Thiamine-induced priming against root-knot nematode infection in rice involves lignification and hydrogen peroxide generation

Abstract

Thiamine (vitamin B1, VB1) can act as a plant defence trigger, or priming agent, leading to a rapid counterattack on pathogen invasion. In this study, the priming effect of thiamine on rice (Oryza sativa cv. Nipponbare) and its activity against root-knot nematode (Meloidogyne graminicola) infection were evaluated. Thiamine treatment and subsequent nematode inoculation activated hydrogen peroxide (H2O2) accumulation and lignin deposition in plant roots, and this correlated with enhanced transcription of OsPAL1 and OsC4H, two genes involved in the phenylpropanoid pathway. The number of nematodes in rice roots was slightly but significantly reduced, and the development of the nematodes was delayed, whereas no direct toxic effects of VB1 on nematode viability and infectivity were observed. The combined application of thiamine with l-2-aminooxy-3-phenylpropionic acid (AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL), significantly hampered the VB1-priming capacity. These findings indicate that thiamine-induced priming in rice involves H2O2 and phenylpropanoid-mediated lignin production, which hampers nematode infection. Further cellular and molecular studies on the mechanism of thiamine-induced defence will be useful for the development of novel nematode control strategies.

Keywords: Meloidogyne graminicola; callose; hydrogen peroxide; induced defence; lignin; priming; thiamine.

Figure 1

Effect of thiamine soaking on nematode viability and infectivity. (a) The percentage of dead juveniles after 24 h and 72 h of incubation in various concentrations of thiamine and water (control). (b) Infectivity and development of thiamine‐incubated and water‐incubated M eloidogyne graminicola in rice roots. (c) thiamine‐incubated (VB1) and water‐incubated nematodes were inoculated on plants and photographed at 7 days pos‐inoculation (dpi) and (d) 14 dpi. The whole experiment was repeated three times, and there were six individual plants in each replicate. Data presented are the means ± standard error (SE) of six replicates.

Figure 2

Effect of 2.5 mM thiamine drenching on the plant and nematode infection. Number of nematodes (a) and root galls (b) in different developmental stages in thiamine‐drenched and non‐drenched Nipponbare roots were counted at 14 days post‐inoculation (dpi). (c) Roots were dyed with acid fuchsin and nematodes in roots were observed under a Leica stereomicroscope. Plant heigth (d) and fresh weigth (e) of the plants at 14 dpi. Mg, M eloidogyne graminicola. The bars in the different graphs represent the means ± SE of the data from three independent biological replicates, each containing six plants. Asterisks indicate significant differences (Duncan's multiple range test with P ≤ 0.05).

Figure 3

Effect of thiamine (VB1) on the behaviour of M eloidogyne graminicola and microscopic observation of giant cells. (a) Attraction of M . graminicola towards the rice root tip after drenching with 2.5 mm thiamine or water. (b) Nematodes within 5 mm from the root tip were counted at 9 h after inoculation. Data presented are the means ± standard error (SE) of six replicates. (c) Giant cells (*) in thiamine‐treated rice root galls and control root galls were stained with toluidine blue and observed under an Olympus BX 51 (Berchem, Belgium) microscope with a ColorView III camera at 7 dpi.

Figure 4

Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis of expression of the callose synthesis gene OsGSL 1 and microscopic observation of callose deposition. (a) The relative transcript levels of the callose synthesis gene (OsGSL 1) were analysed using qRT‐PCR at 6, 24 and 72 h post‐inoculation (hpi). Gene expression levels were normalized using three internal reference genes, OsEXP, OsEif5C and OsEXP narsai. Data are shown as relative transcript levels normalized to the control roots (expression level set at 1). The bars represent the mean expression levels ± standard error (SE) from two independent biological replicates and three technical replicates, each containing a pool of six plants. Asterisks indicate significant differential expression (Duncan's multiple range test with P ≤ 0.05). Mg, M eloidogyne graminicola. (b) Callose deposition in root galls at 7 days post‐inoculation (dpi) was examined under UV light using a Nikon (Amsterdam, The Netherlands) Eclipse Ti‐E epifluorescence microscope. (c) Quantification of callose deposition was performed using Image J software. Data presented are the means ± SE of two independent experiments, each performed using 10 galls.

Figure 5

Quantification of hydrogen peroxide (H₂O ₂) and relative expression analysis of the H₂O ₂ synthesis gene OsRbohB. (a) The H₂O ₂ content per gram of roots was measured using a CLARIOstar (BMG Labtech, Ortenberg, Germany) Microplate Reader at 390 nm. The bars represent the means ± standard error (SE) of four replicates, each containing a pool of six roots. Different letters indicate significant differences (Duncan's multiple range test with P ≤ 0.05). Mg, M eloidogyne graminicola. (b) The relative transcript levels of the H₂O ₂ synthesis gene (OsRbohB) were analysed using quantitative reverse transcription polymerase chain reaction (qRT‐PCR) at 6, 24 and 72 h post‐inoculation (hpi). Gene expression levels were normalized using three internal reference genes, i.e. OsEXP, OsEif5C and OsEXP narsai. Data are shown as relative transcript levels in comparison with the control roots (expression level set at 1). The bars represent the mean expression levels ± SE from two independent biological replicates and three technical replicates, each containing a pool of six plants. Asterisks indicate significant differential expression (P ≤ 0.05).

Figure 6

Quantitative reverse transcription polymerase chain reaction (RT‐PCR) analysis of the defence‐related genes in the phenylpropanoid pathway and the effect of an inhibitor of phenylpropanoid biosynthesis on nematode infection. (a) The relative expression levels of OsPAL 1, OsC4H, OsCAD 6 and OsWRKY 45, which are involved in lignin biosynthesis, were analysed using qRT‐PCR at 6, 24 and 72 h post‐inoculation (hpi). Gene expression levels were normalized using three internal reference genes, OsEXP, OsEif5C and OsEXP narsai. Data are shown as relative transcript levels in comparison with the control roots (expression level set at unity). The bars represent the mean expression levels ± standard error (SE) from two independent biological replicates and three technical replicates, each containing a pool of six plants. Asterisks indicate significant differential expression (P ≤ 0.05). VB1, thiamine drench alone; Mg, M eloidogyne graminicola inoculation alone; VB1 + Mg, thiamine drench plus M . graminicola inoculation; control, water drench and non‐inoculation. WRKY, a transcription factor with the conserved amino acid sequence WRKYGQK at its N‐terminal domain; PAL 1, phenylalanine ammonia‐lyase; C4H, cinnamate 4‐hydroxylase; CAD 6, cinnamyl alcohol dehydrogenase. (b) Effect of an inhibitor (l‐2‐aminooxy‐3‐phenylpropionic acid, AOPP) of phenylpropanoid biosynthesis on nematode infection. AOPP (100 μm) was combined in a drench with or without 2.5 mm thiamine on 2‐week‐old rice roots, 1 day before inoculation. Nematodes in the roots were counted at 14 days post‐inoculation (dpi). The bars represent the means of the data from two independent biological replicates, each containing six plants. Different letters indicate significant differences (Duncan's multiple range test with P ≤ 0.05). (c) The lignin content in the cell wall residue (CWR) of roots of rice was determined using the Acetyl Bromide (AcBr) assay. Measurements were conducted 1 day after thiamine treatment (VB1 + Nipponbare) or water treatment (Nipponbare). The bars represent the means ± SE of the lignin content of six plants. Different letters indicate significant differences.