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

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

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

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

A novel <i>Meloidogyne graminicola</i> effector, MgGPP, is secreted into host cells and undergoes glycosylation in concert with proteolysis to suppress plant defenses and promote parasitism

<div><p>Plant pathogen effectors can recruit the host post-translational machinery to mediate their post-translational modification (PTM) and regulate their activity to facilitate parasitism, but few studies have focused on this phenomenon in the field of plant-parasitic nematodes. In this study, we show that the plant-parasitic nematode <i>Meloidogyne graminicola</i> has evolved a novel effector, MgGPP, that is exclusively expressed within the nematode subventral esophageal gland cells and up-regulated in the early parasitic stage of <i>M</i>. <i>graminicola</i>. The effector MgGPP plays a role in nematode parasitism. Transgenic rice lines expressing MgGPP become significantly more susceptible to <i>M</i>. <i>graminicola</i> infection than wild-type control plants, and conversely, <i>in planta</i>, the silencing of MgGPP through RNAi technology substantially increases the resistance of rice to <i>M</i>. <i>graminicola</i>. Significantly, we show that MgGPP is secreted into host plants and targeted to the ER, where the <i>N</i>-glycosylation and C-terminal proteolysis of MgGPP occur. C-terminal proteolysis promotes MgGPP to leave the ER, after which it is transported to the nucleus. In addition, <i>N</i>-glycosylation of MgGPP is required for suppressing the host response. The research data provide an intriguing example of <i>in planta</i> glycosylation in concert with proteolysis of a pathogen effector, which depict a novel mechanism by which parasitic nematodes could subjugate plant immunity and promote parasitism and may present a promising target for developing new strategies against nematode infections.</p></div

Expression patterns of <i>MgGPP</i> in <i>Meloidogyne graminicola</i>.

<p>(A) Schematic representation of <i>M</i>. <i>graminicola</i> pre-J2. (B) Localization of <i>MgGPP</i> in the subventral esophageal glands of <i>M</i>. <i>graminicola</i> pre-J2s by <i>in situ</i> hybridization. Fixed nematodes were hybridized with (left) sense and (right) antisense cDNA probes from <i>MgGPP</i>. Scale bars, 10 μm. (C) The developmental expression pattern of MgGPP by RT-qPCR analysis in five different life stages of <i>M</i>. <i>graminicola</i>. The fold change values were calculated using the 2^-ΔΔCT method and presented as the change in mRNA level at various nematode developmental stages relative to that of the egg stage. The data shown are the means of three repeats plus standard deviation (SD), and three independent experiments were performed with similar results. dpi, days post-infection; pre-J2, pre-parasitic second-stage juvenile; par-J2, par-J3 and par-J4, parasitic second-, third- and fourth-stage juveniles, respectively.</p

Assays for glycosylation of MgGPP.

<p>(A) Using an anti-MgGPP antibody, western blot analysis of proteins from pre-J2s, par-J3s/J4s and females of <i>Meloidogyne graminicola</i> treated with or without PNGase F all showed the ~25 kDa size band, indicating that MgGPP is not glycosylated in nematodes. (B) Using an anti-GFP antibody, western blot analysis of proteins from the transformed cells of rice and tobacco showed two protein forms of ~43 and ~39 kDa of eGFP:MgGPP^Δsp, the ~39 kDa size of MgGPP^Δsp:eGFP treated with PNGase A, and ~39 kDa size of the point mutation eGFP:MgGPP^Δsp_N110Q, indicating that <i>N</i>-glycosylation of MgGPP occurred in host plants.</p