Decline of the North American avifauna.

Species extinctions have defined the global biodiversity crisis, but extinction begins with loss in abundance of individuals that can result in compositional and functional changes of ecosystems. Using multiple and independent monitoring networks, we report population losses across much of the North American avifauna over 48 years, including once-common species and from most biomes. Integration of range-wide population trajectories and size estimates indicates a net loss approaching 3 billion birds, or 29% of 1970 abundance. A continent-wide weather radar network also reveals a similarly steep decline in biomass passage of migrating birds over a recent 10-year period. This loss of bird abundance signals an urgent need to address threats to avert future avifaunal collapse and associated loss of ecosystem integrity, function, and services.

Directed Evolution of FLS2 towards Novel Flagellin Peptide Recognition.

Microbe-associated molecular patterns (MAMPs) are molecules, or domains within molecules, that are conserved across microbial taxa and can be recognized by a plant or animal immune system. Although MAMP receptors have evolved to recognize conserved epitopes, the MAMPs in some microbial species or strains have diverged sufficiently to render them unrecognizable by some host immune systems. In this study, we carried out in vitro evolution of the Arabidopsis thaliana flagellin receptor FLAGELLIN-SENSING 2 (FLS2) to isolate derivatives that recognize one or more flagellin peptides from bacteria for which the wild-type Arabidopsis FLS2 confers little or no response. A targeted approach generated amino acid variation at FLS2 residues in a region previously implicated in flagellin recognition. The primary screen tested for elevated response to the canonical flagellin peptide from Pseudomonas aeruginosa, flg22. From this pool, we then identified five alleles of FLS2 that confer modest (quantitatively partial) recognition of an Erwinia amylovora flagellin peptide. Use of this Erwinia-based flagellin peptide to stimulate Arabidopsis plants expressing the resulting FLS2 alleles did not lead to a detectable reduction of virulent P. syringae pv. tomato growth. However, combination of two identified mutations into a single allele further increased FLS2-mediated responses to the E. amylovora flagellin peptide. These studies demonstrate the potential to raise the sensitivity of MAMP receptors toward particular targets.

LRR conservation mapping to predict functional sites within protein leucine-rich repeat domains.

Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs) are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM), a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP) receptors, EF-TU RECEPTOR (EFR) and FLAGELLIN-SENSING 2 (FLS2). In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area), Consurf, and PAML (positive selection) analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains.

Identification and mutational analysis of Arabidopsis FLS2 leucine-rich repeat domain residues that contribute to flagellin perception.

Mutational, phylogenetic, and structural modeling approaches were combined to develop a general method to study leucine-rich repeat (LRR) domains and were used to identify residues within the Arabidopsis thaliana FLAGELLIN-SENSING2 (FLS2) LRR that contribute to flagellin perception. FLS2 is a transmembrane receptor kinase that binds bacterial flagellin or a flagellin-based flg22 peptide through a presumed physical interaction within the FLS2 extracellular domain. Double-Ala scanning mutagenesis of solvent-exposed beta-strand/beta-turn residues across the FLS2 LRR domain identified LRRs 9 to 15 as contributors to flagellin responsiveness. FLS2 LRR-encoding domains from 15 Arabidopsis ecotypes and 20 diverse Brassicaceae accessions were isolated and sequenced. FLS2 is highly conserved across most Arabidopsis ecotypes, whereas more diversified functional FLS2 homologs were found in many but not all Brassicaceae accessions. flg22 responsiveness was correlated with conserved LRR regions using Conserved Functional Group software to analyze structural models of the LRR for diverse FLS2 proteins. This identified conserved spatial clusters of residues across the beta-strand/beta-turn residues of LRRs 12 to 14, the same area identified by the Ala scan, as well as other conserved sites. Site-directed randomizing mutagenesis of solvent-exposed beta-strand/beta-turn residues across LRRs 9 to 15 identified mutations that disrupt flg22 binding and showed that flagellin perception is dependent on a limited number of tightly constrained residues of LRRs 9 to 15 that make quantitative contributions to the overall phenotypic response.