Antisense suppression of S-RNase expression in Nicotiana using RNA polymerase II- and III-transcribed gene constructs

Abstract

In the Solanaceae, self-incompatibility is controlled by a single, multi-allelic ('S') locus. One product of this locus is a ribonuclease, the S-RNase, which is expressed predominantly in mature pistils and has recently been shown to cause allele-specific pollen rejection in transgenic plants. Hybrid Nicotiana plumbaginifolia x N. alata plants were used to test the effects of antisense suppression of the SA2-RNase from N. alata using three different gene constructs: two driven by RNA polymerase II-transcribed promoters, and the third, containing a truncated soybean tRNA (met-i) gene, transcribed by RNA polymerase III. All three constructs caused suppression of S-RNase activity in the transgenic plants. Unexpectedly, the CaMV 35S promoter was more effective for antisense suppression than the tissue specific tomato ChiP promoter. Antisense suppression of S-RNase correlated with low sense SA2 transcript levels and high antisense SA2 transcript levels. Untransformed hybrids that contained the N. alata SA2 allele were incompatible with N. alata SA2 pollen, while transgenic plants with suppressed SA2 gene expression accepted the pollen. The utility of this hybrid plant system for studying some aspects of antisense gene suppression is discussed.

S RNase and Interspecific Pollen Rejection in the Genus Nicotiana: Multiple Pollen-Rejection Pathways Contribute to Unilateral Incompatibility between Self-Incompatible and Self-Compatible Species.

“…As shown in Figure 4A, plants that express S A2 RNase at moderate levels reject St& pollen in some but not all crosses. The data in Figure 4B confirm that, as expected, antisense inhibition of S^ RNase expression specifically suppresses the ability to reject S A2 pollen (see also Murfett et al, 1995). Similar results have been reported in Petunia inflata .…”

“…We have shown previously that S allele-specific pollen rejection in transgenic plants resembles the SI response in N. alata (Murfett et al, 1994(Murfett et al, , 1995. Figure 5 shows the morphology of N. plumbaginifolia pollen tubes in transformed and control (N. plumbaginifolia x N. alata) hybrids.…”

“…We used cloned S RNase genes from N. alata and plant transformation to address the question of whether S locus products can cause rejection of pollen from SC species. The S RNase expression system and the antisense constructs used to suppress SA2 RNase have been described previously (Murfett et al, 1994(Murfett et al, ,1995. The results in Figures 3 and 4 show that S RNase expression was altered in the transgenic plants.…”

“…A comparison was made between the efficiency of a tissue-specific promoter (i.e., the ChiP promoter in pHAKA) and a nonspecific promoter (i.e., the cauliflower mosaic virus 35S promoter in pBAGA) for antisense suppression of S A2 RNase expression. Plants carrying the pBAGA construct showed suppression of S A2 RNase expression most frequently, although plants containing pHAKA also showed suppression (Murfett et al, 1995).…”

“…A comparison was made between the efficiency of a tissue-specific promoter (i.e., the ChiP promoter in pHAKA) and a nonspecific promoter (i.e., the cauliflower mosaic virus 35S promoter in pBAGA) for antisense suppression of S A2 RNase expression. Plants carrying the pBAGA construct showed suppression of S A2 RNase expression most frequently, although plants containing pHAKA also showed suppression (Murfett et al, 1995).The most straightforward test of the hypothesis that S RNase causes rejection of pollen from SC species is to introduce a cloned gene directly into an SC genetic background and test for a new pollination phenotype. We employed two such SC genetic backgrounds.…”