Image_2_The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.JPEG

<p>The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.</p

Table_2_The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.xlsx

<p>The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.</p

Image_5_The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.JPEG

<p>The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.</p

Image_4_The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.JPEG

<p>The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.</p

Image_3_The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.JPEG

<p>The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.</p

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

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

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

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