Expression of a Fungal Lectin in Arabidopsis Enhances Plant Growth and Resistance Toward Microbial Pathogens and a Plant-Parasitic Nematode

Abstract

Coprinopsis cinerea lectin 2 (CCL2) is a fucoside-binding lectin from the basidiomycete C. cinerea that is toxic to the bacterivorous nematode Caenorhabditis elegans as well as animal-parasitic and fungivorous nematodes. We expressed CCL2 in Arabidopsis to assess its protective potential toward plant-parasitic nematodes. Our results demonstrate that expression of CCL2 enhances host resistance against the cyst nematode Heterodera schachtii. Surprisingly, CCL2-expressing plants were also more resistant to fungal pathogens including Botrytis cinerea, and the phytopathogenic bacterium Pseudomonas syringae. In addition, CCL2 expression positively affected plant growth indicating that CCL2 has the potential to improve two important agricultural parameters namely biomass production and general disease resistance. The mechanism of the CCL2-mediated enhancement of plant disease resistance depended on fucoside-binding by CCL2 as transgenic plants expressing a mutant version of CCL2 (Y92A), compromised in fucoside-binding, exhibited wild type (WT) disease susceptibility. The protective effect of CCL2 did not seem to be direct as the lectin showed no growth-inhibition toward B. cinerea in in vitro assays. We detected, however, a significantly enhanced transcriptional induction of plant defense genes in CCL2- but not CCL2-Y92A-expressing lines in response to infection with B. cinerea compared to WT plants. This study demonstrates a potential of fungal defense lectins in plant protection beyond their use as toxins.

Keywords: Arabidopsis; Botrytis cinerea; Coprinopsis cinerea lectin 2; Heterodera schachtii; Pseudomonas syringae.

FIGURE 1

Characterization of CCL2-expressing Arabidopsis lines. (A) qPCR analysis of relative CCL2 and CCL2-Y92A transcript levels in 4-week-old plants. Transcript levels were normalized to expG gene (AT4G26410). Mean values ± SE of three independent experiments. (B) Immunoblot visualizing the expression level of CCL2 and CCL2-Y92A proteins. FLAG-tagged proteins were detected with anti-FLAG antibodies. Ponceau-S stained Rubisco large subunit served as loading control. Size bar = 1 cm (C) Growth phenotype of transgenic lines compared to WT. Two independent lines (L1 and L2) are shown for each construct. (D) Fresh weight (FW) and (E) dry weight (DW) of shoots of 4-week-old plants (n = 12; three independent experiments). Boxplots represent median and 1.5 times the interquartile range. Asterisks show significant differences between transgenic lines compared to the WT (^∗∗∗P ≤ 0.001, ^∗∗P ≤ 0.01) determined by one-way ANOVA followed by post hoc analysis with Dunnett’s multiple-comparison test.

FIGURE 2

Partial resistance of CCL2-expressing plants toward the cyst nematode H. schachtii. 12-day-old Arabidopsis seedlings (WT, CCL2, and CCL2-Y92A lines) were inoculated with 30 freshly hatched juveniles per plant and evaluated 14 dpi for number of female nematodes per root centimeter. Boxplots represent median and 1.5 times the interquartile range (WT and CCL2 lines n = 16; CCL2-Y92A n = 15; three independent experiments). Asterisks above columns indicate statistically significant differences (^∗∗∗P ≤ 0.001, ns, not significant) between CCL2 lines and WT plants, analyzed by one-way ANOVA and post hoc analysis with Dunnett’s multiple-comparison test.

FIGURE 3

Resistance of CCL2-expressing plants toward fungal pathogens. (A) Necrotic lesions caused by B. cinerea infection on leaves of 4-week-old WT, CCL2- and CCL2-Y92A lines inoculated with 6 μL droplets of a spore suspension (5 × 10⁴ spores mL^–1). Plants were photographed 3 dpi. Size bar = 1 cm. (B) Trypan Blue-staining of Arabidopsis leaves 60 hpi. The right-side shows close-up images. Black or red size bares are 1 mm and 50 μm, respectively. (C) Quantification of lesion size at 3 dpi. Boxplots represent median and 1.5 times the interquartile range (n = 80 from three independent experiments). (D) Quantification of fungal DNA by qPCR at 0, 1, and 2 dpi. The fungal Cutinase A gene (Genebank: Z69264) was quantified relative to expG gene (AT4G26410) of Arabidopsis. Bars represent mean values ± SE from three independent experiments. (E) Analysis of lesion size of 5-week-old WT and transgenic CCL2 lines droplet-inoculated with C. higginsianum (10 μL of 2 × 10⁶ spores mL^–1 per leaf). Plants were analyzed 10 dpi. (F) Analysis of lesion size of 4-week-old WT and CCL2 lines, droplet-inoculated with P. cucumerina (10 μL of 5 × 10⁶ spores mL^–1 per leaf). Plants were analyzed 5 dpi. Boxplots (E,F) represent median and 1.5 times the interquartile range (n = 30 from three independent experiments). The data was analyzed by one-way ANOVA and post hoc analysis by Dunnett’s multiple-comparison test. Asterisks show a statistically significant difference between the CCL2 expressing lines and WT plants (^∗∗∗P ≤ 0.001, ^∗∗P ≤ 0.01, ns, not significant). The letters a and b signify a between-group difference at the P ≤ 0.05 level.

FIGURE 4

CCL2 enhances induction of Arabidopsis defense gene expression in response to B. cinerea. Four-week-old Arabidopsis plants (WT, CCL2, and CCL2-Y92A lines) were spray-inoculated with B. cinerea (5 × 10⁵ spores mL^–1). Leaves were harvested at 0 and 1 dpi for RNA extraction. Transcript levels of OBP2 (A), PDF1.2 (B), and PR-1 (C) were determined by qPCR. Data were normalized with regard to the Arabidopsis reference gene expG. Data represent mean values ± SE of three independent experiments. The letters a and b signify a between-group difference at the P ≤ 0.05 level. Two-way ANOVA and post hoc analysis by Tukey’s multiple-comparison test were used to calculate significant differences between transgenic lines and WT plants.

FIGURE 5

Increased resistance of CCL2-lines toward the bacterial pathogen P. syringae. Growth of virulent Pst DC3000 in WT plants and CCL2 lines was analyzed at 3 dpi. The bacterial oprF gene was quantified by qPCR using DNA extracted from inoculated leaves. Ten leaf discs from six plants were sampled per replicate. The plant expG gene served as reference. Data represent mean values ± SE of three independent experiments (n = 18). Asterisks indicate statistically significant differences (^∗P ≤ 0.05, ^∗∗P ≤ 0.01, ns: not significant; one-way ANOVA and post hoc analysis with Dunnett’s multiple-comparison test) between transgenic lines and wild type.

FIGURE 6

Exogenous application of purified CCL2 induces defense gene expression and SAR toward P. syringae. Three leaves of 4-week-old wild type plants were infiltrated with 10 mM MgCl₂ (negative mock control), Pst DC3000 (10⁶ CFU mL^–1) as positive SAR control, or 500 μg mL^–1 of purified CCL2 or CCL2-Y92A protein, respectively. (A) Three distal leaves were challenge-inoculated with Pst DC3000 (10⁵ CFU mL^–1) at 48 h after treatment. Ten leaf discs per treatment were sampled from distal leaves of Ten plants at 3 dpi to quantify by qPCR the abundance of the bacterial oprF gene as a proxy for bacterial biomass. (B–F) Transcript levels relative to expG gene in local leaves 48 h after treatment (B) GLI1 (AT1G80460), (C) GLY1 (AT2G40690), (D) PR-1 (AT2G14610), (E) RBOHD (AT5G47910), and (F) RBOHF (AT1G64060). Asterisks indicate statistically significant differences (^∗P ≤ 0.05, ^∗∗P ≤ 0.01, ns: not significant; one-way ANOVA and post hoc analysis with Dunnett’s multiple-comparison test) between treatments and mock control. Data represent mean ± SD of three biological replicates.