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The early endocytic patch protein Sla2 is important for morphogenesis and growth rates inSaccharomyces cerevisiaeandCandida albicans,but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2or ScSLA2are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other components of early endocytic patches (Sla1 and Abp1) with those in strains lacking Sla2. Onlysla2strains had defects in actin cables, a known trigger of the morphogenesis checkpoint, yet all three strains exhibited Swe1-dependent phenotypes. Thus, Swe1 appears to monitor actin patch in addition to actin cable function. Furthermore, Swe1 contributed to virulence in a mouse model of disseminated candidiasis, implying a role for the morphogenesis checkpoint during the pathogenesis ofC. albicansinfections.

The classical genetic map of Arabidopsis contains 462 genes with mutant phenotypes. Chromosomal locations of these genes have been determined over the past 25 years based on recombination frequencies with visible and molecular markers. The most recent update of the classical map was published in a special genome issue ofScience that dealt with Arabidopsis (D.W. Meinke, J.M. Cherry, C. Dean, S.D. Rounsley, M. Koornneef [1998] Science 282: 662–682). We present here a comprehensive list and sequence-based map of 620 cloned genes with mutant phenotypes. This map documents for the first time the exact locations of large numbers of Arabidopsis genes that give a phenotype when disrupted by mutation. Such a community-based physical map should have broad applications in Arabidopsis research and should serve as a replacement for the classical genetic map in the future. Assembling a comprehensive list of genes with a loss-of-function phenotype will also focus attention on essential genes that are not functionally redundant and ultimately contribute to the identification of the minimal gene set required to make a flowering plant.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.

Tubular structures containing bacilliform virions were observed in cell-free extracts of Commelina diffusa infected with Commelina yellow mottle badnavirus (CoYMV). The exterior of the tubule reacted with antibodies to CoYMV movement protein, but not with antibodies to virus coat protein. Similar tubular structures containing bacilliform particles were also observed in ultrathin sections of CoYMV-infected C. diffusa. These tubular structures traversed the cell wall at points where this was thickened or protruded. No similar structures were observed in healthy C. diffusa. These observations support the hypothesis that the virion-containing tubular structures observed in cell-free extracts are the same as those observed in situ, that these structures are composed, at least in part, of virus movement protein, and that they play a role in the cell-to-cell trafficking of virions of CoYMV.

Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity. The genomic region spanning nucleotides 5999-7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana. In A. sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive. In A. thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique. The transgene was inherited and active in progeny plants of both A. sativa and A. thaliana.

Commelinayellow mottle virus (CoYMV) is the type member of the badnaviruses, a genus of plant pararetroviruses. The N-terminus of the polyprotein encoded by ORF III has limited similarity to known cell-to-cell movement proteins. To test the hypothesis that the N-terminus is required for viral movement, the phenotypes caused by mutations constructed in this region were determined. Similar to mutants affected in the reverse transcriptase, mutants affected in the putative movement protein were unable to cause a systemic infection. However, when the abilities of the mutated viral genomes to direct virion assembly and replication were tested using anin vitrostem-culture system, the mutants affected in the putative movement protein were found to assemble virions, whereas the reverse transcriptase mutants were unable to do so. Moreover, the putative movement protein mutants were shown to be replication competent by detection and mapping of one of the genomic discontinuities that are the hallmark of replication by reverse transcription. Thus the N-terminal region of ORF III is required for the systemic movement but not for the replication of CoYMV.

A long-term goal of Arabidopsis research is to define the minimal gene set needed to produce a viable plant with a normal phenotype under diverse conditions. This will require both forward and reverse genetics along with novel strategies to characterize multigene families and redundant biochemical pathways. Here we describe an initial dataset of 250 EMB genes required for normal embryo development in Arabidopsis. This represents the first large-scale dataset of essential genes in a flowering plant. When compared with 550 genes with other knockout phenotypes, EMB genes are enriched for basal cellular functions, deficient in transcription factors and signaling components, have fewer paralogs, and are more likely to have counterparts among essential genes of yeast (Saccharomyces cerevisiae) and worm (Caenorhabditis elegans). EMB genes also represent a valuable source of plant-specific proteins with unknown functions required for growth and development. Analyzing such unknowns is a central objective of genomics efforts worldwide. We focus here on 34 confirmed EMB genes with unknown functions, demonstrate that expression of these genes is not embryo-specific, validate a strategy for identifying interacting proteins through complementation with epitope-tagged proteins, and discuss the value of EMB genes in identifying novel proteins associated with important plant processes. Based on sequence comparison with essential genes in other model eukaryotes, we identify 244 candidate EMB genes without paralogs that represent promising targets for reverse genetics. These candidates should facilitate the recovery of additional genes required for seed development.

The titan mutants of Arabidopsis exhibit striking defects in seed development. The defining feature is the presence of abnormal endosperm with giant polyploid nuclei. SeveralTTN genes encode structural maintenance of chromosome proteins (condensins and cohesins) required for chromosome function at mitosis. Another TTN gene product (TTN5) is related to the ARL2 class of GTP-binding proteins. Here, we identify four additional TTN genes and present a general model for the titan phenotype. TTN1 was cloned after two tagged alleles were identified through a large-scale screen of T-DNA insertion lines. The predicted gene product is related to tubulin-folding cofactor D, which interacts with ARL2 in fission yeast (Schizosaccharomyces pombe) and humans to regulate tubulin dynamics. We propose that TTN5 and TTN1 function in a similar manner to regulate microtubule function in seed development. The titan phenotype can therefore result from disruption of chromosome dynamics (ttn3, ttn7, andttn8) or microtubule function (ttn1 andttn5). Three other genes have been identified that affect endosperm nuclear morphology. TTN4 andTTN9 appear to encode plant-specific proteins of unknown function. TTN6 is related to the isopeptidase T class of deubiquitinating enzymes that recycle polyubiquitin chains following protein degradation. Disruption of this gene may reduce the stability of the structural maintenance of chromosome complex. Further analysis of the TITAN network should help to elucidate the regulation of microtubule function and chromosome dynamics in seed development.

The titan (ttn) mutants of Arabidopsis exhibit striking alterations in chromosome dynamics and cell division during seed development. Endosperm defects include aberrant mitoses and giant polyploid nuclei. Mutant embryos differ in cell size, morphology and viability, depending on the locus involved. Here we demonstrate that three TTN genes encode chromosome scaffold proteins of the condensin (SMC2) and cohesin (SMC1 and SMC3) classes. These proteins have been studied extensively in yeast and animal systems, where they modulate chromosome condensation, chromatid separation, and dosage compensation. Arabidopsis contains single copies of SMC1 and SMC3 cohesins. We used forward genetics to identify duplicate T-DNA insertions in each gene. These mutants (ttn7 and ttn8) have similar titan phenotypes: giant endosperm nuclei and arrested embryos with a few small cells. A single SMC2 knockout (ttn3) was identified and confirmed by molecular complementation. The weak embryo phenotype observed in this mutant may result from expression of a related gene (AtSMC2) with overlapping functions. Further analysis of titan mutants and the SMC gene family in Arabidopsis should provide clues to chromosome mechanics in plants and insights into the regulation of nuclear activity during endosperm development.