The soybean Rhg1 amino acid transporter gene alters glutamate homeostasis and jasmonic acid-induced resistance to soybean cyst nematode

Abstract

Rhg1 (resistance to Heterodera glycines 1) is an important locus that contributes to resistance against soybean cyst nematode (SCN; Heterodera glycines Ichinohe), which is the most economically damaging disease of soybean worldwide. Simultaneous overexpression of three genes encoding a predicted amino acid transporter, an α-soluble N-ethylmaleimide-sensitive factor attachment protein (α-SNAP) and a predicted wound-induced protein resulted in resistance to SCN provided by this locus. However, the roles of two of these genes (excluding α-SNAP) remain unknown. Here, we report the functional characterization of Glyma.18G022400, a gene at the Rhg1 locus that encodes the predicted amino acid transporter Rhg1-GmAAT. Although the direct role of Rhg1-GmAAT in glutamate transport was not demonstrated, multiple lines of evidence showed that Rhg1-GmAAT impacts glutamic acid tolerance and glutamate transportation in soybean. Transcriptomic and metabolite profiling indicated that overexpression of Rhg1-GmAAT activated the jasmonic acid (JA) pathway. Treatment with a JA biosynthesis inhibitor reduced the resistance provided by the Rhg1-containing PI88788 to SCN, which suggested that the JA pathway might play a role in Rhg1-mediated resistance to SCN. Our results could be helpful for the clarification of the mechanism of resistance to SCN provided by Rhg1 in soybean.

Keywords: Rhg1; amino acid transporter; glutamate; jasmonic acid; soybean; soybean cyst nematode.

Figure 1

Expression and subcellular localization of Rhg1‐GmAAT. (a) The expression patterns of Rhg1‐GmAAT were determined by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) and normalized to SKIP16. The values are the means ± standard deviations (SDs) (n = 3). (b–g) Histochemical analysis of β‐glucuronidase (GUS) activity from Arabidopsis plants expressing P_GmAAT‐GUS. GUS staining in a 2‐week‐old cotyledon (b), 2‐week‐old stem (c), 2‐week‐old root (d), leaf of a 35‐day‐old plant (e), flower of a 35‐day‐old plant (scale bar, 10 mm) (f) and silique of a 35‐day‐old plant (g). (h, i) Histochemical analysis of GUS activity in soybean hairy roots expressing P_GmAAT‐GUS. GUS staining in the stele (h) and within a root cross‐section (i). (j) Subcellular localization via a P_35S‐GmAAT‐GFP fusion protein in tobacco epidermal leaf cells. From left to right: 4,6‐diamidino‐2‐phenylindole (DAPI)‐stained nuclear DNA, green fluorescent protein (GFP) fluorescence, bright‐field and overlay panels. Nucleus (N) and plasma membrane (P) are indicated by white and yellow arrows, respectively. Unless otherwise specified, scale bar = 200 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Growth of plants subjected to toxic levels of glutamic acid (Glu) or the toxic Glu analogue N‐methyl sulfoximine (MSX). (a) Col‐0 and Rhg1‐GmAAT‐OX (Rhg1‐GmAAT‐overexpressing) Arabidopsis lines were grown for 3 weeks on half‐strength Murashige and Skoog medium containing 75 mm Glu. (b) The wild‐type (cultivar Tianlong 1) and Rhg1‐GmAAT‐OX soybean lines were grown for 3 days in quarter‐strength Murashige and Skoog liquid medium containing 50 mm Glu. (c, d) Two soybean near‐isogenic lines (NIL‐S and NIL‐R) were grown for 3 days in quarter‐strength Murashige and Skoog liquid medium containing either 50 mm Glu (c) or 10 μm MSX (d). Fresh weights were measured. The values are the means ± standard deviations (SDs) (n = 4). *0.01 < P < 0.05, **P < 0.01 (multiple t‐test followed by the Holm–Sidak post hoc test). The values above the columns provide the percentage of inhibition compared with that of untreated plants. WT, wild‐type.

Figure 3

Expression of the glutamate receptor‐like genes and glutamine synthetase genes in wild‐type and Rhg1‐GmAAT‐overexpressing (Rhg1‐GmAAT‐OX) lines in Arabidopsis and soybean. (a) AtGLR2.7 and AtGSR1 expressed in the roots of 75 mm glutamic acid (Glu) ‐treated Col‐0 and Rhg1‐GmAAT‐OX line at‐3. The expression levels were normalized to ATACT2. (b) Glyma.06G233600 and Glyma.07G104500 expressed in the roots of 50 mm Glu‐treated wild‐type (cultivar Tianlong 1) and Rhg1‐GmAAT‐OX line gm‐3. The expression levels were normalized to SKIP16. The values are the means ± standard deviations (SDs) (n = 3). Asterisks indicate a statistically significant difference of Rhg1‐GmAAT‐OX lines compared with the wild‐type under the same conditions. *0.01 < P < 0.05, **P < 0.01; multiple t‐test followed by the Holm–Sidak post hoc test. WT, wild‐type.

Figure 4

Amino acid contents in the leaf phloem exudates, root xylem exudates, leaves and roots of the wild‐type (cultivar Tianlong 1) and Rhg1‐GmAAT‐overexpressing (Rhg1‐GmAAT‐OX) line gm‐3. (a) Amino acid analysis of the phloem exudates of leaves. (b) Amino acid analysis of the sap flow of stems (n = 4). (c) Free amino acid contents of 21‐day‐old roots. (d) Free glutamic acid (Glu) contents of 21‐day‐old leaves. Leaf measurements were calculated using the dry weight (DW) in (a) and (d). Root measurements were calculated using the DW in (c). Four plants constituted a sample; three samples per line were used for (c) and (d). The values are the means ± standard deviations (SDs) (n = 3). *0.01 < P < 0.05, **P < 0.01 (multiple t‐test followed by the Holm–Sidak post hoc test). The experiments were repeated two or three times, each producing similar results. WT, wild‐type; OX, Rhg1‐GmAAT‐OX line gm‐3.

Figure 5

Expression of a set of jasmonic acid (JA) biosynthesis and JA‐responsive genes. (a) JA biosynthesis genes expressed in the roots of glutamic acid (Glu) ‐treated soybean. Roots were treated with quarter‐strength Murashige and Skoog medium that either lacked nitrogen (control) or was supplemented with 5 mm Glu for 24 h. JA biosynthesis genes (b) and JA‐responsive genes (c) expressed in the roots of the wild‐type (cultivar Tianlong 1) and Rhg1‐GmAAT‐overexpressing (Rhg1‐GmAAT‐OX) line gm‐3. (d) JA biosynthesis genes expressed in the roots of Williams 82 and PI88788. The expression levels of the selected genes were assayed by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) and were normalized to SKIP16. The values are the means ± standard deviations (SDs) (n = 3). **0.01 < P < 0.05, **P < 0.01 (multiple t‐test followed by the Holm–Sidak post hoc test). WT, wild‐type; OX, Rhg1‐GmAAT‐OX line gm‐3.

Figure 6

Jasmonic acid (JA) content in the roots of the wild‐type (cultivar Tianlong 1) and Rhg1‐GmAAT‐overexpressing (Rhg1‐GmAAT‐OX) line gm‐3. Seedlings were cultured in quarter‐strength Murashige and Skoog medium for 4 weeks. Endogenous JA and jasmonoyl isoleucine (JA‐Ile) of roots were quantified using high‐performance liquid chromatography coupled to tandem mass spectrometry. The values are the means ± standard deviations (SDs) (n = 3). **0.01 < P < 0.05, **P < 0.01 (multiple t‐test followed by the Holm–Sidak post hoc test). WT, wild‐type; OX, Rhg1‐GmAAT‐OX line gm‐3.

Figure 7

Responses of n‐propyl gallate (nPG)‐treated PI88788 and Rhg1‐GmAAT‐overexpressing (Rhg1‐GmAAT‐OX) line to soybean cyst nematodes (SCNs). (a) PI88788 and Hutcheson soybean seedlings were pretreated with either water containing 0.02% ethanol (mock) or a 100 μm nPG solution for 3 days prior to inoculation with nematodes. The values are the means ± standard deviations (SDs) (n = 6). Asterisks indicate a statistically significant difference of nPG‐treated PI88788 and mock‐treated Hutcheson compared with mock‐treated PI88788. *0.01 < P < 0.05, **P < 0.01 (multiple t‐test followed by the Holm–Sidak post hoc test). (b) The wild‐type (cultivar Tianlong 1), Rhg1‐GmAAT‐OX line gm‐3 and Hutcheson soybean seedlings were inoculated with nematodes. The values are the means ± SDs (n = 6). Female cysts were quantified after 30 days. The experiments were repeated at least three times, each producing similar results. WT, wild‐type; OX, Rhg1‐GmAAT‐OX line gm‐3.