Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene

Abstract

Secreted parasitism proteins encoded by parasitism genes expressed in esophageal gland cells mediate infection and parasitism of plants by root-knot nematodes (RKN). Parasitism gene 16D10 encodes a conserved RKN secretory peptide that stimulates root growth and functions as a ligand for a putative plant transcription factor. We used in vitro and in vivo RNA interference approaches to silence this parasitism gene in RKN and validate that the parasitism gene has an essential function in RKN parasitism of plants. Ingestion of 16D10 dsRNA in vitro silenced the target parasitism gene in RKN and resulted in reduced nematode infectivity. In vivo expression of 16D10 dsRNA in Arabidopsis resulted in resistance effective against the four major RKN species. Because no known natural resistance gene has this wide effective range of RKN resistance, bioengineering crops expressing dsRNA that silence target RKN parasitism genes to disrupt the parasitic process represents a viable and flexible means of developing novel durable RKN-resistant crops and could provide crops with unprecedented broad resistance to RKN.

Fig. 1.

RNAi silencing of 16D10 in preparasitic M. incognita J2. (A) Fluorescence microscopy showing ingestion of FITC in the treated J2. (Scale bar, 10 μm.) (B) Real-time RT-PCR analysis of 16D10 transcript abundance in the FITC-labeled, transgenic J2 after soaking with short or full-length dsRNA molecules (16D10i-1 RNA or 16D10i-2 RNA) of 16D10 and Res. Controls are J2 soaked in H₂O only, dsRNA without Res (bars labeled 16D10i-1 RNA and 16D10i-2 RNA), and Res without dsRNA (bar labeled Res), respectively. 2^−ΔΔCt represents the amount of 16D10 that is normalized to an endogenous reference (actin) and relative to a calibrator (16D10) from the adult female stage, which has the lowest expression level of 16D10. ΔΔC_t = (ΔC_t-16D10 − ΔC_t-16D10adult); ΔC_t-16D10 = (C_t-16D10 − C_t-actin); ΔC_t-16D10adult = (C_t-16D10adult − C_t-actin). Each bar value represents the mean ± SD of triplicate experiments (Student's t test; ∗, P < 0.001 versus controls). (C) ELISA analysis of 16D10 protein in the treated J2 using the purified 16D10 peptide antiserum (11). Ten micrograms of total extracts of the treated J2 is used in each bar. Each bar value represents the mean ± SD of triplicate experiments (Student's t test; ∗∗, P < 0.01 versus controls). (D) Wild-type Arabidopsis roots inoculated with control J2 (Upper) or full-length 16D10 dsRNA treated J2 (Lower) showing numerous larger galls (Upper) or fewer small galls (Lower) 7 weeks after inoculation, respectively. RKN infection sites are indicated by arrows. (Scale bars, 10 mm.) (E) Reproduction (eggs per gram root) of each of treated M. incognita on wild-type Arabidopsis roots. Each bar value represents the mean ± SD of n = 36 (Student's t test; ∗∗∗, P < 0.01 versus controls).

Fig. 2.

Overexpression of 16D10 dsRNA in Arabidopsis. (A) RNA blots for the expression of 16D10 dsRNA in two 16D10 dsRNA transgenic homozygous T₂ lines (16D10i-1 and 16D10i-2) and the absence of 16D10 dsRNA expression in one vector-transformed homozygous T₂ line (Vector). R, root; S, stem; L, leaf. An ≈21-nt 16D10 siRNA was detected in the two 16D10 dsRNA transgenic lines. Arrowhead indicates the position of a 25-base DNA oligonucleotide. Ethidium bromide-stained gel (before transfer) in the zone corresponding to 5S RNA and tRNA is shown at the bottom. (B) RNAi inhibition of M. incognita infection of A. thaliana. Control vector-transformed line with numerous galls (Upper) and 16D10 dsRNA transgenic line (16D10i-1) showing no galls (Lower) 8 weeks after inoculation. (Scale bars, 10 mm.) (C) Reproduction (eggs per gram root) of four Meloidogyne species (Mi, M. incognita; Mj, M. javanica; Ma, M. arenaria; Mh, M. hapla) on transgenic A. thaliana expressing 16D10 dsRNA is significantly decreased compared with control plants. Each bar value represents the mean ± SD of n = 24–48 (Student's t test, P < 0.001 versus control).

Fig. 3.

RNAi silencing of 16D10 in Arabidopsis. (A) RNA-blot analysis of silencing of 16D10 mRNA in A. thaliana hybrid line. The maternal plant (M, the 16D10 transgenic homozygous T₂ line L17) expressing 16D10 mRNA and the paternal plant (P, the 16D10 dsRNA transgenic homozygous T₂ line 16D10i-1) expressing 16D10 dsRNA (siRNA) are shown, but the expression of 16D10 mRNA in the hybrid line (F₁) is silenced. R, root; S, stem; L, leaf. Arrowhead indicates the position of a 25-nt DNA oligonucleotide. Ethidium bromide staining in the zone corresponding to 5S RNA and tRNA is shown. (B) Seedlings of the 16D10 hybrid line (Right), the 16D10 transgenic homozygous T₂ line L17 (Left), and the 16D10 dsRNA transgenic homozygous T₂ line 16D10i-1 (Center) 12 days after germination. (Scale bar, 10 mm.) (C) Primary root length 12 days after germination. Each bar value represents the mean ± SD of n = 30 (Student's t test, P < 0.01 versus the maternal plant; ∗, P > 0.1).