M6 Mutant Research Articles

The GDSL-Lipolytic Enzyme Lip1 Is Required for Full Virulence of the Cucurbit Pathogenic Bacterium Acidovorax citrulli.

Bacterial fruit blotch caused by Acidovorax citrulli is a serious disease of cucurbit crops. Here we report characterization of a mutant strain of A. citrulli M6 defective in lip1, a gene encoding a lipolytic enzyme. The M6-lip1- mutant was detected in a mutant library screen aimed at identifying M6 mutants with altered levels of twitching motility. In this screen M6-lip1- was the only mutant that showed significantly larger twitching motility haloes around colonies than wild-type M6. Sequence analyses indicated that lip1 encodes a member of the GDSL family of secreted lipolytic enzymes. In line with this finding, lipolytic assays showed that the supernatants of M6-lip1- had lower lipolytic activity as compared with those of wild-type M6 and a lip1-complemented strain. The mutant was also affected in swimming motility and had compromised virulence on melon seedlings and on Nicotiana benthamiana leaves relative to wild-type and complemented strains. Lip1 contains a predicted N-terminal signal sequence for type II secretion. Evidence from our study confirms Lip1 is indeed secreted in a type II secretion-dependent manner, and this is required for full virulence of A. citrulli. To the best of our knowledge this is the first study reporting contribution of lipolytic activity to virulence of a plant-pathogenic Acidovorax species.

Mutation breeding for heat and drought tolerance in tepary bean (Phaseolus acutifolius A. Gray)

Tepary bean (Phaseolus acutifolius A. Gray) is more heat and drought tolerant than common bean (P. vulgaris L.). Four hundred mutant lines of two tepary accessions (G40068 and G40159) were generated by ethyl methane sulfonate (EMS) treatment. In preliminary studies of the M5 mutant lines under abiotic stress, three mutant lines (CMT 38, CMT 109, CMT 187) were selected from six mutated lines based on morpho-physiological traits and superior yield and advanced to the M6 generation. The M6 mutant lines were uniform and genetically stable. These mutant lines and their original (M0) parents were evaluated for heat and drought tolerance under greenhouse conditions. Their performance was evaluated for morpho-physiological attributes, seed yield and yield components. Under high temperature and drought conditions, the CMT 38 mutant (M6 line) and its original tepary (M0) accession (G40068) showed greater values of pod biomass, pod number and 100-seed biomass than the other lines tested. The CMT 109 and CMT 187 mutant lines and their G40159 original accession (M0) also showed the highest value of seed number under high temperature and drought conditions. This suggests that the previous screening performed during the population advancement of these mutant lines, based on morphological traits like growth habit, was not detrimental to the yield variables evaluated here. Under combined heat and drought conditions, different parameters could be incorporated into tepary breeding programmes, as selection criteria to screen genotypes for tolerance to heat and drought stress. These parameters included: chlorophyll (SPAD) readings, seed biomass, 100-seed biomass and seed number because they explain the observed variance in the principal component analysis. Two additional traits (root biomass and stem diameter) were also identified as useful attributes, based on univariate analysis. The mutant lines evaluated here offer potential for further improvement of tepary bean to high temperature and drought

A HSV1 mutant leads to an attenuated phenotype and induces immunity with a protective effect.

Herpes simplex virus type 1 (HSV1) is a complicated structural agent with a sophisticated transcription process and a high infection rate. A vaccine against HSV1 is urgently needed. As multiple viral-encoded proteins, including structural and nonstructural proteins, contribute to immune response stimulation, an attenuated or deficient HSV1 vaccine may be relatively reliable. Advances in genomic modification technologies provide reliable means of constructing various HSV vaccine candidates. Based on our previous work, an M6 mutant with mutations in the UL7, UL41, LAT, Us3, Us11 and Us12 genes was established. The mutant exhibited low proliferation in cells and an attenuated phenotype in an animal model. Furthermore, in mice and rhesus monkeys, the mutant can induce remarkable serum neutralizing antibody titers and T cell activation and protect against HSV1 challenge by impeding viral replication, dissemination and pathogenesis.

Analysis of heritability and correlation of agronomic character towards the yield of several m6 generation of wheat mutants (Triticum aestivum L.) in the lowlands

This research aims to find out the lowland and high production adaptive M6 generation of mutant wheat, to know the positive correlation between growth parameters and production components on yields, and to determine the parameters that have high heritability. The research was carried out in the Bonto Parang which is located in Kelara Village, Tolo Selatan District, Jeneponto Regency at an altitude of 135 m asl, with coordinates of 5 ° 24’58.0 “LS 119 ° 54’58.2” BT, Climate type E (2 wet months and 5 months dry) andosol soil type. The research was conducted from July to October 2018. This research used a Randomized Block Design with 16 lines as genetic material resulting from mutations and four varieties (Dewata, Selayar, Nias, and Munal) used as a comparison so that there were 20 genotypes. The research was conducted with 3 replications so that there were 60 experimental units. The results showed that the M6 mutant wheat is adaptive to lowland and it has a high number of products which is N 350 3.8.9 (3.48 tons.ha-1), N 200 2.4.B.6 (3.37 tons.ha-1) and N 300 4.3.6 (2.71 ton.ha-1). Growth parameters and production components that have a significant positive correlation and bring significant results are plant height, number of tillers, productive tillers, number of stomata, stomatal density, chlorophyll index, panicle length, number of spikelet scales, percentage of empty floret, weight of seed scales and of 1000 seeds, high heritability and high genetic diversity followed by the characteristics of productive tillers, number of seedlings, scum production, clump production, production and flowering age.

Cyclic adenosine monophosphate signal pathway is involved in regulation of triacylglycerol biosynthesis following nitrogen deprivation in Chlamydomonas reinhardtii

The unicellular green alga, Chlamydomonas reinhardtii is a model organism for studying various biological processes, such as photosynthesis, flagellar motility, and lipid metabolism. To find some novel genes regulating the lipid metabolism under various stress conditions, the paromomycin resistance gene aphVIII was transferred into the genome of C. reinhardtii to establish a mutant library. Two genes mutated in two of the TAG-reduced mutants (Cre06.g278111 in M2 mutant, Cre06.g278110 in M6 mutants) were neighboring in the genome, and their expression levels were down-regulated in their corresponding mutants in parallel with their reduced TAG levels following N deprivation. The proteins encoded by these two genes (KCN11 by Cre06.g278111, ACYC3 by Cre06.g278110) contained a conversed cyclic mononucleotide phosphate (cNMP) binding protein and an adenylate domain, respectively. Since cNMP binding protein and adenylate domain have been known as important components of cyclic adenosine monophosphate (cAMP) signaling pathway, suggesting that these two genes might affect cellular TAG biosynthesis through cAMP signal pathway.

A combined association mapping and t-test analysis of SNP loci and candidate genes involving in resistance to low nitrogen traits by a wheat mutant population.

Crop productivity is highly dependent on the application of N fertilizers, but ever-increasing N application is causing serious environmental impacts. To facilitate the development of new wheat cultivars that can thrive in low N growth conditions, key loci and genes associated with wheat responses to low N must be identified. In this GWAS and t-test study of 190 M6 mutant wheat lines (Jing 411-derived) based on genotype data from the wheat 660k SNP array, we identified a total of 221 significant SNPs associated four seedling phenotypic traits that have been implicated in resistance to low N: relative root length, relative shoot length, relative root weight, and relative shoot weight. Notably, we detected large numbers of significantly associated SNP in what appear to be genomic ‘hotspots’ for resistance to low N on chromosomes 2A and 6B, strongly suggesting that these regions are functionally related to the resistance phenotypes that we observed in some of the mutant lines. Moreover, the candidate genes, including genes encoding high-affinity nitrate transporter 2.1, gibberellin responsive protein, were identified for resistance to low N. This study raises plausible mechanistic hypotheses that can be evaluated in future applied or basic efforts by breeders or plant biologists seeking to develop new high-NUE wheat cultivars.

Association Between Loss of Type IV Pilus Synthesis Ability and Phenotypic Variation in the Cucurbit Pathogenic Bacterium Acidovorax citrulli.

Acidovorax citrulli is the causal agent of bacterial fruit blotch of cucurbits. We have shown that functional type IV pili (T4P) are required for full virulence of this bacterium. To identify A. citrulli genes required for T4P activity, we screened a library of about 10,000 transposon mutants of A. citrulli M6 for altered T4P-mediated twitching motility. This screen led to the identification of 50 mutants impaired in twitching ability due to transposon insertions into 20 different genes. Representative mutants with disruptions in these genes were further characterized. All mutants were compromised in their virulence in seed transmission and stem inoculation assays and had reduced biofilm formation ability relative to wild-type M6. When grown on nutrient agar, most mutants produced colonies with a translucent and fuzzy appearance, in contrast to the opaque and smooth appearance of wild-type colonies. The colony morphology of these mutants was identical to that of previously reported phenotypic variants of strain M6. The exceptions were M6 mutants disrupted in genes tonB, pilT, pilW, and pilX that exhibited typical wild-type colony morphology, although lacking twitching haloes surrounding the colony. Transmission electron microscopy revealed that most mutants lacked the ability to produce T4P. The exceptions were mutants with disruptions in tonB, pilT, pilW, and pilX genes that were shown to produce these appendages. These findings support the idea that colony phenotypic variation in A. citrulli is determined by the lack of ability to synthesize T4P but not by lack of T4P functionality.

A role for the membrane protein M6 in the Drosophila visual system

BackgroundMembers of the proteolipid protein family, including the four-transmembrane glycoprotein M6a, are involved in neuronal plasticity in mammals. Results from our group previously demonstrated that M6, the only proteolipid protein expressed in Drosophila, localizes to the cell membrane in follicle cells. M6 loss triggers female sterility, which suggests a role for M6 in follicular cell remodeling. These results were the basis of the present study, which focused on the function and requirements of M6 in the fly nervous system.ResultsThe present study identified two novel, tissue-regulated M6 isoforms with variable N- and C- termini, and showed that M6 is the functional fly ortholog of Gpm6a. In the adult brain, the protein was localized to several neuropils, such as the optic lobe, the central complex, and the mushroom bodies. Interestingly, although reduced M6 levels triggered a mild rough-eye phenotype, hypomorphic M6 mutants exhibited a defective response to light.ConclusionsBased on its ability to induce filopodium formation we propose that M6 is key in cell remodeling processes underlying visual system function. These results bring further insight into the role of M6/M6a in biological processes involving neuronal plasticity and behavior in flies and mammals.

Kinetics and thermodynamics of high level β-glucosidase production by mutant derivative of Aspergillus niger under submerged growth conditions

Gamma rays mediated mutagenesis and subsequent selection on 2-deoxy-D-glucose developed a highly catabolite de-repressed and stable β-glucosidase (BGL) hyper-producer mutant of local Aspergillus niger NIBGE, which has high potential for industrial applications. The M6 mutant showed highest constitutive production of BGL (14,900 IU/L) on glucose as compared to control (5,900 IU/L). Wheat bran was the best carbon source and BGL production by parent and mutant strain was 16,200 and 29,500 IU/L, respectively. Effect of inoculum density, pH, temperature and wheat bran concentration on the BGL production was determined in terms of kinetic growth parameters, that is, specific growth rate (m), biomass doubling time (td), product yield coefficient with respect to cell mass (Yp/x) and specific rate of product formation (qp). Optimal conditions for BGL production by parent and mutant strain were same: inoculum level= 0.184 mg cells/mL (10% v/v); optimum temperature= 30°C and pH= 5.0; wheat bran= 3% (w/v). The apparent subunit molecular mass of BGL from both strains was also same (130 kDa). Determination of thermodynamic parameters led to consider that the hyper-production of BGL by the mutant was due to decrease in: activation energy (Ea(p)), change in enthalpy (ΔH*(p)) and Gibbs free energy (ΔG*(p)) for product (BGL) formation. Key words: β-glucosidase, Aspergillus niger, activation energy, enthalpy, Gibbs free energy.

The resistance response of sunflower genotypes to black stem disease under controlled conditions

Phoma black stem, caused by Phoma macdonaldii , is one of the most important diseases of sunflower in the world. The sources of resistance to Phoma black stem were investigated. A total of 184 genotypes, including some recombinant inbred lines (RILs), several M6 mutant lines obtained by gamma irradiation of seed of the genotype AS 613, and other genotypes from different countries, were evaluated against an aggressive French isolate (MP6) in controlled conditions. The study was carried out in a randomized complete block design with three replicates. Each replicate consisted of 10–12 seedlings. Twenty μL of spore suspension (106 pycnidiospores mL-1) were deposited on the intersection of the cotyledon petiole and the hypocotyl of sunflower plantlets at the two-leaf stage. The percentage of the area exhibiting disease symptoms was scored on the two cotyledon petioles of each of the plantlets three, five and seven days after inoculation. The disease progress rate (rd), as the slope of the regression line for disease severity against time, was also calculated. Analysis of variance detected significant differences among sunflower genotypes for disease severity 7 days after inoculation, as well as for the disease progress rate. A strong correlation (r=0.96, P <0.01) was found between disease severity 7 days after inoculation and the disease progress rate. The inbred lines F1250/03 (origin: Hungary), M5-54-1, M6-862-1 (mutant lines), SDR 18 (origin: USA) and two wild Helianthus accessions, 1012 Nebraska and 211 Illinois, (wild type) were highly resistant to Phoma black stem. These findings will assist breeders in choosing parent plants for breeding durable resistance to Phoma black stem.

Genotype‐isolate interaction for resistance to black stem in sunflower (Helianthus annuus)

Two experiments were undertaken to determine the partial resistance of sunflower genotypes to seven isolates of Phoma macdonaldii. In the first experiment, 28 genotypes, including recombinant inbred lines and their parents, M6 mutant lines developed by gamma irradiation, and some genotypes from different geographical origins, were used. The experiment consisted of a split‐plot design with three replications, each with 12 seedlings per genotype per isolate, in controlled conditions. Seven days after inoculation, plantlets were scored on a 1–9 scale for percentage necrotic area. Highly significant differences were observed among genotypes, isolates and their interactions. The presence of a differential interaction between genotypes and P. macdonaldii isolates was confirmed in a second experiment using 12 genotypes representing large variability for partial resistance to P. macdonaldii isolates, as identified in the first experiment. Inbred lines B454/03, ENSAT‐B5 and LC1064C were the most susceptible sunflower genotypes, whereas two American lines SDR19 and SDR18 presented high partial resistance to all P. macdonaldii isolates studied. The least and most aggressive isolates were MA6 and MP3, respectively. Isolates interacted differentially with sunflower genotypes. This study identified two genotypes (AS613 and PAC2) presenting specific resistance to isolate MP8. The results also showed a wide range of isolate‐nonspecific partial resistances among the lines tested. The information presented here could assist sunflower breeders to choose parents of crosses for breeding of durable resistance to phoma black stem disease.

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD(P)H-dependent flavoprotein aromatic hydroxylases. These enzymes share a conserved FAD-binding domain but lack a recognizable fold for binding the pyridine nucleotide. We have switched the coenzyme specificity of strictly NADPH-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis. To that end, we altered the solvent exposed helix H2 region (residues 33-40) of the FAD-binding domain. Non-conservative selective replacements of Arg33 and Tyr38 weakened the binding of NADPH without disturbing the protein architecture. Introduction of a basic residue at position 34 increased the NADPH binding strength. Double (M2) and quadruple (M4) substitutions in the N-terminal part of helix H2 did not change the coenzyme specificity. By extending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADPH. It is concluded that specificity in P. fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2’-phosphate moiety of bound NADPH, and that introduction of an acidic group at position 38 potentially enables the recognition of the 2′-hydroxy group of NADH. This is the first report on the coenzyme reversion of a flavoprotein aromatic hydroxylase.

A Radiation-induced Mutant with Resistance to Common Bacterial Blight Disease in Common Beans

The leaf reaction of the Phaseolus vulgaris L. germplasm—UNECA (M6 mutant derived from the cultivar Chimbolito, Costa Rica), `Chimbolito', BAC-6 (Brazil), XAN-159 (Centro Internacional de Agricultura Tropical, Cali, Colombia), and `PC-50' (Domican Republic)—to Xanthomonas campestris pv. phaseoli strain V4S1 (Dominican Republic) were determined in two replicated trials conducted in a greenhouse in Lincoln, Neb. (Feb.–Mar. and July–Aug. 1993). `PC-50' and `Chimbolito' were susceptible to Xcp strain V4S1 in both tests. UNECA, BAC-6, and XAN-159 had similar levels of resistance to Xcp in the July to August trial. However, in the February to March trial, the resistance of UNECA was greater than that of BAC-6 but less than that of XAN-159.

Modification of host–parasite interactions through mutagenesis in quantitative resistance of rice to bacterial leaf blight

SummaryA number of mutant lines showing different levels of improved quantitative resistance to bacterial leaf blight (BLB) were obtained through selection of plants or lines derived from seeds of the rice cultivar Harebare exposed to thermal neutrons and gamma-rays. The selections were made on M2–M4 materials under natural BLB-epidemic conditions and/or by artificial inoculation of one of the BLB isolates. Bacterial isolates predominating under the natural conditions were identified as belonging to the differential BLB isolate group I, and the isolates of this group were used in the artificial inoculation for selection of mutants. The selected M4 mutant lines were tested in M5 for BLB resistance by artificial inoculations of four differential bacterium isolates, groups I, II, III and IV. The M6 mutant lines showed quantitatively elevated resistances to the isolate of bacterium group I and also to the other three differential isolates. Analysis of variance of the data demonstrated that the observed elevated quantitative resistances of the mutants to the isolates I, II, III and IV were isolatespecific. Host-parasite interactions in quantitative resistance to BLB and their modification by induced mutations are discussed.

Energy transduction in photosynthetic bacteria X. Composition and function of the branched oxidase system in wild type and respiration deficient mutants of Rhodopseudomonas capsulata

The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems “in vivo”. Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b ( E′ 0 = +413 mV ) is sensitive to low concentrations of KCN (5 · 10 −5 M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10 −3 M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type ( E′ 0 = +413, +260, +47 mV ) and two cytochromes of c type ( E′ 0 = +342, +94 mV ). A clear indication of the involvement in respiration of cytochrome b 413 ), cytochrome c 342 and cytochrome b 47 has been obtained. Only 50 % of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b 47 . A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented.