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. 2006 Sep;142(1):6-20.
doi: 10.1104/pp.106.084517. Epub 2006 Jul 21.

RNA interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat

Silvia Travella  1 , Theres E Klimm, Beat Keller
Affiliations

RNA interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat

Silvia Travella et al. Plant Physiol. 2006 Sep.

Abstract

Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function. In contrast to gain- or loss-of-function approaches, RNA interference (RNAi)-induced gene silencing can possibly silence multigene families and homoeologous genes in polyploids. This is of great importance for functional studies in hexaploid wheat (Triticum aestivum), where most of the genes are present in at least three homoeologous copies and conventional insertional mutagenesis is not effective. We have introduced into bread wheat double-stranded RNA-expressing constructs containing fragments of genes encoding Phytoene Desaturase (PDS) or the signal transducer of ethylene, Ethylene Insensitive 2 (EIN2). Transformed plants showed phenotypic changes that were stably inherited over at least two generations. These changes were very similar to mutant phenotypes of the two genes in diploid model plants. Quantitative real-time polymerase chain reaction revealed a good correlation between decreasing mRNA levels and increasingly severe phenotypes. RNAi silencing had the same quantitative effect on all three homoeologous genes. The most severe phenotypes were observed in homozygous plants that showed the strongest mRNA reduction and, interestingly, produced around 2-fold the amount of small RNAs compared to heterozygous plants. This suggests that the effect of RNAi in hexaploid wheat is gene-dosage dependent. Wheat seedlings with low mRNA levels for EIN2 were ethylene insensitive. Thus, EIN2 is a positive regulator of the ethylene-signaling pathway in wheat, very similar to its homologs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Our data show that RNAi results in stably inherited phenotypes and therefore represents an efficient tool for functional genomic studies in polyploid wheat.

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Figures

Figure 1.
Figure 1.
Location of the wheat ESTs used to construct the RNAi vectors in their corresponding wheat consensus sequence and in the corresponding full-length cDNAs identified in rice. A, Wheat EST BG908924 aligned with its consensus sequence TC236658 and the full-length cDNA of PDS of rice (OsPDS; AF049356). B, Wheat EST AL816731 aligned with its consensus sequence TC257467 and the full-length cDNA of EIN2 of rice (OsEIN2; AY396568). Base pairs, percentage of identity, and overlapping areas (shaded boxes) are indicated (not on scale). C, Self-complementary hp construct derived from hp transgene used in the bombardment experiments. Gene-specific sequences (black arrows indicating the orientation) in the antisense and sense orientations were cloned with a 548-bp fragment of the TAK14 (AF325198) wheat intron (white box) and were controlled by the constitutive ubi promoter (hatched box) and the nopaline synthase terminator (dotted box). Restriction enzymes HindIII (H) and EcoRI (E) used for Southern-blot hybridization analysis are indicated.
Figure 2.
Figure 2.
An RNAi construct expressing a wPDS cDNA fragment induces photobleaching in hexaploid wheat. The PDS-RNAi transgenic T0 lines were arranged based on the severity of photobleaching. A, Strong photobleached phenotype resulting in albino plants and lethality. B, Intermediate phenotype with patterns of streaks where photobleaching is affecting one-half of the leaf surface. C, Intermediate phenotype with patterns of streaks where photobleaching is affecting the central part of the leaves with the margins still green. D, Weak phenotype where only a small sector of the leaves is affected by photobleaching.
Figure 3.
Figure 3.
RNAi-mediated specific silencing of the wPDS genes in hexaploid wheat. A, Quantitative real-time PCR of eight wPDS-RNAi transgenic T0 primary transformants with primers designed within the conserved nt sequence region identified on wheat EST CK163183 and with primers specific to each of the homoeologous wPDS genes (see Supplemental Fig. 1). Relative mRNA levels of wPDS were normalized to the mRNA level of wild-type (Wt) plants (=1). The GAPDH gene was used as an internal standard. Data are the average of triplicate samples (±sd). The numbering of the transgenic lines analyzed by quantitative real-time PCR refers to the Southern-blot data shown in Supplemental Figure 4, and the Southern analysis is summarized at the bottom of A. Transgenic (+) and non transgenic (−) T0 lines. In A, the photobleached phenotype is indicated. A, Albino; S, streaks; and –, wild-type phenotype. B, C, D, and E, T1 generation analysis of the T0 PDS-RNAi transgenic line 5. B, Detection of the hp transgene by PCR. The top band corresponds to the transgene and the bottom band to the wheat homolog of the barley Mlo gene used here as an internal control. pPDS, hp construct used in the transformation experiments. C, Relative mRNA levels of wPDS, which were normalized to the mRNA level of wild-type (Wt) plants (=1). Line T0 from which this population is derived is shown. Plants 7, 11, and 15 were not analyzed, because they did not produce enough leaf material for RNA extraction. Data are the average of triplicate samples (±sd). D, Phenotypes. S, Streaks; P, photobleaching; B, bleaching only at the base of the leaf; n.a., not analyzed; and –, wild type. E, Detection of small RNAs in T1 heterozygous plants (plants 3, 5, and 17 from C) and T1 homozygous plants (plants 4, 8, 10, and 20 from C) of the T0 PDS-RNAi transgenic line 5. Low Mr RNA fractions were hybridized with a mixture of 11 DNA oligos complementary to the sequence of interest (Supplemental Fig. 6A). The 20-nt, 26-nt, and 29-nt DNA oligos were used as size controls (size indicated in nts); Wt, wild type. The same blot was hybridized with the housekeeping GAPDH gene as a control (top segment). The relative intensity of the hybridization signals in the transgenics versus wild-type plants was determined with a phosphoimager (Cyclone gene array system, Perkin-Elmer). The relative mean value of wPDS small RNAs per plant ± sd is indicated by the thick arrowhead.
Figure 4.
Figure 4.
RNAi-mediated specific silencing of the wEIN2 genes in hexaploid wheat. A, Quantitative real-time PCR of eight wEIN2-RNAi transgenic T0 primary transformants with primers designed within the conserved nt sequence region identified on wheat EST CD925940 and with primers specific to each of the homoeologous wEIN2 genes (see Supplemental Fig. 3). Relative mRNA levels of wEIN2 were normalized to the mRNA level of wild-type (Wt) plants (=1). The GAPDH gene was used as an internal standard. Data are the average of triplicate samples (±sd). The numbering of the transgenic lines analyzed by quantitative real-time PCR refers to the Southern-blot data shown in Supplemental Figure 4, and the Southern analysis is summarized at the bottom of A. +, Transgenic; –, nontransgenic T0 lines. B, C, and D, Analysis of the ethylene response in the T1 generation of the T0 EIN2-RNAi transgenic line 10. B, Wild-type seeds germinated in presence and in absence of ACC. C, T1 seeds germinated in presence of ACC. Bars in B and C = 4 cm. D, Relative mRNA levels of wEIN2 that were normalized to the mRNA level of wild-type (Wt) plants (=1). Line T0 from which this population is derived is also shown. Length measurements of each T1 plant are shown on the same diagram. PCR analysis for the presence/absence of the transgene is shown in D.
Figure 5.
Figure 5.
T2 generation analysis of the homozygous T1 EIN2-RNAi transgenic plant 1 generated from the T0 line 10. A, Detection of the hp transgene by PCR; the top band corresponds to the transgene and the bottom band to the wheat homolog of the barley Mlo gene used here as an internal control. pEIN2, hp construct used in the transformation experiments. B, Relative mRNA levels of wEIN2 that were normalized to the mRNA level of wild-type (Wt) plants (=1). The T1 plant from which this population is derived is also shown. Length measurements of each T2 plants are shown on the same diagram. C, Phenotype of the T2 plants which are all ethylene insensitive. Bar in C = 7 cm.
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