DataSheet_1_The rootstock shape microbial diversity and functionality in the rhizosphere of Vitis vinifera L. cultivar Falanghina.docx

The rhizosphere effect occurring at the root-soil interface has increasingly been shown to play a key role in plant fitness and soil functionality, influencing plants resilience. Here, for the first time, we investigated whether the rootstock genotype on which Vitis vinifera L. cultivar Falanghina is grafted can influence the rhizosphere microbiome. Specifically, we evaluated to which extent the 5BB and 1103P rootstocks are able to shape microbial diversity of rhizosphere environment. Moreover, we explored the potential function of microbial community and its shift under plant genotype influence. We investigated seven vineyards subjected to the same pedo-climatic conditions, similar age, training system and management and collected twelve rhizosphere soil samples for metagenomic analyses and composite soil samples for physical-chemical properties. In this study, we used 16S rRNA gene-based metagenomic analysis to investigate the rhizosphere bacterial diversity and composition. Liner discriminant analysis effect size (LEFSe) was conducted for metagenomic biomarker discovery. The functional composition of sampled communities was determined using PICRUSt, which is based on marker gene sequencing profiles. Soil analyses involved the determination of texture, pH, Cation Exchange Capacity (CSC), Organic Carbon (OC), electrical conductivity (EC), calcium (Ca), magnesium (Mg), potassium (K) content, Phosphorous (P), nitrogen (N). The latter revealed that soil features were quite homogenous. The metagenomic data showed that the bacterial alpha-diversity (Observed OTUs) significantly increased in 1103P rhizosphere microbiota. Irrespective of cultivar, Pseudomonadota was the dominant phylum, followed by Actinomycetota > Bacteroidota > Thermoproteota. However, Actinomycetota was the major marker phyla differentiating the rhizosphere microbial communities associated with the different rootstock types. At the genus level, several taxa belonging to Actinomycetota and Alphaproteobacteria classes were enriched in 1103P genotype rhizosphere. Investigating the potential functional profile, we found that most key enzyme-encoding genes involved in N cycling were significantly more abundant in 5BB rootstock rhizosphere soil. However, we found that 1103P rhizosphere was enriched in genes involved in C cycle and Plant Growth Promotion (PGP) functionality. Our results suggest that the different rootstocks not only recruit specific bacterial communities, but also specific functional traits within the same environment.</p

OP-A induced PINK1 accumulation, Bax and Bak translocation and cytochrome <i>c</i> release.

<p>HaCaT, CHL-1 and A375 cell lines were incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h and whole cell extracts (WCE), mitochondrial-enriched and cytosolic fractions were analysed by western blot (A) with antibodies directed against PINK1, Bak, Bax, and cytochrome <i>c</i>. Panels B, C and D, densitometric analysis of PINK1, Bak and Bax levels present in the mitochondrial fraction. β-tubulin was used as loading control and standard for densitometry. Data, expressed as %, or fold variation with respect to β-tubulin, were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.</p

OP-A induced cell cycle alteration.

<p>HaCaT, CHL-1 and A375 cell lines, incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h, were stained with propidium iodide and, after over-night incubation at 4°C, the percentage of cells in each phase of the cycle measured by flow cytometry. *, p<0.05, **, p<0.01, ***, p<0.001.</p

OP-A effect on mitochondrial membrane potential, mitochondrial mass, lysosome content and mitochondrial ROS production.

<p>HaCaT, CHL-1 and A375 cell lines incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h, were analysed to evaluate: A, mitochondrial membrane potential (MMP), (2 μM JC-1 staining); B, mitochondrial mass (100 nM MitoTracker Green staining); C, lysosome content (100 nM LysoTracker Red staining); D, mitochondrial ROS (5 μM MitoSox Red staining). Cells were incubated for 30 min at 37°C and then acquired by means of a FACSCalibur. Data, expressed as %, or fold increase with respect to controls, were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.</p

OP-A induced autophagy and cell death by apoptosis.

<p>A, flow cytometry analysis of cell death. HaCaT, CHL-1 and A375 cell lines were incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 h and cell death analysis performed by propidium iodide staining and flow cytometry evaluation of the sub-G1 population. Where indicated, 20 μM necrostatin-1 was added to evaluate the occurrence of necrotic cell death. B, western blot of PARP, caspase-9 and -3, and LC3II proteins. C, D and E. Densitometric analysis of immunorecognized protein bands of caspase-3, caspase-9 and LC3II, respectively. Data were analysed with FlowJo software. *, p<0.05, **, p<0.01, ***, p<0.001.</p

OP-A reduced A375 melanoma cells viability.

<p>Cells were incubated with 0.3, 0.6 and 1.2 μM OP-A for 24 and 48 h. Panel A: metabolic activity assayed by MTS test. Data are expressed as % of cell survival with respect to control. ***, p<0.0001. Panel B: mitochondrial and nuclear morphology. Mitochondrial network imaging was performed by incubating untreated and treated cells for 20 min at 37°C, with 1 μM MitoTracker Red CMXRos reagent in RPMI medium and nuclei counterstained with 1 μM HOECHST 33342. Images were captured by means of a Floid instrument. Arrowheads indicated picnotic and fragmented nuclei.</p

Identification of proteins differentially represented in OP-A-treated and control A375 melanoma cells as revealed by 2-DE and nanoLC-ESI-LIT-MS/MS analysis.

<p>Spot numbering corresponds to that reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167672#pone.0167672.g006" target="_blank">Fig 6</a>. Spot number, NCBI accession, protein name, MASCOT score, theoretical mass and pI values, peptides and unique peptides identified during analysis, sequence coverage, constitutive amino acids, emPAI score and fold change in treated cells with respect to control are shown.</p