Plant E3 ligases SNIPER1 and SNIPER2 broadly regulate the homeostasis of sensor NLR immune receptors.

In both plants and animals, nucleotide-binding leucine-rich repeat (NLR) immune receptors perceive pathogen-derived molecules to trigger immunity. Global NLR homeostasis must be tightly controlled to ensure sufficient and timely immune output while avoiding aberrant activation, the mechanisms of which are largely unclear. In a previous reverse genetic screen, we identified two novel E3 ligases, SNIPER1 and its homolog SNIPER2, both of which broadly control the levels of NLR immune receptors in Arabidopsis. Protein levels of sensor NLRs (sNLRs) are inversely correlated with SNIPER1 amount and the interactions between SNIPER1 and sNLRs seem to be through the common nucleotide-binding (NB) domains of sNLRs. In support, SNIPER1 can ubiquitinate the NB domains of multiple sNLRs in vitro. Our study thus reveals a novel process of global turnover of sNLRs by two master E3 ligases for immediate attenuation of immune output to effectively avoid autoimmunity. Such unique mechanism can be utilized in the future for engineering broad-spectrum resistance in crops to fend off pathogens that damage our food supply.

SCFSNIPER4 controls the turnover of two redundant TRAF proteins in plant immunity.

In mammals, tumor necrosis factor receptor associated factors (TRAFs) are signaling adaptors that regulate diverse physiological processes, including immunity and stress responses. In Arabidopsis, MUSE13 and MUSE14 are redundant TRAF proteins serving as adaptors in the SCFCRP1 complex to facilitate the turnover of nucleotide-binding domain and leucine-rich repeats (NLR) immune receptors. Degradation of MUSE13 is inhibited by proteasome inhibitor, suggesting that the MUSE13 stability is controlled by the 26S proteasome. However, the E3 ligase that regulates MUSE13 level is unknown. Here we report the identification of an F-box protein, SNIPER4 that regulates the turnover of MUSE13 and MUSE14. Protein levels of MUSE13 and MUSE14 are reduced by SNIPER4 overexpression, while higher accumulation of MUSE13 and MUSE14 is observed when dominant-negative SNIPER4 is expressed. Furthermore, SNIPER4 associates with MUSE13 or MUSE14. Taken together, the SCFSNIPER4 complex controls the turnover of TRAF proteins for an optimum immune output.

Autoimmunity conferred by chs3-2D relies on CSA1, its adjacent TNL-encoding neighbour.

Plant innate immunity depends on the function of a large number of intracellular immune receptor proteins, the majority of which are structurally similar to mammalian nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) proteins. CHILLING SENSITIVE 3 (CHS3) encodes an atypical Toll/Interleukin 1 Receptor (TIR)-type NLR protein with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C-terminus. The gain-of-function mutant allele chs3-2D exhibits severe dwarfism and constitutively activated defense responses, including enhanced resistance to virulent pathogens, high defence marker gene expression, and salicylic acid accumulation. To search for novel regulators involved in CHS3-mediated immune signaling, we conducted suppressor screens in the chs3-2D and chs3-2D pad4-1 genetic backgrounds. Alleles of sag101 and eds1-90 were isolated as complete suppressors of chs3-2D, and alleles of sgt1b were isolated as partial suppressors of chs3-2D pad4-1. These mutants suggest that SAG101, EDS1-90, and SGT1b are all positive regulators of CHS3-mediated defense signaling. Additionally, the TIR-type NLR-encoding CSA1 locus located genomically adjacent to CHS3 was found to be fully required for chs3-2D-mediated autoimmunity. CSA1 is located 3.9 kb upstream of CHS3 and is transcribed in the opposite direction. Altogether, these data illustrate the distinct genetic requirements for CHS3-mediated defense signaling.

Arabidopsis HSP90 protein modulates RPP4-mediated temperature-dependent cell death and defense responses.

Plant defense responses are regulated by temperature. In Arabidopsis, the chilling-sensitive mutant chs2-1 (rpp4-1d) contains a gain-of-function mutation in the TIR-NB-LRR (Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat) gene, RPP4 (RECOGNITION OF PERONOSPORA PARASITICA 4), which leads to constitutive activation of the defense response at low temperatures. Here, we identified and characterized two suppressors of rpp4-1d from a genetic screen, hsp90.2 and hsp90.3, which carry point mutations in the cytosolic heat shock proteins HSP90.2 and HSP90.3, respectively. The hsp90 mutants suppressed the chilling sensitivity of rpp4-1d, including seedling lethality, activation of the defense responses and cell death under chilling stress. The hsp90 mutants exhibited compromised RPM1 (RESISTANCE TO PSEUDOMONAS MACULICOLA 1)-, RPS4 (RESISTANCE TO P. SYRINGAE 4)- and RPP4-mediated pathogen resistance. The wild-type RPP4 and the mutated form rpp4 could interact with HSP90 to form a protein complex. Furthermore, RPP4 and rpp4 proteins accumulated in the cytoplasm and nucleus at normal temperatures, whereas the nuclear accumulation of the mutated rpp4 was decreased at low temperatures. Genetic analysis of the intragenic suppressors of rpp4-1d revealed the important functions of the NB-ARC and LRR domains of RPP4 in temperature-dependent defense signaling. In addition, the rpp4-1d-induced chilling sensitivity was largely independent of the WRKY70 or MOS (modifier of snc1) genes. [Correction added after online publication 11 March 2013: the expansions of TIR-NB-LRR and RPS4 were amended] This study reveals that Arabidopsis HSP90 regulates RPP4-mediated temperature-dependent cell death and defense responses.

Identification and expression profile analysis of odorant binding proteins in the oriental fruit fly Bactrocera dorsalis.

Olfaction is crucial in many insects for critical behaviors, including those regulating survival and reproduction. Insect odorant-binding proteins (OBPs) function in the first step of the olfactory system and play an essential role in the perception of odorants, such as pheromones and host chemicals. The oriental fruit fly, Bactrocera dorsalis, is a destructive fruit-eating pest, due to its wide host range of up to 250 different types of fruits and vegetables, and this fly causes severe economic damage to the fruit and vegetable industry. However, OBP genes have not been largely identified in B. dorsalis. Based on our previously constructed B. dorsalis cDNA library, ten OBP genes were identified in B. dorsalis for the first time. A phylogenetic tree was generated to show the relationships among the 10 OBPs of B. dorsalis to OBP sequences of two other Dipteran species, including Drosophila melanogaster and the mosquito Anopheles gambiae. The expression profiles of the ten OBPs in different tissues (heads, thoraxes, abdomens, legs, wings, male antennae and female antenna) of the mated adults were analyzed by real-time PCR. The results showed that nine of them are highly expressed in the antenna of both sexes, except BdorOBP7. Four OBPs (BdorOBP1, BdorOBP4, BdorOBP8, and BdorOBP10) are also enriched in the abdomen, and BdorOBP7 is specifically expressed in leg, indicating that it may function in other biological processes. This work will provide insight into the roles of OBPs in chemoreception and help develop new pest-control strategies.

Odorant receptor co-receptor Orco is upregulated by methyl eugenol in male Bactrocera dorsalis (Diptera: Tephritidae).

Bactrocera dorsalis is a destructive fruit-eating pest that causes severe economic damage to the fruit and vegetable industry. Methyl eugenol (ME) has been widely used as an effective sexual attractant for male fruit flies through olfactory perception. However, the molecular mechanism underlying the olfactory perception of ME remains unknown. Here, we report the characterization and functional analysis of a newly discovered cDNA that encodes a Drosophila melanogaster odorant receptor co-receptor Orco ortholog in B. dorsalis. qRT-PCR analysis revealed that it was abundantly expressed in the antenna of adult B. dorsalis. Notably, Orco was upregulated by ME in the antenna of male flies. Mature males of B. dorsalis showed significant taxis toward ME within 0.5h, and Orco was significantly upregulated in the attracted adults within the same period. Silencing Orco through the ingestion of dsRNA reduced the attractive effects of ME. These data suggest that Orco may play an essential role in ME attraction in the olfactory signal transduction pathway.