Dataset Information

Deep Learning Neural Network Prediction Method Improves Proteome Profiling of Vascular Sap of Grapevines during Pierce's Disease Development.

ABSTRACT: Plant secretome studies highlight the importance of vascular plant defense proteins against pathogens. Studies on Pierce's disease of grapevines caused by the xylem-limited bacterium Xylella fastidiosa (Xf) have detected proteins and pathways associated with its pathobiology. Despite the biological importance of the secreted proteins in the extracellular space to plant survival and development, proteome studies are scarce due to methodological challenges. Prosit, a deep learning neural network prediction method is a powerful tool for improving proteome profiling by data-independent acquisition (DIA). We explored the potential of Prosit's in silico spectral library predictions to improve DIA proteomic analysis of vascular leaf sap from grapevines with Pierce's disease. The combination of DIA and Prosit-predicted libraries increased the total number of identified grapevine proteins from 145 to 360 and Xf proteins from 18 to 90 compared to gas-phase fractionation (GPF) libraries. The new proteins increased the range of molecular weights, assisted in the identification of more exclusive peptides per protein, and increased identification of low-abundance proteins. These improvements allowed identification of new functional pathways associated with cellular responses to oxidative stress, to be investigated further.

SUBMITTER: Helena Duarte Sagawa C

PROVIDER: S-EPMC7565608 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Deep Learning Neural Network Prediction Method Improves Proteome Profiling of Vascular Sap of Grapevines during Pierce's Disease Development.

Helena Duarte Sagawa Cíntia C Zaini Paulo A PA de A B Assis Renata R Saxe Houston H Salemi Michelle M Jacobson Aaron A Wilmarth Phillip A PA Phinney Brett S BS M Dandekar Abhaya A

Biology 20200901 9

Plant secretome studies highlight the importance of vascular plant defense proteins against pathogens. Studies on Pierce's disease of grapevines caused by the xylem-limited bacterium <i>Xylella fastidiosa</i> (<i>Xf</i>) have detected proteins and pathways associated with its pathobiology. Despite the biological importance of the secreted proteins in the extracellular space to plant survival and development, proteome studies are scarce due to methodological challenges. Prosit, a deep learning ne ...[more]

PMID: 32882865

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Project description:Grapevine is a pillar of the California state economy and agricultural identity. This study provides a comprehensive culture-independent microbiome analysis from the sap of grapevine overtime and in a context of a vascular disease. The vascular system plays a key role by transporting nutrient, water and signals throughout the plant. The negative pressure in the xylem conduits, and low oxygen and nutrient content of its sap make it a unique and underexplored microbial environment. We hypothesized that grapevine hosts in its sap, microbes that have a beneficial impact on plant health by protecting against pathogen attack and supporting key biological processes. To address this hypothesis, we chose a vineyard under high Pierce's disease (PD). PD is caused by the xylem-dwelling pathogenic bacterium Xylella fastidiosa. We selected ten grapevines within this vineyard with a range of disease phenotypes, and monitored them over 2 growing seasons. We sampled each vines at key phenological stages (bloom, veraison, and post-harvest) and used an amplicon metagenomics approach to profile the bacterial (16S -V4) and fungal (ITS) communities of the sap. We identified a core microbiome of the sap composed of seven bacterial (Streptococcus, Micrococcus, Pseudomonas, Bacteroides, Massilia, Acinetobacter and Bacillus) and five fungal (Cladosporium, Mycosphaerella, Alternaria, Aureobasidium, and Filobasidium) taxa that were present throughout the growing season. Overall, the sap microbial makeup collected from canes was more similar to the root microbial profile. Alpha diversity metrics indicated a microbial enrichment at bloom and in vines with moderate PD severity suggesting a host-driven microbial response to environmental cues. Beta diversity metrics demonstrated that disease condition and plant phenology impacted microbial community profiles. Our study identified several potential taxonomic targets with antimicrobial and plant growth promoting capabilities that inhabit the grapevine sap and that should be further tested as potential biological control or biofertilizer agents.

Project description:Pierce's disease (PD) is a significant threat to grape cultivation and industry. The disease caused by bacterium Xylella fastidiosa clogs xylem vessels resulting in wilting of the plant. PD-tolerant grape genotypes are believed to produce certain novel components in xylem tissue that help them to combat invading pathogens. Research has been aimed at characterizing the uniquely expressed xylem proteins by PD-tolerant genotypes. The objectives were to i) compare and characterize Vitis xylem proteins differentially expressed in PD-tolerant and PD-susceptible cultivars and, ii) identify xylem proteins uniquely expressed in PD-tolerant genotypes. A high throughput two-dimensional gel electrophoresis of xylem proteins from three Vitis species identified more than 200 proteins with pls 3.0 to 9.0 and molecular weights of 20 to 75 kDa. The differentially expressed proteins were then excised and analyzed with MALDI/TOF mass spectrometer. The mass spectra were collected and protein identification was performed against the Viridiplantae database using Matrix Science algorithm. Proteins were mapped to the universal protein resource to study gene ontology. Comparative analysis of the xylem proteome of three species indicated the highest number of proteins in muscadine grape, followed by Florida hybrid bunch and bunch grape. These proteins were all associated with disease resistance, energy metabolism, protein processing and degradation, biosynthesis, stress related functions, cell wall biogenesis, signal transduction, and ROS detoxification. Furthermore, ?-1, 3-glucanase, 10-deacetyl baccatin III-10-O-acetyl transferase-like, COP9, and aspartyl protease nepenthesin precursor proteins were found to be uniquely expressed in PD-tolerant muscadine grape, while they are absent in PD-susceptible bunch grape. Data suggests that muscadine and Florida hybrid bunch grapes express novel proteins in xylem to overcome pathogen attack while bunch grape lacks this capability, making them susceptible to PD.

Project description:BackgroundBlood-based protein measurement is a routine practice for detecting biomarkers in human disease. Comprehensive profiling of blood/plasma/serum proteome is a challenge due to an extremely large dynamic range, as exemplified by a small subset of highly abundant proteins. Antibody-based depletion of these abundant proteins alleviates the problem but introduces experimental variations. We aimed to establish a method for direct profiling of undepleted human serum and apply the method toward biomarker discovery for Alzheimer's disease (AD), as AD is the most common form of dementia without available blood-based biomarkers in clinic.MethodsWe present an ultra-deep analysis of undepleted human serum proteome by combining the latest 11-plex tandem-mass-tag (TMT) labeling, exhaustive two-dimensional liquid chromatography (LC/LC) fractionation (the 1st LC: 3 h for 180 fractions, and the 2nd LC: 3 h gradient per fraction), coupled with high resolution tandem mass spectrometry (MS/MS). AD (n = 6) and control (n = 5) sera were analyzed in this pilot study. In addition, we implemented a multiplexed targeted LC-MS3 method (TOMAHAQ) for the validation of selected target proteins.ResultsThe TMT-LC/LC-MS/MS platform is capable of analyzing 4826 protein components (4368 genes), covering at least 6 orders of magnitude in dynamic range, representing one of the deepest serum proteome analysis. We defined intra- and inter- group variability in the AD and control groups. Statistical analysis revealed differentially expressed proteins in AD (26 decreased and 4 increased). Notably, these altered proteins are enriched in the known pathways of mitochondria, fatty acid beta oxidation, and AGE/RAGE. Finally, we set up a TOMAHAQ method to confirm the decrease of PCK2 and AK2 in our AD samples.ConclusionsOur results show an ultra-deep serum discovery study by TMT-LC/LC-MS/MS, and a validation experiment by TOMAHAQ targeted LC-MS3. The MS-based discovery and validation methods are of general use for biomarker discovery from complex biofluids (e.g. serum proteome). This pilot study also identified deregulated proteins, in particular proteins associated with mitochondrial function in the AD serum samples. These proteins may serve as novel AD candidate biomarkers.

Dataset Information

Deep Learning Neural Network Prediction Method Improves Proteome Profiling of Vascular Sap of Grapevines during Pierce's Disease Development.

Publications

Deep Learning Neural Network Prediction Method Improves Proteome Profiling of Vascular Sap of Grapevines during Pierce's Disease Development.

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