Plasmodesmata play pivotal role in sucrose supply to Meloidogyne graminicola-caused giant cells in rice

Abstract

On infection, plant-parasitic nematodes establish feeding sites in roots from which they take up carbohydrates among other nutrients. Knowledge on how carbohydrates are supplied to the nematodes' feeding sites is limited. Here, gene expression analyses showed that RNA levels of OsSWEET11 to OsSWEET15 were extremely low in both Meloidogyne graminicola (Mg)-caused galls and noninoculated roots. All the rice sucrose transporter genes, OsSUT1 to OsSUT5, were either down-regulated in Mg-caused galls compared with noninoculated rice roots or had very low transcript abundance. OsSUT1 was the only gene up-regulated in galls, at 14 days postinoculation (dpi), after being highly down-regulated at 3 and 7 dpi. OsSUT4 was down-regulated at 3 dpi. No noticeable OsSUTs promoter activities were detected in Mg-caused galls of pOsSUT1 to -5::GUS rice lines. Loading experiments with carboxyfluorescein diacetate (CFDA) demonstrated that symplastic connections exist between phloem and Mg-caused giant cells (GCs). According to data from OsGNS5- and OsGSL2-overexpressing rice plants that had decreased and increased callose deposition, respectively, callose negatively affected Mg parasitism and sucrose supply to Mg-caused GCs. Our results suggest that plasmodesmata-mediated sucrose transport plays a pivotal role in sucrose supply from rice root phloem to Mg-caused GCs, and OsSWEET11 to -15 and OsSUTs are not major players in it, although further functional analysis is needed for OsSUT1 and OsSUT4.

Keywords: Meloidogyne graminicola; OsSUT s; callose deposition; giant cells; plasmodesmata; sucrose transport.

FIGURE 1

Expression patterns of OsSUT family genes in galls and giant cells (GCs) during Meloidogyne graminicola (Mg) parasitism. Using noninoculated rice roots as control, expression levels of OsSUT1 to ‐5 in Mg‐caused galls were analysed through quantitative reverse transcription PCR, where the expression levels of OsSUTs were analysed with the 2^{−∆∆ C t} method using both OseIF4α and OsUBQ5 as internal reference genes for normalization, and the calibrator was the transcript level of the corresponding OsSUT in control roots at each time point (a–d). The transcript abundance of OsSUT3 was too low to be detected. Expression levels of OsSUT1 to ‐5 in Mg‐caused galls were further validated by pOsSUTs::GUS lines, using a 35S promoter::GUS rice line as positive control (e). *** indicates significant differences (Student's t test, p < .001). Values are mean ± SE, n = 3. There were three independent biological replicates for each treatment. Bar = 500 μm

FIGURE 2

Loading experiments with carboxyfluorescein diacetate (CFDA). Phloem loading with CFDA or with sterile distilled water (mock) was performed on leaves of Meloidogyne graminicola (Mg)‐infected rice plants at parasitic J2s (par‐J2) and adult stages, respectively. Green fluorescent signals in par‐J2s (a) and adult females (b) isolated from galls, as well as their corresponding galls were tested 12 hr after phloem loading with CFDA. For each sample, there are bright field (a), fluorescent pictures (b), and their composite images (c). Arrows indicate galls. At least 15 galls from seven plants and nematodes isolated from the galls were observed for each experiment, and there were three independent biological replicates of the whole experiment

FIGURE 3

Assessment of root growth and callose deposition inside Meloidogyne graminicola (Mg)‐caused galls of OsGNS5‐ and OsGSL2‐overexpressing (OE) rice plants. Relative expression levels of OsGNS5 and OsGSL2 in OsGNS5‐ and OsGSL2‐overexpressing rice plants (OsGNS5‐OE and OsGSL2‐OE) were assessed by quantitative reverse transcription PCR (a). The longest length (b) and fresh weight (c) of roots from 15‐day‐old seedlings of OsGNS5‐ and OsGSL2‐overexpressing rice plants as well as the wild type (WT) were assessed. Callose depositions inside Mg‐caused galls of OsGNS5‐ and OsGSL2‐overexpressing rice plants as well as the WT were photographed under a stereomicroscope and quantified using ImageJ software (d) at 7 days postinoculation. Values are means ± SE. There were three independent biological replicates. The different uppercase letters (analysis of variance and Tukey's test) and *** indicate significant differences (Student's t test) at p < .001. ns, not significant

FIGURE 4

Investigation of Meloidogyne graminicola (Mg) parasitism on both OsGNS5‐ and OsGSL2‐overexpressing (OE) rice. Numbers of galls (a, b) and juveniles (c, d) in roots of OsGNS5‐ and OsGSL2‐overexpressing rice (OsGNS5‐OE and OsGSL2‐OE) and their wild types (WT) were recorded at 7 days postinoculation (dpi). In addition, giant cells (GCs) induced by Mg were observed by laser scanning confocal microscope at 7 dpi (e), and the largest size area of each GC was recorded using ImageJ software (f). Red and white dotted lines indicate GCs and juveniles, respectively. Values are means ± SE, n = 10. There were three independent biological replicates. The different lowercase letters (analysis of variance and Tukey's test), **, and *** (Student's t test) indicate significant differences at p < .05, p < .01, and p < .001, respectively. Bar = 200 μm

FIGURE 5

Gas chromatography‐mass spectrometry analysis of soluble sugars extracted from different rice root tissues. Sucrose contents in whole noninoculated rice roots and Meloidogyne graminicola (Mg)‐caused galls of wild type (WT), OsGNS5‐ and OsGSL2‐overexpressing (OE) rice lines were analysed at 7 days postinoculation. Bars with the same lowercase letter are not significantly different (analysis of variance and Tukey's test) at p < .05. Values are means ± SE. There were three independent biological replicates