Self-recognition between pollen and stigma during pollination in Brassica olracea is genetically controlled by the multiallelic self-incompatibility locus (S). We describe the S receptor kinase (SRK) gene, a previously uncaracterize gene that resides at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threoninespecific protein kinase activity. Its extracellular domain exhibits king homology to the secreted product of the S-locus glycoprotein (SLG) gene and is connected via a single pass trausmembrane domain to a protein kina catalytic center.SRK alleles derived from different S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLW. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.Pollination and the subsequent invasive growth of pollen tubes into the female stigmatic and pistil tissues prior to fertilization provide an opportunity to study cell-cell interactions in flowering plants. In crucifers such as Brassica oleracea, self-recognition between pollen and stigma is controlled by the multiallelic self-incompatibility, or S, locus (1). In general, pollen germination and/or tube growth are arrested at the stigma surface if the pollen and stigma are borne by plants having identical S-locus genotypes. This arrest prevents self-fertilization and is termed the self-incompatibility (SI) response. Two related genes have been identified at the S locus by molecular methods (2-4). Of these, only one gene, the S-locus glycoprotein (SLG) gene has been characterized extensively. SLG encodes a secreted glycoprotein that is highly polymorphic in different S-locus genotypes (2) and may therefore be involved in determining the recognition specificity displayed in SI. Furthermore, SLG is expressed in stigmatic papillae (3) and anthers (5, 6), consistent with models for SI in which both pollen and stigma bear recognition determinants derived from the S locus.In this study, we show that the second S-locus-linked gene (4) encodes a putative receptor protein kinase, and we have therefore designated it SRK, for S receptor kinase.t Its structure is similar to that predicted in a recently described maize root cDNA clone, ZmPKJ (7), and is analogous to the growth factor receptor tyrosine kinases in animals. The putative ligand-binding domain is homologous to SLG and displays genotype-specific sequence polymorphisms that parallel those of SLG. SRK transcripts were detected only in the male and female reproductive organs, thus showing a pattern of expression similar to that of SLG. These findings offer foundation to the hypothesis that SI is mediated by receptor-ligand interactions between pollen and pistil and provide a ...

Plant cells respond to low concentrations of auxin by cell expansion, and at a slightly higher concentration, these cells divide. Previous work revealed that null mutants of the ␣ -subunit of a putative heterotrimeric G protein ( GPA1 ) have reduced cell division. Here, we show that this prototypical G protein complex acts mechanistically by controlling auxin sensitivity toward cell division. Loss-of-function G protein mutants have altered auxin-mediated cell division throughout development, especially during the auxin-induced formation of lateral and adventitious root primordia. Ectopic expression of the wild-type G ␣ -subunit phenocopies the G ␤ mutants (auxin hypersensitivity), probably by sequestering the G ␤␥ -subunits, whereas overexpression of G ␤ reduces auxin sensitivity and a constitutively active (Q222L) mutant G ␣ behaves like the wild type. These data are consistent with a model in which G ␤␥ acts as a negative regulator of auxin-induced cell division. Accordingly, basal repression of approximately one-third of the identified auxin-regulated genes (47 of 150 upregulated genes among 8300 quantitated) is lost in the G ␤ transcript-null mutant. Included among these are genes that encode proteins proposed to control cell division in root primordia formation as well as several novel genes. These results suggest that although auxin-regulated cell division is not coupled directly by a G protein, the G ␤ -subunit attenuates this auxin pathway upstream of the control of mRNA steady state levels.

With the completion of the Arabidopsis genome sequencing project, the next major challenge is the large-scale determination of gene function. As a model organism for agricultural biotechnology, Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production. In an attempt to aid in the rapid discovery of gene function, we have established a high throughput phenotypic analysis process based on a series of defined growth stages that serve both as developmental landmarks and as triggers for the collection of morphological data. The data collection process has been divided into two complementary platforms to ensure the capture of detailed data describing Arabidopsis growth and development over the entire life of the plant. The first platform characterizes early seedling growth on vertical plates for a period of 2 weeks. The second platform consists of an extensive set of measurements from plants grown on soil for a period of approximately 2 months. When combined with parallel processes for metabolic and gene expression profiling, these platforms constitute a core technology in the high throughput determination of gene function. We present here analyses of the development of wild-type Columbia (Col-0) plants and selected mutants to illustrate a framework methodology that can be used to identify and interpret phenotypic differences in plants resulting from genetic variation and/or environmental stress.

Disease resistance in plants is often controlled by a gene-for-gene mechanism in which avirulence (avr) gene products encoded by pathogens are specifically recognized, either directly or indirectly, by plant disease resistance (R) gene products. Members of the NBS-LRR class of R genes encode proteins containing a putative nucleotide binding site (NBS) and carboxyl-terminal leucine-rich repeats (LRRs). Generally, NBS-LRR proteins do not contain predicted transmembrane segments or signal peptides, suggesting they are soluble cytoplasmic proteins. RPM1 is an NBS-LRR protein from Arabidopsis thaliana that confers resistance to Pseudomonas syringae expressing either avrRpm1 or avrB. RPM1 protein was localized by using an epitope tag. In contrast to previous suggestions, RPM1 is a peripheral membrane protein that likely resides on the cytoplasmic face of the plasma membrane. Furthermore, RPM1 is degraded coincident with the onset of the hypersensitive response, suggesting a negative feedback loop controlling the extent of cell death and overall resistance response at the site of infection.Disease resistance in plants often hinges on the ability of the host to recognize specific avirulence (avr) determinants presented by invading microorganisms. This recognition event is controlled by plant disease resistance (R) genes, which are proposed to act as receptors for specific avr-encoded determinants (1). Avr recognition by the plant initiates an elaborate defense response including changes in membrane ion flux, the production of extracellular reactive oxygen intermediates, irreversible plasma membrane (PM) damage, and changes in gene expression and metabolite production (2). This set of responses typically includes localized cell death at the site of pathogen infection, termed the hypersensitive response (HR). In the absence of either R or avr gene, no recognition occurs and the pathogen is able to colonize the host and cause disease.R genes can be classified by the putative motifs contained in the proteins they encode (1). The largest class encodes a centrally located nucleotide binding site (NBS) and a carboxylterminal block of leucine-rich repeats (LRRs). NBS-LRR genes conferring resistance to a number of bacterial, fungal, and viral pathogens have been cloned from a variety of plant species, suggesting that the general NBS-LRR structure is well adapted to recognize a wide range of signals. Subclassification of NBS-LRR sequences is based on the N-terminal domain, which contains either a leucine zipper (LZ) motif or homology with the cytoplasmic domains of the Toll and interleukin-1 receptors (TIR) domain. Computer analysis of predicted NBS-LRR proteins has failed to identify likely transmembrane segments. A potential exception was the prediction of a putative single-pass transmembrane domain in the RPS2 protein. However, site-directed alteration of this sequence to one not predicted to be membrane associated had no effect on RPS2 function (3). Furthermore, with the exception of the predicted flax L6 protein (4),...

With the completion of the Arabidopsis genome sequencing project, the next major challenge is the large-scale determination of gene function. As a model organism for agricultural biotechnology, Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production. In an attempt to aid in the rapid discovery of gene function, we have established a high throughput phenotypic analysis process based on a series of defined growth stages that serve both as developmental landmarks and as triggers for the collection of morphological data. The data collection process has been divided into two complementary platforms to ensure the capture of detailed data describing Arabidopsis growth and development over the entire life of the plant. The first platform characterizes early seedling growth on vertical plates for a period of 2 weeks. The second platform consists of an extensive set of measurements from plants grown on soil for a period of ‫ف‬ 2 months. When combined with parallel processes for metabolic and gene expression profiling, these platforms constitute a core technology in the high throughput determination of gene function. We present here analyses of the development of wild-type Columbia (Col-0) plants and selected mutants to illustrate a framework methodology that can be used to identify and interpret phenotypic differences in plants resulting from genetic variation and/or environmental stress.

Plants recognize many pathogens through the action of a diverse family of proteins called disease resistance (R) genes. The Arabidopsis R gene RPM1 encodes resistance to specific Pseudomonas syringae strains. We describe an RPM1-interacting protein that is an ortholog of TIP49a, previously shown to interact with the TATA binding protein (TBP) complex and to modulate c-myc- and beta-catenin-mediated signaling in animals. Reduction of Arabidopsis TIP49a (AtTIP49a) mRNA levels results in measurable increases of two R-dependent responses without constitutively activating defense responses, suggesting that AtTIP49a can act as a negative regulator of at least some R functions. Further, AtTIP49a is essential for both sporophyte and female gametophyte viability. Thus, regulators of R function overlap with essential modulators of plant development.

SummaryThe Arabidopsis RPM1 protein confers resistance to disease caused by Pseudomonas syringae strains delivering either the AvrRpm1 or AvrB type III effector proteins into host cells. We characterized two closely related RPM1-interacting proteins, RIN2 and RIN3. RIN2 and RIN3 encode RING-finger type ubiquitin ligases with six apparent transmembrane domains and an ubiquitin-binding CUE domain. RIN2 and RIN3 are orthologs of the mammalian autocrine motility factor receptor, a cytokine receptor localized in both plasma membrane caveolae and the endoplasmic reticulum. RIN2 is predominantly localized to the plasma membrane, as are RPM1 and RPS2. The C-terminal regions of RIN2 and RIN3, including the CUE domain, interact strongly with an RPM1 N-terminal fragment and weakly with a similar domain from the Arabidopsis RPS2 protein. RIN2 and RIN3 can dimerize through their C-terminal regions. The RING-finger domains of RIN2 and RIN3 encode ubiquitin ligases. Inoculation with P. syringae DC3000(avrRpm1) or P. syringae DC3000(avrRpt2) induces differential decreases of RIN2 mobility in SDS-PAGE and disappearance of the majority of RIN2. A rin2 rin3 double mutant expresses diminished RPM1-and RPS2-dependent hypersensitive response (HR), but no alteration of pathogen growth. Thus, the RIN2/RIN3 RING E3 ligases apparently act on a substrate that regulates RPM1-and RPS2-dependent HR.

In Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S2 and S6. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S6 genotype and 350 kb in the S2 genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.