.gitattributes

 **/dataset/** filter=lfs diff=lfs merge=lfs -text
 /_publication/Chandrasekar[[:space:]]et[[:space:]]al.[[:space:]]-[[:space:]]2022[[:space:]]-[[:space:]]Fungi[[:space:]]hijack[[:space:]]a[[:space:]]ubiquitous[[:space:]]plant[[:space:]]apoplastic[[:space:]]endoglu.pdf filter=lfs diff=lfs merge=lfs -text
 _publication/koac114-suppl_data/tpc.21.00790_Supplemental[[:space:]]Data.pdf filter=lfs diff=lfs merge=lfs -text
+assays/Microscopy[[:space:]]of[[:space:]]S[[:space:]]indica[[:space:]]EPS[[:space:]]matrix[[:space:]]and[[:space:]]CW/dataset/Figure[[:space:]]1.png filter=lfs diff=lfs merge=lfs -text
+assays/Proteome[[:space:]]analysis[[:space:]]of[[:space:]]S[[:space:]]indica[[:space:]]EPS[[:space:]]matrix[[:space:]]CW[[:space:]]and[[:space:]]cullture[[:space:]]filtrate/dataset/Figure[[:space:]]2.pdf filter=lfs diff=lfs merge=lfs -text
+assays/Proteome[[:space:]]analysis[[:space:]]of[[:space:]]S[[:space:]]indica[[:space:]]EPS[[:space:]]matrix[[:space:]]CW[[:space:]]and[[:space:]]cullture[[:space:]]filtrate/dataset/Figure[[:space:]]2.png filter=lfs diff=lfs merge=lfs -text

assays/Isolation of extracellular polysaccharide matrix/protocols/Isolation of extracellular polysaccharide matrix.md

+## Microbial strains and culture conditions for EPS matrix production in S. indica and B. sorokiniana
+
+The EPS matrix was isolated from the S. indica strain
+expressing GFP in the dikaryotic wild-type background
+DSM11827 (Wawra et al., 2016) and from the B. sorokiniana
+wild-type strain ND90r. Serendipita indica spores were iso-
+lated from 3-week-old cultures grown on solid complex me-
+dium (Si_CM) using 0.002% (v/v) Tween water (Zuccaro
+et al., 2011). For the preculture, 2 mL of S. indica spores at a
+concentration of 500,000 mL–1 were inoculated in 100 mL
+of TSB medium containing 1% (w/v) sucrose and shaking at
+120 rpm at 28	C. After 48 h, the pre-cultures were trans-
+ferred to 400 mL of TSB containing 1% sucrose and shaken
+at 120 rpm at 28	Cfor 72 h. Bipolaris sorokiniana spores
+were isolated from 10-day-old cultures grown on solid com-
+plete medium (Bs_CM), using 0.002% (v/v) Tween water
+(Sarkar et al., 2019). The spores were inoculated at a final
+concentration of 250 spores	mL–1 in 250 mL of YPD me-
+dium and these samples were shaken at 28	C for 36 h.
+
+## Isolation of EPS matrix from S. indica and B. sorokiniana culture supernatant
+
+Culture supernatants from axenically grown S. indica or B.
+sorokiniana were collected by filtering the mycelia using
+Miracloth (Merck Millipore). The EPS matrix was isolated
+from the culture media using cryogelation. Briefly, the cul-
+ture media were frozen overnight at –20°C and slowly
+thawed at room temperature for 16 h. The precipitated EPS matrix (Supplemental Figure S1) present in the culture me-
+dium was isolated using a pipette controller and washed
+four times with 30 mL of Milli-Q water and either used for
+proteome analyses (see section “Proteome analysis of
+S.indica EPS matrix, CW, and culture filtrate”) or washed
+one more time with 30 mL of 8.3-mM EDTA (pH 8.0) to re-
+move metal ions potentially present in the EPS. The proteins
+present in the EPS matrix were removed by treatment with
+30 mL of protein denaturation solution (containing 8-M
+urea, 2-M thiourea, 4% [w/v] sodium dodecyl sulfate [SDS],
+100-mM Tris–HCl, pH 7.5) and boiling at 95°C for 15 min.
+The SDS present in the EPS matrix was removed by boiling
+the material with 30 mL of Milli-Q water at 95°C for 10 min
+and centrifuging at 10,000g for 10 min at room temperature.
+The latter step was repeated approximately 15 times until
+no further foaming occurred. The resulting protein-free EPS
+matrix was lyophilized and used for glycosyl linkage, TLC or
+MALDI-TOF analyses.
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assays/Microscopy of S indica EPS matrix and CW/protocols/FITC488 labeling and confocal microscopy.md

+## FITC488 labeling and confocal microscopy
+
+FITC488 labeling of SiWSC3 and SiFGB1 and confocal laser
+scanning microscopy was done as described previously
+(Wawra et al., 2019) using the KPL SureLink Fluorescein-X
+(FAM-X) labeling kit (#82-00-02) by Sera Care with the fol-
+lowing modification for SiWSC3 labeling: 20-min incubation
+time at 20°C with half of the recommended concentration.
+The reaction was stopped by adding 1-M TRIS (pH 7.5) to a
+final concentration of 50 mM and then dialyzed overnight
+against 3 L of Milli-Q water. Modifications to the standard
+labeling protocol were necessary because over labeling re-
+duced the ability of the SiWSC3 to bind to its substrate
+(Wawra et al., 2019).
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assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/README.md

+Data is visualized in Figure 2 A-B. 
+
+Raw data is provided in Table S1 for A (total proteins) and Table S2 for A (Proteins with predicted signal peptide)
+

assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/LC-MSMS.md

+## Liquid chromatography coupled mass spectrometric
+protein identification and quantification
+
+The proteins isolated from the EPS matrix, CW and culture
+filtrate were separated using a 10% (v/v) SDS–polyacryl-
+amide gel electrophoresis gel for 15 min and subsequently
+stained with Coomassie Brilliant Blue. Protein-containing
+bands from Coomassie-stained gels were prepared for mass
+spectrometric analysis as described elsewhere (Poschmann
+et al., 2014). Briefly, bands were destained and the proteins
+were reduced with dithiothreitol and alkylated with iodoace-
+tamide and subjected to tryptic digestion. The resulting pep-
+tides were extracted and reconstituted in 0.1% (v/v)
+trifluoroacetic acid in water. Peptides were separated on an
+Ultimate 3000 Rapid Separation Liquid Chromatography sys-
+tem (Thermo Fisher Scientific) on a 25-cm length C18 col-
+umn using a 1-h gradient and subsequently analyzed by a
+QExactive Plus mass spectrometer (Thermo Fisher Scientific)
+as described with minor modifications (Poschmann et al.,
+2014). First, survey scans were carried out at a resolution of
+140,000 and up to 22- and 3-fold charged precursors se-
+lected by the quadrupole (4 m/z isolation window), frag-
+mented by higher energy collisional dissociation and
+fragment spectra recorded at a resolution of 17,500. Mass
+spectrometric data were further processed by MaxQuant
+1.6.12.0 (Max-Planck Institute for Biochemistry, Planegg,
+Germany) with standard parameters if not otherwise stated.
+Label-free quantification, “match between runs” and iBAQ
+quantification were enabled. Searches were carried out based
+on S. indica reference protein entries (UP000007148), down-
+loaded on 15 May 2020 from the UniProt Knowledge Base.
+Carbamidomethylation at cysteines was considered as fixed
+and methionine oxidation and proteins N-terminal acetyla-
+tion as variable modifications. Peptides and proteins were
+accepted at a false discovery rate of 1% and only proteins
+further considered were identified with at least two different
+peptides. The identified proteins were grouped into families
+using the Pfam database (Mistry et al., 2021). The mass spec-
+trometry proteomics data have been deposited to the
+ProteomeXchange Consortium via the PRIDE partner reposi-
+tory with the dataset identifier PXD025640.
+
+## Proteome analysis of S. indica EPS matrix, CW, and culture filtrate
+
+Further calculations were done on label-free quantification
+(LFQ) intensity values. Only proteins were considered show-
+ing at least two valid intensity values at least in one group.
+LFQ intensity values were log2 transformed and missing val-
+ues imputed with values drawn from a downshifted normal
+distribution (width 0.3, downshift 1.8) before statistical and
+enrichment analysis. Statistical analysis was done for selected
+protein groups using the significance analysis of microarrays
+method (Tusher et al., 2001;5% false discovery
+rate, S0 = 0.1) and Student’s t test. To identify proteins contain-
+ing certain domains showing a higher abundance in the EPS
+matrix, we performed an annotation enrichment analysis
+(Cox and Mann, 2012) based on the differences of mean val-
+ues of log2 transformed LFQ intensities.
+The percentage relative abundance of signal peptide con-
+taining proteins detected in the three components (EPS ma-
+trix, CW, and culture filtrate) was calculated using LFQ
+intensities.
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assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/Protein isolation of S indica EPS matrix CW and culture filtrate.md

+# Protein isolation of S. indica EPS matrix, CW, and culture filtrate
+
+The proteins were isolated from the EPS matrix, CW, and
+the culture filtrate obtained from axenic cultures of S. indica
+strain expressing GFP grown in different media (CM, YPD,
+and TSB).
+
+## Protein isolation from the CW
+
+Mycelium collected from the S. indica GFP strain was
+ground in liquid N2 and resuspended in PBS buffer contain-
+ing 1-mM PMSF and 1% (v/v) NP-40 using an ULTRA-
+TURRAX (IKA, Staufen, Germany). The resuspended mixture
+was incubated at 4°C in a rotating wheel for 30 min. The
+pellet obtained after centrifugation at 8,000g for 15 min at
+4°C was resuspended in PBS buffer containing 1-mM PMSF
+and 0.1% (v/v) IGEPAL using an ULTRA-TURRAX. The pellet
+obtained after centrifugation at 8,000g for 15 min at 4°C
+was washed three times with Milli-Q water. Finally, the pel-
+let was resuspended in Laemmli buffer containing 8-M urea,
+2-M thiourea, and b-mercaptoethanol and boiled at 95°C
+for 10 min.
+
+## Protein isolation from the EPS matrix
+
+The EPS matrix obtained from the culture media by cryoge-
+lation was washed four times with Milli-Q water and was di-
+rectly boiled in Laemmli buffer containing 8-M urea, 2-M
+thiourea, and b-mercaptoethanol at 95°C for 10 min.
+
+## Protein isolation from the culture filtrate
+
+The culture supernatant left over from the EPS matrix isola-
+tion step was first filtered using a Whatman filter paper and
+then using a 0.22-mM syringe filter. Approximately 30 mL of
+EPS matrix depleted culture supernatant was treated with 5
+mL of 95% (v/v) trichloroacetic acid and incubated over-
+night at 4°C. The precipitated proteins were collected by
+centrifugation (10,000g) for 1 h at 4°C. The isolated proteins
+were washed at least three times with 100% (v/v) acetone.
+The dried protein pellet was resuspended in Laemmli buffer
+containing 8-M urea, 2-M thiourea, and b-mercaptoethanol
+andboiledat 95°C for 10 min.
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