The role of AtPP2-A3 and AtPP2-A8 genes encoding Nictaba-related lectin domains in the defense response of Arabidopsis thaliana to Heterodera schachtii

Abstract

Expression levels of AtPP2-A3 and AtPP2-A8 are reduced in syncytia induced by Heterodera schachtii and decline of their expression levels decreases host susceptibility, whereas their overexpression promotes susceptibility to parasite. Plant-parasitic nematodes cause huge crop losses worldwide. Heterodera schachtii is a sedentary cyst-forming nematode that induces a feeding site called a syncytium via the delivery of secreted chemical substances (effectors) to host cells, which modulate host genes expression and phytohormone regulation patterns. Genes encoding the Nictaba-related lectin domain have been found among the plant genes with downregulated expression during the development of syncytia induced by H. schachtii in Arabidopsis thaliana roots. To investigate the role of two selected Nictaba-related genes in the plant response to beet cyst nematode parasitism, mutants and plants overexpressing AtPP2-A3 or AtPP2-A8 were infected, and promoter activity and protein localization were analyzed. In wild-type plants, AtPP2-A3 and AtPP2-A8 were expressed only in roots, especially in the cortex and rhizodermis. After nematode infection, their expression was switched off in regions surrounding a developing syncytium. Astonishingly, plants overexpressing AtPP2-A3 or AtPP2-A8 were more susceptible to nematode infection than wild-type plants, whereas mutants were less susceptible. Based on these results and changes in AtPP2-A3 and AtPP2-A8 expression patterns after treatments with different stress phytohormones, we postulate that the AtPP2-A3 and AtPP2-A8 genes play important roles in the defense response to beet cyst nematode infection.

Keywords: Agglutinin; Beet cyst nematode; Phloem protein (P-protein); Plant-parasitic nematode; Plant–nematode interaction; Resistance; Susceptibility; Syncytium.

Fig. 1

Relative expression levels of AtPP2-A3 and AtPP2-A8 genes in syncytia induced by H. schachtii in wild-type A. thaliana. a Relative transcript levels of AtPP2-A3 in roots containing syncytia at 5 and 15 dpi. b Relative transcript levels of AtPP2-A8 in roots containing syncytia at 5 and 15 dpi. Bars show mean values ± SD. Asterisks indicate means ± SD (n = 3), which are significantly different at *P < 0.05 according to one-way ANOVA and post hoc Fisher’s least significant difference (LSD)

Fig. 2

Relative expression levels of AtPP2-A3 (a) and AtPP2-A8 (b) genes in uninfected roots of wild-type A. thaliana treated with jasmonic acid (JA), (–)-methyl jasmonate (MeJA), salicylic acid (SA), or abscisic acid (ABA) (24 h treatment with 100 µM concentration each). Bars show mean values ± SD (n = 3). Different letters on bars indicate statistically significant differences according to one-way ANOVA (P < 0.05) and post hoc Fisher’s least significant difference (LSD)

Fig. 3

H. schachtii development test comparing the susceptibility of wild-type, mutant, and AtPP2-overexpressing A. thaliana lines at 5 and 15 dpi. a Average numbers of sedentary second-stage juveniles developed on mutant roots at 5 dpi. b Average numbers of females developed on mutant roots at 15 dpi. c Average numbers of males developed on mutant roots at 15 dpi. d Average numbers of sedentary second-stage juveniles developed on overexpressing roots at 5 dpi. e Average numbers of females developed on overexpressing roots at 15 dpi. f Average numbers of males developed on overexpressing roots at 15 dpi. Bars show means ± SD (n > 30). Different letters on bars indicate statistically significant differences according to one-way ANOVA (P < 0.05) and post hoc Fisher’s least significant difference (LSD)

Fig. 4

Microscopic analyses of AtPP2-A3 and AtPP2-A8 expression in Arabidopsis plants. Stereo-light microscope (a–l) and confocal laser scanning (m–t) images of transgenic plants expressing the GUS reporter gene (bluish coloration) (a–l) or GFP reporter gene (greenish coloration) (m-t) under control of the AtPP2-A3 (a, b, e, f, i, j, m, n, q and r) or AtPP2-A8 (c, d, g, h, k, l, o, p, s and t) promoter. a and c Expression in 12-day-old seedlings. b and d Expression in inflorescence and siliques. e, g and m–p Expression in uninfected roots. f, i–l and q–t Expression in roots and around syncytia induced by beet cyst nematode. (f and h–l 15 dpi; r-t 5 dpi). F flower, N nematode, Nu nucleus, RT root tip, S syncytium, Si silique. Scale bars: 1 mm (a–d); 250 µm (e–h, o); 100 µm (i–n, p, q, s); 20 µm (r, t)