Transcriptomic and histological responses of African rice (Oryza glaberrima) to Meloidogyne graminicola provide new insights into root-knot nematode resistance in monocots

Abstract

Background and aims: The root-knot nematode Meloidogyne graminicola is responsible for production losses in rice ( Oryza sativa ) in Asia and Latin America. The accession TOG5681 of African rice, O. glaberrima , presents improved resistance to several biotic and abiotic factors, including nematodes. The aim of this study was to assess the cytological and molecular mechanisms underlying nematode resistance in this accession.

Methods: Penetration and development in M. graminicola in TOG5681 and the susceptible O. sativa genotype 'Nipponbare' were compared by microscopic observation of infected roots and histological analysis of galls. In parallel, host molecular responses to M. graminicola were assessed by root transcriptome profiling at 2, 4 and 8 d post-infection (dpi). Specific treatments with hormone inhibitors were conducted in TOG5681 to assess the impact of the jasmonic acid and salicylic acid pathways on nematode penetration and reproduction.

Key results: Penetration and development of M. graminicola juveniles were reduced in the resistant TOG5681 in comparison with the susceptible accession, with degeneration of giant cells observed in the resistant genotype from 15 dpi onwards. Transcriptome changes were observed as early as 2 dpi, with genes predicted to be involved in defence responses, phenylpropanoid and hormone pathways strongly induced in TOG5681, in contrast to 'Nipponbare'. No specific hormonal pathway could be identified as the major determinant of resistance in the rice-nematode incompatible interaction. Candidate genes proposed as involved in resistance to M. graminicola in TOG5681 were identified based on their expression pattern and quantitative trait locus (QTL) position, including chalcone synthase, isoflavone reductase, phenylalanine ammonia lyase, WRKY62 transcription factor, thionin, stripe rust resistance protein, thaumatins and ATPase3.

Conclusions: This study provides a novel set of candidate genes for O. glaberrima resistance to nematodes and highlights the rice- M. graminicola pathosystem as a model to study plant-nematode incompatible interactions.

Keywords: Meloidogyne graminicola; Oryza glaberrima; Oryza sativa; QTL; RNA-Seq; differentially expressed genes; histology; hormones; monocots; resistance responses; root-knot nematode; transcriptomics.

Fig. 1. Meloidogyne graminicola penetration in rice roots (Oryza sativa ‘Nipponbare’ and Oryza glaberrima TOG5681) at 1 or 7 dpi. (A) Light microscope photographs of nematode juveniles after fuchsin staining of infested rice roots. Bar indicates 1 mm. (B) Mean nematode number in rice roots after plant inoculation with 400 second-stage juveniles (J2). Bars are the mean numbers (± s.d.) of nematodes per plant in three independent biological replicates, with 20 plants per condition (n = 5;3). Stars indicate statistical differences determined by one-way ANOVA (**P < 0·01; *P < 0·5, n = 5).

Fig. 2.

Histological analysis of galls induced by Meloidogyne graminicola in resistant rice (Oryza glaberrima TOG5681) roots. Gall cross-sections (10 µm) obtained at 11, 15 and 19 dpi were observed under UV light (middle panel) or were stained with toluidine blue and observed with white light (left and right panels). (A, B, C) Multiple nematode feeding on giant cells within the same gall. Note degraded nematode bodies and empty giant cells at 19 dpi (C). (D, E, F) Autofluorescence of sectioned galls. Note the absence of fluorescence accumulation around giant cells and nematodes. (G, H, I) Time course of giant cell degradation. (G) 11 dpi: dense cytoplasm and a large number of nuclei encircled by numerous neighbouring parenchymatic cells. (H) 15 dpi: vacuolated cytoplasm. (I) 19 dpi: degraded, devoid of cytoplasm and nuclei. Asterisk, giant cell; N, nematode.

Fig. 3.

Role of PAL activity and JA biosynthesis in the resistance response of Oryza glaberrima TOG5681. (A, B) Number of galls and nematodes in rice roots 14 d after infection with 250 J2 Meloidogyne graminicola. Effects of (A) PAL inhibitor l-2-aminooxy-3-phenylpropionic acid (AOPP, 100 µm) and (B) lipoxygenase inhibitor salicyl hydroxamic acid (SHAM, 200 µm) on plant susceptibility to nematode infection. Leaves of eight plants were sprayed with inhibitor or water (as control) and plants were inoculated with nematodes 24 h later. The experiment was repeated three times. (C) Effect of treatments on gene expression levels in rice roots 24 h after foliar treatment with the inhibitors. Gene expression levels were measured by RT-qPCR assays and normalized using three internal reference genes: OsEIF5C, OsREF3 and OsEXPNAR. Data shown are relative transcript levels compared with control roots of water-sprayed plants (expression level set at 1). Bars represent the mean expression level ± s.e. from two independent biological replicates and three technical replicates, each containing a pool of six plants. Asterisks indicate significantly different expression levels (*P < 0·1; **P ≤ 0·05).