diff --git a/.gitattributes b/.gitattributes index 43b1c61a40561f4990d5883943b1d1bf77f98d60..a14d9acb2132f3227f8c85ff0814c02ff47ba439 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 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 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 0000000000000000000000000000000000000000..fe90b9087a14e4b1da86574b7341a57e6ff8bb88 --- /dev/null +++ b/assays/Microscopy of S indica EPS matrix and CW/dataset/Figure 1.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:45338cb2bb79f55eecfbda8974f067e4773fd77057ca916d16990ee35f8814bd +size 1643251 diff --git a/assays/Microscopy of S indica EPS matrix and CW/isa.assay.xlsx b/assays/Microscopy of S indica EPS matrix and CW/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..a97acb2ded9ebe81eaa16733945b2c893c47ac27 Binary files /dev/null and b/assays/Microscopy of S indica EPS matrix and CW/isa.assay.xlsx differ 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 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 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 0000000000000000000000000000000000000000..6e13cdc48e173a7224f1543147f28a993db4a8ac --- /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 e69de29bb2d1d6434b8b29ae775ad8c2e48c5391..25fcee57878eaed09c940a03cc5c27fb8a6ec562 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 0000000000000000000000000000000000000000..22aa52634b2feed0672f7052fe2bf88afeb7deb3 --- /dev/null +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.pdf @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:31b1579e2e0e45556a59fc149e38abcc51107f610eef2e97215bf74cf605f536 +size 1814993 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 0000000000000000000000000000000000000000..32168b03450893d91125994fc2ffd1a35ee13606 --- /dev/null +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/Figure 2.png @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:16f3e1ef14fc39296faf943869230903c21985df1f9c1ceebcbc44aba158a892 +size 1084936 diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS1_all_proteins.xlsx b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS1_all_proteins.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..73fdc1d51b4a28803caf5c00e6c65fb2776b61d4 --- /dev/null +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS1_all_proteins.xlsx @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:a2e57f204ffc1eaeaa3a9ffe48fdc5dfe80fd2037670f4a6959440bb9d28f855 +size 663068 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 0000000000000000000000000000000000000000..b6a8b651f0e5489e785fbb716c11ceaae08a7511 --- /dev/null +++ b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/dataset/TableS2_SP_proteins.xlsx @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:c09d42a7c863366af8180052c1f0e8eae2c1f9477a7959895dc9af86e2058855 +size 85809 diff --git a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/isa.assay.xlsx b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/isa.assay.xlsx index f75b32f9697784b2e5a47fde0d3b559c36cea043..09d97d00925201c93bda5c8c0b2da50e769108c6 100644 Binary files a/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/isa.assay.xlsx and b/assays/Proteome analysis of S indica EPS matrix CW and cullture filtrate/isa.assay.xlsx differ 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 0000000000000000000000000000000000000000..c845d5c63f4ada165b4fb9d9bd8f305431d23ae0 --- /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 0000000000000000000000000000000000000000..40d831676c9d19baa588ec5a08da6ca8df955146 --- /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