No abstract

Multi-color BiFC is a useful technique to determine interactions simultaneously between a given" bait" protein and multiple "prey" proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

SummaryPlant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

Successful transformation of plants by Agrobacterium tumefaciens requires that the bacterial T-complex actively escorts T-DNA into the host's nucleus. VirD2 and VirE2 are virulence proteins on the T-complex that have plant-functional nuclear localization signal sequences that may recruit importin a proteins of the plant for nuclear import. In this study, we evaluated the involvement of seven of the nine members of the Arabidopsis thaliana importin a family in Agrobacterium transformation. Yeast two-hybrid, plant bimolecular fluorescence complementation, and in vitro protein-protein interaction assays demonstrated that all tested Arabidopsis importin a members can interact with VirD2 and VirE2. However, only disruption of the importin IMPa-4 inhibited transformation and produced the rat (resistant to Agrobacterium transformation) phenotype. Overexpression of six importin a members, including IMPa-4, rescued the rat phenotype in the impa-4 mutant background. Roots of wild-type and impa-4 Arabidopsis plants expressing yellow fluorescent protein-VirD2 displayed nuclear localization of the fusion protein, indicating that nuclear import of VirD2 is not affected in the impa-4 mutant. Somewhat surprisingly, VirE2-yellow fluorescent protein mainly localized to the cytoplasm of both wild-type and impa-4 Arabidopsis cells and to the cytoplasm of wild-type tobacco (Nicotiana tabacum) cells. However, bimolecular fluorescence complementation assays indicated that VirE2 could localize to the nucleus when IMPa-4, but not when IMPa-1, was overexpressed.

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants. The Arabidopsis rat1 mutant, previously characterized as resistant to Agrobacterium tumefaciens root transformation, is due to a mutation in the gene for the Lys-rich AGP, AtAGP17. We show that the phenotype of rat1 correlates with down-regulation of AGP17 in the root as a result of a T-DNA insertion into the promoter of AGP17. Complementation of rat1 plants by a floral dip method with either the wild-type AGP17 gene or cDNA can restore the plant to a wild-type phenotype in several independent transformants. Based on changes in PR1 gene expression and a decrease in free salicylic acid levels upon Agrobacterium infection, we suggest mechanisms by which AGP17 allows Agrobacterium rapidly to reduce the systemic acquired resistance response during the infection process.Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants. They are found in the extracellular matrix associated with the plasma membrane and cell wall (Knox, 1995;Du et al., 1996). Although the precise function(s) that AGPs perform is unknown, they have been implicated in diverse developmental roles, including differentiation, cell-cell recognition, and embryogenesis (Knox, 1996;Schultz et al., 1998;Majewski-Sawka and Nothnagel, 2000).Most studies investigating AGP expression and function have used an AGP-binding dye, b-D-glucosyl (b-D-Glc) Yariv reagent, and/or antibodies that recognize the carbohydrate epitopes of AGPs (for review, see Gaspar et al., 2001). These previous studies support a role for AGPs in plant cell growth and development; however, they do not inform us of the function of individual AGPs. The identification of AGP genes from Arabidopsis provides us with a wide range of tools to determine the function(s) of individual AGPs.To date, almost 50 genes encoding putative AGP protein backbones (hereafter referred to as AGP genes) have been identified in Arabidopsis (Schultz et al., 2002). These include the classical AGPs, those with Lys-rich domains, the arabinogalactan (AG)-peptides with short protein backbones, and the fasciclin-like AGPs ). Fasciclin-like AGPs are a class of chimeric AGPs that, in addition to AGP motifs, have fasciclin-like domains Johnson et al., 2003a). In addition, another approximately 50 glycosylphosphatidylinositol (GPI)-anchored proteins are likely to contain AG chains as part of larger proteins based on the presence of short Pro-, Ser-, Thr-, and Ala-rich regions containing noncontiguous Pro residues (Borner et al., 2002. Pro-, Ser-, Thr-, and Ala-rich regions contain noncontiguous Pro residues are referred to as AG-glycomodules because there is increasing evidence that these motifs direct the O-glycosylation of Hyp with type II AG chains (Goodrum et al., 2000;Zhao et al., 2002;Tan et al., 2003).Only a few AGP mutants have been identified to date. The haploinsufficient mutant, rat1 (resistant to Agrobacterium transformation), is resistant to transient and stable transforma...