Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.
After a cell wall protein fraction (CWP) of Pythium oligandrum was sprayed on sugar beet leaves, we screened leaves for induced expression of defence-related genes and for resistance against Cercospora leaf spot. In a western blot analysis, the CWP was primarily retained on the surface of leaves without degradation for at least 48 h after spraying. In northern blot analyses, four defencerelated genes (b-1, 3-glucanase, acidic class III chitinase, 5-enol-pyruvylshikimate-phosphate synthase and oxalate oxidase-like germin) were expressed more rapidly in CWPtreated leaves compared to control leaves treated with distilled water (DW). When CWP was applied to a suspension of cultured cells of sugar beet, an oxidative burst was observed that did not occur after the DW treatment. In growth chamber trials after inoculation with Cercospora beticola, the severity of Cercospora leaf spot was significantly reduced in CWP-treated plants compared to the DW-treated controls. In a field experiment, CWP treatment was also effective against the disease. CWP did not reduce growth rate of the pathogen in plate tests. The results together suggest that the CWP from P. oligandrum can be retained on the leaf surface and induce expression of disease resistance genes, thereby reducing Cercospora leaf spot on sugar beet.
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