Two decades after the first report of the plant homolog of the Receptor for Activated C Kinase 1 (RACK1) in cultured tobacco BY2 cells, a significant advancement has been made in the elucidation of its cellular and molecular role. The protein is now implicated in many biological functions including protein translation, multiple hormonal responses, developmental processes, pathogen infection resistance, environmental stress responses, and miRNA production. Such multiple functional roles are consistent with the scaffolding nature of the plant RACK1 protein. A significant advance was achieved when the β-propeller structure of the Arabidopsis RACK1A isoform was elucidated, thus revealing that its conserved seven WD repeats also assembled into this typical topology. From its crystal structure, it became apparent that it shares the structural platform for the interaction with ligands identified in other systems such as mammals. Although RACK1 proteins maintain conserved Protein Kinase C binding sites, the lack of a bona fide PKC adds complexity and enigma to the nature of the ligand partners with which RACK1 interacts in plants. Nevertheless, ligands recently identified using the split-ubiquitin based and conventional yeast two-hybrid assays, have revealed that plant RACK1 is involved in several processes that include defense response, drought and salt stress, ribosomal function, cell wall biogenesis, and photosynthesis. The information acquired indicates that, in spite of the high degree of conservation of its structure, the functions of the plant RACK1 homolog appear to be distinct and diverse from those in yeast, mammals, insects, etc. In this review, we take a critical look at the novel information regarding the many functions in which plant RACK1 has been reported to participate, with a special emphasis on the information on its currently identified and missing ligand partners.

Pro®lin in Phaseolus vulgaris is encoded by two genes (only one expressed in root nodules) but multiple isoforms are generated in vivo by phosphorylation on tyrosine residues Gabriel Guille  n, Võ Âctor Valde  s-Lo  pez, Rau  l Noguez, Juan Olivares, Luis Carlos Rodrõ Âguez-Zapata, He  ctor Pe  rez ³ , Luis Vidali ² , Marco A. Villanueva and Federico Sa  nchez* Plant Molecular Biology Department, Institute of Biotechnology UNAM, PO Box 510±3, Cuernavaca, Morelos 62250, Mexico Summary Actin-binding proteins such as pro®lins participate in the restructuration of the actin cytoskeleton in plant cells. Pro®lins are ubiquitous actin-, polyproline-, and inositol phospholipid-binding proteins, which in plants are encoded by multigene families. By 2D-PAGE and immunoblotting, we detected as much as ®ve pro®lin isoforms in crude extracts from nodules of Phaseolus vulgaris. However, by immunoprecipitation and gel electrophoresis of in vitro translation products from nodule RNA, only the most basic isoform of those found in nodule extracts, was detected. Furthermore, a bean pro®lin cDNA probe hybridised to genomic DNA digested with different restriction enzymes, showed either a single or two bands. These data indicate that pro®lin in P. vulgaris is encoded by only two genes. In root nodules only one gene is expressed, and a single pro®lin transcript gives rise to multiple pro®lin isoforms by post-translational modi®cations of the protein. By in vivo 32 P-labelling and immunoprecipitation with both, antipro®lin and antiphosphotyrosine-speci®c antibodies, we found that pro®lin is phosphorylated on tyrosine residues. Since chemical (TLC) and immunological analyses, as well as plant tyrosine phosphatase (AtPTP1) treatments of pro®lin indicated that tyrosine residues were phosphorylated, we concluded that tyrosine kinases must exist in plants. This ®nding will focus research on tyrosine kinases/tyrosine phosphatases that could participate in novel regulatory functions/ pathways, involving not only this multifunctional cytoskeletal protein, but other plant proteins.

Plant-targeted pCB302 plasmids containing sequences encoding gfp fusions with a microtubule-binding domain; gfp with the fimbrin actin-binding domain 2; and gfp with AtRACK1C from Arabidopsis thaliana, all harbored in Agrobacterium tumefaciens, were used to assay heterologous expression on three different clades of the photosynthetic dinoflagellate, Symbiodinium. Accessibility to the resistant cell wall and through the plasma membrane of these dinoflagellates was gained after brief but vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection of the cells expressing the hybrid proteins, which showed a characteristic green fluorescence, although they appeared to lose their photosynthetic pigments and did not further divide. Cell GFP expression frequency measured as green fluorescence emission yielded 839 per every 106 cells for Symbiodinium kawagutii, followed by 640 and 460 per every 106 cells for Symbiodinium microadriaticum and Symbiodinium sp. Mf11, respectively. Genomic PCR with specific primers amplified the AtRACK1C and gfp sequences after selection in all clades, thus revealing their presence in the cells. RT-PCR from RNA of S. kawagutii co-incubated with A. tumefaciens harboring each of the three vectors with their respective constructs, amplified products corresponding to the heterologous gfp sequence while no products were obtained from three distinct negative controls. The reported procedure shows that mild abrasion followed by co-incubation with A. tumefaciens harboring heterologous plasmids with CaMV35S and nos promoters can lead to expression of the encoded proteins into the Symbiodinium cells in culture. Despite the obvious drawbacks of the procedure, this is an important first step towards a stable transformation of Symbiodinium.

Reproducible and reliable genetic transformation methods are a key tool for understanding the physiology and cell biology of Symbiodinium. Nevertheless, transformation methods previously applied to cells such as microalgae, including those utilizing glass beads, have not been tested on these microorganisms. Here, we report a simple, transient transformation method, which allowed plasmid incorporation into three distinct clades of cultured Symbiodinium cells with the plant-targeted plasmid pCB302 harboring sequences encoding a fusion of green fluorescent protein (gfp) with RACK1C from Arabidopsis thaliana (AtRACK1C). Accessibility of the plasmid to the resistant cell wall and through the plasma membrane of the dinoflagellates was achieved through vigorous shaking in the presence of glass beads and polyethylene glycol. A resistance gene to the herbicide Basta allowed appropriate selection in the photosynthetic cells. The transformation frequency per every 10 6 cells was 107 ± 7 transformants for Symbiodinium kawagutii, 74 ± 8 for Symbiodinium microadriaticum ssp. microadriaticum, and 65 ± 5 for Symbiodinium sp. Mf11. Moreover, Symbiodinium pulchrorum cultures were successfully transformed with a different vector (pCAMBIA-FABD2-gfp) under the same conditions, further validating our procedure. The observation of green fluorescent emission from the cell cytoplasm in all performed transformations indicated that the procedure allowed the heterologous plasmids to enter and undergo expression in the Symbiodinium cells. The success of this transient transformation method opens interesting possibilities for functional genomics studies in Symbiodinium spp.

Receptor for activated C kinase (RACK1) is a highly conserved, eukaryotic protein of the WD-40 repeat family. Its peculiar β-propeller structure allows its interaction with multiple proteins in various plant signal-transduction pathways, including those arising from hormone responses, development, and environmental stress. During Phaseolus vulgaris root development, RACK1 (PvRACK1) mRNA expression was induced by auxins, abscissic acid, cytokinin, and gibberellic acid. In addition, during P. vulgaris nodule development, PvRACK1 mRNA was highly accumulated at 12 to 15 days postinoculation, suggesting an important role after nodule meristem initiation and Rhizobium nodule infection. PvRACK1 transcript accumulation was downregulated by a specific RNA interference construct which was expressed in transgenic roots of composite plants of P. vulgaris inoculated with Rhizobium tropici. PvRACK1 downregulated transcript levels were monitored by quantitative reverse-transcription polymerase chain reaction analysis in individual transgenic roots and nodules. We observed a clear phenotype in PvRACK1-knockdown nodules, in which nodule number and nodule cell expansion were impaired, resulting in altered nodule size. Microscopic analysis indicated that, in PvRACK1-knockdown nodules, infected and uninfected cells were considerably smaller (80 and 60%, respectively) than in control nodules. In addition, noninfected cells and symbiosomes in silenced nodules showed significant defects in membrane structure under electron microscopy analysis. These findings indicate that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development.