A novel, two‐component system for cell lethality and its use in engineering nuclear male‐sterility in plants

Abstract

SummaryAblation of cells by the controlled expression of a lethal gene can be used to engineer plant traits such as male sterility and disease resistance. However, it may not be possible to achieve suf®cient speci®city of expression to prevent secondary effects in non-targeted tissues. In this paper we demonstrate that the extracellular ribonuclease, barnase, can be engineered into two complementary fragments, allowing overlapping promoter speci®city to be used to enhance targeting speci®city. Using a transient system, we ®rst show that barnase can be split into two inactive peptide fragments, that when co-expressed can complement each other to reconstitute barnase activity. When a luciferase reporter gene was introduced into plant cells along with genes encoding both partial barnase peptides, a substantial reduction in luciferase activity was seen. Cytotoxicity of the reconstituted barnase was demonstrated by crossing together parents constitutively expressing each of the barnase fragments, then assaying their progeny for the presence of both partial barnase genes. None of over 300 tomato seeds planted resulted in a viable progeny that inherited both transgenes. When expression of the partial barnase genes was instead targeted to the tapetum, male sterility resulted. All 13 tomato progeny that inherited both transgenes were male sterile, whereas the three progeny inheriting only the N-terminal barnase gene were male fertile. Finally, we describe how male sterility generated by this type of two-component system can be used in hybrid seed production.

Two similar but distinct second intron fragments from tobacco AGAMOUS homologs confer identical floral organ-specific expression sufficient for generating complete sterility in plants

“…Another approach to address this leaky expression is to take advantage of the reconstitutable function of truncated, complementary Barnase peptide fragments. For example, when truncated Barnase gene fragments driven by diVerent promoters sharing overlapping Xoral-speciWc activity are simultaneously expressed in tomato plants, the truncated, non-functional Barnase peptides are biochemically reconstituted in Xoral tissues as functional Barnase proteins that quickly ablate their resident cells or tissues through the degradation of cellular RNAs (Burgess et al 2002). This is a sophisticated tactic, but whether it is feasible and practical for crop species remains to be demonstrated.…”

Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 Are Essential for Tapetum Development and Microspore Maturation

“…Anthers in Arabidopsis thaliana have a four-lobed structure, each containing a sporangium in which, early in development, two cell lines differentiate: (1) the germ line is a mass of cells that through sporogenesis and gametogenesis produces the male gametophytes and (2) the surrounding sporophytic tissues differentiate into four cell layers named from outside to inside, the epidermis, the endothecium, the middle cell layer, and the tapetum (Sanders et al, 1999;Ma, 2005). Several studies have shown that the surrounding four cell layers are crucial for the development of microsporocytes into mature pollen (Mariani et al, 1990;Burgess et al, 2002). Thus, mature anthers are highly organized structures.…”

A tapetal ablation transgene induces stable male sterility and slows field growth in Populus

“…These results suggest that barnase toxicity can vary widely depending on species and growth environment. It also supports the need for steps to reduce barnase toxicity due to misexpression, which could include the use of spacers (Jagannath et al 2001), separation of subunits among plants following crossing (Burgess et al 2002;Bihao et al 2012), or attenuated versions of the protein (Zhang et al 2012).…”