From 1034020dd4520f005b5b05b38346b52c797eb01e Mon Sep 17 00:00:00 2001
From: Pia Saake <psaake@uni-koeln.de>
Date: Wed, 16 Oct 2024 20:58:53 +0200
Subject: [PATCH] add microscopy and proteome analysis

---
 .gitattributes                                |   3 +
 .../README.md}                                |   0
 .../dataset/.gitkeep                          |   0
 .../dataset/Figure 1.png                      |   3 +
 .../isa.assay.xlsx                            | Bin 0 -> 6919 bytes
 .../protocols/.gitkeep                        |   0
 ...ITC488 labeling and confocal microscopy.md |  14 ++++
 .../README.md                                 |   4 ++
 .../dataset/Figure 2.pdf                      |   3 +
 .../dataset/Figure 2.png                      |   3 +
 .../dataset/TableS1_all_proteins.xlsx         |   3 +
 .../dataset/TableS2_SP_proteins.xlsx          |   3 +
 .../isa.assay.xlsx                            | Bin 6933 -> 7066 bytes
 .../protocols/LC-MSMS.md                      |  62 ++++++++++++++++++
 ...dica EPS matrix CW and culture filtrate.md |  43 ++++++++++++
 15 files changed, 141 insertions(+)
 rename assays/{Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/Proteome analysis of S. indica EPS matrix, CW, and culture filtrate => Microscopy of S indica EPS matrix and CW/README.md} (100%)
 create mode 100644 assays/Microscopy of S indica EPS matrix and CW/dataset/.gitkeep
 create mode 100644 assays/Microscopy of S indica EPS matrix and CW/dataset/Figure 1.png
 create mode 100644 assays/Microscopy of S indica EPS matrix and CW/isa.assay.xlsx
 create mode 100644 assays/Microscopy of S indica EPS matrix and CW/protocols/.gitkeep
 create mode 100644 assays/Microscopy of S indica EPS matrix and CW/protocols/FITC488 labeling and confocal microscopy.md
 create mode 100644 assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.pdf
 create mode 100644 assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.png
 create mode 100644 assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS1_all_proteins.xlsx
 create mode 100644 assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS2_SP_proteins.xlsx
 create mode 100644 assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/LC-MSMS.md
 create mode 100644 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

diff --git a/.gitattributes b/.gitattributes
index 43b1c61..a14d9ac 100644
--- a/.gitattributes
+++ b/.gitattributes
@@ -1,3 +1,6 @@
 **/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
diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/Proteome analysis of S. indica EPS matrix, CW, and culture filtrate b/assays/Microscopy of S indica EPS matrix and CW/README.md
similarity index 100%
rename from assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/Proteome analysis of S. indica EPS matrix, CW, and culture filtrate
rename to assays/Microscopy of S indica EPS matrix and CW/README.md
diff --git a/assays/Microscopy of S indica EPS matrix and CW/dataset/.gitkeep b/assays/Microscopy of S indica EPS matrix and CW/dataset/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Microscopy of S indica EPS matrix and CW/dataset/Figure 1.png b/assays/Microscopy of S indica EPS matrix and CW/dataset/Figure 1.png
new file mode 100644
index 0000000..fe90b90
--- /dev/null
+++ b/assays/Microscopy of S indica EPS matrix and CW/dataset/Figure 1.png	
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diff --git a/assays/Microscopy of S indica EPS matrix and CW/protocols/.gitkeep b/assays/Microscopy of S indica EPS matrix and CW/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/Microscopy of S indica EPS matrix and CW/protocols/FITC488 labeling and confocal microscopy.md b/assays/Microscopy of S indica EPS matrix and CW/protocols/FITC488 labeling and confocal microscopy.md
new file mode 100644
index 0000000..6e13cdc
--- /dev/null
+++ b/assays/Microscopy of S indica EPS matrix and CW/protocols/FITC488 labeling and confocal microscopy.md	
@@ -0,0 +1,14 @@
+## 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).
\ No newline at end of file
diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/README.md b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/README.md
index e69de29..25fcee5 100644
--- a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/README.md	
+++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/README.md	
@@ -0,0 +1,4 @@
+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)
+
diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.pdf b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.pdf
new file mode 100644
index 0000000..22aa526
--- /dev/null
+++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.pdf	
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diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.png b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.png
new file mode 100644
index 0000000..32168b0
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new file mode 100644
index 0000000..73fdc1d
--- /dev/null
+++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS1_all_proteins.xlsx	
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diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS2_SP_proteins.xlsx b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS2_SP_proteins.xlsx
new file mode 100644
index 0000000..b6a8b65
--- /dev/null
+++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS2_SP_proteins.xlsx	
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diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/LC-MSMS.md b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/LC-MSMS.md
new file mode 100644
index 0000000..c845d5c
--- /dev/null
+++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/protocols/LC-MSMS.md	
@@ -0,0 +1,62 @@
+## 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.
\ No newline at end of file
diff --git a/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 b/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
new file mode 100644
index 0000000..40d8316
--- /dev/null
+++ b/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	
@@ -0,0 +1,43 @@
+# 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.
\ No newline at end of file
-- 
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