The Rx1 protein, as many resistance proteins of the nucleotide binding–leucine-rich repeat (NB-LRR) class, is predicted to be cytoplasmic because it lacks discernable nuclear targeting signals. Here, we demonstrate that Rx1, which confers extreme resistance to Potato virus X, is located both in the nucleus and cytoplasm. Manipulating the nucleocytoplasmic distribution of Rx1 or its elicitor revealed that Rx1 is activated in the cytoplasm and cannot be activated in the nucleus. The coiled coil (CC) domain was found to be required for accumulation of Rx1 in the nucleus, whereas the LRR domain promoted the localization in the cytoplasm. Analyses of structural subdomains of the CC domain revealed no autonomous signals responsible for active nuclear import. Fluorescence recovery after photobleaching and nuclear fractionation indicated that the CC domain binds transiently to large complexes in the nucleus. Disruption of the Rx1 resistance function and protein conformation by mutating the ATP binding phosphate binding loop in the NB domain, or by silencing the cochaperone SGT1, impaired the accumulation of Rx1 protein in the nucleus, while Rx1 versions lacking the LRR domain were not affected in this respect. Our results support a model in which interdomain interactions and folding states determine the nucleocytoplasmic distribution of Rx1.

Ectotherms rely for their body heat on surrounding temperatures. A key question in biology is why most ectotherms mature at a larger size at lower temperatures, a phenomenon known as the temperature–size rule. Since temperature affects virtually all processes in a living organism, current theories to explain this phenomenon are diverse and complex and assert often from opposing assumptions. Although widely studied, the molecular genetic control of the temperature–size rule is unknown. We found that the Caenorhabditis elegans wild-type N2 complied with the temperature–size rule, whereas wild-type CB4856 defied it. Using a candidate gene approach based on an N2 × CB4856 recombinant inbred panel in combination with mutant analysis, complementation, and transgenic studies, we show that a single nucleotide polymorphism in tra-3 leads to mutation F96L in the encoded calpain-like protease. This mutation attenuates the ability of CB4856 to grow larger at low temperature. Homology modelling predicts that F96L reduces TRA-3 activity by destabilizing the DII-A domain. The data show that size adaptation of ectotherms to temperature changes may be less complex than previously thought because a subtle wild-type polymorphism modulates the temperature responsiveness of body size. These findings provide a novel step toward the molecular understanding of the temperature–size rule, which has puzzled biologists for decades. Biologists are fascinated by variation in body size, which is hardly surprising, considering that the range of body sizes spans orders of magnitude from bacteria to blue whales. Even within species, body sizes can vary dramatically. This intraspecies variation is intriguing because it suggests strong associations between body size and environment. Already in 1847, Bergmann noticed that mammals tend to be larger in colder environments. More recently similar relationships were found for ectotherms, which rely for their body heat on the temperature of their surroundings, where more than 85% of the species studied grew larger at lower temperatures. This phenomenon, dubbed the temperature–size rule, has caused a renewed interest to understand how temperature affects body size. Yet the control of the temperature–size rule remains enigmatic, and the hypotheses proposed have been inconclusive. In this paper the authors show that a single nucleic acid change in one gene is required for regulation of the temperature–size rule in the nematode C. elegans. Using protein modelling they also show that this subtle change in DNA decreases the function of the encoded protein. The data suggest that temperature adaptation can be simple and far less complex than previously thought.

Esophageal gland secretions from nematodes are believed to include effectors that play important roles in plant parasitism. We have identified a novel gene family encoding secreted proteins specifically expressed in the dorsal esophageal gland of Globodera rostochiensis early in the parasitic cycle, and which contain the B30.2/SPRY domain. The secondary structure of these proteins, named the secreted SPRY domain-containing proteins (SPRYSEC), includes highly conserved regions folding into β-strands interspersed with loops varying in sequence and in length. Mapping sequence diversity onto a three-dimensional structure model of the SPRYSEC indicated that most of the variability is in the extended loops that shape the so-called surface A in the SPRY domains. Seven of nine amino acid sites subjected to diversifying selection in the SPRYSEC are also at this surface. In both yeast-two-hybrid screening using a library from a susceptible tomato and in an in vitro pull-down assay, one of the SPRYSEC interacted with the leucine-rich repeat (LRR) region of a novel coiled-coil nucleotide-binding LRR protein, which is highly similar to members of the SW5 resistance gene cluster. Given that the tomato cultivar used is susceptible to nematode infection, this SPRYSEC could be an evolutionary intermediate that binds to a classical immune receptor but does not yet, or no longer, triggers a resistance response. Alternatively, this SPRYSEC may bind to the immune receptor to downregulate its activity.

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.

The ability to induce a defense response after pathogen attack is a critical feature of the immune system of any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. However, the mechanism by which defense signaling is triggered by these NLRs in plants is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, the complexes are highly heterometric. Also, in contrast to metazoan NLRs, the N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions in plants is proposed to provide resilience to perturbation by pathogens. The ability to induce defenses in response to pathogen attack is a critical feature of immunity in any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and have evolved to perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. The mechanism by which defense signaling is triggered by these NLRs is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, their complexes are highly heterometric. Also, in contrast to metazoan NLRs, their N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions is proposed to provide resilience to perturbation by pathogens.