Leucine-rich Repeat Domain Protein Research Articles

The unconventional resistance protein PTR recognizes the Magnaporthe oryzae effector AVR-Pita in an allele-specific manner.

Blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating rice diseases. Disease resistance genes such as Pi-ta or Pi-ta2 are critical in protecting rice production from blast. Published work reports that Pi-ta codes for a nucleotide-binding and leucine-rich repeat domain protein (NLR) that recognizes the fungal protease-like effector AVR-Pita by direct binding. However, this model was challenged by the recent discovery that Pi-ta2 resistance, which also relies on AVR-Pita detection, is conferred by the unconventional resistance gene Ptr, which codes for a membrane protein with a cytoplasmic armadillo repeat domain. Here, using NLR Pi-ta and Ptr RNAi knockdown and CRISPR/Cas9 knockout mutant rice lines, we found that AVR-Pita recognition relies solely on Ptr and that the NLR Pi-ta has no role in it, indicating that it is not the Pi-ta resistance gene. Different alleles of Ptr confer different recognition specificities. The A allele of Ptr (PtrA) detects all natural sequence variants of the effector and confers Pi-ta2 resistance, while the B allele of Ptr (PtrB) recognizes a restricted set of AVR-Pita alleles and, thereby, confers Pi-ta resistance. Analysis of the natural diversity in AVR-Pita and of mutant and transgenic strains identified one specific polymorphism in the effector sequence that controls escape from PtrB-mediated resistance. Taken together, our work establishes that the M. oryzae effector AVR-Pita is detected in an allele-specific manner by the unconventional rice resistance protein Ptr and that the NLR Pi-ta has no function in Pi-ta resistance and the recognition of AVR-Pita.

New recognition specificity in a plant immune receptor by molecular engineering of its integrated domain

Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that recognize pathogen effectors. Here, we show that molecular engineering of the integrated decoy domain (ID) of an NLR can extend its recognition spectrum to a new effector. We relied for this on detailed knowledge on the recognition of the Magnaporthe oryzae effectors AVR-PikD, AVR-Pia, and AVR1-CO39 by, respectively, the rice NLRs Pikp-1 and RGA5. Both receptors detect their effectors through physical binding to their HMA (Heavy Metal-Associated) IDs. By introducing into RGA5_HMA the AVR-PikD binding residues of Pikp-1_HMA, we create a high-affinity binding surface for this effector. RGA5 variants carrying this engineered binding surface perceive the new ligand, AVR-PikD, and still recognize AVR-Pia and AVR1-CO39 in the model plant N. benthamiana. However, they do not confer extended disease resistance specificity against M. oryzae in transgenic rice plants. Altogether, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs.

Tartan underlies the evolution of Drosophila male genital morphology

Male genital structures are among the most rapidly evolving morphological traits and are often the only features that can distinguish closely related species. This process is thought to be driven by sexual selection and may reinforce species separation. However, while the genetic bases of many phenotypic differences have been identified, we still lack knowledge about the genes underlying evolutionary differences in male genital organs and organ size more generally. The claspers (surstyli) are periphallic structures that play an important role in copulation in insects. Here, we show that divergence in clasper size and bristle number between Drosophila mauritiana and Drosophila simulans is caused by evolutionary changes in tartan (trn), which encodes a transmembrane leucine-rich repeat domain protein that mediates cell-cell interactions and affinity. There are no fixed amino acid differences in trn between D. mauritiana and D. simulans, but differences in the expression of this gene in developing genitalia suggest that cis-regulatory changes in trn underlie the evolution of clasper morphology in these species. Finally, analyses of reciprocal hemizygotes that are genetically identical, except for the species from which the functional allele of trn originates, determined that the trn allele of D. mauritiana specifies larger claspers with more bristles than the allele of D. simulans Therefore, we have identified a gene underlying evolutionary change in the size of a male genital organ, which will help to better understand not only the rapid diversification of these structures, but also the regulation and evolution of organ size more broadly.

Differential regulation of TNL-mediated immune signaling by redundant helper CNLs.

Higher plants utilize nucleotide-binding leucine-rich repeat domain proteins (NLRs) as intracellular immune receptors to recognize pathogen-derived effectors and trigger a robust defense. The Activated Disease Resistance 1 (ADR1) family of coiled-coil NLRs (CNLs) have evolved as helper NLRs that function downstream of many TIR-type sensor NLRs (TNLs). Close homologs of ADR1s form the N REQUIREMENT GENE 1 (NRG1) family in Arabidopsis, the function of which is unclear. Through CRISPR/Cas9 gene editing methods, we discovered that the tandemly repeated NRG1A and NRG1B are functionally redundant and operate downstream of TNLs with differential strengths. Interestingly, ADR1s and NRG1s function in two distinct parallel pathways contributing to TNL-specific immunity. Synergistic effects on basal and TNL-mediated defense were detected among ADR1s and NRG1s. An intact P-loop of NRG1s is not required for mediating signals from sensor TNLs, whereas auto-active NRG1A exhibits autoimmunity. Importantly, NRG1s localize to the cytosol and endomembrane network regardless of the presence of effectors, suggesting a cytosolic activation mechanism. Taken together, different sensor TNLs differentially use two groups of helper NLRs, ADR1s and NRG1s, to transduce downstream defense signals.

The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance.

Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.

Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

Global gene expression analysis of Anopheles gambiae responses to microbial challenge

Anopheles gambiae transcript responses to experimental challenge with heat inactivated Salmonella typhimurium, Staphylococcus aureus and Beauveria bassiana have been analyzed with an Affymetrix GeneChip comprising the entire predicted mosquito transcriptome. Significant up- or down-regulation (greater than 2-fold) can be assayed for approximately 2% of the mosquito transcriptome and affected genes represent a variety of functional classes that include immunity, apoptosis, stress response, detoxification, metabolism, blood digestion, olfaction and others. Transcript responses to the 3 microbial elicitors exhibit an exceptionally high degree of specificity and only a few genes are significantly regulated by more than 1 of the tested elicitors. This study identifies several transcripts that have not been linked directly to immune response in A. gambiae previously; their infection responsiveness and sequence features do however suggest implication in defence reactions; examples are genes encoding leucine-rich repeat domain proteins, cuticle domain proteins and proteins containing immunoglobulin and fibronectin domains.