Arabidopsis leucine-rich repeat receptor–like kinase NILR1 is required for induction of innate immunity to parasitic nematodes

Plant-parasitic nematodes are destructive pests causing losses of billions of dollars annually. An effective plant defence against pathogens relies on the recognition of pathogen-associated molecular patterns (PAMPs) by surface-localised receptors leading to the activation of PAMP-triggered immunity (PTI). Extensive studies have been conducted to characterise the role of PTI in various models of plant-pathogen interactions. However, far less is known about the role of PTI in roots in general and in plant-nematode interactions in particular. Here we show that nematode-derived proteinaceous elicitor/s is/are capable of inducing PTI in Arabidopsis in a manner dependent on the common immune co-receptor BAK1. Consistent with the role played by BAK1, we identified a leucine-rich repeat receptor-like kinase, termed NILR1 that is specifically regulated upon infection by nematodes. We show that NILR1 is essential for PTI responses initiated by nematodes and nilr1 loss-of-function mutants are hypersusceptible to a broad category of nematodes. To our knowledge, NILR1 is the first example of an immune receptor that is involved in induction of basal immunity (PTI) in plants or in animals in response to nematodes. Manipulation of NILR1 will provide new options for nematode control in crop plants in future

Arabidopsis leucine-rich repeat receptor–like kinase NILR1 is required for induction of innate immunity to parasitic nematodes

Plant-parasitic nematodes are destructive pests causing losses of billions of dollars annually. An effective plant defence against pathogens relies on the recognition of pathogen-associated molecular patterns (PAMPs) by surface-localised receptors leading to the activation of PAMP-triggered immunity (PTI). Extensive studies have been conducted to characterise the role of PTI in various models of plant-pathogen interactions. However, far less is known about the role of PTI in roots in general and in plant-nematode interactions in particular. Here we show that nematode-derived proteinaceous elicitor/s is/are capable of inducing PTI in Arabidopsis in a manner dependent on the common immune co-receptor BAK1. Consistent with the role played by BAK1, we identified a leucine-rich repeat receptor-like kinase, termed NILR1 that is specifically regulated upon infection by nematodes. We show that NILR1 is essential for PTI responses initiated by nematodes and nilr1 loss-of-function mutants are hypersusceptible to a broad category of nematodes. To our knowledge, NILR1 is the first example of an immune receptor that is involved in induction of basal immunity (PTI) in plants or in animals in response to nematodes. Manipulation of NILR1 will provide new options for nematode control in crop plants in future

NemaWater treatment induced PTI responses were reduced strongly upon proteinase K, heat treatment, and in <i>bak1-5</i> plants.

<p>(<b>A</b>) Effect of Proteinase K and heat on production of ROS burst in root segments from Col-0 plants treated with water, <i>Hs</i>NemaWater or flg22. ROS burst was measured by using L-012 based assay from 0 to 120 min. PK, Proteinase K. Bars represent mean ± SE for two independent biological replicates. Data were analysed using single-factor ANOVA and Tukey’s post hoc test (P<0.05). Columns sharing same letter are not statistically different. (<b>B</b>) 5-day-old Col-0 seedlings were incubated in water, <i>Hs</i>NemaWater, or flg22 with or without Proteinase K for seven days. Fresh weight was measured at 12 days after germination. Bars represent mean ± SE for two independent biological replicates. Data were analysed using single-factor ANOVA and Tukey’s post hoc test (P<0.05). Columns sharing same letter are not statistically different. (<b>C</b>) Average number of female nematodes per plant in Col-0, <i>bak1-5</i> and <i>bak1-5 bkk1</i>. (<b>D</b>) Root segments from Col-0 and <i>bak1-5</i> plants were treated with water, <i>Hs</i>NemaWater or flg22 and ROS burst was measured using L-012 based assay from 0 to 120 min. (<b>E</b>) 5-days-old Col-0 and <i>bak1-5</i> seedlings were incubated in water, <i>Hs</i>NemaWater or flg22 for seven days. Fresh weight was measured at 12 days after germination. (<b>C-E</b>) Bars represent mean ± SE for three independent biological replicates. Data were analyzed using single-factor ANOVA and Dunnet post hoc test. Asterisks represent significant difference to control (P<0.05).</p

NemaWater treatment induced defense responses in plants that are characteristics of PTI.

<p><b>(A)</b> Expression of PTI marker genes in microarray analysis upon <i>Hs</i>NemaWater treatment. Root segments from uninfected roots were used as control. Asterisk indicates significant difference to control <i>(FDR</i> <0.05; Fold change >1.5). (<b>B</b>) A heatmap showing expression of PTI marker genes upon nematode infection or upon <i>Hs</i>NemaWater treatment. (<b>A-B</b>) Values represent fold change compared with control. (<b>C-E</b>) Expression of glucuronidase (GUS) driven by <i>pCYP71A12</i> in control (C), <i>H</i>. <i>schachtii</i> infection at migratory stage (D) and <i>Hs</i>NemaWater treated plants (E) (<b>F</b>) A Venn diagram showing distribution of upregulated genes in Arabidopsis upon nematode infection or upon <i>Hs</i>NemaWater treatment.</p

Validation of changes in gene expression upon <i>Hs</i>NemaWater treatment via qRT-PCR.

<p>The values represent relative fold change in response to NemaWater treatment as compared with control roots. 18S was used as housekeeping gene to normalize the data. All values are means of three biological replicates +/- SD.</p

Knock-out <i>nilr1-1</i> enhances susceptibility to nematodes.

<p>(<b>A</b>) Average number of female nematodes induced by <i>H</i>. <i>schachtii</i> per plant in Col-0, <i>nilr1-1</i> and <i>nilr2-1</i>. Bars represent mean ± SE for three biological replicates. (<b>B</b>) Average number of galls induced <i>by M</i>. <i>incognita</i> per plants in Col-0, <i>nilr1-1</i> and <i>nilr2-1</i>. Bars represent mean ± SE for three biological replicates. (<b>C</b>) Root segments from Col-0, and <i>nilr1-1</i> plants were treated with water, <i>Hs</i>NemaWater or flg22 and ROS burst was measured using L-012 based assay from 0 to 120 min. Bars represent mean ± SE for sixteen biological replicates. (<b>D</b>) 5-day-old Col-0 and <i>nilr1-1</i> seedlings were incubated in water, <i>Hs</i>NemaWater, or flg22 for seven days. Fresh weight was measured at 12 days after germination. Bars represent mean ± SE for three independent biological replicates. (<b>E</b>) 5-day-old Col-0 and <i>nilr2-1</i> seedlings were incubated in water, <i>Hs</i>NemaWater, or flg22 for seven days. Fresh weight was measured at 12 days after germination. Bars represent mean ± SE for three independent biological replicates. (<b>F)</b> Root segments from Col-0 and <i>nilr2-1</i> plants were treated with water, <i>Hs</i>NemaWater or flg22 and ROS burst was measured using L-012 based assay from 0 to 120 min. Bars represent mean ± SE for sixteen biological replicates (<b>A-E</b>) Data were analysed using single-factor ANOVA and Tukey’s post hoc test (P<0.05). Columns sharing same letter are not statistically different.</p

Pre-treatment with NemaWater induces resistance to pathogens.

<p>(A-B) Roots of Col-0 plants were treated with water or <i>Hs</i>NemaWater prior to infection and number of females were counted at 14 dai for cyst nematodes and number of galls were counted at 19 dai for root-knot nematodes. Bars represent mean ± SE for three independent biological replicates. (C-D) Plants were sprayed with flg22 or <i>Hs</i>NemaWater prior to inoculation and C. F.U/cm² was counted at 4 dai. Bars represent mean ± SE. Experiments were repeated three times with similar results. Asterisks represent significant difference to water-treated control root segments (P<0.05).</p

NILR1 is localised in plasma membrane.

<p>(A) Confocal microscopy of epidermis of <i>Nicotianna benthamiana</i> transiently expressing <i>35S</i>:<i>NILR1-GFP</i> and plasma membrane marker <i>35S</i>:<i>PIP2A-mCherry</i>. Scale, 50 μm. (B-E) Leaf discs from tomato (B), <i>N</i>. <i>benthamiana</i> (C), sugarbeet (D) and rice plants were treated with water, <i>Hs</i>NemaWater or flg22 and ROS burst was measured using L-012 based assay from 0 to 120 min. Bars represent mean ± SE for three technical replicates. Experiment was repeated three times with same results. RLU, relative light units.</p

Nematode infection induced defense responses in plants that are characteristics of PTI.

<p><b>(A)</b> Expression of PTI marker genes in microarray analysis upon nematode infection in migratory stage. Root segments from uninfected roots were used as control. Values indicate fold change compared with control. Asterisk indicates significant difference to control <i>(FDR</i> <0.05; Fold change >1.5). (<b>B</b>) Root segments from Col-0 plants were treated with water, <i>Hs</i>NemaWater or flg22 and ROS burst was measured using L-012 based assay from 0 to 120 min. (<b>C</b>) Root segments from Col-0 plants were treated with water, different dilutions of <i>Hs</i>NemaWater or flg22 and ROS burst was measured using L-012 based assay from 0 to 120 min. (<b>D</b>) Root segments from Col-0 plants were incubated with <i>Hs</i>NemaWater for 1 hour and then this <i>Hs</i>NemaWater was used for production of ROS burst on fresh root segments. Water, fresh <i>Hs</i>NemaWater or flg22, were used as controls. (<b>E</b>) Root segments from Col-0 plants were treated with water, <i>Mi</i>NemaWater, or flg22 and ROS burst was measured using L-012 based assay from 0 to 120 min. (<b>B-E</b>) Bars represent mean ± SE for three technical replicates. Experiment was repeated three times with same results. RLU, relative light units. (<b>F</b>) 5-day-old Col-0 seedlings were incubated in water, <i>Hs</i>NemaWater or flg22 for seven days. Fresh weight was measured at 12 days after germination. Data were analysed using <i>t-test</i>. Asterisk represent significant difference to water-treated control root segments (P<0.05). Hs, <i>Heterodera schachtii</i>. Mi, <i>Meloidogyne incognita</i>.</p