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Genome annotation. (XLSX 9 kb

Silencing <i>ACS</i> genes in tomato does not compromise <i>Mi-1</i>-mediated resistance to root-knot nematodes.

<p>Two-week-old tomato plants cvs. Moneymaker (<i>mi/mi</i>) and Motelle (<i>Mi-1/Mi-1</i>) were used in agroinfiltration of tobacco rattle virus (TRV) empty vector, and cv. Motelle was used with TRV containing a portion of <i>Mi-1</i> (TRV-Mi-1) or containing <i>1-aminocyclopropane-1-carboxylic acid synthase</i> (<i>ACS</i>) constructs (TRV-ACSI and TRV-ACSII), which were either individually or simultaneously agroinfiltrated (TRV-ACSI+II). Three weeks after agroinfiltration, plants were infected with 10,000 second-stage juvenile root-knot-nematodes and evaluated for nematodes reproduction 8 weeks later. Dots represent the number of egg masses counted on a single root system (<i>n</i> = 18–25). Two independent experiments were performed with similar results and data from one are presented.</p

Root-knot nematodes (<i>Meloidogyne incognita</i>) induce the expression of ethylene biosynthetic genes in tomato.

<p><i>In vitro</i> grown seedlings of near isogenic tomato cvs. Moneymaker and Motelle were infected with 100–150 second-stage juvenile root-knot-nematodes in sterile conditions. The infected root tips were sampled at 0, 12, 24 and 36 h post infection (hpi). Expression of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase genes (<i>ACO</i>) and ACC synthase genes (<i>ACS</i>) was determined by semi-quantitative RT-PCR using gene-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063281#pone.0063281.s002" target="_blank">Table S1</a>) in two biological replicates with similar results. PCR amplification from a single sample is presented for each time point and genotype. Amplification of the tomato ubiquitin <i>Ubi3</i> gene was used as internal control. PCR cycles are indicated on the right side of the panel.</p

Effect of MCP treatment on ethylene response and resistance to root-knot nematode in tomato roots.

<p>(<b>A</b>) Efficiency of the 1-methylcyclopropene (MCP)-blocking of ethylene (ET) perception was assessed by monitoring the expression of <i>E4</i> after induction by ET. Seven-week-old cv. Moneymaker plants (+MCP/+ET) were pre-treated with MCP, and two plants were treated daily for 18 h with 10 µl/l ET prior to harvest. Root tissues were pooled and frozen. Tissues from untreated plants (−MCP/−ET) or plants only induced by ET (−MCP/+ET) were used as control. Total RNA (25 µg) for each sample was used for RNA blot analysis. The blot was hybridized sequentially with <i>E4</i> and an 18S rDNA probe used to normalize expression. (<b>B</b>, <b>C</b>) Five-week-old tomato plants cvs. Moneymaker and Motelle were treated with MCP (+MCP) or untreated (−MCP) prior root-knot nematode (RKN) infection with 3,000 second-stage juvenile. During the first 2 weeks following RKN infection, the plants (+MCP) were repeatedly treated with MCP every 2 days. RKN reproduction was evaluated 7 weeks after infection as (<b>B</b>) egg masses and (<b>C</b>) egg production. Results are presented relative to the fresh weight (FW) of roots. Error bars indicate standard error of the mean (<i>n</i> = 16), where bars with different letters denote significant difference at <i>P</i><0.05. The bioassay was performed twice with both tomato cultivars tested and twice more with cv. Moneymaker only. In all experiments, results from the same genotypes were similar. Data from one representative experiment are presented.</p

Root-knot nematodes reproduction on tomato is affected by the <i>Nr</i> mutation in compatible host only.

<p>Root-knot nematodes (RKN) reproduction was evaluated on <i>Never ripe (Nr)</i> mutant, wild type tomato cvs. Pearson and VFN, and the <i>Nr</i> introgressed line VFNx<i>Nr</i>. Four-week-old plants were infected with 3,000 second-stage juvenile RKN. (<b>A</b>) Egg masses and (<b>B</b>) eggs production were evaluated 6 weeks after RKN infection. Results are presented relative to the fresh weight (FW) of roots. Error bars indicate standard error of the mean (<i>n</i> = 20–30), where bars with different letters denote significant difference at <i>P</i><0.05. Two independent experiments were performed with similar results and data from one are presented.</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

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