Relative abundance of race 4-enriched genes at four different stages in race 4 compared with that in race 3.

a<p>The expression level of genes was analyzed with the 2^−ΔΔCt method using the GAPDH gene as an internal reference gene for normalization. The calibrator was the transcript level of the corresponding gene in race 3 at the same developmental stage.</p>b<p>The relative abundance of the gene indicated was very close to zero.</p

Comparative Transcriptome Analysis of Two Races of <i>Heterodera glycines</i> at Different Developmental Stages

<div><p>The soybean cyst nematode, <i>Heterodera glycines</i>, is an important pest of soybeans. Although resistance is available against this nematode, selection for virulent races can occur, allowing the nematode to overcome the resistance of cultivars. There are abundant field populations, however, little is known about their genetic diversity. In order to elucidate the differences between races, we investigated the transcriptional diversity within race 3 and race 4 inbred lines during their compatible interactions with the soybean host Zhonghuang 13. Six different race-enriched cDNA libraries were constructed with limited nematode samples collected from the three sedentary stages, parasitic J2, J3 and J4 female, respectively. Among 689 putative race-enriched genes isolated from the six libraries with functional annotations, 92 were validated by quantitative RT-PCR (qRT-PCR), including eight putative effector encoding genes. Further race-enriched genes were validated within race 3 and race 4 during development in soybean roots. Gene Ontology (GO) analysis of all the race-enriched genes at J3 and J4 female stages showed that most of them functioned in metabolic processes. Relative transcript level analysis of 13 selected race-enriched genes at four developmental stages showed that the differences in their expression abundance took place at either one or more developmental stages. This is the first investigation into the transcript diversity of <i>H. glycines</i> races throughout their sedentary stages, increasing the understanding of the genetic diversity of <i>H. glycines</i>.</p></div

Morphology of <i>H. glycines</i> at different developmental stages.

<p>The sedentary <i>H. glycines</i> J2s, J3s and J4 females were isolated from soybean roots at 5 dpi, 7 dpi, and 12 dpi, respectively. The morphology of <i>H. glycines</i> at parasitic J2 stage (<b>A</b>), J3 stage (<b>B</b>) and J4 female stage (<b>C</b>) was examined with a microscope. Bar = 200 µm.</p

Characteristics of race 3- and race 4-enriched cDNA libraries at three sedentary stages.

a<p>Total number of clones isolated from different race-enriched cDNA libraries at three sedentary stages.</p>b<p>Unigenes with functional annotation.</p>c<p>Unigenes without functional annotation.</p

Gene Ontology analysis of race-enriched genes at sedentary stages J3 and J4 female.

<p>The gene annotation of induced genes found in race 3 (<b>A</b>) and race 4 (<b>B</b>) at J3 stage, and the ones in race 3 (<b>C</b>) and race 4 (<b>D</b>) at J4 female stage was carried out with BLAST2GO. The horizontal number represents the percentage of genes involved in different GO categories in all the induced genes found in both the two races at J3 and J4 stages, respectively.</p

Gene expression profiles of 13 race-enriched genes in both race 3 and race 4.

<p>RNA samples were isolated from preparasitic J2s, parasitic J2s (i-J2), J3s (J3) and J4 females (J4 female) of SCN race 3 and race 4, respectively. The expression profiles of the 13 genes categorized into similar (<b>A</b>) and uncoordinated (<b>B</b>) expression profiles in both the races. Preparasitic J2 stage was considered as the calibration stage, and the expression level of these 13 genes at preparasitic J2 stage was arbitrarily set to 1.0. The expression level was analyzed with the 2^−ΔΔCt method using the GAPDH gene as an internal reference gene for normalization. Mean and standard errors were determined with data from three independent replicates.</p

Developmental expression of <i>hg-pel-3</i>, <i>hg-pel-4</i>, <i>hg-pel-6</i> and <i>hg-pel-7</i> in different stages of <i>Heterodera glycines</i>.

<p>Pre-J2, pre-parasitic second stage juveniles; J2, parasitic second stage juveniles; J3, parasitic third stage juveniles; J4, parasitic forth stage juveniles; Female, adult females: eggs; N, negative control (genomic DNA of soybean roots). Maker, DL2000 DNA ladder. Actin, the <i>H</i>. <i>glycines</i> β-actin genem was used as positive control for each cDNA templates.</p

Alignment of the putative protein sequences of the pectate lyases that belong to the polysaccharide lyase family 3.

<p>HG-PEL-1 [AAK08974], HG-PEL-2 [AAM74954] and HG-PEL-5 [ADW77534] correspond to <i>Heterodera glycines</i>; HS-PEL-1 [ABN14273] and HS-PEL-2 [ABN14272] correspond to <i>H</i>. <i>schachtii</i>; GR-PEL-1 [AAF80747] and GR-PEL-2 [AAM21970] correspond to <i>Golobedera rostochiensis</i>; GP-PEL-2 [ACU64826] corresponds to <i>G</i>. <i>pallida</i>; BX-PEL-1 [BAE48369] and BX-PEL-2 [BAE48370] correspond to <i>Bursaphelenchus xylophilus</i>; BM-PEL-1 [BAE48373] and BM-PEL-2 [BAE48375] correspond to <i>B</i>. <i>mucronatus</i>; AA-PEL-1 [BAI44999] and AA-PEL-2 [BAI44997] correspond to <i>Aphelenchus avenae</i>; MI-PEL-1 [AAQ09004] and MI-PEL-2 [AAQ97032] correspond to <i>Meloidogyne incognita</i>. Identical residues are highlighted in black. Black bars (I to IV) indicate the conserved regions characteristic of the PL3 pectate lyases. The asterisk (*) indicates conserved cysteine residues. The positions of the intron <i>hg-pel-3</i>, <i>hg-pel-4</i> and <i>hg-pel-7</i>are indicated by Hg, that of <i>hg-pel-6</i> by Hg6, that of <i>Bx-pel-1</i>, <i>Bx-pel-2</i>, <i>Bm-pel-1</i> and <i>Bm-pel-2</i> by B, that of <i>Gr-pel-1</i> by Gr1, that of <i>Mi-pel-1</i> by Mi1and that of <i>Mi-pel-2</i> by Mi2. Diamonds, black triangles and white triangles represent phase 0, 1 and 2 introns, respectively.</p

Novel Pectate Lyase Genes of <i>Heterodera glycines</i> Play Key Roles in the Early Stage of Parasitism

<div><p>Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated <i>Hg-pel-3</i>, <i>Hg-pel-4</i>, <i>Hg-pel-6</i> and <i>Hg-pel-7</i>, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, <i>Heterodera glycines</i>. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of <i>H</i>. <i>glycines</i> (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of <i>H</i>. <i>glycines</i> are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of <i>H</i>. <i>glycines</i> may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple <i>Hg-pel-6</i>-like genes were present in the <i>H</i>. <i>glycines</i> genome. <i>In situ</i> hybridization showed that four novel pectate lyases (<i>Hg-pel-3</i>, <i>Hg-pel-4</i>, <i>Hg-pel-6</i> and <i>Hg-pel-7)</i> were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down <i>Hg-pel-6</i> using <i>in vitro</i> RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of <i>H</i>. <i>glycines</i> females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the <i>Hg-pel-6</i>gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests the important roles of this gene in the early stages of parasitism. Our combined data suggest that two types of pectate lyases are present in the <i>H</i>. <i>glycines</i> genome and may have different roles during infection.</p></div

Southern blotting of <i>Hg-pel-6</i>.

<p>Southern blot of the genomic DNA from <i>H</i>. <i>glycines</i> digested with <i>Eco</i>RI, <i>Eco</i>RV and <i>Hind</i>III as well as hybridized with an <i>hg-pel-6</i> cDNA probe. The DNA size markers are indicated in kb.</p