Pathogen-associated molecular pattern (PAMP)-trigged immunity (PTI) is the first defensive line of plant innate immunity and is mediated by pattern recognition receptors. Here, we show that a mutation in BR-SIGNALING KINASE1 (BSK1), a substrate of the brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE1, suppressed the powdery mildew resistance caused by a mutation in ENHANCED DISEASE RESISTANCE2, which negatively regulates powdery mildew resistance and programmed cell death, in Arabidopsis thaliana. A loss-of-function bsk1 mutant displayed enhanced susceptibility to virulent and avirulent pathogens, including Golovinomyces cichoracearum, Pseudomonas syringae, and Hyaloperonospora arabidopsidis. The bsk1 mutant also accumulated lower levels of salicylic acid upon infection with G. cichoracearum and P. syringae. BSK1 belongs to a receptor-like cytoplasmic kinase family and displays kinase activity in vitro; this kinase activity is required for its function. BSK1 physically associates with the PAMP receptor FLAGELLIN SENSING2 and is required for a subset of flg22-induced responses, including the reactive oxygen burst, but not for mitogen-activated protein kinase activation. Our data demonstrate that BSK1 is involved in positive regulation of PTI. Together with previous findings, our work indicates that BSK1 represents a key component directly involved in both BR signaling and plant immunity.

Arabidopsis () immune receptor FLAGELLIN SENSING2 (FLS2) rapidly forms a complex to activate pathogen-associated molecular pattern-triggered immunity (PTI) upon perception of the bacterial protein flagellin. The receptor-like cytoplasmic kinase BRASSINOSTEROID-SIGNALINGKINASE1 (BSK1) interacts with FLS2 and is critical for the activation of PTI. However, it is unknown how BSK1 transduces signals to activate downstream immune responses. We identified MEK Kinase5 (MAPKKK5) as a potential substrate of BSK1 by whole-genome phosphorylation analysis. In addition, we demonstrated that BSK1 interacts with and phosphorylates MAPKKK5. In the - mutant, the Ser-289 residue of MAPKKK5 was not phosphorylated as it was in the wild type. Similar to the mutant, the mutant displayed enhanced susceptibility to virulent and avirulent strains of the bacterial pathogen pv DC3000, and to the fungal powdery mildew pathogen Phosphorylation of the Ser-289 residue is not involved in MAPKKK5-triggered cell death but is critical for MAPKKK5-mediated resistance to both bacterial and fungal pathogens. Furthermore, MAPKKK5 interacts with multiple MAPK kinases, including MKK1, MKK2, MKK4, MKK5, and MKK6. Overall, these results indicate that BSK1 regulates plant immunity by phosphorylating MAPKKK5 and suggest a direct regulatory mode of signaling from the immune complex to the MAPK cascade.

RNA aptamers selected against proteins can be used to modulate specific protein function. Expression of such reagents in cells and whole organisms could provide a means of dissecting and controlling molecular mechanisms in vivo. We demonstrate that Drosophila B52 protein can be specifically inhibited in vitro and in vivo by a multivalent RNA aptamer. This inhibitory aptamer RNA binds B52 avidly and inhibits B52-stimulated pre-mRNA splicing. It can be expressed in cultured cells and whole animals in a stable form that accumulates up to 10% of total mRNA. It binds B52 in vivo and suppresses all phenotypes caused by B52 overexpression. The strategies presented here should prove general in design and expression of functional and therapeutic RNAs.Multiprotein assemblies drive a variety of highly regulated biological processes. To better understand and control such processes, novel reagents are needed to modulate functions of specific proteins in cells and whole organisms. An ideal reagent would, (i) like an antibody, be made to order specifically for a particular protein, (ii) like a small organic molecule, be able to rapidly target a specific protein domain within cells, and (iii) like a conditional allele, be able to exert its effect in whole organisms, but (iv) also be targetable to specific tissues, cells, or stages of development.Although many strategies exist for inactivating genes or gene products, use of RNA aptamers (1) presents several compelling advantages. Extremely rare RNAs that have high affinity for specific proteins can be selected in vitro (SELEX) (2, 3). Genetically controlled induction of such high affinity RNA aptamers could provide a means of rapidly inactivating a specific domain of a protein and thereby assessing its primary function and mechanism of action in vivo. Alternatively, continuous expression of specific RNA aptamers after stable gene transfer could achieve a long-term alteration in the activity of a target protein. In yeast, the constitutive expression of an aptamer against RNA polymerase II caused cell growth defects under conditions in which the RNA polymerase level was artificially reduced (4). Although this study demonstrated the potential of RNA aptamers as inhibitors of protein function in vivo, several advances are needed to apply this approach to multicellular organisms.Previously, we selected and characterized RNA aptamers to B52, a regulator of RNA splicing in Drosophila melanogaster. B52, also known as SRp55, is a member of the Drosophila SR protein family, a group of nuclear proteins that are essential for pre-mRNA splicing and influence splice site choice (5, 6). B52 contains two RNA recognition motifs in its N-terminal half and a domain rich in serine-arginine dipeptide repeats in its C-terminal half (7). RNA aptamers that bind B52 with high affinity (K d ϭ 20-50 nM) and specificity were selected from a large pool of RNAs (8). The B52 binding sites (BBS) on members of this nonclonally derived family of RNA aptamers not only have a ''conserved'' consensus sequen...