamiana leaves. Plants were incubated at 23 °C for 2–3 d. YFP fluorescence was detected with a Zeiss microscope (Zeiss LSM710) and analysed using ZEN software. All experiments comprised three biological replicates.All constructs were transformed into m were cloned into pET-GST, and the CDS of GhWDR was fused with pET-28a. These constructs were transformed into E.coli to express glutathione S-transferase (GST)–GhMML4_D12, GST–GhMML4_D12m, and His–GhWDR proteins, respectively. The pull-down assay was performed as described previously . GhMML4_D12, GhMML4_D12m, and GhWDR promoters were ligated into the pAbAi vector (Clotech). All primers used are listed in TM Gold Yeast One-Hybrid Library Screening System kit .The CDSs of GhMML4_D12, GhMML4_D12N. benthamiana leaves. The nuclear localization site (NLS) was ligated to the 3′ end of GhMML4_D12 and GhMML4_D12m promoters, then GFP was fused with the 3′ end of NLS to construct GhMML4_D12pro:NLS-GFP and GhMML4_D12mpro:NLS-GFP, respectively. The full length CDSs of GhMML4_D12, GhMML4_D12m, GhWDR, and GUS were driven by the Cauliflower mosaic virus (CaMV) 35S promoter to construct 35Spro:GhMML4_D12, 35Spro:GhMML4_D12m, 35Spro:GhWDR, and 35Spro:GUS. These constructs were then introduced into A. tumefaciens (strain GV3101). Infected tissues were analysed 48 h after inﬁltration. The GFP signal was observed under a confocal laser scanning microscope (Zeiss LSM710). All experiments were repeated with three independent biological replicates with similar results.A transient expression assay was performed in GhMML4_D12, GhMML4_D12m, and GhWDR promoters were inserted into pGreenII 0800-LUC to drive the firefly LUC reporter gene, with Renilla (REN) luciferase controlled by the constitutive 35S promoter on the same plasmid as a reference to normalize infection efficiency. The CDSs of GhMML4_D12, GhMML4_D12m, and GhWDR were inserted into the pGreenII62-SK vector under the control of the 35S promoter. All primers used are listed in A. tumefaciens (strain GV3101). The transformed Agrobacterium cells were mixed with the Agrobacterium strains harboring the effectors and reporters, in a volume ratio of 1:2.The Agrobacterium mixtures into the abaxial side of N. benthamiana leaves using a syringe. After culturing for 3 d, ﬁreﬂy LUC and REN activities were measured using the Dual-Luciferase Reporter Assay System , and the LUC/REN ratio was determined . Three biological repeats were measured for each combination.Transient transformation was conducted by inﬁltration of the GhWDR were fused with pBINGFP4, under cotton native promoter. Binary construct (GhWDRpro:GhWDR) was introduced into A. tumefaciens strain GV3101 and subsequently transferred into Arabidopsis using the floral dip method (−1) added with kanamycin (50 µg ml−1) for 7 d. Kanamycin-resistant seedlings were moved onto fresh kanamycin plates for 10 d before transfer to soil. Progeny from self-fertilized primary transformants were examined for complementation of other ttg1 mutant phenotypes. Trichome restoration was examined by growing on plates containing MS salts and 1.2% phytagel for about 2 weeks. Root hair position was studied in seedlings grown vertically on plates containing MS salts and 1.2% phytagel for about 5–6 d. Anthocyanin synthesis was investigated in seedlings grown on plates containing MS salts and 1.2% phytagel for about 10 d. Seed coat color was examined by stereoscope after harvest.The full length CDS of the p method . SelectiGhWDR-R was fused with GFP driven by CaMV35S (35Spro:GhWDR-R) into ttg1 Arabidopsis mutants. Selection of transformants and phenotype examination were as above.GhMML4_D12 regulates lint fiber development, we used a Y2H assay to identify its potential interaction partners. Full-length GhMML4_D12 CDS was isolated and fused with bait vector (pGBKT7-GhMML4_D12) to screen the cotton fiber specific yeast library. We did not observe any bHLH genes in the Y2H screening, probably due to both GhMML genes lacking the core amino acids needed in the interaction with bHLH as reported before , but GhWDR-A at the A subgenome had a seven amino acid insertion in its N-terminus . Bait and prey vectors were co-transformed into yeast and the GhMML4–GhWDR interaction was reconstructed. However, no interaction was observed between GhMML4_D12m and GhWDR , and GhMML4_D12 and GhMML4_D12nd GhWDR .m, and GhWDR revealed that all these proteins were mainly present in the cell nucleus  into the ttg1 mutant. Of the 19 primary kanamycin-resistant T1 transgenic lines, four  with ectopic expression of GhWDR showed the wild type phenotype, whereas no trichomes were observed in non-transformed (indicating as ttg1) and empty-vector controls (indicating as 35Spro:GFP)  . qRT-PCRhenotype .ttg1 mutant phenotypes. We transformed GhWDR-R fused to GFP driven by CaMV35S (35Spro:GhWDR-R) into ttg1 Arabidopsis mutants. Phenotype examination revealed that no T1GhWDR-R overexpression plants had wild type anthocyanin synthesis or trichome development. After all seeds were harvested, we were surprised to find that some of the mature seeds from GhWDR-R overexpression T1 lines were brown, similar to the wild type  were ligated into pAbAi. Sequence comparison of these two promoters showed no notable difference between them  and GhMML4_D12 or GhMML4_D12m promoter-driven firefly luciferase  reporter approach. ciferase . The expEN ratio , while cUS alone . Co-exprUS alone . All them and GhWDR on GhWDR was also investigated. We found that neither GhMML4_D12 nor GhMML4_D12m was able to activate the expression of GhWDR, and GhWDR was also unable to activate its own transcriptional activity  associated with other types of trichome or root hairs. So, these MML transcription factors have likely evolved independent ovule-specific functions to direct the expansion of epidermal cells to produce the cotton fiber. These findings will also be helpful to illustrate the roles of MIXTA genes in regulating epidermal cell differentiation such as production of a ‘pulpy layer’ secreted from the teguments surrounding cacao seeds, and mucilages in other Malvaceae fruit (Abelmoschus (okra), and Cola (kola)).The MYB gene family members, which largely function as transcription factors, have several conserved binding domains. Of these, R2R3 MYB is the largest subgroup. Subgroup 9 of the R2R3 MYBs contains TTG1 locus regulates several developmental and biochemical pathways in Arabidopsis, including the formation of hairs on leaves, stems, and roots, as well as the production of seed mucilage and anthocyanin pigments  and the WD dipeptide (Trp-Asp) at the C- and N-termini . WD40 preen them . GhWDR celopment . Furthercue ttg1 . FurtherThe MBW ternary complex consists of MYB, bHLH, and WD40 transcription factors, and has important roles in anthocyanin and proanthocyanin biosynthesis and trichome initiation in a variety of plant species. Usually, WD40 proteins do not interact with MYB proteins directly; they just serve as scaffolding molecules to interact with bHLH factors, and then bHLH factors interact with different kinds of MYB factors to regulate different cell fates . The MYBMML factor, GhMML4_D12, could directly interact with a newly found WD40 factor, GhWDR, but not with any bHLH genes. And no interaction between GhMML4_D12m and GhWDR was observed  gene AtMYB106 was a negative regulator of branching in Arabidopsis, while its homologue GhMYB25 is reported to be a key regulator for cotton ﬁber initiation (We believe cotton fiber (seed trichome) and leaf/root hair trichomes have a similar but different regulatory network. Both Arabidopsis trichomes and cotton fibers are unicellular structures of epidermal origin. And they may share similar molecular machinery of regulation. Several genes implicated in cotton fiber development were shown to functionally substitute for their homologues in Arabidopsis. Ectopic expression of itiation , and genDue to the similarity and difference between the GhMML4–GhWDR complex and MBW complex, therefore, we report here that cotton fibers and Arabidopsis leaf/hair trichomes may have similar but different regulatory networks in plants. Further investigation is needed to uncover the mechanisms underlying cotton fiber development and trichome formation in other organs of the plant.2NSM. GhWDR simply serves as a scaffolding molecule in protein–protein interactions to assist the function of GhMML4_D12. However, the mutated GhMML4_D12m has lost the C-terminal domain, and therefore could not interact with GhWDR in XZ142FLM. GhMML4_D12 bound to its own promoter to enhance its transcriptional activity, and this may help in the function of GhMML4_D12 in n2NSM, but GhMML4_D12m did not do this in XZ142FLM (2NSM.In summary, the C-terminal domain of GhMML4_D12 and WD40 repeat domain of GhWDR determine their interaction in nXZ142FLM . These tJXB online.Supplementary data are available at GhWDR.Fig. S1. Cloning and characterization of Fig. S2. Expression pattern analysis of WD40 genes in fiber mutants.m, and GhWDR protein in leaf cells of tobacco.Fig. S3. Subcellular localization of GhMML4_D12, GhMML4_D12GhMMLs genes in cotton.Fig. S4. Alignment of amino acids from 10 m and GhTTG1–GhTTG4, AtTTG1 in yeast.Fig. S5. Interaction between GhMML4_D12, GhMML4_D12GhMML4_D12 promoters from TM-1, n2NSM, and XZ142FLM.Fig. S6. Sequence comparison of Table S1. All primers developed and used in present research.Table S2. Proteins interacting with GhMML4_D12 identified by Y2H screening.eraa104_suppl_supplementary_figures_S1-S7_tables_S1-S2Click here for additional data file.


E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from  Entamoeba histolytica is an enteric protozoan parasite. It causes a wide range of infections such as diarrhoea, liver abscess, and in severe condition, invades the brain. For the successful completion of the amoebic pathogenesis, there is a remarkable change in amoebic cytoskeleton as soon as the parasite adheres to the host extracellular matrix. However, till date, only very few amoebic proteins have been reported to regulate the cytoskeleton dynamics and EhRab21 is one among them. In this study, for the first time, we have identified an effector of EhRab21, namely EhC2B, and have established its role in phagocytosis and actin nucleation. EhC2B is a member of C2 superfamily and binds to the lipids in the presence of calcium. In the amoebic trophozoites, EhC2B localises to pseudopodal membrane protrusions as well as advancing tips of the phagocytic cup. It also catalyses the actin polymerization via nucleation of actin monomer, thereby, promoting membrane deformation. Thus, EhC2B acts like a link between calcium signalling and cytoskeleton remodelling with implication on phagocytosis. Entamoeba histolytica, a highly motile and pathogenic parasite, is an etiological agent of amoebic dysentery and liver abscesses in humans. It invades the mucosal barriers of the intestinal epithelium and, in turn, spreads into the extraintestinal organs, resulting in amoebiasis. This infectious disease is characterized by extensive destruction of human tissues caused by killing and phagocytosis of human cells . The cells were lysed, and the lysate was clarified by centrifugation at 50,000 × g for 30 min at 4°C. Proteins were purified using affinity chromatography, followed by size exclusion chromatography .The His-tagged C2 domain of wild-type EhC2B (His-EhC2B121)  containing an excess of nucleotide. The binding of GSTEhRab21to HisEhC2B121 or MBPEhC2BHis was detected by immunoblotting using anti-His and anti-MBP antibody, respectively. Similarly, to detect the actin-binding, one micromolar of MBPEhC2BHis and HisEhC2B121 protein was allowed to bind with the Ni-NTA bead and incubated with 1μM of purified rabbit muscles actin (Thermo scientific) for one hour. The binding was detected by immunoblotting using an anti-actin antibody.Fluorescence measurements were performed with a fluoromax-4 spectrofluorometer (Horiba Jobin). Fluorescence of MantGppNHp (Sigma-Aldrich) was measured directly at 360nm excitation and 440nm emission wavelength. Experiments were performed in a buffer containing 25 mM HEPES (pH 7.3), 100 mM NaCl, and 2 mM DTT at 25°C. EhRab21 was loaded with MantGppNHp, GDP and GppNHp and passed through NAP-5 column to remove unbound nucleotide. All the reactions were performed separately in a cuvette containing 100μl reaction volume, and the fluorescence intensity of each scan was recorded and overlaid.2+ and EhC2B121/EhC2A or EhRab21 and full-length of EhC2B or its C2 domain (EhC2B121). Proteins were dissolved in 25 mM HEPES (pH7.4), 100 mM NaCl, 1 mM DTT and 5% glycerol. 76 μM of EhRab21 solution was titrated into 10 μM of HisEhC2B121 or MBPEhC2B in the presence of GDP and GppNHp at 25°C. Similarly, 700μM Ca2+ was titrated into 50 μM of wild-type and calcium mutant D72N, of EhC2B121. All the experiments were performed using Nano ITC (TA Instruments). Data were processed using nano analyze software to extract the thermodynamic parameters ΔH, Kd (1/Ka) and the stoichiometry (n), ΔG and ΔS were derived from the relationship: ΔG = -RTlnKa and ΔG = ΔH—TΔS.ITC experiments were carried out to determine the thermodynamic parameters for interaction between CaLipid overlay assay was performed using PIP strips according to the manufacturer's instructions (Thermo Scientific). The membrane was incubated with 0.5 mg/ml EhC2B for 4 hours in blocking buffer at room temperature. The HisEhC2B121 protein bound to the lipids was detected by anti-His antibody [1:10000 (Thermo Scientific)].3), dried under argon. The remaining moisture was removed by keeping the lipid in a desiccator for 1 hour followed by resuspending it into a mixture of CHCl3: Methanol (MeOH): 1N hydrochloric acid in a 2:1:0.01 molar ratio. The lipids were dried once again and allowed to desiccate followed by resuspending in CHCl3: MeOH in a 3:1 ratio and dried again under argon. The dried lipids were resuspended in CHCl3 and stored at -20°C. The control liposomes were prepared by mixing POPC and POPE in a 90:10 molar ratio. The phosphoinositides containing liposomes were prepared by mixing POPC, POPE and PIPs in an 80:10:10 molar ratio respectively. 30% of POP was used to prepare the control, as well as phosphoinositides containing liposome. A suspension of multilamellar liposomes containing sucrose was prepared by hydrating the dried lipid in 25 mM HEPES (pH 7.2) containing 220 mM sucrose. This solution was then freeze-thawed to produce unilamellar liposomes. Liposomes were diluted 1:5 in 25 mM HEPES (pH 7.2), and 125 mM NaCl solution. The solution was centrifuged at 250,000 g to remove sucrose from the medium. The pelleted liposomes were resuspended in a solution containing 25 mM HEPES (pH 7.2), and 125 mM NaCl to the desired concentration of 0.5 mM. All liposomes were used within one day of preparation.All the phosphoinositides were protonated before usage. Briefly, powdered lipids were resuspended in chloroform  containing 125 mM NaCl. The samples were then collected for analysis on a precast 4–12% bis-tris gel (Novex) by Coomassie staining.Twenty micromolars of the protein of interest wild-type, calcium  and lipid  mutants of HisEhC2B121 was added to a final volume of 100 μl of the liposome solution both in the presence and absence of Ca2)] and subjected to high-speed centrifuge at 90,000 rpm for 40 minutes. The upper part of the solution was taken carefully into a separate tube without touching the bottom of the tube.Pyrene-labelled rabbit muscles actin was thawed on the ice for overnight, diluted with the G buffer  and with rabbit polyclonal anti-Cysteine Synthase (1:1000 dilution) wherever necessary. Further, it was washed and incubated with HRP-conjugated anti-mouse  or anti-rabbit  respectively and eventually developed by enhanced chemiluminescence.E. histolytica grown in glass culture tubes were chilled on ice for 10 min. Trophozoites were pelleted down at 900xg for 3mins, counted by haemocytometer, resuspended in BI-S-33 medium and transferred into 8-well chamber slides  at 37°C having 5x104 cells/well. After 15 mins, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton X-100 in 1XPBS. Trophozoites were then blocked using 5% foetal bovine serum in 1XPBS for 45 mins. Next, trophozoites were co-incubated with Alexa-conjugated Phalloidin568  secondary antibody (1:50 dilution) and DAPI  for 1 hr at room temperature. Following three washes with 1XPBS solution, coverslips were mounted on the glass slide using Mowiol. Slides were then examined using LSM-780 laser scanning confocal microscope  with a 63X/1.4 NA oil immersion objective lens. Fluorescence signals were captured in individual face (XY axes) planes throughout the cellular z-axis at 1μm intervals.The gene knock-down pKT3M vector carrying the EhC2B gene was transfected into the trophozoites. These transfected cells, as well as untransfected trophozoites, were incubated in glass wells for 10 mins and then fixed by 4% PFA. These trophozoites were then co-incubated with Alexa-conjugated Phalloidin568  secondary antibody (1:50 dilution). Following confocal microscopy, the numbers of the cellular protrusions filled with Phalloidin568 labelled-actin were then quantified in each cell. The analysis was performed for n = 195 cells from N = 4 set of experiments inclusive of both technical as well as biological repeats.Five millilitres of human blood was collected to extract RBCs which were then briefly washed with 1% bovine serum albumin (BSA) (in 1XPBS) and immediately incubated with Cell Tracker Orange (50 μg/ml)  for 45 min at 37°C in the dark. Afterwards, the RBCs were subsequently washed four times with 1% BSA and then resuspended in the BI-S-33 medium for experiments.E. histolytica trophozoites and Cell-Tracker Orange labelled RBCs were resuspended separately in incomplete BI-S-33 medium. Equal numbers of amoebic cells were then incubated with RBCs in 1:50 ratio or 1:100 ratio for assays involving EhC2B-knockdown strain, into 8-well chamber, slides for 10 min at 37°C. Cells were then fixed with 4% PFA and further processed for fluorescence microscopy.The trophozoites expressing GFP-tagged fusion proteins were grown in 20μg/ml G418, harvested in log phase, washed, and resuspended in BI-S-33 medium. Trophozoites were transferred to glass-bottom dishes (MatTekCorp.), allowed to settle for 10 min, and observed using an Olympus inverted confocal microscope equipped with a 60X/1.4NA oil immersion objective inverted microscope. Mercury Arc laser (488nm) was used for excitation of GFP. At least 10 examples were observed in each experiment, and one representative set of images are shown.1⁄2, the FRAP curve from each of the experiments was normalised and fitted to an exponential recovery curve. The curve fit was done with help from CellSens software for the Olympus microscope.FRAP was performed using the FRAP module on Olympus Confocal microscope Software. For FRAP, the amoebic trophozoites harbouring GFPEhC2Bwere observed 48 hours after addition of 20 μg/ml G418 using Olympus inverted confocal microscope equipped with 60x/1.4NA oil immersion objective and a 488 nm Mercury Arc laser. Bleaching was performed during fly forward using ROI scan features and high laser power. In the FRAP experiments, spherical areas of approximately 5 μm diameter were photobleached for 500msec, and subsequently, images of the area were collected approximately every 4 frames/s. To calculate the time of 50% signal recovery, τTrophozoites were washed twice with BI-S-33 media followed by resuspension in Complete BI-S-33 media (Incomplete BI-S-33 media supplemented with 15% Albumin Bovine Serum). BAPTA-AM (dissolved in DMSO) was also added and raised to respective final working concentrations (0μM-500μM). These cells were immediately incubated into 8-well plates for 30 mins at 37°C. Afterwards, the cells were quickly fixed with 4% paraformaldehyde and processed further for fluorescence analysis.Statistical significance was determined by comparing the differences between different conditions and involving the use of unpaired two-tailed Student's t-test with the help of GraphPad Prism version 6.0.S1 Fig(A) MBPEhC2BHis full-length protein purification. The SDS-PAGE analysis of MBPEhC2BHis purification using Ni-NTA based affinity chromatography. (B) Size-exclusion chromatography of MBPEhC2BHis. The fraction from the affinity purification was subjected to superdex 200 prep grade column (16/60) . The recombinant MBPEhC2BHis was eluted at the elution volume of 76 ml. (C) SDS-PAGE analysis of the fractions from the size-exclusion chromatography.(TIF)Click here for additional data file.S2 Fig(A) The SDS-PAGE analysis of HisEhC2B121, purified using Ni-NTA affinity chromatography. (B) The protein bound to the Ni-NTA beads was eluted, concentrated and subjected to size exclusion chromatography using superdex75 prep grade column (16/60) . The HisEhC2B121 was eluted at an elution volume of 85 ml, and the protein present in different fractions was detected by Coomassie staining.(TIF)Click here for additional data file.S3 Fig(A) Fluorescence-based assay for EhRab21 and EhC2B binding. 1μM of Mant-GppNHp bound EhRab21 was incubated with HisEhC2B121 in the cuvette. The arrows indicate different time points where the latter protein was added to the final concentration of 2μM, 4μm, 6μM and 8 μM, respectively. The fluorescence emission intensity was measured at 360nm excitation and 440nm emission wavelength. To assess the nucleotide specific interaction of EhC2B with EhRab21, GppNHp-GSTEhRab21 and GDP-EhRab21 were added at the indicated time points to the final concentration of 20μM in the reaction mixture. (B) The overlay of the fluorescence emission spectrum of mantGppNHp-GSTEhRab21 obtained with 2μM (black), 4μM (violet), (green) and 8μM (maroon) final concentrations of the C2 domain of EhC2B. The spectrum was obtained with 20μM of GppNHp-EhRab21 (brown) and GDP-EhRab21 (red) in the reaction mixture, respectively.(TIF)Click here for additional data file.S4 Fig2 at room temperature in the ITC cell. The lower panel represents the best least-square fit of the integrated heat obtained after subtracting the heat of dilution.The top panel shows the raw data obtained from the titration of 200μM of HisEhC2A (C2 domain) against 2 mM CaCl(TIF)Click here for additional data file.S5 FigNitrocellulose membrane with different lipids spots was blocked by 5% BSA prepared in TBST and then incubated with (A) wild-type and (B) lipid-mutants HisEhC2B121 for 4 hours at room temperature. The binding of the C2 domain protein with lipids was detected by immunoblotting by using an anti-His antibody.(TIF)Click here for additional data file.S6 FigThe fluorescence intensity of GFPEhC2B was analysed by drawing a line across multiple amoebic trophozoites and change in the fluorescence intensity with the distance is shown by line intensity plot.(TIF)Click here for additional data file.S7 FigThe montage shows the abundance of GFPEhC2B beneath the membrane as indicated by white colour arrows.(TIF)Click here for additional data file.S8 Fig(A) Total cell lysates (100μg) from pEhEx-HA (Vector control) and pEhEX-HAEhRab21 transfected amoebic trophozoites were analysed on SDS-PAGE followed by immunoblotting using anti-HA (1:1000) antibody. For loading control anti-EhCS1 (1:1000) antibody was used. The blot was then probed with secondary antibodies, anti-mouse (1:10000) and anti-rabbit (1:1000) respectively. (B) Amoebic trophozoites were stably expressing pEhEx-HAEhRab21were incubated with Cell Tracker Orange-labelled RBCs (1:50) for 10 mins at 37°C. Trophozoites were then fixed, permeabilised, stained and later studied by confocal microscopy. The RBCs ingested per cell were enumerated manually for n = 48 cells from N = 4 set of biological repeats. The data represents Mean±SEM; ns-non significant.(TIF)Click here for additional data file.S9 FigPyrene labelled monomeric rabbit muscles actin (0.250 μM) was allowed to polymerize in the presence of different concentrations of EhC2B or EhC2B121, mDia1, and MBP as mentioned in detail in “Materials and methods” section. The fluorescence intensity of polymerization was measured at 360 and 407nm of excitation or emission spectra, respectively.(TIF)Click here for additional data file.S1 MovieThese trophozoites were then observed using an Olympus inverted confocal microscope equipped with 100x/1.4NA oil immersion objective. The scale bar represents 5μm. The arrows have been marked to highlight the transient enrichment of GFPEhC2B underneath membranes and pseudopod-like extensions over time.(AVI)Click here for additional data file.S2 Movie(MP4)Click here for additional data file.S3 MovieIn the FRAP experiments, an area of approximately 5μm diameter was photobleached for 500ms, and subsequently, images of the bleached area were collected approximately every 4 frames/s.(MP4)Click here for additional data file.S4 MovieThe trophozoites were then incubated with 1XPBS washed RBCs for 2 minutes, and immediately observed using an Olympus inverted confocal microscope equipped with a 60X/1.4NA oil immersion objective inverted microscope.(MP4)Click here for additional data file.S5 Movie(MP4)Click here for additional data file.S6 Movie(MP4)Click here for additional data file.S7 Movie(MP4)Click here for additional data file.S8 Movie(MP4)Click here for additional data file.S9 Movie(MP4)Click here for additional data file.S10 Movie(MP4)Click here for additional data file.S11 Movie(MP4)Click here for additional data file.


The conserved, immuno-subdominant influenza virus hemagglutinin (HA) stalk region is a potential universal group-specific influenza virus vaccine epitope. We analyzed antibody responses to H1 hemagglutinin stalk domain  following trivalent influenza inactivated vaccine (IIV3) immunization in pregnant women, and association with protection against influenza virus illness.One hundred forty-five human immunodeficiency virus (HIV)–uninfected pregnant women (68 IIV3 and 77 placebo recipients) and 140 pregnant women with HIV infection (72 IIV3 and 68 placebo recipients) were independently randomized in placebo-controlled efficacy trials of IIV3. Plasma samples were tested for H1/stalk immunoglobulin G (IgG) and hemagglutination inhibition (HAI) antibodies prevaccination and 1 month postvaccination. Women had weekly surveillance for influenza illness, confirmed by polymerase chain reaction.P = .001). H1/stalk IgG concentration ≥215 AU/mL was associated with 90% lower odds  of A/H1N1 illness. Also, H1/stalk IgG was significantly lower among women with influenza B illness (93.9 AU/mL) than among their counterparts (215.5 AU/mL) (P = .04); however, no association was observed after adjusting for HAI titers.Increases in H1/stalk IgG (and HAI) antibody levels were elicited post-IIV3, with responses being higher in HIV-uninfected women than in women living with HIV. Among HIV-uninfected vaccinees, there was no correlation (postvaccination) between H1/stalk and HAI antibody responses, whereas a strong correlation was observed in vaccinees with HIV. The H1/stalk IgG concentration was lower among women developing A/H1N1 illness (85.3 arbitrary units [AU]/mL) than those without A/H1N1 illness (219.6 AU/mL; H1/stalk IgG concentration was associated with lower odds for A/H1N1 influenza virus illness, indicating its potential as an epitope for a universal vaccine against group 1 influenza virus. This study showed an association of H1 hemagglutinin stalk-specific immunoglobulin G in broader protection from influenza virus infections in pregnant women in a placebo-controlled trial of an inactivated influenza vaccine trial. The findings have implications on designing more-effective influenza vaccine candidates. The effectiveness of current seasonal influenza virus vaccines is unpredictable, including low or no effectiveness when there is antigenic mismatch between the seasonal vaccine and circulating strains . ImmunogWe previously reported, in 2 separate randomized controlled trials in people living with human immunodeficiency virus (HIV), of the disconnect between trivalent IIV (IIV3)–induced HAI titers and the vaccine efficacy observed against confirmed influenza illness , 11. In Efforts are under way to identify conserved influenza virus epitopes as targets for broadly protective or universal influenza virus vaccines , 13. ThiThe objective of this study was to investigate immunoglobulin G (IgG) response to the H1 hemagglutinin stalk domain  following IIV immunization in pregnant women with or without HIV infection, and evaluate the association of H1/stalk IgG and odds for developing influenza virus illness.We retrospectively tested plasma samples from women enrolled as part of an immunogenicity study in 2 parallel randomized, placebo-controlled trials on IIV3 in pregnant women with or without HIV infection, as described previously . In brieFor detection of H1/stalk IgG, chimeric H6/1 recombinant protein was utilized containing an H6 head domain (to which humans are naive) linked with an H1 stalk domain, to which humans are known to have preexisting immunity . Plasma t test for data sets in Gaussian distribution. Antibody concentrations between groups were compared using Mann-Whitney test. For all analyses, P values, geometric means, and 95% CIs were reported. For categorical variables, groups were compared using Fisher exact test reporting the odds ratio (OR). Reverse cumulative plots were constructed and threshold concentrations were determined.We pooled data from participants with HIV and those without HIV to assess the association between H1/stalk antibody concentrations and risk for influenza virus illness, using the 1-month postimmunization levels IgG in IIV3 recipients and the baseline visit IgG concentration in placebo recipients. The baseline and postvaccination antibody concentrations were compared using Wilcoxon matched-pairs signed-rank test for skewed data sets or Student P value <.05 was considered statistically significant.Logistic regression reporting of the OR was used to identify associations between antibody concentrations and influenza illness with H1/stalk IgG and HAI titers as covariates. The association was further adjusted  and Stata version 13 software . Correlation analysis was performed using Spearman correlation test. For all analyses, a The study was approved by the Human Research Ethics Committee of the University of the Witwatersrand . All study participants provided written informed consent for inclusion into the parent study.P = .02; P = .009; Demographic and baseline clinical characteristics were similar between IIV3 and placebo recipients among HIV-uninfected women and women living with HIV, except for mean age being higher among the HIV-infected placebo group (28.8 ± 5.2 years) than the IIV3 group (26.9 ± 4.9 years) P = .02; . There wP < .0001) in HIV-uninfected women; and by 1.79-fold in women living with HIV (116.5 vs 209.3 AU/mL)  increased 2.24-fold between prevaccination (204.2 AU/mL) and postvaccination (457.9 AU/mL) (P < .0001) and women living with HIV   .P < .0001 for all strains; We previously reported on HAI titers in the HIV-uninfected women (2011 and 2012 cohort) and women living with HIV (2011 only) [r = 0.51, P < .0001) and women living with HIV , but not so for H1/stalk IgG vs A/H3N2 or B/Victoria HAI titers , whereas the correlation was stronger in women living with HIV  than before vaccination. Also, a significant correlation was observed 1 month following vaccination in women living with HIV who received IIV3 between H1/stalk IgG and A/H3N2 HAI titers  and B/Victoria HAI titers , as well as a modest correlation for HAI titers to B/Victoria  in HIV-uninfected IIV3 recipients  than the corresponding analyses of HAI titers to A/H1N1 , A/H3N2 , or B/Victoria   as well as for A/H1N1 , A/H3N2 , and B/Victoria  HAI titers  . In contI titers .P = .001; P = .002), which remained significant after adjusting for HIV status  or vaccine status .There were 23 confirmed influenza illnesses overall, including 19  in women living with HIV and 4 cases  in HIV-uninfected women. Overall, H1/stalk IgG GMC was lower among women developing A/H1N1 illness (85.3 AU/mL) than those who did not develop A/H1N1 illness (219.6 AU/mL) (P = .005) of women with and without A/H1N1 illness, respectively  of developing A/H1N1 illness was ≥215 AU/mL, which was prevalent among 9% and 52.1% , but not significant for A/H3N2 illness . In the logistic regression analysis, the H1/stalk IgG concentration was associated with significantly lower odds of influenza B illness , including after adjusting for HIV infection status . For influenza illness due to non–group 1 viruses, there was a trend of lower odds for acquiring illness with increasing H1/stalk IgG concentration, albeit not significant at any threshold  (121.4 AU/mL) compared to their counterparts (217 AU/mL) P = .04; . This dihreshold .P = .02). Furthermore, women developing A/H1N1 illness were 84%  less likely than those without A/H1N1 illness to have A/H1N1 HAI titers ≥40  , however, this was not significant after adjusting for HIV  or vaccine status . No difference was observed in homotypic HAI GMT in women with and without illness due to A/H3N2  or B/Victoria . Furthermore, the percentage of women with HAI titers ≥40 to the homotypic virus was not different between those with and without illness due to A/H3N2 . None of the women with B/Victoria illness compared to 46.2% who did not develop illness had B/Victoria HAI titers ≥40, albeit not significant (P = .50).The homotypic HAI GMT (1 month postvaccination in IIV3 recipients and baseline in placebo recipients) was lower in women who developed A/H1N1 illness compared with those who did not , A/H3N2 (r = 0.46), and B/Victoria (r = 0.52)  after adjusting for HAI titers (P = .62). Also, H1/stalk IgG or B/Victoria HAI titer association with protection against influenza B illness was not significant after adjusting for the other as covariates (P = .21), more so than for A/H1N1 HAI titers . However, neither of these associations was significant, most likely due to reduced study power for these analyses  illness. An H1/stalk IgG threshold of ≥215 was associated with 90% lower risk for A/H1N1 illness. Although A/H1N1 HAI titers were also associated with lower odds of A/H1N1 illness, this was no longer significant after adjusting for H1/stalk IgG concentrations. Although the stratified analysis restricted to women living with HIV was similar to that of the composite population in terms of association of H1/stalk IgG, the lack of statistical significance is probably due to reduction in the study power for such an analysis. Nevertheless, as the majority of the A/H1N1 cases (10/11) were in women living with HIV, these data suggest that protection against A/H1N1 illness in the women living with HIV is more likely associated with H1/stalk antibody responses than that mediated by H1N1 HAI antibody.We also observed an association of H1/stalk IgG and lower odds for non–group 1 viruses (A/H3N2 or influenza B) illness (the majority of cases had HIV infection), and significantly so for influenza B illness. This association was, however, no longer significant in the multivariate logistic regression after adjusting for strain-specific HAI titers. Hence, the causal association of either HAI or H1 stalk antibody in lowering the odds for influenza B illness is equivocal. Nevertheless, 4 of the 6 influenza B cases were due to infection with B/Yamagata lineage virus, which was a mismatch to the B/Victoria lineage strain in the vaccine. Speculatively, H1/stalk IgG may have possibly played a role in conferring cross-protection against illness from the vaccine mismatched circulating influenza B virus . This isThe neutralization breadth of stalk-specific antibodies is typically group-specific , whereinAntibody responses following IIV3 were predominantly elicited toward the immune-dominant HA head domain as compared to the immune-subdominant stalk domain in IIV3 vaccinees, both those HIV uninfected and those living with HIV. This is consistent with previous studies on responses against seasonal influenza virus vaccine , 30. TheLimitations of our study include it being an exploratory study and not being adequately powered to address the association of H1/stalk and protection against A/H1N1 separately in women living with HIV and HIV-uninfected women.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciz927_suppl_Supplementary_MaterialClick here for additional data file.