Copyright 0 1995 by the Genetics Society of America Pollen Fertility Restoration by Nuclear Gene Fr in CMS Bean: Nuclear-Directed Alteration of a Mitochondrial Population Shichuan He, Anna Lyznik and Sally Mackenzie Department o f Agronomy, Purdue University, West Lafayette, Indiana 47907 Manuscript received June 21, 1994 Accepted for publication November 2, 1994 ABSTRACT Two nuclear genes, Fr and Fr2, have been identified that restore pollen fertility to cytoplasmic male sterile (CMS) common bean (Phmeolus vulgaris L.) by apparently distinct mechanisms. Whereas Fr2 appears to suppress the expression of a male sterility associated mitochondrial sequence (designated pus) , Fr restores pollen fertility by causing the elimination of this unusual mitochondrial DNA segment. To further investigate the mechanism of Fr action, Fr and Fr2 were cointroduced into the nucleus of a bean line containing the sterility inducing cytoplasm. When the effect of pus was suppressed by Fr2, the presence of Fr no longer directed the elimination of the mitochondrial pus sequence. This result suggests that the Fr function is dependent on proper expression of the pus sequence. To evaluate the temporal and spatial patterns of Fr action, we undertook a polymerase chain reaction-based approach to trace the fate of the pus sequence in different tissues of F2 and F3 fertile-restored plants derived from a genetic cross between a cytoplasmic male sterile line of common bean, CMSSprite (frfr) , and fertility restorer line R351 (FrFr) . We demonstrate that the Frdirected disappearance of pus sequence occurs during flower development. Elimination of the pus sequence from developing megaspores results in permanent fertility restoration in the following generations. Genetic analysis demonstrated that permanent fertility restoration, that is, the complete elimination of pus from reproductive tissues requires two doses of the Fr allele or the absence of fr in F2 individuals. The effect of Fr was reversible until full fertility was achieved. On the basis of these results, we propose a model for the mechanism of pus elimination by the Fr gene and discuss the dynamics of puscontaining mitochondrial transmission in the presence of the Fr gene. N UCLEAR genes play essential roles in mitochon- drial biogenesis and function (TZAGALOFF and MYERS 1986; A~TARDI and SCHATZ 1988). Although much progress has been made in understanding how nuclear and mitochondrial genes interact in yeast and mammals ( CHOMYN and ATTARDI 1992),little is known about the mechanism by which the nuclear genome regulates mitochondrial gene expression in higher plants. Because mitochondrial genes encode proteins that are required for essential cellular functions in every phase of plant growth and development, mutations in either the nuclear or mitochondrial genome that lead to mitochondrial dysfunction will likely be lethal. As a result, genetic approachesto investigating this complex intracellular communication are hindered due to the relatively small number of available mutants. Four promising plant nuclear-mitochondrial interaction sys- tems have been investigated at a molecular level. They are cytoplasmic male sterility (CMS) andnuclear fertil- ity restoration ( HANSON and CONDE 1985), the “teo- sinte cytoplasmic-associated miniature” ( tcm) resulting from nuclear-cytoplasmic incompatibility in maize (COOPER et al. 1990), nonchromosomal stripe muta- Corresponding authe-r: Sally A. Mackenzie, Department of Agronomy, Purdue University, West Lafayette, IN 47907-1150. Genetics 139: 955-962 (February, 1995) tions (NCS ) of maize (NEWTON 1988; NEWTON et al. 1990) and a leaf variegation phenotype associated with mitochondrial genome rearrangements due to a nu- clear mutation in Arabidopsis ( MARTINEZ-ZAPATER et al. 1992). CMS, a maternally inherited trait characterized by the inability of the plant to produce viable pollen, has been studied extensively. In all cases investigated to date, it is associated with mitochondrial mutations ( HANSON 1991 ) . These mutations often create novel open reading frames that are expressed. Unusual mito- chondrial sterility associated sequences apparently are derived by DNA recombinations, insertions, and re- arrangements ( DEWEY et al. 1986; YOUNG and HANSON 1987; SINGH and BROWN 1991; KOHLER et al. 1991; LA- VER et al. 1991). In most of these cases, nuclear genes have been identified that apparently suppress expres- sion of the mitochondrial sterility associated mutations. However, the mechanisms by which these nuclear re- storer genes act to alter mitochondrial gene expression remain to be defined. CMS common bean (Phuseolus vulgaris L.) provides a different genetic system for the study of plant nuclear- mitochondrial interaction. A 3.7-kb transcriptionally ac- tive CMSassociated sequence, designated pus, was iden- tified in the mitochondrial genome (CHASE and OR-