Additional file 2: of Evolution and functional characterization of pectate lyase PEL12, a member of a highly expanded Clonostachys rosea polysaccharide lyase 1 family
Creators: name: Atanasova, Lea
name: Dubey, Mukesh
name: Grujić, Marica
name: Gudmundsson, Mikael
name: Lorenz, Cindy
name: Sandgren, Mats
name: Kubicek, Christian
name: Jensen, Dan
name: Karlsson, Magnus
Publisher: name: figshare
Year: 2018
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Ecology
subject: 69999 Biological Sciences not elsewhere classified
subject: Marine Biology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology

description: Figure S1. Partial protein alignment of the closest neighbors of PEL12 and separation of the group A and B for further reverse conservation analysis. C. rosea paralogs BN869_T0006915 (PEL12) and BN869_T00007653 (PEL13) align to the group B, whereas the protein BN869_T0008859 (PEL2), Fusaria and some members of Sordariales are separated by several hallmarks forming the group A. Figure S2. SDS-PAGE gel showing the putative expression of the PEL12 protein (arrows) at 37 kDa in the fermentation broths of two independent T. reesei PEL12 overexpression strains pel12OE-1 and pel12OE-2. The strains were cultivated for 48 h in Mandels-Anderotti medium supplemented with 1% glucose. Figure S3. Carbon source utilization of T. reesei WT and pel12OE mutants on 95 different carbon sources based on BIOLOG Phenotypic assay for filamentous fungi. Mycelial density is shown for 96 h of incubation at 28 °C in darkness. Black, gray and white circles represent mycelial density of T. reesei QM9414 (wt) and the mutants pel12OE-1 and − 2, respectively. Figure S4. Validation of pel12 mutant strains using PCR and RT-PCR. A: PCR verification of Δpel12 using primers located within the hygB cassette (Hyg F /Hyg R) in combination with primers located upstream and downstream regions from the deletion cassette (6915 ko F / 6915 ko R). A PCR product of ~ 2.5 kb using primers 6915 ko F / Hyg R and 6915 ko R / Hyg F were expected from a correct gene replacement. M, gene ruler DNA ladder mix; 1–3, independent Δpel12 mutants; 4, WT strain. B: RT-PCR analysis of pel12 gene expression in WT and deletion strains using pel12 specific primers. A PCR product of 92 bp was expected from the WT. M, gene ruler DNA ladder mix; 1, WT; 2–4, independent deletion strains. Primer combinations used for PCR and RT-PCR are given above the images. Figure S5. Carbon source utilization of C. rosea WT and ∆pel12 mutants (∆pel12 KOA, KOB, KOC) on 95 different carbon sources based on BIOLOG Phenotypic assay for filamentous fungi. Mycelial density is shown for 144 h of incubation at 28 °C in darkness. Figure S6. Measurement of B. cinerea necrotic lesions area on detached leaves of Arabidopsis thaliana. The leaves were inoculated with C. rosea strains (A) or T. reesei (B) strains spores 30 min before application of B. cinerea spores. Necrotic lesion area was measured under the microscope using a DeltaPix camera and software 56 h post inoculations. Only B. cinerea inoculated leaves were used as control. Error bars represent standard deviation based on three biological replicates. Different letters indicate statistically significant differences (P ≤ 0.05) based on the Fisher test. (PDF 633 kb)