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The Root-Knot Nematode Effector MiEFF18 Targets the Core Spliceosomal Protein SmD1 and Facilitates Giant Cell Formation

ABSTRACT: The goals of this study is understand the effect of the MiEFF18 nematode effector on alternative splicing. Methods: PolyA RNA from WT, MiEFF18 overexpressor and smd1b mutant seedlings were sequenced using the illumina NextSeq500 sequencer, in triplicate. Total RNA was extracted from the roots of the three Arabidopsis lines (Col0, OE-MiEFF18#13.6 and smd1b) with TriZol (Invitrogen), according to the Invitrogen protocol. The RNA was treated with DNAse treatment (Ambion), and its quality and integrity were assessed with a Bioanalyzer (Agilent). Libraries were constructed with the Tru-Seq Stranded mRNA Sample Prep kit (Illumina®). Paired-end sequencing with 75-bp reads was performed on a NextSeq500 perform. A minimum of 30 million paired-end reads per sample was generated. RNA-seq preprocessing included the trimming of library adapters and quality controls with Trimmomatic. Paired-end reads with a Phred Quality Score Qscore > 20 and a read length > 30 bases were retained, and ribosomal RNA sequences were removed with SortMeRNA. Processed reads were aligned using Tophat2 with the following arguments: --max-multihits 1 -i 20 --min-segment-intron 20 --min-coverage-intron 20 --library-type fr-firststrand --microexon-search -I 1000 --max-segment-intron 1000 --max-coverage-intron 1000 --b2-very-sensitive. Conclusions: MiEFF18 overexpression modifies the expression of genes important for the ontogenesis of giant cells, notably those involved in microtubule cytoskeleton reorganization and cell cycle regulation, strongly suggesting that MiEFF18-mediated SmD1 inactivation in plants leads to a loss of susceptibility to root-knot nematodes.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE153171 | GEO | 2020/06/25

REPOSITORIES: GEO

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Project description:This data displays both known and unknown extra-cellular proteins from 13 species of Lactic Acid bacteria found in the honey-crop of the honeybee Apis. mellifera mellifera. The tryptic peptides from the secreted proteins were run on an Agilent HPLC on a C18 reverse phase column (75 µm x 150 mm, particle size 3 µm). Total run time was 90 min and flow rate 300 nl/min. Buffers used for gradient was 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B). The buffer mixing was 5 min 5% buffer B, followed by 5%-45% buffer B in a linear gradient for 50 min, followed by 45%-80% buffer B in a linear gradient for 5 min. The 80% of buffer B was then kept for 15 min and then rapidly back to 5% buffer B for the final 15 min. The fractions from HPLC were loaded on an LCQ Deca XP Plus Ion trap mass spectrometer (ThermoScientific). Genomic DNA were prepared from all 13 LAB strains depicted earlier and sequenced at MWG Eurofins Operon (Ebensburg, Germany) using Roche GS FLX Titanium technology from Roche (Basel, Switzerland). For each genome a shotgun library was constructed with up to 700,000 reads per segment and was generated by sequencing in 2x half segment of a full FLX+ run. Each genome had an 8 kpb long-paired end library constructed. Approximately 300,000 true paired end reads, sequence tags, and scaffolds with GS FLX+ chemistry using 2x half segment of a full run were generated. Clonal amplification was performed by emPCR in both library types. The sequencing was continued until 15-20 fold coverage was reached. The obtained reads were assembled by the software Newbler 2.6 from Roche (Basel, Switzerland). ORF prediction and automated annotation was performed at Integrated Genomics Assets Inc. (Mount Prospect, Illinois, USA). In ORF prediction three different software were used, GLIMMER, Critica, and Prokpeg. Automated annotation was performed with the ERGOTM algorithms (Integrated Genomics Assets Inc. Mount Prospect, Illinois, USA). The resulting mass spectra-files obtained from the mass spectrometry analysis were searched using MASCOT against a local database containing the predicted proteome of the 13 LAB. We used a cut off Ions score of 38 as a value for determining that the protein was identified. Individual ion scores that were greater than 38 indicated identity or extensive homology (p<0.05) of the protein. Protein sequence similarity searches were performed with software BLASTP in the software package BLAST 2.27+ against a non-redundant protein database at NCBI. Pfam (default database), and InterProScan (default databases). Expressed proteins identified by peptide mass fingerprinting were manually re-annotated.

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The Root-Knot Nematode Effector MiEFF18 Targets the Core Spliceosomal Protein SmD1 and Facilitates Giant Cell Formation

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