Activation of Plant Immune Responses by a Gain-of-Function Mutation in an Atypical Receptor-Like Kinase

Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases.Receptor-like kinases (RLKs) are a large group of kinases with a variable extracellular domain and a cytoplasmic kinase domain linked by a single transmembrane motif. RLKs have been shown to play diverse roles in regulating plant innate immunity as well as growth and development (Morillo and Tax, 2006). The extracellular domains of RLKs are believed to bind directly to ligands to perceive extracellular signals, whereas the cytoplasmic kinase domains transduce these signals into the cell. There are over 600 RLKs (Shiu and Bleecker, 2001) in Arabidopsis (Arabidopsis thaliana). The biological functions of most RLKs are unknown.Several RLKs have been identified to be receptors of microbe-associated molecular patterns (MAMPs). FLS2 and EFR are two well-characterized RLKs with extracellular Leu-rich repeats (LRRs) that recognize bacterial flagellin and translation elongation factor EF-Tu, respectively (Gomez-Gomez and Boller, 2000; Zipfel et al., 2006). BAK1 is also an RLK with extracellular LRRs. BAK1 seems to function as an adaptor protein for multiple RLKs including BRI1, FLS2, and BIR1 (Li et al., 2002; Nam and Li, 2002; Chinchilla et al., 2007; Heese et al., 2007; Gao et al., 2009). Interestingly, knocking out BIR1 activates cell death and defense responses mediated by another RLK, SOBIR1 (Gao et al., 2009). Recently, the rice (Oryza sativa) RLK Xa21 was also suggested to be a MAMP receptor. Xa21 recognizes a peptide derived from the secreted effector protein AvrXa21, which is conserved among different Xanthomonas species (Lee et al., 2009). Unlike FLS2, EFR, and Xa21, the putative receptor for chitin is an RLK with three extracellular LysM domains instead of LRRs that are required for the perception of chitin as well as resistance against bacterial pathogens (Miya et al., 2007; Wan et al., 2008; Gimenez-Ibanez et al., 2009).Perception of MAMPs by receptors leads to the rapid activation of mitogen-activated protein (MAP) kinases including MAP KINASE3 (MPK3), MPK4, and MPK6 (Boller and Felix, 2009). MAP KINASE SUBSTRATE1 (MKS1) was identified as an MPK4-interacting protein that positively regulates defense responses (Andreasson et al., 2005). Silencing of MKS1 compromises basal resistance to Pseudomonas syringae pv tomato DC3000, whereas overexpression of MKS1 leads to enhanced pathogen resistance. Activation of MAMP receptors also induces a number of responses such as oxidative burst, callose deposition, and increased salicylic acid synthesis (Boller and Felix, 2009). Defense responses induced by different MAMPs seem similar, suggesting that they may share common signaling components. Identification of the signaling components downstream of RLK receptors remains a major task in understanding MAMP-triggered immunity.Here we report the identification and characterization of suppressor of npr1-1, constitutive4-1D (snc4-1D), a gain-of-function mutant of an atypical RLK that is autoactivated by a mutation in its kinase domain. The snc4-1D mutant plants constitutively express defense maker genes PR1, PR2, and PDF1.2, and display enhanced resistance to Hyaloperonospora arabidopsidis (H. a.) Noco2. Epistasis analysis showed that snc4-1D-mediated defense responses are dependent on multiple factors including NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), MKS1, and OPR3.     

Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line

A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine- and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty-four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated.     

Mito‐nuclear discordance across a recent contact zone for California voles

To examine the processes that maintain genetic diversity among closely related taxa, we investigated the dynamics of introgression across a contact zone between two lineages of California voles (Microtus californicus). We tested the prediction that introgression of nuclear loci would be greater than that for mitochondrial loci, assuming ongoing gene flow across the contact zone. We also predicted that genomic markers would show a mosaic pattern of differentiation across this zone, consistent with genomes that are semi‐permeable. Using mitochondrial cytochrome b sequences and genome‐wide loci developed via ddRAD‐seq, we analyzed genetic variation for 10 vole populations distributed along the central California coast; this transect included populations from within the distributions of both parental lineages as well as the putative contact zone. Our analyses revealed that (1) the two lineages examined are relatively young, having diverged ca. 8.5–54 kya, (2) voles from the contact zone in Santa Barbara County did not include F1 or early generation backcrossed individuals, and (3) there appeared to be little to no recurrent gene flow across the contact zone. Introgression patterns for mitochondrial and nuclear markers were not concordant; only mitochondrial markers revealed evidence of introgression, putatively due to historical hybridization. These differences in genetic signatures are intriguing given that the contact zone occurs in a region of continuous vole habitat, with no evidence of past or present physical barriers. Future studies that examine specific isolating mechanisms, such as microhabitat use and mate choice, will facilitate our understanding of how genetic boundaries are maintained in this system.     

Genetic profiling of cancer with circulating tumor DNA analysis

Circulating cell-free tumor DNA(ctDNA) in the blood is DNA released from apoptotic, circulating, and living tumor cells. ctDNA is about 140 nt in length and has a half-life of about 1.5 h. ctDNA analysis provides a noninvasive means to assess the genetic profile of cancer in real time. With the advent of molecular technologies, including digital PCR and massively parallel sequencing(MPS), ctDNA analysis has shown promise as a highly sensitive and specific alternative to conventional tissue biopsy in cancer detection, longitudinal monitoring, and precision therapy. This review provides an overview of the latest development in our understanding of the biologic characteristics, detection methodologies, and potential clinical implications of ctDNA, as well as the challenges in translating ctDNA analysis from the research arena to patient care.     

Novel antitumor agent family of 1H-benzo[c,d]indol-2-one with flexible basic side chains: synthesis and biological evaluation

A series of mono-1H-benzo[c,d]indol-2-one with different amine side chains and bis-1H-benzo[c,d]indol-2-one as novel family of DNA intercalators were designed and synthesized, the contributions of aromatic chromophores and amine side chains for DNA binding properties, for example, intercalation and electrostatic binding, respectively, were evaluated. Among them, A3 tailed with N,N-dimethylamino-ethyl-ethane-1,2-diamine showed selective anti-tumor activities against cell lines A549 and P388 with IC(50) 0.428microm and 1.69microm.     

Gastrin regulates the heparin-binding epidermal-like growth factor promoter via a PKC/EGFR-dependent mechanism

Gastrin is a known growth/differentiation factor for the gastric mucosa. Its effects are likely mediated by the induction of heparin-binding epidermal-like growth factor (HB-EGF), a member of the EGF family of growth factors that is expressed by gastric parietal cells. In this study, we investigated the regulation of the HB-EGF promoter by gastrin in a human gastric cancer cell line. Serial human HB-EGF promoter-luciferase reporter deletion constructs and heterologous promoter constructs were transfected into AGS-E cells and stimulated with gastrin (10(-7) M) with or without various signal transduction inhibitors. EMSA were also performed. Gastrin stimulation resulted in a fivefold increase in HB-EGF-luciferase activity. The cis-acting element mediating gastrin responsiveness was mapped to the -69 to -58 region of the HB-EGF promoter. Gastrin stimulation was PKC dependent and at least partially mediated by activation of the EGF receptor.     

CUGBP1, a crucial factor for heart regeneration in mice

The mammalian heart is capable of achieving perfect regeneration following cardiac injury through sustained cardiomyocyte proliferation during the early period after birth. However, this regenerative capacity is lost by postnatal day 7 and throughout adulthood. CUGBP1 is critical for normal cardiac development but its role in heart regeneration remains unclear. Cardiac CUGBP1 levels are high in the early postnatal period and soon downregulate to adult levels within 1 week following birth in mice. The simultaneously diminished regenerative capacity and CUGBP1 levels by postnatal day lead us to hypothesize that CUGBP1 may be beneficial in heart regeneration. In this study, the function of CUGBP1 in heart regeneration was tested by a heart apex resection mouse model. We demonstrate that cardiac inactivation of CUGBP1 impairs neonatal heart regeneration at P1, in turn, replenishment of CUGBP1 levels prolong regenerative potential at P8 and P14. Furthermore, our results imply that the Wnt/β-catenin signaling and GATA4 involve in the CUGBP1 modulated neonatal heart regeneration. Altogether, our findings support CUGBP1 as a key factor promoting post-injury heart regeneration and provide a potential therapeutic method for heart disease.Subject terms: Cell proliferation, Self-renewal     

Isolation and characterization of ftsZ alleles that affect septal morphology.

The ftsZ gene encodes an essential cell division protein that specifically localizes to the septum of dividing cells. In this study we characterized the effects of the ftsZ2(Rsa) mutation on cell physiology. We found that this mutation caused an altered cell morphology that included minicell formation and an increased average cell length. In addition, this mutation caused a temperature-dependent effect on cell lysis. During this investigation we fortuitously isolated a novel temperature-sensitive ftsZ mutation that consisted of a 6-codon insertion near the 5' end of the gene. This mutation, designated ftsZ26(Ts), caused an altered polar morphology at the permissive temperature and blocked cell division at the nonpermissive temperature. The altered polar morphology resulted from cell division and correlated with an altered geometry of the FtsZ ring. An intragenic cold-sensitive suppressor of ftsZ26(Ts) that caused cell lysis at the nonpermissive temperature was isolated. These results support the hypothesis that the FtsZ ring determines the division site and interacts with the septal biosynthetic machinery.