The Complete Mitochondrial Genome of <i>Meloidogyne graminicola</i> (Tylenchina): A Unique Gene Arrangement and Its Phylogenetic Implications

<div><p><i>Meloidogyne graminicola</i> is one of the most economically important plant parasitic-nematodes (PPNs). In the present study, we determined the complete mitochondrial (mt) DNA genome sequence of this plant pathogen. Compared with other PPNs genera, this genome (19,589 bp) is only slightly smaller than that of <i>Pratylenchus vulnus</i> (21,656 bp). The nucleotide composition of the whole mtDNA sequence of <i>M. graminicola</i> is significantly biased toward A and T, with T being the most favored nucleotide and C being the least favored. The A+T content of the entire genome is 83.51%. The mt genome of <i>M. graminicola</i> contains 36 genes (lacking <i>atp8</i>) that are transcribed in the same direction. The gene arrangement of the mt genome of <i>M. graminicola</i> is unique. A total of 21 out of 22 tRNAs possess a DHU loop only, while <i>tRNA^Ser(AGN)</i> lacks a DHU loop. The two large noncoding regions (2,031 bp and 5,063 bp) are disrupted by <i>tRNA^Ser(UCN)</i>. Phylogenetic analysis based on concatenated amino acid sequences of 12 protein-coding genes support the monophylies of the three orders Rhabditida, Mermithida and Trichinellida, the suborder Rhabditina and the three infraorders Spiruromorpha, Oxyuridomorpha and Ascaridomorpha, but do not support the monophylies of the two suborders Spirurina and Tylenchina, and the three infraorders Rhabditomorpha, Panagrolaimomorpha and Tylenchomorpha. The four Tylenchomorpha species including <i>M. graminicola</i>, <i>P. vulnus</i>, <i>H. glycines</i> and <i>R. similis</i> from the superfamily Tylenchoidea are placed within a well-supported monophyletic clade, but far from the other two Tylenchomorpha species <i>B. xylophilus</i> and <i>B. mucronatus</i> of Aphelenchoidea. In the clade of Tylenchoidea, <i>M. graminicola</i> is sister to <i>P</i>. <i>vulnus</i>, and <i>H. glycines</i> is sister to <i>R. similis</i>, which suggests root-knot nematodes has a closer relationship to Pratylenchidae nematodes than to cyst nematodes.</p></div

Arrangement of the mitochondrial genome of <i>Meloidogyne graminicola</i>.

<p>Gene scaling is only approximate. All genes are coding by the same DNA strand, and the arrow indicates the direction of transcription. All protein-coding genes have standard nomenclature. All tRNA genes follow the one-letter amino acid code; L1/L2 and S1/S2 indicate tRNA genes for <i>tRNA^Leu(CUN)</i>/<i>tRNA^Leu(UUR)</i> and <i>tRNA^Ser(AGN)</i>/<i>tRNA^Ser(UCN)</i>, respectively. “NCR1” refers to a small noncoding region and “NCR2” refers to a large noncoding region.</p

Phylogenetic tree from maximum likelihood analysis of amino sequences for 12 protein-coding genes for 50 nematode mitochondrial genomes.

<p><i>Lithobius forficatus</i> and <i>Limulus polyphemus</i> were used as the outgroups. Bootstrap percentage (BP) values are indicated at the nodes. Classification according to De Ley and Blaxter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098558#pone.0098558-DeLey1" target="_blank">[6]</a>.</p

Organization of the <i>Meloidogyne graminicola</i> mitochondrial genome.

<p>a: Indicates gap nucleotides (positive value) or overlapping nucleotides (negative value) between two adjacent genes;</p><p>NCR: Noncoding region.</p

Comparison of mitochondrial gene arrangements between <i>Meloidogyne graminicola</i> and <i>Pratylenchus vulnus</i>.

<p>Gene and genome size are not to scale. The noncoding region (NCR) is not indicated. Arrows below the gene order map indicate the direction of transcription of genes. Genes involved in the rearrangements are shown in dashed boxes.</p

Codon usage pattern and relative synonymous codon usage (RSCU) of mtDNA of <i>Meloidogyne graminicola</i>.

<p>Numbers on the Y-axis refer to the total number of codons (A) and the RSCU value (B). Codon families are provided on the X-axis. Codons that are not present in the mitochondrial genome are indicated in red at the tops of the columns.</p

Phylogenetic tree from Bayesian analysis of amino sequences for 12 protein-coding genes for 50 nematode mitochondrial genomes.

<p><i>Lithobius forficatus</i> and <i>Limulus polyphemus</i> were used as the outgroups. Numbers along the branches indicate Bayesian posterior probability (BPP) values. Classification according to De Ley and Blaxter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098558#pone.0098558-DeLey1" target="_blank">[6]</a>.</p

Image_1_A Bursaphelenchus xylophilus effector BxICD1 inducing plant cell death, concurrently contributes to nematode virulence and migration.tif

The migratory endoparasitic phytonematodes Bursaphelenchus xylophilus is the causal agent of pine wilt disease and causes significant economic damage to pine forests in China. Effectors play a key role in the successful parasitism of plants by phytonematodes. In this study, 210 genes obtained by transcriptomics analyses were found to be upregulated in B. xylophilus infecting Pinus massoniana that were not functionally annotated nor reported previously in B. xylophilus infecting P. thunbergii. Among these differentially expressed genes, a novel effector, BxICD1, that could induce cell death in the extracellular space of Nicotiana benthamiana was identified. BxICD1 was upregulated in the early stages of infection, as shown by RT-qPCR analyses. In situ hybridization analysis showed that BxICD1 was expressed in the esophageal gland of nematodes. The yeast signal sequence trap system indicated that BxICD1 possessed an N-terminal signal peptide with secretion functionality. Using an Agrobacterium-mediated transient expression system, it was demonstrated that the cell death-inducing activity of BxICD1 was dependent on N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1). Finally, BxICD1 contributed to B. xylophilus virulence and migration in host pine trees, as demonstrated by RNAi silencing assays. These findings indicate that BxICD1 both induces plant cell death and also contributes to nematode virulence and migration in P. massonian.</p

Table_1_A Bursaphelenchus xylophilus effector BxICD1 inducing plant cell death, concurrently contributes to nematode virulence and migration.xlsx

The migratory endoparasitic phytonematodes Bursaphelenchus xylophilus is the causal agent of pine wilt disease and causes significant economic damage to pine forests in China. Effectors play a key role in the successful parasitism of plants by phytonematodes. In this study, 210 genes obtained by transcriptomics analyses were found to be upregulated in B. xylophilus infecting Pinus massoniana that were not functionally annotated nor reported previously in B. xylophilus infecting P. thunbergii. Among these differentially expressed genes, a novel effector, BxICD1, that could induce cell death in the extracellular space of Nicotiana benthamiana was identified. BxICD1 was upregulated in the early stages of infection, as shown by RT-qPCR analyses. In situ hybridization analysis showed that BxICD1 was expressed in the esophageal gland of nematodes. The yeast signal sequence trap system indicated that BxICD1 possessed an N-terminal signal peptide with secretion functionality. Using an Agrobacterium-mediated transient expression system, it was demonstrated that the cell death-inducing activity of BxICD1 was dependent on N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1). Finally, BxICD1 contributed to B. xylophilus virulence and migration in host pine trees, as demonstrated by RNAi silencing assays. These findings indicate that BxICD1 both induces plant cell death and also contributes to nematode virulence and migration in P. massonian.</p

Image_3_A Bursaphelenchus xylophilus effector BxICD1 inducing plant cell death, concurrently contributes to nematode virulence and migration.tif

The migratory endoparasitic phytonematodes Bursaphelenchus xylophilus is the causal agent of pine wilt disease and causes significant economic damage to pine forests in China. Effectors play a key role in the successful parasitism of plants by phytonematodes. In this study, 210 genes obtained by transcriptomics analyses were found to be upregulated in B. xylophilus infecting Pinus massoniana that were not functionally annotated nor reported previously in B. xylophilus infecting P. thunbergii. Among these differentially expressed genes, a novel effector, BxICD1, that could induce cell death in the extracellular space of Nicotiana benthamiana was identified. BxICD1 was upregulated in the early stages of infection, as shown by RT-qPCR analyses. In situ hybridization analysis showed that BxICD1 was expressed in the esophageal gland of nematodes. The yeast signal sequence trap system indicated that BxICD1 possessed an N-terminal signal peptide with secretion functionality. Using an Agrobacterium-mediated transient expression system, it was demonstrated that the cell death-inducing activity of BxICD1 was dependent on N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1). Finally, BxICD1 contributed to B. xylophilus virulence and migration in host pine trees, as demonstrated by RNAi silencing assays. These findings indicate that BxICD1 both induces plant cell death and also contributes to nematode virulence and migration in P. massonian.</p