Simple Bioassay for PAMP-Triggered Immunity in Rice Seedlings Based on Lateral Root Growth Inhibition

doi:10.1016/j.rsci.2021.12.006

Abstract

Abstract:

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is an essential layer of plant disease resistance. Robust bioassays for PTI are pre-required to dissect its molecular mechanism. In this study, we established that lateral root growth inhibition as a simple and robust measurement of PTI in rice seedlings. Specifically, flg22, a well-characterized PAMP from bacterial flagellin, was used to induce PTI in rice seedlings. While flg22 treatment induced PR gene expression and mitogen-activated protein kinase activation in the roots of rice seedlings to support the PTI triggered, this treatment substantially repressed lateral root growth, but it did not alter primary root growth. Moreover, treatments with chitin (i.e., a fungal PAMP) and oligogalacturonides (i.e., classical damage-associated molecular pattern) clearly inhibited the lateral root growth, although a priming step involving ulvan was required for the chitin treatment. The bioassay developed was applicable to various rice cultivars and wild species. Thus, lateral root growth inhibition represents a simple and reliable assay for studying PTI in rice plants.

Key words: pathogen-associated molecular pattern (PAMP), PAMP-triggered immunity, lateral root growth, mitogen-activated protein kinase, rice, flg22

Wang Rui, Zhang Dandan, Li Shengnan, Gao Jinlan, Han Liebao, Qiu Jinlong. Simple Bioassay for PAMP-Triggered Immunity in Rice Seedlings Based on Lateral Root Growth Inhibition[J]. Rice Science, 2022, 29(1): 67-75.

Figures/Tables 5

Fig. 1. Pathogen-associated molecular pattern (PAMP) flg22 inhibits lateral root growth of rice seedlings. A, Primary root growth following flg22 treatment. Oryza sativa ssp. japonica cultivar Nipponbare seedlings with a primary root length of 1.5?2.0 cm were transferred to half-strength Murashige and Skoog (MS) medium supplied with 10 μmol/L flg22 or without (mock) and grown vertically for 3 d before the data were collected. Scale bars, 1 cm. B, Primary root length of rice seedlings treated without (mock) or with 10 μmol/L flg22. C, Lateral root growth of rice seedlings treated without (mock) or with 10 μmol/L flg22. The primary root (1.5-4.5 cm) used for analyzing lateral root length and density is outlined in red. Scale bars, 1 cm. D and E, Lateral root length (D) and density (E) of rice seedlings treated without (mock) or with 10 μmol/L flg22. F, Effect of flg22 on lateral root growth inhibition is dose dependence. Lateral root length of rice seedlings was measured 3 d after grown vertically on half-strength MS medium with indicated flg22 concentrations. G, Lateral root length of the osfls2 mutant seedlings treated without (mock) or with 10 μmol/L flg22. Bars indicate Mean ± SD of n > 8 seedlings per treatment. The significance of the differences was determined by a one-way ANOVA with the Tukey’s multiple comparisons test. Different lowercase letters above the bars indicate significant differences at P < 0.01.

Fig. 2. flg22-induced responses in lateral roots of rice seedlings. A, Activation of mitogen-activated protein kinases (MAPKs) upon the 5 μmol/L flg22 treatment. MAPK activity was analyzed by immunoblotting with Phospho-p44/42 MAPK (Erk1/2) antibody (top panel), and ACTIN was used as a protein loading control (bottom panel). The intensity of the osfls2 mutant before the flg22 treatment was identified as ‘1.0’. WT, Wild type. B?D, Defense gene expression induced upon the flg22 treatment. The relative expression levels of OsPAL1 (B), OsPBZ1 (C) and OsPR10 (D) in the roots of rice seedlings treated with 5 μmol/L flg22 were determined by quantitative real-time PCR. OsActin2 was used as an internal reference. Data are presented as Mean ± SD of three biological replicates. Different lowercase letters above the bars indicate significant differences at P < 0.01.

Fig. 3. Oligogalacturonides (OGs) inhibit lateral root growth of rice seedlings. A , Primary root length of rice seedlings following the OG treatment. B, Lateral root length of rice seedlings after the OG treatment. C, Lateral root density of rice seedlings after the OG treatment. Bars indicate Mean ± SD of n > 8 seedlings per treatment. The significance of the differences was determined by a one-way ANOVA with the Tukey’s multiple comparisons test. Different lowercase letters above the bars indicate significant differences at P < 0.01.

Fig. 4. Ulvan priming of rice seedlings is required for inhibition of lateral root growth by chitin. A , Lateral root length of rice seedlings following the chitin treatment. B, Lateral root length of rice seedlings primed with ulvan and treated with chitin. C, Lateral root length of rice seedlings treated with indicated concentrations of chitin and primed with 100 μg/mL ulvan. D, Lateral root length of oscerk1 mutant seedlings treated without (mock) or with 0.5 mg/mL chitin and 100 μg/mL ulvan. Bars indicate Mean ± SD of n ≥ 4 seedlings per treatment. The significance of the differences was determined by a one-way ANOVA with the Tukey’s multiple comparison test. Different lowercase letters above the bars indicate significant differences at P < 0.01.

Fig. 5. Pathogen-associated molecular pattern (PAMP)-induced lateral root growth inhibition in various rice cultivars and wild species. A , Lateral root length of japonica rice Zhonghua 11 seedlings treated without (mock) or with 5 μmol/L flg22. B, Lateral root length of indica rice Huanghuazhan seedlings treated without (mock) or with 5 μmol/L flg22. C, Lateral root length of O. rufipogon Griff. seedlings treated without (mock) or with 5 μmol/L flg22. Bars indicate Mean ± SD of n > 8 seedlings per treatment. The significance of the differences was determined by a one-way ANOVA with the Tukey’s multiple comparison test. Different lowercase letters above the bars indicate significant differences at P < 0.01.

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