The expression of Gr-VAP1 coincides with host invasion and migration of <i>Globodera rostochiensis</i>.

<p>The expression of <i>Gr-VAP1</i>, as shown by semi-quantitative reverse transcription PCR, is highly up-regulated in the migratory stages of <i>G. rostochiensis</i> (ppJ2, J2, and males (♂)), while it declines after initiation of the permanent feeding site in the sedentary juvenile stages (J3 and J4, and adult females (♀). Changes in expression of <i>Gr-VAP1</i> were assessed using the constitutively expressed <i>cAMP-</i>dependent protein kinase (<i>cAMP</i>) gene in <i>G. rostochiensis</i> as reference. Reactions using uninfected tomato roots as template (Root) and without reverse transcriptase (-RT) were included as controls.</p

The venom allergen-like protein Gr-VAP1 is required for the onset of parasitism in host plants.

<p>(A) RNA interference specifically knocked down <i>Gr-VAP1</i> expression in pre-parasitic second stage juveniles of <i>G. rostochiensis</i>. Semi-quantitative reverse transcription-PCR of <i>Gr-VAP1</i> and a reference gene (<i>60S rib. gene</i>) in pre-parasitic second juveniles in double stranded RNA either matching the <i>Gr-VAP1</i> sequence or the sequence of the <i>NAU</i> gene of <i>Drosophila melanogaster</i> as control. Numbers indicate the cycles in the PCR. (B) The knockdown of <i>Gr-VAP1</i> expression significantly reduces the number of infective juveniles of <i>G. rostochiensis</i> inside roots of tomato plants (<i>S. lycopersicum</i>). Pre-parasitic second juveniles were either treated with double stranded RNA matching the <i>Gr-VAP1</i> or the <i>Nau</i> sequence. Bars represent standard error of mean of number of nematodes per plant at 7 days after inoculation over 10 replicates. Asterisk marks significance in a Student's t-test (with <i>P</i>-value <0.05).</p

Ectopic venom allergen-like proteins from cyst and root-knot nematodes selectively suppress defense-related programmed cell death.

<p>(A) Agroinfiltration assays in <i>Nicotiana benthamiana</i> showing the transient co-expression in the apoplast of cell death inducing elicitin <i>INF1</i> of <i>Phytophthora infestans</i> and venom allergen-like proteins from <i>G. rostochiensis</i> (Gr-VAP1), <i>H. schachtii</i> (Hs-VAP1 and Hs-VAP2), and <i>Meloidogyne incognita</i> (Mi-VAP1). Co-expressions with the corresponding empty binary vector (EV) and green fluorescent protein (GFP) were included as controls. (B and C) Transient co-expression of receptor-like proteins Cf-4 and Cf-9 from tomato and their cognate elicitors Avr4 and Avr9 from <i>C. fulvum</i> with venom allergen-like proteins and controls as described above. Photographs were taken 4 days post infiltration for INF1, and 7 days post infiltration for Cf-4/Avr4 and Cf9/Avr9. The bars represent the mean number of events in which cell death suppression was observed for a total of 60 inoculation spots over 5 biological replicates (with standard error of mean). Different letters indicate a significant difference when using <i>P</i>-value <0.05 (in an ANOVA).</p

Ectopic venom allergen-like proteins suppress basal immunity in <i>Arabidopsis thaliana</i>.

<p>(A) Heterologous expression of the venom allergen-like protein Gr-VAP1 from <i>G. rostochiensis</i>, and Hs-VAP1 and Hs-VAP2 from <i>Heterodera schachtii</i> in the apoplast of transgenic Arabidopsis lines enhances their susceptibility to <i>H. schachtii</i>. Two independent transgenic lines per construct (-A and -B) were compared with corresponding transgenic line harboring the T-DNA of the empty vector (EV) and wild type <i>A. thaliana</i> (Col-0). Bars represent mean number of nematodes per plants with standard errors of the means. Letters indicate statistical significance when using <i>P</i>-value <0.05 as threshold. (B) Ectopic Hs-VAP1 and Hs-VAP2 enhance development of disease symptoms of fungal and oomycete pathogens in leaves of transgenic Arabidopsis lines. Pictures show symptoms on leaves inoculated with <i>Botrytis cinerea</i>, <i>Plectosphaerella cucumeria</i>, and two isolates of <i>Phytophthora brassicae</i>, or mock inoculated. (C) Ectopic Hs-VAP1 and Hs-VAP2 suppress seedling growth response of Arabidopsis to the immunogenic peptide flg22. Bars represent mean root length of transgenic lines with standard error of mean after 10 days in the presence or absence of 10 µM flg22.</p

Apoplastic Gr-VAP1 suppresses immunity of potato plants to <i>G. rostochiensis</i>.

<p>(A) Transgenic potato plants stably overexpressing Gr-VAP1 in the apoplast show enhanced susceptibility to <i>G. rostochiensis</i>. The number of nematodes per plant was compared at 6 weeks post inoculation for two independent transgenic potato lines harboring either Gr-VAP1 (<i>Gr-VAP1-A</i> and <i>Gr-VAP1-B</i>) or the corresponding T-DNA insert of the empty binary expression vector (<i>EV</i>). The expression constructs included native signal peptide for secretion of Gr-VAP1. Bars represent standard errors of the means. Different letters indicate statistically significant differences between plant genotypes as determined with ANOVA (with <i>P</i>-values <0.05). (B) Apoplastic Gr-VAP1 perturbs the active site of the extracellular defense-related papain-like cysteine protease C14^tub of potato (<i>S. tuberosum</i>). Image shows binding of the fluorescent activity-based probe DCG-04 to the active site of C14^tub and C14^lyc of tomato (<i>S. lycopersicum</i>) following treatment with Gr-VAP1 isolated from apoplastic fluids of agroinfiltrated leaves. Treatments with the Avr2, egg white cystatin, and apoplastic fluids from agroinfiltrations with the empty binary expression vector (Empty vector), and with buffer alone (Buffer) were included as controls.</p

A plant cell wall-associated subtilase and non-photochemical quenching in chloroplasts regulate immunity to plant-parasitic nematodes.

<p>The lack of the subtilisin-like serine protease AtSBT3.13 and the chlorophyll-associated Photosystem II subunit S protein in homozygous Arabidopsis mutants (<i>sbt3.14</i> and <i>npq4-1</i>, respectively) significantly alters their susceptibility to <i>H. schachtii</i>. Bars represent mean number of nematodes per plant with standard error of mean. Different letters indicate statistically significant differences between homozygous knockout mutants and corresponding wild type Arabidopsis at four weeks after inoculation (determined with ANOVA (with <i>P</i>-values <0.05).</p

Ten most down-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis.

<p>Differentially expressed genes in transgenic <i>Arabidopsis thaliana</i> overexpressing <i>Hs-VAP1</i> and <i>Hs-VAP2</i> relative to the corresponding transgenic empty vector control plants (for full lists see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004569#ppat.1004569.s008" target="_blank">S1 Table</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004569#ppat.1004569.s009" target="_blank">S2 Table</a>).</p><p>* The fold change (FC) is calculated as standardized log2-transformed counts per million relative to transgenic plants harboring the corresponding empty vector.</p><p>Ten most down-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis.</p

Defense-related extracellular papain-like cysteine proteases regulate immunity to cyst nematodes in Arabidopsis.

<p>(A) Inhibition of papain-like cysteine proteases by heterologous expression of the apoplastic effector Avr2 from <i>Cladosporium fulvum</i> in transgenic Arabidopsis lines suppresses immunity to <i>H. schachtii</i>. The bars represent mean number of nematodes per plant with standard error at four weeks post inoculation in transgenic line harboring ectopic Avr2 in the apoplast (Avr2) and the corresponding wild type Arabidopsis. Asterisk indicates statistical significance when using <i>P</i>-value <0.05 as threshold (Student's t-test). (B) Members of the extracellular papain-like cysteine protease family AtPAP in Arabidopsis are required for immunity to <i>H. schachtii</i>. Bars represent mean number of nematodes per plant with standard error of mean. Different letters indicate statistically significant differences between homozygous knockout mutants <i>pap1</i>, <i>pap4</i>, <i>pap5</i> and corresponding wild type Arabidopsis at four weeks after inoculation determined with ANOVA (with <i>P</i>-values <0.05).</p

Ten most up-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis.

<p>Differentially expressed genes in transgenic <i>Arabidopsis thaliana</i> overexpressing <i>Hs-VAP1</i> and <i>Hs-VAP2</i> relative to the corresponding transgenic empty vector control plants (for full lists see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004569#ppat.1004569.s010" target="_blank">S3 Table</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004569#ppat.1004569.s011" target="_blank">S4 Table</a>).</p><p>* The fold change (FC) is calculated as standardized log2-transformed counts per million relative to transgenic plants harboring the corresponding empty vector.</p><p>Ten most up-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis.</p