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Pattern‐triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad‐spectrum disease resistance. PTI is activated upon perception of microbe‐associated molecular patterns (MAMPs) by pattern‐recognition receptors (PRRs). We have recently demonstrated that the L‐type lectin receptor kinase‐VI.2 (LecRK‐VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull‐down, bimolecular fluorescence complementation and co‐immunoprecipitation analyses that LecRK‐VI.2 associates with the PRR FLS2. We also demonstrated that LecRK‐VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK‐VI.2 were indeed more resistant to virulent hemi‐biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK‐VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK‐VI.2 in N. benthamiana primed PTI‐mediated reactive oxygen species production, mitogen‐activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK‐VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.  相似文献   

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Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial‐derived α‐ketoacids and α‐hydroxyacids that are produced by long‐lived Mit mutants but not by other long‐lived mutants or by short‐lived mitochondrial mutants. We show that accumulation of these compounds is dependent on concerted inhibition of three α‐ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer's disease. When the expression of DLD in wild‐type animals was reduced using RNA interference we observed an unprecedented effect on lifespan – as RNAi dosage was increased lifespan was significantly shortened, but, at higher doses, it was significantly lengthened, suggesting that DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype.  相似文献   

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Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. The P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe‐associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms for suppression of two branches of MAMP‐activated MAP kinase (MAPK) cascades. In addition to blocking MKK5 (MAPK kinase 5) activation in the MEKK1 (MAPK kinase kinase 1)/MEKKs–MKK4/5–MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1–MKK1/2–MPK4 cascade and the plasma membrane‐localized receptor‐like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane‐localized receptor‐like kinase that is involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 and between two other P. syringae effectors, AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild‐type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to show that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells.  相似文献   

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Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.  相似文献   

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Low temperature is an environmental factor that affects plant growth and development and plant–pathogen interactions. How temperature regulates plant defense responses is not well understood. In this study, we characterized chilling‐sensitive mutant 1 (chs1), and functionally analyzed the role of the CHS1 gene in plant responses to chilling stress. The chs1 mutant displayed a chilling‐sensitive phenotype, and also displayed defense‐associated phenotypes, including extensive cell death, the accumulation of hydrogen peroxide and salicylic acid, and an increased expression of PR genes: these phenotypes indicated that the mutation in chs1 activates the defense responses under chilling stress. A map‐based cloning analysis revealed that CHS1 encodes a TIR‐NB‐type protein. The chilling sensitivity of chs1 was fully rescued by pad4 and eds1, but not by ndr1. The overexpression of the TIR and NB domains can suppress the chs1–conferred phenotypes. Interestingly, the stability of the CHS1 protein was positively regulated by low temperatures independently of the 26S proteasome pathway. This study revealed the role of a TIR‐NB‐type gene in plant growth and cell death under chilling stress, and suggests that temperature modulates the stability of the TIR‐NB protein in Arabidopsis.  相似文献   

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Genome editing facilitated by Cas9‐based RNA‐guided nucleases (RGNs) is becoming an increasingly important and popular technique for reverse genetics in both model and non‐model species. So far, RGNs were mainly applied for the induction of point mutations, and one major challenge consists in the detection of genome‐edited individuals from a mutagenized population. Also, point mutations are not appropriate for functional dissection of non‐coding DNA. Here, the multiplexing capacity of a newly developed genome editing toolkit was exploited for the induction of inheritable chromosomal deletions at six different loci in Nicotiana benthamiana and Arabidopsis. In both species, the preferential formation of small deletions was observed, suggesting reduced efficiency with increasing deletion size. Importantly, small deletions (<100 bp) were detected at high frequencies in N. benthamiana T0 and Arabidopsis T2 populations. Thus, targeting of small deletions by paired nucleases represents a simple approach for the generation of mutant alleles segregating as size polymorphisms in subsequent generations. Phenotypically selected deletions of up to 120 kb occurred at low frequencies in Arabidopsis, suggesting larger population sizes for the discovery of valuable alleles from addressing gene clusters or non‐coding DNA for deletion by programmable nucleases.  相似文献   

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Proteins belonging to the enhancer of RNA interference‐1 subfamily of 3′–5′ exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE‐1‐LIKE‐1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3′–5′ exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus‐encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.  相似文献   

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Plant immunity against pathogens is achieved through rapid activation of defense responses that occur upon sensing of microbe‐ or damage‐associated molecular patterns, respectively referred to as MAMPs and DAMPs. Oligogalacturonides (OGs), linear fragments derived from homogalacturonan hydrolysis by pathogen‐secreted cell wall‐degrading enzymes, and flg22, a 22‐amino acid peptide derived from the bacterial flagellin, represent prototypical DAMPs and MAMPs, respectively. Both types of molecules induce protection against infections. In plants, like in animals, calcium is a second messenger that mediates responses to biotic stresses by activating calcium‐binding proteins. Here we show that simultaneous loss of calcium‐dependent protein kinases CPK5, CPK6 and CPK11 affects Arabidopsis thaliana basal as well as elicitor‐ induced resistance to the necrotroph Botrytis cinerea, by affecting pathogen‐induced ethylene production and accumulation of the ethylene biosynthetic enzymes 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase 2 (ACS2) and 6 (ACS6). Moreover, ethylene signaling contributes to OG‐triggered immunity activation, and lack of CPK5, CPK6 and CPK11 affects the duration of OG‐ and flg22‐induced gene expression, indicating that these kinases are shared elements of both DAMP and MAMP signaling pathways.  相似文献   

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Nonsense‐mediated mRNA decay (NMD) is a eukaryotic process that targets selected mRNAs for destruction, for both quality control and gene regulatory purposes. SMG1, the core kinase of the NMD machinery in animals, phosphorylates the highly conserved UPF1 effector protein to activate NMD. However, SMG1 is missing from the genomes of fungi and the model flowering plant Arabidopsis thaliana, leading to the conclusion that SMG1 is animal‐specific and questioning the mechanistic conservation of the pathway. Here we show that SMG1 is not animal‐specific, by identifying SMG1 in a range of eukaryotes, including all examined green plants with the exception of A. thaliana. Knockout of SMG1 by homologous recombination in the basal land plant Physcomitrella patens reveals that SMG1 has a conserved role in the NMD pathway across kingdoms. SMG1 has been lost at various points during the evolution of eukaryotes from multiple lineages, including an early loss in the fungal lineage and a very recent observable gene loss in A. thaliana. These findings suggest that the SMG1 kinase functioned in the NMD pathway of the last common eukaryotic ancestor.  相似文献   

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TANK‐binding kinase 1 (TBK1) activation is a central event in type I interferon production in anti‐virus innate immunity. However, the regulatory mechanism underlying TBK1 activation remains unclear. Here we report that Raf kinase inhibitory protein (RKIP) is essential for TBK1 activation and type I interferon production triggered by viral infection. Upon viral infection, RKIP is phosphorylated at serine 109 (S109) by TBK1. Phosphorylation of RKIP enhances its interaction with TBK1 and in turn promotes TBK1 autophosphorylation. Mutation of RKIP S109 to alanine abrogates the interaction between RKIP and TBK1, and the anti‐viral function of RKIP. RKIP deficiency inhibits intracellular double‐stranded RNA‐ or DNA‐induced type I interferon production. Consistently, RKIP deficiency renders the mice more susceptible to vesicular stomatitis virus (VSV) and herpes simplex virus (HSV) infections. This study reveals a previously unrecognized positive feedback loop between RKIP and TBK1 that is essential for type I interferon production in anti‐viral innate immunity.  相似文献   

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Functional genomic studies of many polyploid crops, including rapeseed (Brassica napus), are constrained by limited tool sets. Here we report development of a gain‐of‐function platform, termed ‘iFOX (inducible Full‐length cDNA OvereXpressor gene)‐Hunting’, for inducible expression of B. napus seed cDNAs in Arabidopsis. A Gateway‐compatible plant gene expression vector containing a methoxyfenozide‐inducible constitutive promoter for transgene expression was developed. This vector was used for cloning of random cDNAs from developing B. napus seeds and subsequent Agrobacterium‐mediated transformation of Arabidopsis. The inducible promoter of this vector enabled identification of genes upon induction that are otherwise lethal when constitutively overexpressed and to control developmental timing of transgene expression. Evaluation of a subset of the resulting ~6000 Arabidopsis transformants revealed a high percentage of lines with full‐length B. napus transgene insertions. Upon induction, numerous iFOX lines with visible phenotypes were identified, including one that displayed early leaf senescence. Phenotypic analysis of this line (rsl‐1327) after methoxyfenozide induction indicated high degree of leaf chlorosis. The integrated B. napuscDNA was identified as a homolog of an Arabidopsis acyl‐CoA binding protein (ACBP) gene designated BnACBP1‐like. The early senescence phenotype conferred by BnACBP1‐like was confirmed by constitutive expression of this gene in Arabidopsis and B. napus. Use of the inducible promoter in the iFOX line coupled with RNA‐Seq analyses allowed mechanistic clues and a working model for the phenotype associated with BnACBP1‐like expression. Our results demonstrate the utility of iFOX‐Hunting as a tool for gene discovery and functional characterization of Brassica napus genome.  相似文献   

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Ent‐kaurenoic acid oxidase (KAO), a class of cytochrome P450 monooxygenases of the subfamily CYP88A, catalyzes the conversion of ent‐kaurenoic acid (KA) to gibberellin (GA) GA12, the precursor of all GAs, thereby playing an important role in determining GA concentration in plants. Past work has demonstrated the importance of KAO activity for growth in various plant species. In Arabidopsis, this enzyme is encoded by two genes designated KAO1 and KAO2. In this study, we used various approaches to determine the physiological roles of KAO1 and KAO2 throughout plant development. Analysis of gene expression pattern reveals that both genes are mainly expressed in germinating seeds and young developing organs, thus suggesting functional redundancy. Consistent with this, kao1 and kao2 single mutants are indistinguishable from wild‐type plants. By contrast, the kao1 kao2 double mutant exhibits typical non‐germinating GA‐dwarf phenotypes, similar to those observed in the severely GA‐deficient ga1‐3 mutant. Phenotypic characterization and quantitative analysis of endogenous GA contents of single and double kao mutants further confirm an overlapping role of KAO1 and KAO2 throughout Arabidopsis development.  相似文献   

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Gibberellins (GAs) play a critical role in fruit‐set and fruit growth. Gibberellin is perceived by its nuclear receptors GA INSENSITIVE DWARF1s (GID1s), which then trigger degradation of downstream repressors DELLAs. To understand the role of the three GA receptor genes (GID1A, GID1B and GID1C) in Arabidopsis during fruit initiation, we have examined their temporal and spatial localization, in combination with analysis of mutant phenotypes. Distinct expression patterns are revealed for each GID1: GID1A is expressed throughout the whole pistil, while GID1B is expressed in ovules, and GID1C is expressed in valves. Functional study of gid1 mutant combinations confirms that GID1A plays a major role during fruit‐set and growth, whereas GID1B and GID1C have specific roles in seed development and pod elongation, respectively. Therefore, in ovules, GA perception is mediated by GID1A and GID1B, while GID1A and GID1C are involved in GA perception in valves. To identify tissue‐specific interactions between GID1s and DELLAs, we analyzed spatial expression patterns of four DELLA genes that have a role in fruit initiation (GAI, RGA, RGL1 and RGL2). Our data suggest that GID1A can interact with RGA and GAI in all tissues, whereas GID1C–RGL1 and GID1B–RGL2 interactions only occur in valves and ovules, respectively. These results uncover specific functions of each GID1–DELLA in the different GA‐dependent processes that occur upon fruit‐set. In addition, the distribution of GA receptors in valves along with lack of expression of GA biosynthesis genes in this tissue, strongly suggests transport of GAs from the developing seeds to promote fruit growth.  相似文献   

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Plants constantly monitor changes in photoperiod and temperature throughout the year to synchronize flowering with optimal environmental conditions. In the temperate zones, both photoperiod and temperature fluctuate in a somewhat predictable manner through the seasons, although a transient shift to low temperature is also encountered during changing seasons, such as early spring. Although low temperatures are known to delay flowering by inducing the floral repressor FLOWERING LOCUS C (FLC), it is not fully understood how temperature signals are coordinated with photoperiodic signals in the timing of seasonal flowering. Here, we show that the cold signaling activator INDUCER OF CBF EXPRESSION 1 (ICE1), FLC and the floral promoter SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) constitute an elaborate signaling network that integrates cold signals into flowering pathways. The cold‐activated ICE1 directly induces the gene encoding FLC, which represses SOC1 expression, resulting in delayed flowering. In contrast, under floral promotive conditions, SOC1 inhibits the binding of ICE1 to the promoters of the FLC gene, inducing flowering with a reduction of freezing tolerance. These observations indicate that the ICE1‐FLC‐SOC1 signaling network contributes to the fine‐tuning of flowering during changing seasons.  相似文献   

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