The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34 . Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1 . Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1 . Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins.

We used conserved domains in the major class (nucleotide binding site plus leucine-rich repeat) of dicot resistance (R) genes to isolate related gene fragments via PCR from the monocot species rice and barley. Peptide sequence comparison of dicot R genes and monocot R-like genes revealed shared motifs but provided no evidence for a monocotspecific signature. Mapping of these genes in rice and barley showed linkage to genetically characterized R genes and revealed the existence of mixed clusters, each harboring at least two highly dissimilar R-like genes. Diversity was detected intraspecifically with wide variation in copy number between varieties of a particular species. Interspecific analyses of R-like genes frequently revealed nonsyntenic map locations between the cereal species rice, barley, and foxtail millet although tight collinear gene order is a hallmark of monocot genomes. Our data suggest a dramatic rearrangement of R gene loci between related species and implies a different mechanism for nucleotide binding site plus leucine-rich repeat gene evolution compared with the rest of the monocot genome.Plant resistance to particular pathogens involves specific recognition events (1). These resistance reactions are racespecific and triggered by corresponding resistance (R) genes in the host and avirulence (Avr) genes in the pathogen. Resistance mechanisms operate in both major classes of flowering plants, dicots and monocots. Several dicot and one monocot R gene to diverse pathogens have been isolated, revealing structural similarities of the deduced proteins (1).One class comprises genes containing both a 5Ј terminal nucleotide binding site (NBS) and 3Ј terminal leucine-rich repeats (LRRs) of various length. The NBS-LRR type includes Rps2, Rpm1, N, L6, M, Rpp5, Prf,

The NBS-LRR (nucleotide-binding site plus leucine-rich repeat) genes represent the major class of disease resistance genes in flowering plants and comprise 166 genes in the ecotype Col-0 of Arabidopsis thaliana. NBS-LRR genes are organized in single-gene loci, clusters, and superclusters. Phylogenetic analysis reveals nine monophyletic clades and a few phylogenetic orphans. Most clusters contain only genes from the same phylogenetic lineage, reflecting their origin from the exchange of sequence blocks as a result of intralocus recombination. Multiple duplications increased the number of NBS-LRR genes in the progenitors of Arabidopsis, suggesting that the present complexity in Col-0 may derive from as few as 17 progenitors. The combination of physical and phylogenetic analyses of the NBS-LRR genes makes it possible to detect relatively recent gene rearrangements, which increased the number of NBS-LRR genes by about 50, but which are almost never associated with large segmental duplications. The identification of 10 heterogeneous clusters containing members from different clades demonstrates that sequence sampling between different resistance gene loci and clades has occurred. Such events may have taken place early during flowering plant evolution, but they generated modules that have been duplicated and remobilized also more recently.

Race-specific resistance in barley to the powdery mildew fungus (Erysiphe graminis f sp hordei) is associated with a cell death reaction (hypersensitive response [HR]). Genetically, it is dependent on dominant resistance genes (Mlx), and in most cases, it is also dependent on Rar1 and Rar2. Non-race-specific resistance to the fungus, which is due to the lack of the Mlo wild-type allele, is dependent on Ror1 and Ror2 and is not associated with an HR in the region of pathogen attack. However, the absence of the Mlo wild-type allele stimulates a spontaneous cell death response in foliar tissue. This response is also controlled by Ror1 and Ror2, as indicated by trypan blue staining patterns. Lack of Mlo enhances transcript accumulation of pathogenesis-related genes upon fungal challenge, and this response is diminished by mutations in Ror genes. Using DNA marker-assisted selection of genotypes, we provide evidence, via gene interaction studies, that Ror1 and Ror2 are not essential components of race-specific resistance and do not compromise hypersensitive cell death. Reciprocal experiments show that neither is Rar1 a component of mlo-controlled resistance nor does it affect spontaneous cell death. We show that mlo- and Ror-dependent resistance is active when challenged with E. g. f sp tritici, a nonhost pathogen of barley. Our observations suggest separate genetic pathways operating in race-specific and non-race-specific resistance; they indicate also a separate genetic control of hypersensitive and spontaneous cell death in foliar tissue.