Showing papers on "Mutant published in 2007"

Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database.

Abstract: The tumor suppressor gene TP53 is frequently mutated in human cancers More than 75% of all mutations are missense substitutions that have been extensively analyzed in various yeast and human cell assays The International Agency for Research on Cancer (IARC) TP53 database (www-p53iarcfr) compiles all genetic variations that have been reported in TP53 Here, we present recent database developments that include new annotations on the functional properties of mutant proteins, and we perform a systematic analysis of the database to determine the functional properties that contribute to the occurrence of mutational "hotspots" in different cancer types and to the phenotype of tumors This analysis showed that loss of transactivation capacity is a key factor for the selection of missense mutations, and that difference in mutation frequencies is closely related to nucleotide substitution rates along TP53 coding sequence An interesting new finding is that in patients with an inherited missense mutation, the age at onset of tumors was related to the functional severity of the mutation, mutations with total loss of transactivation activity being associated with earlier cancer onset compared to mutations that retain partial transactivation capacity Furthermore, 80% of the most common mutants show a capacity to exert dominant-negative effect (DNE) over wild-type p53, compared to only 45% of the less frequent mutants studied, suggesting that DNE may play a role in shaping mutation patterns These results provide new insights into the factors that shape mutation patterns and influence mutation phenotype, which may have clinical interest

CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis

Abstract: Chitin is a major component of fungal cell walls and serves as a microbe-associated molecular pattern (MAMP) for the detection of various potential pathogens in innate immune systems of both plants and animals. We recently showed that chitin elicitor-binding protein (CEBiP), plasma membrane glycoprotein with LysM motifs, functions as a cell surface receptor for chitin elicitor in rice. The predicted structure of CEBiP does not contain any intracellular domains, suggesting that an additional component(s) is required for signaling through the plasma membrane into the cytoplasm. Here, we identified a receptor-like kinase, designated CERK1, which is essential for chitin elicitor signaling in Arabidopsis. The KO mutants for CERK1 completely lost the ability to respond to the chitin elicitor, including MAPK activation, reactive oxygen species generation, and gene expression. Disease resistance of the KO mutant against an incompatible fungus, Alternaria brassicicola, was partly impaired. Complementation with the WT CERK1 gene showed cerk1 mutations were responsible for the mutant phenotypes. CERK1 is a plasma membrane protein containing three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain with autophosphorylation/myelin basic protein kinase activity, suggesting that CERK1 plays a critical role in fungal MAMP perception in plants.

Genetic Characterization and Functional Analysis of the GID1 Gibberellin Receptors in Arabidopsis

Abstract: We investigated the physiological function of three Arabidopsis thaliana homologs of the gibberellin (GA) receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) by determining the developmental consequences of GID1 inactivation in insertion mutants. Although single mutants developed normally, gid1a gid1c and gid1a gid1b displayed reduced stem height and lower male fertility, respectively, indicating some functional specificity. The triple mutant displayed a dwarf phenotype more severe than that of the extreme GA-deficient mutant ga1-3. Flower formation occurred in long days but was delayed, with severe defects in floral organ development. The triple mutant did not respond to applied GA. All three GID1 homologs were expressed in most tissues throughout development but differed in expression level. GA treatment reduced transcript abundance for all three GID1 genes, suggesting feedback regulation. The DELLA protein REPRESSOR OF ga1-3 (RGA) accumulated in the triple mutant, whose phenotype could be partially rescued by loss of RGA function. Yeast two-hybrid and in vitro pull-down assays confirmed that GA enhances the interaction between GID1 and DELLA proteins. In addition, the N-terminal sequence containing the DELLA domain is necessary for GID1 binding. Furthermore, yeast three-hybrid assays showed that the GA-GID1 complex promotes the interaction between RGA and the F-box protein SLY1, a component of the SCFSLY1 E3 ubiquitin ligase that targets the DELLA protein for degradation.

A Pseudo-Response Regulator is misexpressed in the photoperiod insensitive Ppd-D1a mutant of wheat ( Triticum aestivum L.)

Abstract: Ppd-D1 on chromosome 2D is the major photoperiod response locus in hexaploid wheat (Triticum aestivum). A semi-dominant mutation widely used in the "green revolution" converts wheat from a long day (LD) to a photoperiod insensitive (day neutral) plant, providing adaptation to a broad range of environments. Comparative mapping shows Ppd-D1 to be colinear with the Ppd-H1 gene of barley (Hordeum vulgare) which is a member of the pseudo-response regulator (PRR) gene family. To investigate the relationship between wheat and barley photoperiod genes we isolated homologues of Ppd-H1 from a 'Chinese Spring' wheat BAC library and compared them to sequences from other wheat varieties with known Ppd alleles. Varieties with the photoperiod insensitive Ppd-D1a allele which causes early flowering in short (SD) or LDs had a 2 kb deletion upstream of the coding region. This was associated with misexpression of the 2D PRR gene and expression of the key floral regulator FT in SDs, showing that photoperiod insensitivity is due to activation of a known photoperiod pathway irrespective of day length. Five Ppd-D1 alleles were found but only the 2 kb deletion was associated with photoperiod insensitivity. Photoperiod insensitivity can also be conferred by mutation at a homoeologous locus on chromosome 2B (Ppd-B1). No candidate mutation was found in the 2B PRR gene but polymorphism within the 2B PRR gene cosegregated with the Ppd-B1 locus in a doubled haploid population, suggesting that insensitivity on 2B is due to a mutation outside the sequenced region or to a closely linked gene.

The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA.

Abstract: Retention of juvenile traits in the adult reproductive phase characterizes a process known as neoteny, and speculation exists over whether it has contributed to the evolution of new species. The dominant Corngrass1 (Cg1) mutant of maize is a neotenic mutation that results in phenotypes that may be present in the grass-like ancestors of maize. We cloned Cg1 and found that it encodes two tandem miR156 genes that are overexpressed in the meristem and lateral organs. Furthermore, a target of Cg1 is teosinte glume architecture1 (tga1), a gene known to have had a role in the domestication of maize from teosinte. Cg1 mutant plants overexpressing miR156 have lower levels of mir172, a microRNA that targets genes controlling juvenile development. By altering the relative levels of both microRNAs, it is possible to either prolong or shorten juvenile development in maize, thus providing a mechanism for how species-level heterochronic changes can occur in nature.

A gain-of-function mutation in a cytokinin receptor triggers spontaneous root nodule organogenesis

Abstract: Legume root nodules originate from differentiated cortical cells that reenter the cell cycle and form organ primordia. We show that perception of the phytohormone cytokinin is a key element in this switch. Mutation of a Lotus japonicus cytokinin receptor gene leads to spontaneous development of root nodules in the absence of rhizobia or rhizobial signal molecules. The mutant histidine kinase receptor has cytokinin-independent activity and activates an Escherichia coli two-component phosphorelay system in vivo. Mutant analysis shows that cytokinin signaling is required for cell divisions that initiate nodule development and defines an autoregulated process where cytokinin induction of nodule stem cells is controlled by shoots.

Circadian and CLOCK-controlled regulation of the mouse transcriptome and cell proliferation

Abstract: Circadian rhythms of cell and organismal physiology are controlled by an autoregulatory transcription-translation feedback loop that regulates the expression of rhythmic genes in a tissue-specific manner. Recent studies have suggested that components of the circadian pacemaker, such as the Clock and Per2 gene products, regulate a wide variety of processes, including obesity, sensitization to cocaine, cancer susceptibility, and morbidity to chemotherapeutic agents. To identify a more complete cohort of genes that are transcriptionally regulated by CLOCK and/or circadian rhythms, we used a DNA array interrogating the mouse protein-encoding transcriptome to measure gene expression in liver and skeletal muscle from WT and Clock mutant mice. In WT tissue, we found that a large percentage of expressed genes were transcription factors that were rhythmic in either muscle or liver, but not in both, suggesting that tissue-specific output of the pacemaker is regulated in part by a transcriptional cascade. In comparing tissues from WT and Clock mutant mice, we found that the Clock mutation affects the expression of many genes that are rhythmic in WT tissue, but also profoundly affects many nonrhythmic genes. In both liver and skeletal muscle, a significant number of CLOCK-regulated genes were associated with the cell cycle and cell proliferation. To determine whether the observed patterns in cell-cycle gene expression in Clock mutants resulted in functional dysregulation, we compared proliferation rates of fibroblasts derived from WT or Clock mutant embryos and found that the Clock mutation significantly inhibits cell growth and proliferation.

A Heat-Inducible Transcription Factor, HsfA2, Is Required for Extension of Acquired Thermotolerance in Arabidopsis

Abstract: The expression of heat shock proteins (Hsps) induced by nonlethal heat treatment confers acquired thermotolerance (AT) to organisms against subsequent challenges of otherwise lethal temperature. After the stress signal is removed, AT gradually decays, with decreased Hsps during recovery. AT of sufficient duration is critical for sessile organisms such as plants to survive repeated heat stress in their environment, but little is known regarding its regulation. To identify potential regulatory components, we took a reverse genetics approach by screening for Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants that show decreased thermotolerance after a long recovery (2 d) under nonstress conditions following an acclimation heat treatment. Among the tested mutants corresponding to 48 heat-induced genes, only the heat shock transcription factor HsfA2 knockout mutant showed an obvious phenotype. Following pretreatment at 37°C, the mutant line was more sensitive to severe heat stress than the wild type after long but not short recovery periods, and this could be complemented by the introduction of a wild-type copy of the HsfA2 gene. Quantitative hypocotyl elongation assay also revealed that AT decayed faster in the absence of HsfA2. Significant reduction in the transcript levels of several highly heat-inducible genes was observed in HsfA2 knockout plants after 4 h recovery or 2 h prolonged heat stress. Immunoblot analysis showed that Hsa32 and class I small Hsp were less abundant in the mutant than in the wild type after long recovery. Our results suggest that HsfA2 as a heat-inducible transactivator sustains the expression of Hsp genes and extends the duration of AT in Arabidopsis.

Adenomatous polyposis coli (APC): a multi-functional tumor suppressor gene

Abstract: The adenomatous polyposis coli (APC) gene is a key tumor suppressor gene. Mutations in the gene have been found not only in most colon cancers but also in some other cancers, such as those of the liver. The APC gene product is a 312 kDa protein that has multiple domains, through which it binds to various proteins, including beta-catenin, axin, CtBP, Asefs, IQGAP1, EB1 and microtubules. Studies using mutant mice and cultured cells have demonstrated that APC suppresses canonical Wnt signalling, which is essential for tumorigenesis, development and homeostasis of a variety of cell types, such as epithelial and lymphoid cells. Further studies have suggested that APC plays roles in several other fundamental cellular processes. These include cell adhesion and migration, organization of the actin and microtubule networks, spindle formation and chromosome segregation. Deregulation of these processes caused by mutations in APC is implicated in the initiation and expansion of colon cancer.

EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana.

Abstract: Shortly after the release of singlet oxygen (1O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all 1O2-responsive genes. Retrograde control of 1O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.

p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM

Abstract: Tp53 is the most commonly mutated tumour-suppressor gene in human cancers. In addition to the loss of tumour-suppression function, some missense mutants gain novel oncogenic activities. To elucidate the nature of the gain of function, we introduced the most common p53 cancer mutations (R248W and R273H) independently into the humanized p53 knock-in (HUPKI) allele in mice. Tumour-suppressor functions of p53 are abolished in p53-mutant mice. Several lines of evidence further indicate gain-of-function of p53 mutants in promoting tumorigenesis. p53(R248W) mice rapidly succumb to certain types of cancers not commonly observed in p53(-/-) mice. Interchromosomal translocations, a type of genetic instability rarely observed in p53(-/-) cells, are readily detectable in p53-mutant pre-tumor thymocytes. Although normal in p53(-/-) mouse cells, the G(2)-M checkpoint is impaired in p53-mutant cells after DNA damage. These acquired oncogenic properties of mutant p53 could be explained by the findings that these p53 mutants interact with the nuclease Mre11 and suppress the binding of the Mre11-Rad50-NBS1 (MRN) complex to DNA double-stranded breaks (DSBs), leading to impaired Ataxia-telangiectasia mutated (ATM) activation. Therefore, p53 gain-of-function mutants promote tumorigenesis by a novel mechanism involving active disruption of critical DNA damage-response pathways.

A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance.

Abstract: MicroRNAs are predicted to regulate ≈30% of all human genes by targeting sequences in their 3′ UTR Polymorphisms in 3′ UTR of several genes have been reported to affect gene expression, but the mechanism is not fully understood Here, we demonstrate that 829C→T, a naturally occurring SNP, near the miR-24 binding site in the 3′ UTR of human dihydrofolate reductase (DHFR) affects DHFR expression by interfering with miR-24 function, resulting in DHFR overexpression and methotrexate resistance miR-24 has a conserved binding site in DHFR 3′ UTR DHFR with WT and 3′ UTR containing the 829C→T mutation were expressed in DG44 cells that lack DHFR Overexpression of miR-24 in cells with WT DHFR resulted in down-regulation of DHFR protein, whereas no effect on DHFR protein expression was observed in the mutant 3′ UTR-expressing cells Inhibition of endogenous miR-24 with a specific inhibitor led to up-regulation of DHFR in WT and not in mutant cells Cells with the mutant 3′ UTR had a 2-fold increase in DHFR mRNA half-life, expressed higher DHFR mRNA and DHFR protein, and were 4-fold more resistant to methotrexate as compared with WT cells SNP-829C→T, therefore, leads to a decrease in microRNA binding leading to overexpression of its target and results in resistance to methotrexate We demonstrate that a naturally occurring miRSNP (a SNP located at or near a microRNA binding site in 3′ UTR of the target gene or in a microRNA) is associated with enzyme overproduction and drug resistance

Two calcineurin B-like calcium sensors, interacting with protein kinase CIPK23, regulate leaf transpiration and root potassium uptake in Arabidopsis.

Abstract: Calcium signalling involves sensor proteins that decode temporal and spatial changes in cellular Ca2+ concentration. Calcineurin B-like proteins (CBLs) represent a unique family of plant calcium sensors that relay signals by interacting with a family of protein kinases, designated as CBL-interacting protein kinases (CIPKs). In a reverse genetic screen for altered drought tolerance, we identified a loss-of-function allele of CIPK23 as exhibiting a drought-tolerant phenotype. In the cipk23 mutant, reduced transpirational water loss from leaves coincides with enhanced ABA sensitivity of guard cells during opening as well as closing reactions, without noticeable alterations in ABA content in the plant. We identified the calcium sensors CBL1 and CBL9 as CIPK23-interacting proteins that targeted CIPK23 to the plasma membrane in vivo. Expression analysis of the CIPK23, CBL1 and CBL9 genes suggested that they may function together in diverse tissues, including guard cells and root hairs. In addition, expression of the CIPK23 gene was induced by low-potassium conditions, implicating a function of this gene product in potassium nutrition. Indeed, cipk23 mutants displayed severe growth impairment on media with low concentrations of potassium. This phenotype correlates with a reduced efficiency of K+ uptake into the roots. In support of the conclusion that CBL1 and CBL9 interact with and synergistically serve as upstream regulators of CIPK23, the cbl1 cbl9 double mutant, but not the cbl1 or cbl9 single mutants, exhibit altered phenotypes for stomatal responses and low-potassium sensitivity. Together with the recent identification of the potassium channel AKT1 as a target of CIPK23, these results imply that plasma membrane-localized CBL1- and CBL9-CIPK23 complexes simultaneously regulate K+ transport processes in roots and in stomatal guard cells.

A Heat-Inducible Transcription Factor, HsfA2, Is Required for Extension of Acquired Thermotolerance

Abstract: The expression of heat shock proteins (Hsps) induced by nonlethal heat treatment confers acquired thermotolerance (AT) toorganisms against subsequent challenges of otherwise lethal temperature. After the stress signal is removed, AT graduallydecays, with decreased Hsps during recovery. ATof sufficient duration is critical for sessile organisms such as plants to surviverepeated heat stress in their environment, but little is known regarding its regulation. To identify potential regulatorycomponents, we took a reverse genetics approach by screening for Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutantsthat show decreased thermotolerance after a long recovery (2 d) under nonstress conditions following an acclimation heattreatment. Among the tested mutants corresponding to 48 heat-induced genes, only the heat shock transcription factor HsfA2knockout mutant showed an obvious phenotype. Following pretreatment at 37 C, the mutant line was more sensitive to severeheat stress than the wild type after long but not short recovery periods, and this could be complemented by the introduction ofa wild-type copy of the HsfA2 gene. Quantitative hypocotyl elongation assay also revealed that AT decayed faster in theabsence of HsfA2. Significant reduction in the transcript levels of several highly heat-inducible genes was observed in HsfA2knockout plants after 4 h recovery or 2 h prolonged heat stress. Immunoblot analysis showed that Hsa32 and class I small Hspwere less abundant in the mutant than in the wild type after long recovery. Our results suggest that HsfA2 as a heat-inducibletransactivator sustains the expression of Hsp genes and extends the duration of AT in Arabidopsis.

Medicago truncatula NIN is essential for rhizobial-independent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase.

Abstract: The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11::GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules.

Structure-function-rescue: the diverse nature of common p53 cancer mutants.

Abstract: The tumor suppressor protein p53 is inactivated by mutation in about half of all human cancers. Most mutations are located in the DNA-binding domain of the protein. It is, therefore, important to understand the structure of p53 and how it responds to mutation, so as to predict the phenotypic response and cancer prognosis. In this review, we present recent structural and systematic functional data that elucidate the molecular basis of how p53 is inactivated by different types of cancer mutation. Intriguingly, common cancer mutants exhibit a variety of distinct local structural changes, while the overall structural scaffold is largely preserved. The diverse structural and energetic response to mutation determines: (i) the folding state of a particular mutant under physiological conditions; (ii) its affinity for the various p53 target DNA sequences; and (iii) its protein-protein interactions both within the p53 tetramer and with a multitude of regulatory proteins. Further, the structural details of individual mutants provide the basis for the design of specific and generic drugs for cancer therapy purposes. In combination with studies on second-site suppressor mutations, it appears that some mutants are ideal rescue candidates, whereas for others simple pharmacological rescue by small molecule drugs may not be successful.

The Arabidopsis MATE Transporter TT12 Acts as a Vacuolar Flavonoid /H+ -Antiporter Active in Proanthocyanidin-Accumulating Cells of the Seed Coat

Abstract: Phenotypic characterization of the Arabidopsis thaliana transparent testa12 (tt12) mutant encoding a membrane protein of the multidrug and toxic efflux transporter family, suggested that TT12 is involved in the vacuolar accumulation of proanthocyanidin precursors in the seed. Metabolite analysis in tt12 seeds reveals an absence of flavan-3-ols and proanthocyanidins together with a reduction of the major flavonol quercetin-3-O-rhamnoside. The TT12 promoter is active in cells synthesizing proanthocyanidins. Using translational fusions between TT12 and green fluorescent protein, it is demonstrated that this transporter localizes to the tonoplast. Yeast vesicles expressing TT12 can transport the anthocyanin cyanidin-3-O-glucoside in the presence of MgATP but not the aglycones cyanidin and epicatechin. Inhibitor studies demonstrate that TT12 acts in vitro as a cyanidin-3-O-glucoside/H+-antiporter. TT12 does not transport glycosylated flavonols and procyanidin dimers, and a direct transport activity for catechin-3-O-glucoside, a glucosylated flavan-3-ol, was not detectable. However, catechin-3-O-glucoside inhibited TT12-mediated transport of cyanidin-3-O-glucoside in a dose-dependent manner, while flavan-3-ol aglycones and glycosylated flavonols had no effect on anthocyanin transport. It is proposed that TT12 transports glycosylated flavan-3-ols in vivo. Mutant banyuls (ban) seeds accumulate anthocyanins instead of proanthocyanidins, yet the ban tt12 double mutant exhibits reduced anthocyanin accumulation, which supports the transport data suggesting that TT12 mediates anthocyanin transport in vitro.

MAX2 participates in an SCF complex which acts locally at the node to suppress shoot branching.

Abstract: The Arabidopsis gene ORE9/MAX2 encodes an F-box leucine-rich repeat protein. F-box proteins function as the substrate-recruiting subunit of SCF-type ubiquitin E3 ligases in protein ubiquitination. One of several phenotypes of max2 mutants, the highly branched shoot, is identical to mutants at three other MAX loci. Reciprocal grafting, double mutant analysis and gene cloning suggest that all MAX genes act in a common pathway, where branching suppression depends on MAX2 activity in the shoot, in response to an acropetally mobile signal that requires MAX3, MAX4 and MAX1 for its production. Here, we further investigate the site and mode of action of MAX2 in branching. Transcript analysis and a translational MAX2-GUS fusion indicate that MAX2 is expressed throughout the plant, most highly in developing vasculature, and is nuclear-localized in many cell types. Analysis of cell autonomy shows that MAX2 acts locally, either in the axillary bud, or in adjacent stem or petiole tissue. Expression of MAX2 from the CaMV 35S promoter complements the max2 mutant, does not affect branching in a wild-type background and partially rescues increased branching in the max1, max3 and max4 backgrounds. Expression of mutant MAX2, lacking the F-box domain, under the CaMV 35S promoter does not complement max2, and dominant-negatively affects branching in the wild-type background. Myc-epitope-tagged MAX2 interacts with the core SCF subunits ASK1 and AtCUL1 in planta. We conclude that axillary shoot growth is controlled locally, at the node, by an SCF(MAX2), the action of which is enhanced by the mobile MAX signal.

Impairment of Cellulose Synthases Required for Arabidopsis Secondary Cell Wall Formation Enhances Disease Resistance

Abstract: Cellulose is synthesized by cellulose synthases (CESAs) contained in plasma membrane-localized complexes. In Arabidopsis thaliana, three types of CESA subunits (CESA4/IRREGULAR XYLEM5 [IRX5], CESA7/IRX3, and CESA8/IRX1) are required for secondary cell wall formation. We report that mutations in these proteins conferred enhanced resistance to the soil-borne bacterium Ralstonia solanacearum and the necrotrophic fungus Plectosphaerella cucumerina. By contrast, susceptibility to these pathogens was not altered in cell wall mutants of primary wall CESA subunits (CESA1, CESA3/ISOXABEN RESISTANT1 [IXR1], and CESA6/IXR2) or POWDERY MILDEW-RESISTANT5 (PMR5) and PMR6 genes. Double mutants indicated that irx-mediated resistance was independent of salicylic acid, ethylene, and jasmonate signaling. Comparative transcriptomic analyses identified a set of common irx upregulated genes, including a number of abscisic acid (ABA)-responsive, defense-related genes encoding antibiotic peptides and enzymes involved in the synthesis and activation of antimicrobial secondary metabolites. These data as well as the increased susceptibility of ABA mutants (abi1-1, abi2-1, and aba1-6) to R. solanacearum support a direct role of ABA in resistance to this pathogen. Our results also indicate that alteration of secondary cell wall integrity by inhibiting cellulose synthesis leads to specific activation of novel defense pathways that contribute to the generation of an antimicrobial-enriched environment hostile to pathogens.

Molecular Interactions of a Soluble Gibberellin Receptor, GID1, with a Rice DELLA Protein, SLR1, and Gibberellin

Abstract: GIBBERELLIN INSENSITIVE DWARF1 (GID1) encodes a soluble gibberellin (GA) receptor that shares sequence similarity with a hormone-sensitive lipase (HSL). Previously, a yeast two-hybrid (Y2H) assay revealed that the GID1-GA complex directly interacts with SLENDER RICE1 (SLR1), a DELLA repressor protein in GA signaling. Here, we demonstrated, by pull-down and bimolecular fluorescence complementation (BiFC) experiments, that the GA-dependent GID1-SLR1 interaction also occurs in planta. GA(4) was found to have the highest affinity to GID1 in Y2H assays and is the most effective form of GA in planta. Domain analyses of SLR1 using Y2H, gel filtration, and BiFC methods revealed that the DELLA and TVHYNP domains of SLR1 are required for the GID1-SLR1 interaction. To identify the important regions of GID1 for GA and SLR1 interactions, we used many different mutant versions of GID1, such as the spontaneous mutant GID1s, N- and C-terminal truncated GID1s, and mutagenized GID1 proteins with conserved amino acids replaced with Ala. The amino acid residues important for SLR1 interaction completely overlapped the residues required for GA binding that were scattered throughout the GID1 molecule. When we plotted these residues on the GID1 structure predicted by analogy with HSL tertiary structure, many residues were located at regions corresponding to the substrate binding pocket and lid. Furthermore, the GA-GID1 interaction was stabilized by SLR1. Based on these observations, we proposed a molecular model for interaction between GA, GID1, and SLR1.

Molecular and Genetic Studies of Fusarium Trichothecene Biosynthesis : Pathways, Genes, and Evolution

Abstract: Trichothecenes are a large family of sesquiterpenoid secondary metabolites of Fusarium species (e.g., F. graminearum) and other molds. They are major mycotoxins that can cause serious problems when consumed via contaminated cereal grains. In the past 20 years, an outline of the trichothecene biosynthetic pathway has been established based on the results of precursor feeding experiments and blocked mutant analyses. Following the isolation of the pathway gene Tri5 encoding the first committed enzyme trichodiene synthase, 10 biosynthesis genes (Tri genes; two regulatory genes, seven pathway genes, and one transporter gene) were functionally identified in the Tri5 gene cluster. At least three pathway genes, Tri101 (separated alone), and Tri1 and Tri16 (located in the Tri1-Tri16 two-gene cluster), were found outside of the Tri5 gene cluster. In this review, we summarize the current understanding of the pathways of biosynthesis, the functions of cloned Tri genes, and the evolution of Tri genes, focusing on Fusarium species.

Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion

Abstract: †Summary ABCG11/WBC11, an ATP binding cassette (ABC) transporter from Arabidopsis thaliana, is a key component of the export pathway for cuticular lipids. Arabidopsis wbc11 T-DNA insertional knock-out mutants exhibited lipidic inclusions inside epidermal cells similar to the previously characterized wax transporter mutant cer5, with a similar strong reduction in the alkanes of surface waxes. Moreover, the wbc11 knock-out mutants also showed defects not present in cer5, including post-genital organ fusions, stunted growth and a reduction in cutin load on the plant surface. A mutant line previously isolated in a forward genetics screen, calledpermeable leaves 1 (pel1), was identified as an allele of ABCG11/WBC11. The double knock-out wbc11 cer5 exhibited the same morphological and biochemical phenotypes as the wbc11 knock-out. A YFP-WBC11 fusion protein rescued a T-DNA knock-out mutant and was localized to the plasma membrane. These results show that WBC11 functions in secretion of surface waxes, possibly by interacting with CER5. However, unlike ABCG12/ CER5, ABCG11/WBC11 is important to the normal process of cutin formation.

BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14.

Abstract: The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifA mutant but does not contribute to the observed swarming defect. The ΔbifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.

Rare cancer-specific mutations in PIK3CA show gain of function

Abstract: Fifteen rare cancer-derived mutants of PIK3CA, the gene coding for the catalytic subunit p110α of phosphatidylinositol 3-kinase (PI3K), were examined for their biological and biochemical properties. Fourteen of these mutants show a gain of function: they induce rapamycin-sensitive oncogenic transformation of chicken embryo fibroblasts, constitutively activate Akt and TOR-mediated signaling, and show enhanced lipid kinase activity. Mapping of these mutants on a partial structural model of p110α suggests three groups of mutants, defined by their location in distinct functional domains of the protein. We hypothesize that each of these three groups induces a gain of PI3K function by a different molecular mechanism. Mutants in the C2 domain increase the positive surface charge of this domain and therefore may enhance the recruitment of p110α to cellular membranes. Mutants in the helical domain map to a contiguous surface of the protein and may affect the interaction with other protein(s). Mutants in the kinase domain are located near the hinge of the activation loop. They may alter the position and mobility of the activation loop. Arbitrarily introduced mutations that have no detectable phenotype map either to the interior of the protein or are positioned on a surface region that lies opposite to the exposed surfaces containing gain-of-function mutants. Engineered mutants that exchange acidic or neutral residues for basic residues on the critical surfaces show a gain of function.

A Chlorophyll-Deficient Rice Mutant with Impaired Chlorophyllide Esterification in Chlorophyll Biosynthesis

Abstract: Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors.

Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice

Abstract: Brassinosteroids (BR) are essential growth hormones found throughout the plant kingdom. BR bind to the receptor kinase BRI1 on the cell surface to activate a signal transduction pathway that regulates nuclear gene expression and plant growth. To understand the downstream BR signaling mechanism in rice, we studied the function of OsBZR1 using reverse genetic approaches and identified OsBZR1-interacting proteins. Suppressing OsBZR1 expression by RNAi resulted in dwarfism, erect leaves, reduced BR sensitivity, and altered BR-responsive gene expression in transgenic rice plants, demonstrating an essential role of OsBZR1 in BR responses in rice. Moreover, a yeast two-hybrid screen identified 14-3-3 proteins as OsBZR1-interacting proteins. Mutation of a putative 14-3-3-binding site of OsBZR1 abolished its interaction with the 14-3-3 proteins in yeast and in vivo. Such mutant OsBZR1 proteins suppressed the phenotypes of the Arabidopsis bri1–5 mutant and showed an increased nuclear distribution compared with the wild-type protein, suggesting that 14-3-3 proteins directly inhibit OsBZR1 function at least in part by reducing its nuclear localization. These results demonstrate a conserved function of OsBZR1 and an important role of 14-3-3 proteins in brassinosteroid signal transduction in rice.