The brassinosteroid insensitive1-like3 signalosome complex regulates Arabidopsis root development.

Brassinosteroid (BR) hormones are primarily perceived at the cell surface by the leucine-rich repeat receptor-like kinase brassinosteroid insensitive1 (BRI1). In Arabidopsis thaliana, BRI1 has two close homologs, BRI1-LIKE1 (BRL1) and BRL3, respectively, which are expressed in the vascular tissues and regulate shoot vascular development. Here, we identify novel components of the BRL3 receptor complex in planta by immunoprecipitation and mass spectrometry analysis. Whereas BRI1 associated kinase1 (BAK1) and several other known BRI1 interactors coimmunoprecipitated with BRL3, no evidence was found of a direct interaction between BRI1 and BRL3. In addition, we confirmed that BAK1 interacts with the BRL1 receptor by coimmunoprecipitation and fluorescence microscopy analysis. Importantly, genetic analysis of brl1 brl3 bak1-3 triple mutants revealed that BAK1, BRL1, and BRL3 signaling modulate root growth and development by contributing to the cellular activities of provascular and quiescent center cells. This provides functional relevance to the observed protein-protein interactions of the BRL3 signalosome. Overall, our study demonstrates that cell-specific BR receptor complexes can be assembled to perform different cellular activities during plant root growth, while highlighting that immunoprecipitation of leucine-rich repeat receptor kinases in plants is a powerful approach for unveiling signaling mechanisms with cellular resolution in plant development.

Peptide aptamers that bind to geminivirus replication proteins confer a resistance phenotype to tomato yellow leaf curl virus and tomato mottle virus infection in tomato.

Geminiviruses constitute a large family of single-stranded DNA viruses that cause serious losses in important crops worldwide. They often exist in disease complexes and have high recombination and mutation rates, allowing them to adapt rapidly to new hosts and environments. Thus, an effective resistance strategy must be general in character and able to target multiple viruses. The geminivirus replication protein (Rep) is a good target for broad-based disease control because it is highly conserved and required for viral replication. In an earlier study, we identified a set of peptide aptamers that bind to Rep and reduce viral replication in cultured plant cells. In this study, we selected 16 of the peptide aptamers for further analysis in yeast two-hybrid assays. The results of these experiments showed that all 16 peptide aptamers interact with all or most of the Rep proteins from nine viruses representing the three major Geminiviridae genera and identified two peptide aptamers (A22 and A64) that interact strongly with different regions in the Rep N terminus. Transgenic tomato lines expressing A22 or A64 and inoculated with Tomato yellow leaf curl virus or Tomato mottle virus exhibited delayed viral DNA accumulation and often contained lower levels of viral DNA. Strikingly, the effect on symptoms was stronger, with many of the plants showing no symptoms or strongly attenuated symptoms. Together, these results established the efficacy of using Rep-binding peptide aptamers to develop crops that are resistant to diverse geminiviruses.

Functional analysis of a novel motif conserved across geminivirus Rep proteins.

Members of the Geminiviridae have single-stranded DNA genomes that replicate in nuclei of infected plant cells. All geminiviruses encode a conserved protein (Rep) that catalyzes initiation of rolling-circle replication. Earlier studies showed that three conserved motifs-motifs I, II, and III-in the N termini of geminivirus Rep proteins are essential for function. In this study, we identified a fourth sequence, designated GRS (geminivirus Rep sequence), in the Rep N terminus that displays high amino acid sequence conservation across all geminivirus genera. Using the Rep protein of Tomato golden mosaic virus (TGMV AL1), we show that GRS mutants are not infectious in plants and do not support viral genome replication in tobacco protoplasts. GRS mutants are competent for protein-protein interactions and for both double- and single-stranded DNA binding, indicating that the mutations did not impair its global conformation. In contrast, GRS mutants are unable to specifically cleave single-stranded DNA, which is required to initiate rolling-circle replication. Interestingly, the Rep proteins of phytoplasmal and algal plasmids also contain GRS-related sequences. Modeling of the TGMV AL1 N terminus suggested that GRS mutations alter the relative positioning of motif II, which coordinates metal ions, and motif III, which contains the tyrosine involved in DNA cleavage. Together, these results established that the GRS is a conserved, essential motif characteristic of an ancient lineage of rolling-circle initiators and support the idea that geminiviruses may have evolved from plasmids associated with phytoplasma or algae.

Isolation of Peptide aptamers to target protein function.

Peptide aptamers are small recombinant proteins typically inserted into a supportive protein scaffold. These short peptide domains can bind to their target proteins with high specificity and affinity, often resulting in an altered target protein. We describe high-throughput protocols that facilitate the selection and characterization of peptide aptamers from yeast dihybrid libraries. These protocols include the preparation and evaluation of the bait fusion and the peptide aptamer screen. They also include confirmation of interaction specificity as well as isolation and sequencing of peptide inserts. Once the amino acid sequence is determined, we describe a protocol for aligning and comparing short peptide sequences and assessing the statistical significance of the alignments.