A natural diversity screen in Arabidopsis thaliana reveals determinants for HopZ1a recognition in the ZAR1-ZED1 immune complex.

Pathogen pressure on hosts can lead to the evolution of genes regulating the innate immune response. By characterizing naturally occurring polymorphisms in immune receptors, we can better understand the molecular determinants of pathogen recognition. ZAR1 is an ancient Arabidopsis thaliana NLR (Nucleotide-binding [NB] Leucine-rich-repeat [LRR] Receptor) that recognizes multiple secreted effector proteins from the pathogenic bacteria Pseudomonas syringae and Xanthomonas campestris through its interaction with receptor-like cytoplasmic kinases (RLCKs). ZAR1 was first identified for its role in recognizing P. syringae effector HopZ1a, through its interaction with the RLCK ZED1. To identify additional determinants of HopZ1a recognition, we performed a computational screen for ecotypes from the 1001 Genomes project that were likely to lack HopZ1a recognition, and tested ~300 ecotypes. We identified ecotypes containing polymorphisms in ZAR1 and ZED1. Using our previously established Nicotiana benthamiana transient assay and Arabidopsis ecotypes, we tested for the effect of naturally occurring polymorphisms on ZAR1 interactions and the immune response. We identified key residues in the NB or LRR domain of ZAR1 that impact the interaction with ZED1. We demonstrate that natural diversity combined with functional assays can help define the molecular determinants and interactions necessary to regulate immune induction in response to pathogens.

A novel protein Moat prevents ectopic epithelial folding by limiting Bazooka/Par3-dependent adherens junctions.

Contractile myosin and cell adhesion work together to induce tissue shape changes, but how they are patterned to achieve diverse morphogenetic outcomes remains unclear. Epithelial folding occurs via apical constriction, mediated by apical contractile myosin engaged with adherens junctions, as in Drosophila ventral furrow formation. While it has been shown that a multicellular gradient of myosin contractility determines folding shape, the impact of multicellular patterning of adherens junction levels on tissue folding is unknown. We identified a novel Drosophila gene moat essential for differential apical constriction and folding behaviors across the ventral epithelium which contains both folding ventral furrow and nonfolding ectodermal anterior midgut (ectoAMG). We show that Moat functions to downregulate polarity-dependent adherens junctions through inhibiting cortical clustering of Bazooka/Par3 proteins. Such downregulation of polarity-dependent junctions is critical for establishing a myosin-dependent pattern of adherens junctions, which in turn mediates differential apical constriction in the ventral epithelium. In moat mutants, abnormally high levels of polarity-dependent junctions promote ectopic apical constriction in cells with low-level contractile myosin, resulting in expansion of infolding from ventral furrow to ectoAMG, and flattening of ventral furrow constriction gradient. Our results demonstrate that tissue-scale distribution of adhesion levels patterns apical constriction and establishes morphogenetic boundaries.

A novel protein Moat prevents ectopic epithelial folding by limiting Bazooka/Par3-dependent adherens junctions.

Cortical myosin contraction and cell adhesion work together to promote tissue shape changes, but how they are modulated to achieve diverse morphogenetic outcomes remains unclear. Epithelial folding occurs via apical constriction, mediated by apical accumulation of contractile myosin engaged with adherens junctions, as in Drosophila ventral furrow formation. While levels of contractile myosin correlate with apical constriction, whether levels of adherens junctions modulate apical constriction is unknown. We identified a novel Drosophila gene moat that maintains low levels of Bazooka/Par3-dependent adherens junctions and thereby restricts apical constriction to ventral furrow cells with high-level contractile myosin. In moat mutants, abnormally high levels of Bazooka/Par3-dependent adherens junctions promote ectopic apical constriction in cells with low-level contractile myosin, insufficient for apical constriction in wild type. Such ectopic apical constriction expands infolding behavior from ventral furrow to ectodermal anterior midgut, which normally forms a later circular invagination. In moat mutant ventral furrow, a perturbed apical constriction gradient delays infolding. Our results indicate that levels of adherens junctions can modulate the outcome of apical constriction, providing an additional mechanism to define morphogenetic boundaries.