HYP effector variation between individuals.

<p>Each lane shows PCR products amplified from discrete nematode samples. Each PCR in lane one corresponds to DNA extracted from the same single <i>G. pallida</i> nematode. PCR on 6 individual nematodes using subfamily specific primers highlights a profound difference between individuals. In particular for subfamily-1 and -3 a reproducible difference can be seen in the complement of HYP effectors between nematodes both within and between subfamilies. The identity of amplification products from various samples was confirmed by sequencing. No two nematodes tested had the same genetic complement of HYP effectors.</p

RNA interference of <i>Gp-hyp</i> genes.

<p>A) An inverted repeat construct targeting a single <i>Gp-hyp</i> 1 gene was transformed into hairy roots of potato. Due to the nature of the conserved regions the construct was expected to target all <i>Gp-hyp</i> genes in all subfamilies. For all <i>Gp-hyp</i>-IR lines a significant reduction in nematode numbers (p<0.05) was seen when compared to the GFP IR hairy roots (n = 8). Error bars indicate the standard error of the mean. B) Semi-quantitative RT-PCR confirms expression of the inverted repeat construct in the RNAi lines. Line 2b was not tested for resistance as the phenotype varied too greatly from the GFP and empty vector controls.</p

Schematic representation of HYP effector domain organisation in <i>G. pallida</i>.

<p>Within the conserved regions, non-synonymous SNPs result in various domains. Combinations of domains give rise to “types”. Certain types are subfamily specific, whereas others are not. For each column only, the same colour indicates the same sequence. By organising the sequences using types as indicators (not in the order they appear in the genes), HYP effectors do not group by subfamily. The three different subfamily specific tandem repeats are indicated in the final column as different shapes, where the size roughly correlates to number of repeats. Sequences encoding similar tandem repeat regions can have different domains at every locus, and similarly sequences encoding identical domains at every locus can have different tandem repeat regions.</p

Immunolocalisation of Gp-HYP proteins to the apoplasm.

<p>A) 2 µm section showing Gp-HYP protein (green) detected between cell walls (blue) in the apoplasm between the feeding site (*) and the anterior end of the nematode (N). Cell walls are stained with calcofluor white (blue). B) Comparison with a schematic highlights the nematode in the context of the syncytium and the root tissue.</p

Pipeline for identification of genes involved in plant-nematode biotrophy.

<p>A) Summarises the process of identifying novel genes, highly up regulated during biotrophy, that encode secreted proteins. B) Example expression data of all candidates with the addition of all other full length or partial HYP effector sequences present in the genome assembly, highlighted in red and green for subfamily-1 and -3 respectively.</p

HYP effectors are expressed in the amphid sheath cells of parasitic females.

<p>A) Schematic representation of <i>Globodera pallida</i> feeding female <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004391#ppat.1004391-Bird1" target="_blank">[64]</a>. <i>In situ</i> hybridisation probe designed to a conserved region common to all HYP effectors identifies extremely strong and specific dark staining in a paired structure anterior to the metacorpal bulb of 14 days post infection females (C). No such staining pattern is observed with the negative control, or the positive control on second stage juvenile (J2) nematodes (B). Comparison of the staining pattern with a schematic identified this structure as the two lobed amphid sheath cells.</p

Comparison of HYP effector variation between UK populations of <i>G. pallida</i> and <i>G. rostochiensis</i>.

<p>For <i>Gp-hyp</i> subfamily -1 and -3 large variation can be seen when amplifying from DNA extracted from a pool of thousands of <i>G. pallida</i> as indicated by multiple bands. Considerably less variation is seen under the same reaction conditions for <i>G. rostochiensis</i>, probably reflecting a more genetically restricted introduction to the UK. Amplicons from each reaction were sequenced to confirm they correspond to HYP effectors.</p

HYP effector gene family schematic and location of primer combinations.

<p>All HYP effector genes identified to date encode a secretion signal followed by two highly conserved regions flanking a region of variable number tandem repeats, and a subfamily specific 3′ UTR. Conserved regions of <i>Gp-hyp</i> genes share >95% identity between subfamilies. Subfamily-2 has a slightly different signal peptide (highlighted). 3′ UTRs are identical within subfamilies, irrespective of number of tandem repeats. Members of all subfamilies can be amplified using the F-ALL and R-ALL primer pair, subfamilies can be distinguished by PCR using a combination of signal peptide and 3′ UTR primers (summarised in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004391#ppat.1004391.s006" target="_blank">Table S1</a>). Solid lines represent introns, while dashed lines represent alignments.</p

Conservation of <i>hyp</i> genes in <i>Globodera</i> and <i>Rotylenchulus</i> species.

<p>Amino acid alignment of HYP-2 sequences from <i>G. pallida, G. rostochiensis</i> and <i>R. reniformis</i>. HYP-2 sequences are highly conserved (>93%) across the full length of the protein in the three species. The tandem repeat region, and true locations of the subfamily independent domains are highlighted.</p

All_transcripts_from_the_same_comp_as_those_containing_cep-like_sequences_in_transcriptome

All transcripts from the same comp as those containing cep-like sequences in transcriptome