The Root-Knot Nematode Effector MiEFF18 Targets the Core Spliceosomal Protein SmD1 and Facilitates Giant Cell Formation
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. Overall design: mRNA profiles of PolyA RNA from WT, MiEFF18 overexpressor and smd1b mutant seedlings PolyA RNA from WT, MiEFF18 overexpressor and smd1b mutant seedlings