Overexpression of GmAKT2 potassium channel enhances resistance to soybean mosaic virus

Abstract

Background: Soybean mosaic virus (SMV) is the most prevalent viral disease in many soybean production areas. Due to a large number of SMV resistant loci and alleles, SMV strains and the rapid evolution in avirulence/effector genes, traditional breeding for SMV resistance is complex. Genetic engineering is an effective alternative method for improving SMV resistance in soybean. Potassium (K+) is the most abundant inorganic solute in plant cells, and is involved in plant responses to abiotic and biotic stresses. Studies have shown that altering the level of K+ status can reduce the spread of the viral diseases. Thus K+ transporters are putative candidates to target for soybean virus resistance.

Results: The addition of K+ fertilizer significantly reduced SMV incidence. Analysis of K+ channel gene expression indicated that GmAKT2, the ortholog of Arabidopsis K+ weak channel encoding gene AKT2, was significantly induced by SMV inoculation in the SMV highly-resistant genotype Rsmv1, but not in the susceptible genotype Ssmv1. Transgenic soybean plants overexpressing GmAKT2 were produced and verified by Southern blot and RT-PCR analysis. Analysis of K+ concentrations on different leaves of both the transgenic and the wildtype (Williams 82) plants revealed that overexpression of GmAKT2 significantly increased K+ concentrations in young leaves of plants. In contrast, K+ concentrations in the old leaves of the GmAKT2-Oe plants were significantly lower than those in WT plants. These results indicated that GmAKT2 acted as a K+ transporter and affected the distribution of K+ in soybean plants. Starting from 14 days after inoculation (DAI) of SMV G7, severe mosaic symptoms were observed on the WT leaves. In contrast, the GmAKT2-Oe plants showed no symptom of SMV infection. At 14 and 28 DAI, the amount of SMV RNA in WT plants increased 200- and 260- fold relative to GmAKT2-Oe plants at each time point. Thus, SMV development was significantly retarded in GmAKT2-overexpressing transgenic soybean plants.

Conclusions: Overexpression of GmAKT2 significantly enhanced SMV resistance in transgenic soybean. Thus, alteration of K+ transporter expression is a novel molecular approach for enhancing SMV resistance in soybean.

Figure 1

SMV resistance and K⁺ concentrations of Williams 82 soybean plants grown under different K supplies. (A) SMV symptoms at 14 or 28 days after inoculation (DAI) with SMV in soybean plants grown in soil pots with low (36.5 mg/kg) or high (200 mg/kg) levels of K⁺. Unrolled unifoliate leaves of 10-day-old soybean plants were mechanically inoculated with SMV strain G7 or buffer (Mock). Photos were taken on the newest leaf of the plants, which were 2^nd trifoliate leaf at 14 DAI and 7^th trifoliate leaf at 28 DAI, respectively. (B) Amount of SMV RNA detected by quantitative RT-PCR (qRT-PCR). The middle leaflets of the leaves of Williams 82 plants which grown in low or high K soil were sampled at 14 and 28 DAI to extract total RNA for qRT-PCR analysis of SMV. Transcript levels were calculated using the formula 2^-ΔCt for the expression levels relative to GmACTIN. (C) K⁺ concentrations in individual leaves of plants grown in either high- or low-K soil. The first through seventh trifoliate leaves from 45-day-old soybean plants were sampled. Data represent means of three biological replicates with error bars indicating SD. Asterisks indicate a significant difference between high- and low-K soil (*P < 0.05).

Figure 2

SMV resistance and GmAKT2 expression in three soybean genotypes. (A) Symptoms of three soybean genotypes at 14 and 28 DAI. Rsmv1, SMV highly-resistant; Williams 82, susceptible; Ssmv1, highly-susceptible. Ten-day-old soybean plants in low-K soil with unrolled unifoliate leaves were mechanically inoculated with SMV G7 or buffer (Mock). (B) Amount of SMV RNA detected by qRT-PCR. Leaves of three soybean genotypes were sampled at 14 and 28 DAI to extract total RNA for qRT-PCR analysis. Transcript levels were calculated using the formula 2^-ΔCt for the expression levels relative to GmACTIN. Data represent means of three biological replicates with error bars indicating SD. (C) Relative GmAKT2 expression in three soybean genotypes at 28 DAI. Leaves of three soybean genotypes were sampled at 28 DAI to extract total RNA for qRT-PCR analysis. Transcript levels were calculated using the formula 2^-ΔΔCt for the expression levels relative to GmACTIN. Data represent means of three biological replicates with error bars indicating SD.

Figure 3

GmAKT2 expression in different tissues and leaves of Williams 82 under various nutrient supply conditions. (A) Relative GmAKT2 expression in roots (R), unifoliate leaves (UL), trifoliate leaves (TL), stems (S), flowers (F), and pods (P). Soybean seedlings were grown hydroponically in growth chambers for 6 weeks. (B) Relative GmAKT2 expression in leaves of plants grown under different nutrient stress. Ten-day-old soybean seedlings were transferred into modified half-strength Hoagland hydroponic solution (CK) or solutions lacking nitrogen (-N), phosphate (-P), or potassium (-K) for 7 days. Transcript levels were calculated using the formula 2^-ΔΔCt for the expression levels relative to GmACTIN. Data represent means of three biological replicates with error bars indicating SD. Asterisks indicate a significant difference between the control and treated samples (***P < 0.001).

Figure 4

Construction and verification of GmAKT2-overexpressing transgenic soybean plants. (A) T-DNA region of the GmAKT2 overexpression vector. LB, left border; RB, right border; bar, phosphinothricin acetyl transferase gene; P35S, CaMV double 35S promoter; T35S, CaMV 35S terminator; RFP, red fluorescence protein gene. (B) Southern blot analysis of GmAKT2-overexpressing transgenic lines. Oe1, Oe2, Oe3, and Oe4 represent four independent GmAKT2 overexpressing lines. Genomic DNA of 2-week-old T₁ transgenic seedlings and the non-transformed recipient soybean genotype Williams 82 was extracted and digested with HindIII. bar gene was digoxigenin labeled and used as the probe for analysis. (C) Relative GmAKT2 expression in the leaves of the transgenic lines. T₂ generations of Oe1, Oe2, Oe3, and Oe4 and WT were cultured in a hydroponic system. RNA was extracted from the leaves of 2-week-old seedlings. GmAKT2 transcript levels were determined by qRT-PCR. Data represent means of three biological replicates with error bars indicating SD. GmACTIN expression was used as the internal control.

Figure 5

Potassium concentrations in the leaves of WT and transgenic soybean plants grown in high- or low-K soil. WT and transgenic lines were germinated and grown in pots with soil containing high K (A, 200 mg/kg) or low K (B, 36.5 mg/kg) for 6 weeks. The first through seventh trifoliate leaves from each treatment were collected and K⁺ contents were measured. Data represent means of four biological replicates with error bars indicating SD. Asterisks indicate a significant difference between the WT and transgenic lines (*P < 0.05; **P < 0.01).

Figure 6

Overexpression of GmAKT2 enhanced SMV resistance. (A) SMV symptoms in the WT and GmAKT2 overexpression plants at 35 DAI. Ten-day-old soybean plants in low-K soil with unrolled unifoliate leaves were mechanically inoculated with SMV or buffer (Mock). (B) SMV symptoms on the newest trifoliate leaves of Williams 82 and GmAKT2-overexpressing transgenic lines Oe1 and Oe2 at 14, 28, and 35 DAI. (C) Amount of SMV RNA detected by qRT-PCR. The middle leaflets of the leaves of Williams 82, and GmAKT2-ovexpression lines Oe1 and Oe2 were sampled at 14 and 28 DAI to extract total RNA for quantitative RT-PCR analysis. Transcript levels were calculated using the formula 2^-ΔCt for the expression levels relative to GmACTIN. Data represent means of three biological replicates with error bars indicating SD.