Molecular mapping of two genes conferring resistance to Phytophthora sojae in a soybean landrace PI 567139B

Phytophthora root and stem rot (PRR), caused by the soil-borne oomycete pathogen Phytophthora sojae, is one of the most destructive diseases of soybean. PRR can be effectively controlled by race-specific genes conferring resistance to P. sojae (Rps). However, the Rps genes are usually non-durable, as populations of P. sojae are highly diverse and quick to adapt, and can be overcome 8–15 years after deployment. Thus, it is important to identify novel Rps genes for development of resistant soybean cultivars. PI 567139B is a soybean landrace carrying excellent resistance to nearly all predominant P. sojae races in Indiana. A mapping population consisting of 245 F₂ individuals and 403 F_2:3 families was developed from a cross between PI 567139B and the susceptible cultivar ‘Williams’, and used to dissect the resistance carried by PI 567139B. We found that the resistance in PI 567139B was conferred by two independent Rps genes, designated RpsUN1 and RpsUN2. The former was mapped to a 6.5 cM region between SSR markers Satt159 and BARCSOYSSR_03_0250 that spans the Rps1 locus on chromosome 3, while the latter was mapped to a 3.0 cM region between BARCSOYSSR_16_1275 and Sat_144, approximately 3.0–3.4 cM upstream of Rps2 on chromosome 16. According to the ‘Williams 82’ reference genome sequence, both regions are highly enriched with NBS-LRR genes. Marker assisted resistance spectrum analyses of these genes with 16 isolates of P. sojae, in combination with the mapping results, suggested that RpsUN1 was likely to be a novel allele at the Rps1 locus, while RpsUN2 was more likely to be a novel Rps gene.     

Diphtheria toxin resistance in human fibroblast cell strains from normal and cancer-prone individuals

Mutants resistant to diphtheria toxin (Dip^r) have been selected from a variety of human fibroblast cell strains derived from both normal subjects and individuals with known genetic predisposition to cancer such as xeroderma pigmentosum, Fanconi anemia and Bloom's syndrome. Treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) led to a marked increase in the frequency of Dip^r mutants in various cell strains. The increase in the frequency of Dip^r mutants occurred in a linear dose-dependent manner in response to MNNG and ethyl methanesulfonate, in one of the cell strains examined. The rate of muation to diphtheria toxin as determined by fluctuation analysis was very similar in various cell strains (1–3 × 10^?7 mutations/cell/generation), except for the strain GM1492 (8.8 × 10^?7 mutations/cell/generation) which is derived from a Bloom syndrome patient.     

Selection and initial characterization of partially nitrate tolerant nodulation mutants of soybean

Since NO₃⁻ availability in the rooting medium seriously limits symbiotic N₂ fixation by soybean (Glycine max [L.] Merr.), studies were initiated to select nodulation mutants which were more tolerant to NO₃⁻ and were adapted to the Midwest area of the United States. Three independent mutants were selected in the M₂ generation from ethyl methanesulfonate or N-nitroso-N-methylurea mutagenized Williams seed. All three mutants (designated NOD1-3, NOD2-4, and NOD3-7) were more extensively nodulated (427 to 770 nodules plant⁻¹) than the Williams parent (187 nodules plant⁻¹) under zero-N growth conditions. This provided evidence that the mutational event(s) affected autoregulatory control of nodulation. Moreover, all three mutants were partially tolerant to NO₃⁻; each retained greater acetylene reduction activity when grown hydroponically with 15 millimolar NO₃⁻ than did Williams at 1.5 millimolar NO₃⁻. The NO₃⁻ tolerance did not appear to be related to an altered ability to take up or metabolize NO₃⁻, based on solution NO₃⁻ depletion and on in vivo nitrate reductase assays. Enhanced nodulation appeared to be controlled by the host plant, being consistent across four Bradyrhizobium japonicum strains tested. In general, the mutant lines produced less dry weight than the control, with root dry weights being more affected than shoot dry weights. The nodulation trait has been stable through the M₅ generation in all three mutants.     

Further evidence that ouabain-resistant variants induced by chemical carcinogens in transformable C3H/10T1/2 Cl 8 mouse fibroblasts are mutants

Ouabain-resistant (Oua^r) variants were induced in C3H/10T1/2 Cl 8 cells by the chemical carcinogens, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF), and benzo[a]pyrene (BaP). The use of the Poisson calculation to determine Oua^r variant frequencies gave more linear dose-response curves than when variant frequencies were calculated from the observed number of Oua^r colonies. Increasing the Oua concentration from 3 to 6 mM decreased the frequency of Oua^r variants. When cloned Oua^r variants were mixed with wild-type cells, there was no metabolic cooperation and no loss of mutants when mock expression-time curves were determined. Oua^r variants remained Oua^r after prolonged cultivation in the absence of Oua. ⁸⁶Rubidium (⁸⁶Rb) uptake was at least 10-fold more resistant to inhibition by Oua in Oua^r variants than in wild-type cells. In one Oua^r clone, one-third of the ⁸⁶Rb uptake was not inhibited by Oua concentrations as high as 10 mM, indicating that C3H/10T1/2 Cl 8 cells might be triploid at the Oua^r locus. The relationship between the inhibition of ⁸⁶Rb uptake and the cytotoxicity caused by the same concentration of Oua was the same for 2 Oua^r clones and wild-type C3H/10T1/2 Cl 8 cells. Therefore, the Oua^r variants detected by this assay are most likely true mutants possessing an altered Na⁺K⁺ transport system, the Na⁺K⁺Mg²⁺-activated adenosine triphosphate (ATPase), that is more resistant to Oua inhibition than the ATPase in wild-type cells.     

Identification and Candidate Gene Analysis of a Novel Phytophthora Resistance Gene Rps10 in a Chinese Soybean Cultivar

Resistance to Phytophthora sojae isolate PsMC1 was evaluated in 102 F_2∶3 families derived from a cross between the resistant soybean cultivar Wandou 15 and the susceptible cultivar Williams and genotyped using simple sequence repeat (SSR) markers. The segregation ratio of resistant, segregating, and susceptible phenotypes in the population suggested that the resistance in Wandou 15 was dominant and monogenic. Twenty-six polymorphic SSR markers were identified on soybean chromosome 17 (Molecular linkage group D2; MLG D2), which were linked to the resistance gene based on bulked segregation analysis (BSA). Markers Sattwd15-24/25 and Sattwd15-47 flanked the resistance gene at a distance of 0.5 cM and 0.8 cM, respectively. Two cosegregating markers, Sattwd15-28 and Sattwd15-32, were also screened in this region. This is the first Rps resistance gene mapped on chromosome 17, which is designated as Rps10. Eight putative genes were found in the mapped region between markers Sattwd15-24/25 and Sattwd15-47. Among them, two candidate genes encoding serine/threonine (Ser/Thr) protein kinases in Wandou 15 and Williams were identified and sequenced. And the differences in genomic sequence and the putative amino acid sequence, respectively, were identified within each candidate gene between Wandou 15 and Williams. This novel gene Rps10 and the linked markers should be useful in developing soybean cultivars with durable resistance to P. sojae.     

Fur mutants of Pseudomonas aeruginosa PAO1: phenotypic characterization in terms of iron-dependent pattern of deregulation of pyoverdin synthesis

Fur mutants (Mn^r) of Pseudomonas aeruginosaFe10 (FF13 and FF28) and PAO1 (FPA12) were evaluated for the pattern of deregulation of pyoverdin synthesis in iron-replete medium with Casamino acids [CAM(Fe)] or succinate [SM(Fe)]. With respect to siderophore synthesis, we found in CAM(Fe) medium two Fur phenotypes: FurA (full deregulation, FF13) and FurB (partial deregulation, FF28 and FPA12). Fur mutants compared to parental strains grew with slower specific growth rates on SM(0) and CAM(0) media in a stirred bioreactor. Fur mutants grew in SM(0) with about half of that in CAM(0).     

High tyrosine-specific protein kinase activity in normal human peripheral blood lymphocytes

Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60^src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent K_m values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg²⁺ to Mn²⁺ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N⁶;O^2′-dibutyryl cyclic AMP (100 μM), N⁶;O^2′-dibutyryl cyclic GMP (100 μM), Ca²⁺ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with M_r 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase.     

Peroxidase Activity in Soybeans Following Inoculation with Phytophthora sojae

The effects of race-specific resistance as conditioned by Rps genes (rps, Rps1-k, Rps2, Rps3, Rps6) in two genetic backgrounds (Williams & Harosoy) on accumulation of soluble peroxidases were determined by a soybean peroxidase capture assay (SPCA) after inoculation with P. sojae races 2, 7, or 25. Peroxidase activity increased in all isolines during the 72 h after inoculation, but reactions varied depending on time after inoculation, genetic background, Rps gene and P. sojae race. Peroxidase activity was higher in race-specific resistant than in susceptible reactions at 72 h. after inoculation, except for plants with the Rps2 gene which confers a unique form of root resistance in addition to the whole plant race-specific resistance. Williams isolines had larger increases in peroxidase activity than Harosoy isolines when data were averaged across Rps genes, and was most evident when plants were inoculated with race 2. When soybeans were inoculated with race 7 Rps1-k resistant plants had the highest increase in peroxidase activity, but Rps2 susceptible plants had a significantly higher peroxidase activity than plants with rps, Rps3, and Rps6 that were also susceptible. Results from inoculations with race 25 were somewhat different, Rps2 resistant plants had the highest increase in peroxidase activity; however, plants with the Rps3 or Rps6 gene that were also resistant did not have a significantly higher peroxidase activity than susceptible plants with the rps or Rps1-k gene.     

Biochemical, Genetic, and Zoosporicidal Properties of Cyclic Lipopeptide Surfactants Produced by Pseudomonas fluorescens

Zoospores play an important role in the infection of plant and animal hosts by oomycetes and other zoosporic fungi. In this study, six fluorescent Pseudomonas isolates with zoosporicidal activities were obtained from the wheat rhizosphere. Zoospores of multiple oomycetes, including Pythium species, Albugo candida, and Phytophthora infestans, were rendered immotile within 30 s of exposure to cell suspensions or cell culture supernatants of the six isolates, and subsequent lysis occurred within 60 s. The representative strain SS101, identified as Pseudomonas fluorescens biovar II, reduced the surface tension of water from 73 to 30 mN m⁻¹. The application of cell suspensions of strain SS101 to soil or hyacinth bulbs provided significant protection against root rot caused by Pythium intermedium. Five Tn5 mutants of strain SS101lacked the abilities to reduce the surface tension of water and to cause lysis of zoospores. Genetic characterization of two surfactant-deficient mutants showed that the transposons had integrated into condensation domains of peptide synthetases. A partially purified extract from strain SS101 reduced the surface tension of water to 30 mN m⁻¹ and reached the critical micelle concentration at 25 μg ml⁻¹. Reverse-phase high-performance liquid chromatography yielded eight different fractions, five of which had surface activity and caused lysis of zoospores. Mass spectrometry and nuclear magnetic resonance analyses allowed the identification of the main constituent as a cyclic lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon hydroxy fatty acid. The other four zoosporicidal fractions were closely related to the main constituent, with molecular massesranging from 1,111 to 1,169 Da.     

Novel quantitative trait loci for partial resistance to Phytophthora sojae in soybean PI 398841

Phytophthora root and stem rot caused by Phytophthora sojae Kaufmann and Gerdemann is one of the most severe soybean [Glycine max (L.) Merr] diseases in the USA. Partial resistance is as effective in managing this disease as single-gene (Rps gene)-mediated resistance and is more durable. The objective of this study was to identify quantitative trait loci (QTL) associated with partial resistance to P. sojae in PI 398841, which originated from South Korea. A population of 305 F_7:8 recombinant inbred lines derived from a cross of OX20-8 × PI 398841 was used to evaluate partial resistance against P. sojae isolate C2S1 using a tray test. Composite interval mapping using a genome-wide logarithm of odd (LOD) threshold detected three QTL on chromosomes 1, 13, and 18, which individually explained 4–16 % of the phenotypic variance. Seven additional QTL, accounting for 2–3 % of phenotypic variance each, were identified using chromosome-wide LOD thresholds. Seven of the ten QTL for resistance to P. sojae were contributed by PI 398841. Seven QTL co-localized with known Rps genes and previously reported QTL for soil-borne root pathogens, isoflavone, and seed oil. Three QTL on chromosomes 3, 13, and 18 co-localized with known Rps genes, but PI 398841 did not exhibit an Rps gene-mediated resistance response following inoculation with 48 different isolates of P. sojae. PI 398841 is potentially a source of novel genes for improving soybean cultivars for partial resistance to P. sojae.     

Mutagenesis in cyanophage LPP-1

Mutagenesis in cyanophage LPP-1 was investigated with physical and chemical mutagens. Rapid lysis (r), host range (h) and temperature sensitivity (ts) were studied. Mutations induced by ultraviolet irradiation in free phages were photoreactivable by visible light. The h and ts markers were efficiently induced by ultraviolet, N-methyl-N′-nitro-N-nitrosoguanidine, 2-aminogpurine and acriflavine under intracellular conditions, and r mutants by ultraviolet and photodynamic action of acriflavine. The conditions required for induction are described.     

Effects of fungicides and surfactants on the zoospores of Olpidium brassicae

Lettuce big-vein disease is transmitted from diseased to healthy plants by zoospores of the lettuce root-infecting fungus Olpidium brassicae. A laboratory technique based on microscopical examination of Olpidium Zoospores is described for assaying the toxicity of chemicals to zoospores. Chemcials found to kill zoospores in <1 h included copper (4 μ/ml), zinc (10μ/ml), diluted preparations of carbendazim (methyl-2-yl-benzimidazole carbamate) as Bavistinand a formulation of Bavisitin containing no carbendazim. Bavistn controlled the disease when introduced at a concentration of 0.6 g/litre into a lettuce crop grown in a re-circulated film of nutrient. Various surfactants inlcuding Agral, Cetrimide, Deciquam, Ethylan CPX, Hyamine 1622, Manoxol/OT and sodium lauryl sulphate were toxic to zoospores at concentrations of 1–10 μ/ml.     

The nature of single-strand breaks in DNA following treatment of L-cells with methylating agents

DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 ± 0.58·10⁸ daltons as measured by sedimentation in an alkaline sucrose gradient. This value was reduced by one half after the cells were treated for 1 h with 8 μg/ml of N-methyl-N-nitrosourea (MNUA), 34 μg/ml of methyl methanesulfonate (MMS) or 0.16 μg/ml of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). That dose of MNUA produced 52 methylations per 5.7·10⁸ daltons DNA. 20% of these were not purine derivatives and were assumed to contain some phosphotriesters. That dose of MMS (above) produced 290 methylations per 5.7·10⁸ daltons DNA and about 14% of these were not purine derivatives. The rates of loss of methylated purines from DNA were 2.3% per hour for 7-methylguanine (7-MeG), 7.4% per hour for 3-methyladenine (3-MeA) and no detectable loss of O⁶-methylguanine (O⁶-MeG) over a 12 h period. Since phosphotriesters are alkali-labile the single-strand breaks probably arose from this structure and did not form within the cell. This conclusion is supported by the following considerations. MNUA was more effective than MMS at reducing the molecular weight of DNA, as measured in alkaline medium. The greater SN₁ character of MNUA would cause a greater formation of phosphotriesters than would MMS.     

Improvement of milbemycin-producing <Emphasis Type="Italic">Streptomyces bingchenggensis</Emphasis> by rational screening of ultraviolet- and chemically induced mutants

Milbemycin antibiotics are produced by Streptomyces hygroscopicus subsp. aureolacrimosus and a newly isolated Streptomyces bingchenggensis, respectively. According to the biosynthetic pathway of milbemycins generated by S. hygroscopicus subsp. aureolacrimosus, a rational screening procedure with UV irradiation and N-methyl-N′-nitroso-N-nitrosoguanidine (NTG) mutation was performed to obtain high milbemycin-producing S. bingchenggensis. Aminoacetic acid (Glycine)-resistant mutants (AA^r), propionate-resistant mutants (PRP^r), streptomycin-resistant mutants (STR^r) and 2-deoxy-d-glucose-resistant mutants (DOG^r) were selected successively. A strain S. bingchenggensis BC-109-6 with AA^r, PRPr, STR^r and DOG^r was obtained and its production of milbemycin A3 and A4 reached 1,450 μg/ml, which was 80% higher than that of the ancestral strain S. bingchenggensis BC-101-4. The subculture experiments indicated that the hereditary characteristic of high productivity of S. bingchenggensis BC-109-6 was stable. The production of milbemycin A3 and A4 by S. bingchenggensis BC-109-6 in a 50-l fermentor could reach 1,380 μg/ml after 360 h batch fermentation.     

Phytophthora nicotianae transformants lacking dynein light chain 1 produce non-flagellate zoospores

Biflagellate zoospores of the highly destructive plant pathogens in the genus Phytophthora are responsible for the initiation of infection of host plants. Zoospore motility is a critical component of the infection process because it allows zoospores to actively target suitable infection sites on potential hosts. Flagellar assembly and function in eukaryotes depends on a number of dynein-based molecular motors that facilitate retrograde intraflagellar transport and sliding of adjacent microtubule doublets in the flagellar axonemes. Dynein light chain 1 (DLC1) is one of a number of proteins in the dynein outer arm multiprotein complex. It is a 22 kDa leucine-rich repeat protein that binds to the catalytic motor domain of the dynein γ heavy chain. We report the cloning and characterization of DLC1 homologues in Phytophthora cinnamomi and Phytophthora nicotianae (PcDLC1 and PnDLC1). PcDLC1 and PnDLC1 are single copy genes that are more highly expressed in sporulating hyphae than in vegetative hyphae, zoospores or germinated cysts. Polyclonal antibodies raised against PnDLC1 locallized PnDLC1 along the length of the flagella of P. nicotianae zoospores. RNAi-mediated silencing of PnDLC1 expression yielded transformants that released non-flagellate, non-motile zoospores from their sporangia. Our observations indicate that zoospore motility is not required for zoospore release from P. nicotianae sporangia or for breakage of the evanescent vesicle into which zoospores are initially discharged.     

DNA mismatch repair MSH2 gene-based SNP associated with different populations

We screened for the major essential single-nucleotide polymorphism (SNP) variant that might be associated with the MSH2 gene based on the data available from three types of human tissue samples [156 lymphoblastoid cell variations (LCL), 160 epidermis, 166 fat]. An association analysis confirmed that the KCNK12 SNP variant (rs748780) was highly associated (p value 9 × 10^?4) with the MSH2 gene for all three samples. Using SNP identification, we further found that the recognized SNP was also relevant among Hapmap populations. Techniques that display specific SNPs associated with the gene of interest or nearby genes provide more reliable genetic associations than techniques that rely on data from individual SNPs. We investigated the MSH2 gene regional linkage association with the determined SNP (rs748780), KCNK12 variant (Allele T>C) in the intronic region, in HapMap3 full dataset populations, Yoruba in Ibadan, Nigeria (YRI), Utah residents with ancestry from northern Europe (CEU), Han Chinese in Beijing, China (CHB), and a population of Mexican ancestry in Los Angeles, California (MEX). A gene-based SNP association analysis analyzes the combined impact of every variant within the gene while creating referrals to linkage disequilibrium or connections between markers. Our results indicated that among the four populations studied, this association was highest in the MEX population based on the r ² value; a similar pattern was also observed in the other three populations. The relevant SNP rs748780 in KCNK12 is related to a superfamily of potassium channel pore-forming P-domain proteins as well as to other non-pore-forming proteins and has been shown to be relevant to neurological disorder predisposition in MEX as well as in other populations.     

Improvement of pristinamycin-producing Streptomyces pristinaespiralis by rational screening

Summary According to the biosynthetic pathway of pristinamycin, a rational selection procedure with u.v. mutation was performed to obtain a high pristinamycin-producing strain. Aminoacetic acid-resistant mutants (AA^r), valine hydroxamate-resistant mutants (VH^r), kitasamycin-resistant mutants (KTM^r) and 2-deoxy-D-glucose-resistant mutants (DOG^r) were selected, successively. A strain Streptomyces pristinaespiralis 12–55 with AA^r, Val^r, KTM^r, and DOG^r was obtained, and its production of pristinamycin reached 3000 u/ml which is 100 times higher than that of the parent strain S. pristinaespiralis ATCC 25486. It is inferred that S. pristinaespiralis 12–55 can alleviate catabolite repression caused by carbon sources, provide more acetic acid and valine for pristinamycin biosynthesis and increase its resistance to pristinamycin produced by itself, all of which are favorable for pristinamycin production. The subculture experiments indicated that the hereditary character of high productivity of S. pristinaespiralis 12–55 is stable. The pristinamycin production of S. pristinaespiralis 12–55 in a 15-l fermentor could reach 3010 u/ml after a 56 h batch fermentation.     

Calcium efflux associated with encystment of Phytophthora palmivora zoospores

Zoospores of the fungus $\text{Phytophthora palmivora}$, pre-labeled with ⁴⁵Ca, excreted up to 30% of their total ⁴⁵Ca when stimulated to encyst. Excretion was essentially completed within 90 sec of the application of the stimulus. Encystment of the population was completed within 5 min. Four different stimuli were used: pectin addition (420 μg ml^?1), Sr²⁺ addition (5 mM), cyclic AMP addition (6.7 mM) and mechanical agitation. The kinetics and amount of Ca excretion were essentially the same in each case. The calcium ionophore A23187 increased the rate of ⁴⁵Ca uptake by motile zoospores, incubated in 100 μM CaCl₂, but did not induce encystment under these conditions. The ionophore did not induce ⁴⁵Ca efflux from pre-labeled zoospores. Incubation in EGTA and in K⁺ failed to induce either encystment or ⁴⁵Ca excretion. We conclude that rapid excretion of a significant proportion of the zoospore calcium is linked to the early stage of stimulus-induced encystment, and that this comes from an intracellularly located, non-cytoplasmic source, such as the peripheral vesicles, but that changes in cellular Ca²⁺ are not necessarily the single controlling factor in the induction of encystment.