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Background Macrolides are recommended for treating the emerging enteropathogen Arcobacter butzleri ; nonetheless, this bacterium often exhibits highly variable resistance rates, and the mechanisms behind this resistance phenotype remain largely unexplored. Objectives To understand the phenotypic and genotypic consequences associated with the acquisition of erythromycin resistance in A. butzleri , as well as the effects on the fitness of this species. Methods Resistant strains resulting from spontaneous mutations and adaptive laboratory evolution under increasing erythromycin concentrations were examined regarding their cross-resistance and collateral susceptibility profiles. Genetic causes of phenotypic antibiotic resistance were analysed by sequencing and bioinformatics, with functional correlation through ethidium bromide accumulation assays. Growth profiles in the presence and absence of erythromycin, motility and biofilm formation abilities were assessed to detect potential changes in fitness and virulence. Results Clones from spontaneous mutation rate evolution demonstrated decreased susceptibility to erythromycin and other classes of antibiotics, associated with mutations in the transcriptional repressor areR , causing overexpression of the AreABC efflux pump. In turn, WGS analysis of the evolved strain showed additional mutations in the ribosomal proteins L4 and L22 and in the areR gene. Furthermore, the acquisition of macrolide resistance altered A. butzleri virulence and entailed a high biological cost. Conclusions The findings of this study have proved that efflux activity contributes synergistically with mutations in the ribosomal proteins L4 and L22 to A. butzleri 's high-level macrolide resistance. The results further suggest an impact on the bacterial physiology and virulence, with the increased fitness cost justifying the low worldwide prevalence of high-level resistant circulating strains. [ABSTRACT FROM AUTHOR]

In the early 1900s, mutation breeding to select varieties with desirable traits using spontaneous mutation was actively conducted around the world, including Japan. In rice, the number of fixed mutations per generation was estimated to be 1.38-2.25. Although this low mutation rate was a major problem for breeding in those days, in the modern era with the development of next-generation sequencing (NGS) technology, it was conversely considered to be an advantage for efficient gene identification. In this paper, we proposed an in silico approach using NGS to compare the whole genome sequence of a spontaneous mutant with that of a closely related strain with a nearly identical genome, to find polymorphisms that differ between them, and to identify the causal gene by predicting the functional variation of the gene caused by the polymorphism. Using this approach, we found four causal genes for the dwarf mutation, the round shape grain mutation and the awnless mutation. Three of these genes were the same as those previously reported, but one was a novel gene involved in awn formation. The novel gene was isolated from Bozu-Aikoku, a mutant of Aikoku with the awnless trait, in which nine polymorphisms were predicted to alter gene function by their whole-genome comparison. Based on the information on gene function and tissue-specific expression patterns of these candidate genes, Os03g0115700/LOC_Os03g02460, annotated as a shortchain dehydrogenase/reductase SDR family protein, is most likely to be involved in the awnless mutation. Indeed, complementation tests by transformation showed that it is involved in awn formation. Thus, this method is an effective way to accelerate genome breeding of various crop species by enabling the identification of useful genes that can be used for crop breeding with minimal effort for NGS analysis. [ABSTRACT FROM AUTHOR]

Abstract Background Over evolutionary timescales, genomic loci can switch between functional and non-functional states through processes such as pseudogenization and de novo gene birth. Particularly, de novo gene birth is a widespread process, and many examples continue to be discovered across diverse evolutionary lineages. However, the general mechanisms that lead to functionalization are poorly understood, and estimated rates of de novo gene birth remain contentious. Here, we address this problem within a model that takes into account mutations and structural variation, allowing us to estimate the likelihood of emergence of new functions at non-functional loci. Results Assuming biologically reasonable mutation rates and mutational effects, we find that functionalization of non-genic loci requires the realization of strict conditions. This is in line with the observation that most de novo genes are localized to the vicinity of established genes. Our model also provides an explanation for the empirical observation that emerging proto-genes are often lost despite showing signs of adaptation. Conclusions Our work elucidates the properties of non-genic loci that make them fertile for adaptation, and our results offer mechanistic insights into the process of de novo gene birth.

A spontaneous mutant of the duckweed Lemna gibba clone no. 7796 (known as strain G3, WT) was discovered. In this mutant clone, L. gibba clone no. 9602 (mt), the morphological parameters (frond length, frond width, root length, root diameter) indicated an enlarged size. A change in the frond shape was indicated by the decreased frond length/width ratio, which could have taxonomic consequences. Several different cell types in both the frond and the root were also enlarged. Flow cytometric measurements disclosed the genome size of the WT as 557 Mbp/1C and that of the mt strain as 1153 Mbp/1C. This represents the results of polyploidisation of a diploid clone to a tetraploid one. The mutant clone flowered under the influence of long day-treatment in half-strength Hutner's medium in striking contrast to the diploid WT. Low concentration of salicylic acid (<1 µM) induced flowering in the tetraploid mutant but not in the diploid plants. The transcript levels of nuclear-encoded genes of the photosynthetic apparatus (CAB, RBCS) showed higher abundance in light and less dramatic decline in darkness in the mt than in WT, while this was not the case with plastid-encoded genes (RBCL, PSAA, PSBA, PSBC). [ABSTRACT FROM AUTHOR]

The spontaneous mutation rate µ is a crucial parameter to understand evolution and biodiversity. Mutation rates are highly variable across species, suggesting that µ is susceptible to selection and drift and that species life cycle and life history may impact its evolution. In particular, asexual reproduction and haploid selection are expected to affect the mutation rate, but very little empirical data are available to test this expectation. Here, we sequence 30 genomes of a parent–offspring pedigree in the model brown alga Ectocarpus sp.7, and 137 genomes of an interspecific cross of the closely related brown alga Scytosiphon to have access to the spontaneous mutation rate of representative organisms of a complex multicellular eukaryotic lineage outside animals and plants, and to evaluate the potential impact of life cycle on the mutation rate. Brown algae alternate between a haploid and a diploid stage, both multicellular and free living, and utilize both sexual and asexual reproduction. They are, therefore, excellent models to empirically test expectations of the effect of asexual reproduction and haploid selection on mutation rate evolution. We estimate that Ectocarpus has a base substitution rate of µ bs = 4.07 × 10−10 per site per generation, whereas the Scytosiphon interspecific cross had µ bs = 1.22 × 10−9. Overall, our estimations suggest that these brown algae, despite being multicellular complex eukaryotes, have unusually low mutation rates. In Ectocarpus , effective population size (N e) could not entirely explain the low µ bs. We propose that the haploid–diploid life cycle, combined with extensive asexual reproduction, may be additional key drivers of the mutation rate in these organisms. [ABSTRACT FROM AUTHOR]

In the entire scenario of Pakistan citrus sector, kinnow (Citrus reticulata Blanco) right from its introduction and successful acclimatization continues to dominate both in area and production besides an exportable commodity. Presence of large seed number in Kinnow is big issue for fresh consumption and as well as for processing industry in the international markets. Visualizing such compulsions of international markets, a systematic study was undertaken by Horticultural Research Station, Sahiwal with identification of five less seeded kinnow strains (1-6 seeds). Results depicted that less seeded kinnow strain, encoded LSKS-2, produced superior external and internal quality attributes. Average fruit weight and size outstripped other contestant strains. It produces average weight per fruit as 170.58gms with length/breadth, as 70.68mm and 68.08 mm respectively. Maximum juice recovery as 46.22% with glucose 48.08gms per liter, fructose 27.72gms per liter was obtained significantly high in less seeded strain LSKS-2. Total soluble solids as 11.70% remained in the same strain (LSKS-2). Vitamin C was18.43 mg/100gms besides total phenolic compounds 94.78 µgms in LSKS-2. However, somewhat tenacious fruit with more peel thickness was observed in LSKS-4 which was 3.31 mm. Highest firmness remained 2.48kg in LSKS-1 of less seeded kinnow. Conclusively less seeded strain LSKS-2 proved best of all the five strains of less seeded kinnow. The study is worthwhile for exploiting natural mutant kinnow strains bearing less seeded fruit of high quality characters through vegetative method of propagation thereby avoiding inevitable heterozygosity and polyembyonic nature of citrus fruits. [ABSTRACT FROM AUTHOR]

‘SCS453 Noninha’ and ‘SCS454 Carvoeira’ are new banana cultivars derived from the subgroup Prata (AAB). In comparison with the latter, ‘SCS453 Noninha’ has a low pseudostem height, ‘SCS454 Carvoeira’ a high yield and both unchanged fruit characteristics. Both also have increased resistance to Panama wilt and ‘SCS454 Carvoeira’ to Sigatoka. [ABSTRACT FROM AUTHOR]

The increasingly widespread use of antibiotics has raised an urgent public health question: how does nonlethal exposure of antibiotics increase rates of bacterial mutations that confer antibiotic resistance? Quantitative studies of this sort are rare, and interested investigators tend to neglect scenarios where the mutation inducer and the selective agent are the same. One possible reason for this puzzling neglect is that such an investigation may lead to results that are prone to a Lamarckian interpretation, but a more tangible reason is that successful estimation of mutation rates in such scenarios requires new computational methods. This study presents computational methods tailored for a mutation model in which some wide-type cells may be killed by nonlethal exposure to an antibiotic, but in which mutants proliferate unimpeded. Methods for computing maximum likelihood estimates of mutation rates and their corresponding confidence intervals are devised and evaluated by simulations. [ABSTRACT FROM AUTHOR]

Mutations are the origin of genetic diversity and are fundamental parameters needed to understand the molecular evolution of species. Estimations of mutation rates have been conducted for many diverse taxa, although rates in several major eukaryotic lineages remain unexplored. Here, the first estimation is reported of the spontaneous mutation rate for the multicellular eukaryote red alga, Pyropia yezoensis , which exhibits a complex life cycle. An estimated mutation rate of 2.97 × 10−8 (95 % CI: 2.16 × 10−8–3.99 × 10−8) per site per generation was generated for the primary life cycle, the sexual cycle, which is the highest sexual mutation rate among published sexual plants. Combined with tetrad analysis, meiosis I was identified as the primary period responsible for the high mutation rate during the complex life cycle of P. yezoensis. This result provides direct evidence for the "meiosis is mutagenic" hypothesis for multicellular organisms. The accurate estimate of the mutation rate of P. yezoensis also informs several immediate applications. Based on the above estimate, the effective population size (N e) of P. yezoensis was estimated at about 19,000, with extensive haploid phases and asexual reproduction through monospores possibly leading to linked selection that may reduce the genome-wide genetic diversity of P. yezoensis and consequently influence N e estimation. Lastly, P. yezoensis was estimated to have diverged from P. haitanensis about 4.2 Ma, representing a more recent date than estimates from fossil-calibrated phylogenies. These findings provide valuable new information for understanding the evolution of red algae, in addition to the underlying mechanism of mutations. • The first spontaneous mutation rate for the multicellular red alga, P. yezoensis , has been reported. • Meiosis I has been identified as the primary period responsible for the high mutation rate of P. yezoensis. • The effective population size of P. yezoensis has been estimated at about 19,000. • P. yezoensis has been estimated to diverge from P. haitanensis about 4.2 Ma. [ABSTRACT FROM AUTHOR]

The process of tooth formation is a series of reciprocal interactions between the ectoderm and mesoderm, and it is believed that many genetic factors are involved in this complex process. More than a dozen genes have been identified in non-syndromic tooth agenesis; however, the genetic etiology underlying tooth agenesis is not fully understood yet. In this study, we identified two novel LRP6 mutations in two non-syndromic oligodontia families. Both probands had 16 and 17 missing teeth in their permanent dentition. Mutational analysis identified a de novo frameshift mutation by a 1-bp insertion in exon 9 (NM_002336.2: c.1870dupA, p.(Met624Asnfs*29)) and a splicing donor site mutation in intron 8 (c.1762+2T>C). An in vitro splicing assay confirmed the deletion of exon 8, and the deletion would result in a frameshift. Due to the premature termination codons introduced by the frameshift, both mutant transcripts would be degraded by nonsense-mediated mRNA decay, resulting in haploinsufficiency. [ABSTRACT FROM AUTHOR]

We revisit our earlier paper, with two of the coauthors, in which we proposed an unbiased and consistent estimator μ ^ n for an unknown mutation rate μ of microorganisms. Previously, we proved that the associated sequence of estimators μ ^ n converges to μ almost surely pointwise on a nonextinct set Ω 0 . Here, we show that this sequence converges also in the mean square with respect to conditional probability measure P 0 · = P · ∩ Ω 0 / P Ω 0 and that, with respect to P 0 , the estimator is asymptotically unbiased. We further assume that a microorganism can mutate or turn to a different variant of one of the two types. In particular, it can mean that bacteria under attack by a virus or chemical agent are either perishing or surviving, turning them to stronger variant. We propose estimators for their respective types and show that they are a.s. pointwise and L 2 -consistent and asymptotically unbiased with respect to measure P 0 . [ABSTRACT FROM AUTHOR]

A spontaneous mutant of the duckweed Lemna gibba clone no. 7796 (known as strain G3, WT) was discovered. In this mutant clone, L. gibba clone no. 9602 (mt), the morphological parameters (frond length, frond width, root length, root diameter) indicated an enlarged size. A change in the frond shape was indicated by the decreased frond length/width ratio, which could have taxonomic consequences. Several different cell types in both the frond and the root were also enlarged. Flow cytometric measurements disclosed the genome size of the WT as 557 Mbp/1C and that of the mt strain as 1153 Mbp/1C. This represents the results of polyploidisation of a diploid clone to a tetraploid one. The mutant clone flowered under the influence of long day-treatment in half-strength Hutner’s medium in striking contrast to the diploid WT. Low concentration of salicylic acid (CAB, RBCS) showed higher abundance in light and less dramatic decline in darkness in the mt than in WT, while this was not the case with plastid-encoded genes (RBCL, PSAA, PSBA, PSBC).

Because errors at the DNA level power pathogen evolution, a systematic understanding of the rate and molecular spectra of mutations could guide the avoidance and treatment of infectious diseases. We thus accumulated tens of thousands of spontaneous mutations in 768 repeatedly bottlenecked lineages of 18 strains from various geographical sites, temporal spread, and genetic backgrounds. Entailing over ∼1.36 million generations, the resultant data yield an average mutation rate of ∼0.0005 per genome per generation, with a significant within-species variation. This is one of the lowest bacterial mutation rates reported, giving direct support for a high genome stability in this pathogen resulting from high DNA-mismatch-repair efficiency and replication-machinery fidelity. Pathogenicity genes do not exhibit an accelerated mutation rate, and thus, elevated mutation rates may not be the major determinant for the diversification of toxin and secretion systems. Intriguingly, a low error rate at the transcript level is not observed, suggesting distinct fidelity of the replication and transcription machinery. This study urges more attention on the most basic evolutionary processes of even the best-known human pathogens and deepens the understanding of their genome evolution. [ABSTRACT FROM AUTHOR]

Genetic background often influences the phenotypic consequences of mutations, resulting in variable expressivity. How standing genetic variants collectively cause this phenomenon is not fully understood. Here, we comprehensively identify loci in a budding yeast cross that impact the growth of individuals carrying a spontaneous missense mutation in the nuclear-encoded mitochondrial ribosomal gene MRP20. Initial results suggested that a single large effect locus influences the mutation's expressivity, with 1 allele causing inviability in mutants. However, further experiments revealed this simplicity was an illusion. In fact, many additional loci shape the mutation's expressivity, collectively leading to a wide spectrum of mutational responses. These results exemplify how complex combinations of alleles can produce a diversity of qualitative and quantitative responses to the same mutation. [ABSTRACT FROM AUTHOR]

Serratia marcescens is a common bacterium well-known for the red secondary metabolite prodigiosin. However, color mutants have long been described. Non-pigmented strains can be found to exist both naturally and under laboratory conditions. It is unclear why S. marcescens loses prodigiosin synthesis capacity in certain conditions. In the present study, we find that the spontaneous color mutants arise within a few generations (about five passages) and rapidly replace the wild-type parent cells (about 24 passages), which indicates a growth advantage of the former. Although, the loss of prodigiosin synthesis genes (pigA-N) is frequently reported as the major reason for pigment deficiency, it was unexpected that the whole gene cluster is completely preserved in the different color morphotypes. Comparative transcriptomic analysis indicates a dramatic variation at the transcriptional level. Most of the pig genes are significantly downregulated in the color morphotypes which directly lead to prodigiosin dyssynthesis. Besides, the transcriptional changes of several other genes have been noticed, of which transcriptional regulators, membrane proteins, and nearly all type VI secretion system (T6SS) components are generally downregulated, while both amino acid metabolite and transport systems are activated. In addition, we delete the transcription regulator slyA to generate a non-pigmented mutant. The Δ slyA strain loses prodigiosin synthesis capacity, but has a higher cell density, and surprisingly enhances the virulence as an entomopathogen. These data indicate that S. marcescens shuts down several high-cost systems and activates the amino acid degradation and transport pathways at the transcriptional level to obtain extra resources, which provides new insights into the competitive growth advantage of bacterial spontaneous color mutants. [ABSTRACT FROM AUTHOR]

Sugarcane mosaic virus (SCMV; genus Potyvirus) induces maize dwarf mosaic disease that has caused serious yield losses of maize in China. Cross-protection is one of the efficient strategies to fight against severe virus strains. Although many mild strains have been identified, the spontaneous mutation is one of the challenging problems affecting their application in cross-protection. In this study, we found that the substitution of cysteine (C) at positions 57 or 60 in the zinc finger-like motif of HC-Pro with alanine (A; C57A or C60A) significantly reduced its RNA silencing suppression activity and SCMV virulence. To reduce the risk of mild strains mutating to virulent ones by reverse or complementary mutations, we obtained attenuated SCMV mutants with double-mutations in the zinc finger-like and FRNK motifs of HC-Pro and evaluated their potential application in cross-protection. The results showed that the maize plants infected with FKNK/C60A double-mutant showed symptomless until 95 days post-inoculation and FKNK/C60A cross-protected plants displayed high resistance to severe SCMV strain. This study provides theoretical and material bases for the control of SCMV through cross-protection. [ABSTRACT FROM AUTHOR]

Serratia marcescens is a common bacterium well-known for the red secondary metabolite prodigiosin. However, color mutants have long been described. Non-pigmented strains can be found to exist both naturally and under laboratory conditions. It is unclear why S. marcescens loses prodigiosin synthesis capacity in certain conditions. In the present study, we find that the spontaneous color mutants arise within a few generations (about five passages) and rapidly replace the wild-type parent cells (about 24 passages), which indicates a growth advantage of the former. Although, the loss of prodigiosin synthesis genes (pigA-N) is frequently reported as the major reason for pigment deficiency, it was unexpected that the whole gene cluster is completely preserved in the different color morphotypes. Comparative transcriptomic analysis indicates a dramatic variation at the transcriptional level. Most of the pig genes are significantly downregulated in the color morphotypes which directly lead to prodigiosin dyssynthesis. Besides, the transcriptional changes of several other genes have been noticed, of which transcriptional regulators, membrane proteins, and nearly all type VI secretion system (T6SS) components are generally downregulated, while both amino acid metabolite and transport systems are activated. In addition, we delete the transcription regulator slyA to generate a non-pigmented mutant. The ΔslyA strain loses prodigiosin synthesis capacity, but has a higher cell density, and surprisingly enhances the virulence as an entomopathogen. These data indicate that S. marcescens shuts down several high-cost systems and activates the amino acid degradation and transport pathways at the transcriptional level to obtain extra resources, which provides new insights into the competitive growth advantage of bacterial spontaneous color mutants.

Sugarcane mosaic virus (SCMV; genus Potyvirus) induces maize dwarf mosaic disease that has caused serious yield losses of maize in China. Cross-protection is one of the efficient strategies to fight against severe virus strains. Although many mild strains have been identified, the spontaneous mutation is one of the challenging problems affecting their application in cross-protection. In this study, we found that the substitution of cysteine (C) at positions 57 or 60 in the zinc finger-like motif of HC-Pro with alanine (A; C57A or C60A) significantly reduced its RNA silencing suppression activity and SCMV virulence. To reduce the risk of mild strains mutating to virulent ones by reverse or complementary mutations, we obtained attenuated SCMV mutants with double-mutations in the zinc finger-like and FRNK motifs of HC-Pro and evaluated their potential application in cross-protection. The results showed that the maize plants infected with FKNK/C60A double-mutant showed symptomless until 95 days post-inoculation and FKNK/C60A cross-protected plants displayed high resistance to severe SCMV strain. This study provides theoretical and material bases for the control of SCMV through cross-protection.

ABSTRACT Trehalose biosynthesis pathway is a potential target for antifungal drug development, and trehalose 6-phosphate (T6P) accumulation is widely known to have toxic effects on cells. However, how organisms maintain a safe T6P level and cope with its cytotoxicity effects when accumulated have not been reported. Herein, we unveil the mechanism by which the rice blast fungus Magnaporthe oryzae avoids T6P accumulation and the genetic and physiological adjustments it undergoes to self-adjust the metabolite level when it is unavoidably accumulated. We found that T6P accumulation leads to defects in fugal development and pathogenicity. The accumulated T6P impairs cell wall assembly by disrupting actin organization. The disorganization of actin impairs the distribution of chitin synthases, thereby disrupting cell wall polymer distribution. Additionally, accumulation of T6P compromise energy metabolism. M. oryzae was able to overcome the effects of T6P accumulation by self-mutation of its MoTPS3 gene at two different mutation sites. We further show that mutation of MoTPS3 suppresses MoTps1 activity to reduce the intracellular level of T6P and partially restore ΔMotps2 defects. Overall, our results provide insights into the cytotoxicity effects of T6P accumulation and uncover a spontaneous mutation strategy to rebalance accumulated T6P in M. oryzae. IMPORTANCE M. oryzae, the causative agent of the rice blast disease, threatens rice production worldwide. Our results revealed that T6P accumulation, caused by the disruption of MoTPS2, has toxic effects on fugal development and pathogenesis in M. oryzae. The accumulated T6P impairs the distribution of cell wall polymers via actin organization and therefore disrupts cell wall structure. M. oryzae uses a spontaneous mutation to restore T6P cytotoxicity. Seven spontaneous mutation sites were found, and a mutation in MoTPS3 was further identified. The spontaneous mutation in MoTPS3 can partially rescue ΔMotps2 defects by suppressing MoTps1 activity to alleviate T6P cytotoxicity. This study provides clear evidence for better understanding of T6P cytotoxicity and how the fungus protects itself from T6P’s toxic effects when it has accumulated to severely high levels.

We revisit our earlier paper, with two of the coauthors, in which we proposed an unbiased and consistent estimator μ^n for an unknown mutation rate μ of microorganisms. Previously, we proved that the associated sequence of estimators μ^n converges to μ almost surely pointwise on a nonextinct set Ω0. Here, we show that this sequence converges also in the mean square with respect to conditional probability measure P0·=P·∩Ω0/PΩ0 and that, with respect to P0, the estimator is asymptotically unbiased. We further assume that a microorganism can mutate or turn to a different variant of one of the two types. In particular, it can mean that bacteria under attack by a virus or chemical agent are either perishing or surviving, turning them to stronger variant. We propose estimators for their respective types and show that they are a.s. pointwise and L2-consistent and asymptotically unbiased with respect to measure P0.

The spontaneous mutation rate is a very variable trait that is subject to drift, selection and is sometimes highly plastic. Consequently, its variation between close species, or even between populations from the same species, can be very large. Here, I estimated the spontaneous mutation rate of Drosophila pseudoobscura and Drosophila persimilis crosses to explore the mutation rate variation within the Drosophila genus. All mutation rate estimations in Drosophila varied fourfold, probably explained by the sensitivity of the mutation rate to environmental and experimental conditions. Moreover, I found a very high mutation rate in the hybrid cross between D. pseudoobscura and D. persimilis, in agreement with known elevated mutation rate in hybrids. This mutation rate increase can be explained by heterozygosity and fitness decrease effects in hybrids. [ABSTRACT FROM AUTHOR]

Continuous passaging in vitro can lead to the accumulation of changes in DNA sequence that potentially affect the properties of microbes, making them different from the original isolates. The identification of such genetic alterations is rare in fungi. A set of insertional mutants in the plant pathogenic fungus Leptosphaeria maculans , all derived from the same transformation experiment, had independent Agrobacterium T-DNA insertions and reduced pathogenicity on canola (Brassica napus). None of the insertions co-segregated in progeny from crosses with the reduction in pathogenicity. Genome sequences of three strains were analysed, and a mutation identified in a gene (ptf1 , for pathogenicity-associated transcription factor 1) encoding a putative Zn 2 (II)Cys 6 transcription factor. Homologs are found in other ascomycetes, and are required for pathogenicity by Fusarium graminearum , Fusarium oxysporum and Magnaporthe oryzae. The mutation in the L. maculans ptf1 gene co-segregates in progeny from crosses with the reduction in pathogenicity, a strain with an independent mutant allele isolated using CRISPR-Cas9 editing has reduced pathogenicity, and addition of wild type copies of the gene restores pathogenicity. Thus, this work defines a base pair substitution that occurred during in vitro passaging of a fungus that contributed to an attenuation of pathogenicity. • Fungal strains from the same transformation experiment had reduced pathogenicity. • T-DNA insertion were not responsible for reduced disease. • Point mutations were found in a new gene, ptf1 , in Leptosphaeria maculans. • ptf1 encodes a transcription factor conserved in other plant pathogens. • The basis behind mutations during in vitro culture can be identified in fungi. [ABSTRACT FROM AUTHOR]

The Photosystem II water-plastoquinone oxidoreductase is a multi-subunit complex which catalyses the light-driven oxidation of water to molecular oxygen in oxygenic photosynthesis. The D1 reaction centre protein exists in multiple forms in cyanobacteria, including D1FR which is expressed under far-red light. We investigated the role of Phe184 that is found in the lumenal cd-loop of D1FR but is typically an isoleucine in other D1 isoforms. The I184F mutant in Synechocystis sp. PCC 6803 was similar to the control strain but accumulated a spontaneous mutation that introduced a Gln residue in place of His252 located on the opposite side of the thylakoid membrane. His252 participates in the protonation of the secondary plastoquinone electron acceptor Q B. The I184F:H252Q double mutant exhibited reduced high-light-induced photodamage and an altered Q B -binding site that impaired herbicide binding. Additionally, the H252Q mutant had a large increase in the variable fluorescence yield although the number of photochemically active PS II centres was unchanged. In the I184F:H252Q mutant the extent of the increased fluorescence yield decreased. Our data indicates substitution of Ile184 to Phe modulates PS II-specific variable fluorescence in cells with the His252 to Gln substitution by modifying the Q B -binding site. • Ile184 in the lumenal cd-loop of D1 was changed to Phe found in far-red forms of D1. • Mutation of Ile184 led to a cytosolic His252 change to Gln and modified Q B -binding. • His252 to Gln reduced ROS and herbicide binding, and increased PS II fluorescence. • The PS II variable fluorescence was attenuated in the I184F:H252Q double mutant. • Substitution at Ile184 in the cd-loop modifies the acceptor side of PS II at His252. [ABSTRACT FROM AUTHOR]

Abstract Background Riemerella anatipestifer (R. anatipestifer) is one of the most important poultry pathogens worldwide, with associated infections causing significant economic losses. Rifampin Resistance is an important mechanism of drug resistance. However, there is no information about rpoB mutations conferring rifampin resistance and its fitness cost in Riemerella anatipestifer. Results Comparative analysis of 18 R.anatipestifer rpoB sequences and the determination of rifampin minimum inhibitory concentrations showed that five point mutations, V382I, H491N, G502K, R494K and S539Y, were related to rifampin resistance. Five overexpression strains were constructed using site-directed mutagenesis to validate these sites. To investigate the origin and fitness costs of the rpoB mutations, 15 types of rpoB mutations were isolated from R. anatipestifer ATCC 11845 by using spontaneous mutation in which R494K was identical to the type of mutation detected in the isolates. The mutation frequency of the rpoB gene was calculated to be 10− 8. A total of 98.8% (247/250) of the obtained mutants were located in cluster I of the rifampin resistance-determining region of the rpoB gene. With the exception of D481Y, I537N and S539F, the rifampin minimum inhibitory concentrations of the remaining mutants were at least 64 μg/mL. The growth performance and competitive experiments of the mutant strains in vitro showed that H491D and 485::TAA exhibit growth delay and severely impaired fitness. Finally, the colonization abilities and sensitivities of the R494K and H491D mutants were investigated. The sensitivity of the two mutants to hydrogen peroxide (H2O2) and sodium nitroprusside (SNP) increased compared to the parental strain. The number of live colonies colonized by the two mutants in the duckling brain and trachea were lower than that of the parental strain within 24 h. Conclusions Mutations of rpoB gene in R. anatipestifer mediate rifampin resistance and result in fitness costs. And different single mutations confer different levels of fitness costs. Our study provides, to our knowledge, the first estimates of the fitness cost associated with the R. anatipestifer rifampin resistance in vitro and in vivo.

In China, a soil-borne virus causing a disease of winter wheat and associated with Polymyxa graminis, has been reported for many years and is now recognized as a new species, Chinese wheat mosaic virus (CWMV). Since the determination of its genomic sequence, more progress has been made in understanding its genomic structure and functions. Molecular and serological methods have been developed to help survey the distribution of the virus and to provide the basic information needed for disease forecasting and control. At present, the best countermeasure is cultivation of resistant wheat varieties. In addition, development and application of some auxiliary countermeasures, such as rotation of non-host crops, delayed seed-sowing, reasonable application of nitrogen fertilizer, and treatment of imported seeds with fungicides before sowing, may be helpful for controlling the disease. The viral distribution and damage, virion properties, genome organization and spontaneous mutation, temperature sensitivity, and disease management options are here reviewed and/or discussed to help in developing more cost-effective countermeasures to control the disease in the future.

In the early 1900s, mutation breeding to select varieties with desirable traits using spontaneous mutation was actively conducted around the world, including Japan. In rice, the number of fixed mutations per generation was estimated to be 1.38-2.25. Although this low mutation rate was a major problem for breeding in those days, in the modern era with the development of next-generation sequencing (NGS) technology, it was conversely considered to be an advantage for efficient gene identification. In this paper, we proposed an in silico approach using NGS to compare the whole genome sequence of a spontaneous mutant with that of a closely related strain with a nearly identical genome, to find polymorphisms that differ between them, and to identify the causal gene by predicting the functional variation of the gene caused by the polymorphism. Using this approach, we found four causal genes for the dwarf mutation, the round shape grain mutation and the awnless mutation. Three of these genes were the same as those previously reported, but one was a novel gene involved in awn formation. The novel gene was isolated from Bozu-Aikoku, a mutant of Aikoku with the awnless trait, in which nine polymorphisms were predicted to alter gene function by their whole-genome comparison. Based on the information on gene function and tissue-specific expression patterns of these candidate genes, Os03g0115700/LOC&#95;Os03g02460, annotated as a short-chain dehydrogenase/reductase SDR family protein, is most likely to be involved in the awnless mutation. Indeed, complementation tests by transformation showed that it is involved in awn formation. Thus, this method is an effective way to accelerate genome breeding of various crop species by enabling the identification of useful genes that can be used for crop breeding with minimal effort for NGS analysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Suganami, Kojima, Yoshida, Mori, Kawamura, Koketsu and Matsuoka.)

SUMMARY: Spontaneous mutants are mainly obtained from tissue culture or natural occurrences in plants. The traditional strategy for identifying spontaneously mutated genes is to continuously backcross these mutants to another variety and develop a near‐isogenic F2 population for map‐based cloning or bulked segregant analysis. However, this strategy is time‐consuming. Here, we have developed a new method to efficiently accelerate the identification process. The chemical mutagen ethyl methanesulfonate was first used to treat the wild type of the spontaneous mutants to induce thousands of neutral mutations. An induced individual without any statistically significant phenotypic changes which was compared with the wild type was chosen as the neutral mutant. The spontaneous mutant was then crossed with the neutral mutant to develop a pseudo‐near‐isogenic F2 population in which only the induced neutral mutations and the causal mutation were segregated in the genome. This population ensures that the variation of the mutated trait is controlled only by the spontaneously mutated gene. Finally, after sequencing the neutral mutant and the mutant‐type DNA pool of the F2 population the spontaneous mutation will be identified quickly by bioinformatics analysis. Using this method, two spontaneously mutated genes were identified successfully. Therefore, the neutral mutant‐bridging method efficiently identifies spontaneously mutated genes in rice, and its value in other plants is discussed. Significance Statement: Ethyl methanesulfonate (EMS) can induce thousands of single nucleotide polymorphisms as molecular markers in plants. By crossing a spontaneous mutant with the neutral mutant induced by EMS, a pseudo‐near‐isogenic F2 population is developed and only the induced neutral mutations and causal mutation are segregated in the genome. After sequencing the neutral mutant and the mutant type DNA pool of the F2 population, spontaneous mutation will be identified quickly by bioinformatics analysis. [ABSTRACT FROM AUTHOR]

Cross-protection is a promising measure to control plant viral diseases. Reverse genetics had been recently adopted to generate attenuated mutants that have potential in cross-protection. But studies on the variability of the progeny viruses of the attenuated mutants are scarce. Sugarcane mosaic virus (SCMV; genus Potyvirus, family Potyviridae) is the prevalent virus inducing maize dwarf mosaic disease in China. Here, we showed that the substitution of arginine with isoleucine in the FRNK motif at position 184 of helper component-proteinase (HC-Pro) abolished its RNA silencing suppression (RSS) activity, drastically reduced the virulence and accumulation level of SCMV, and impaired the synergism between SCMV and maize chlorotic mottle virus. The attenuated mutant could protect maize plants from a severe infection of SCMV. However, a spontaneous mutation of glycine at position 440 to arginine in HC-Pro rescued the virulence and synergism with maize chlorotic mottle virus of SCMV and the RSS activity of HC-Pro. Similar results were obtained with tobacco vein banding mosaic virus and watermelon mosaic virus. These results provide novel evidence for the complementary mutation of potyviruses in maintaining the HC-Pro RSS activity and potyviral virulence and remind us of evaluating the potential risk of attenuated mutants thoroughly before applying for the control of plant viral diseases via cross-protection.

In general, for chemical reactions occurring in systems, where fluctuations are not negligibly small, it is necessary to introduce a master equation for distribution of probability of fluctuations. It has been established that the monomolecular reactions of a type as A ↔ X are described by the master equation, which leads to a Poisson distribution with the variance equal to the average value N0. However, the consideration of the Löwdin mechanism as autocatalytic non-linear chemical reactions such as A + X ↔ 2X and the corresponding master equation lead to a non-Poissonian probability distribution of fluctuations. In the presented work, first-order autocatalysis has been applied to the Löwdin's mechanism of spontaneous mutations in DNA. Describing double proton transfers between complimentary nucleotide bases along the chain by first-order autocatalytic reactions, the corresponding master equation for protons in tautomeric states becomes non-linear, and at non-equilibrium conditions this leads to the non-Poissonian distribution of spontaneous mutations in DNA. It is also suggested that the accumulation of large fluctuations of successive cooperative concerted protons along the chain may produce higher non-linearities which could have a significant impact on some biochemical processes, occurring in DNA.

Cross-protection is a promising measure to control plant viral diseases. Reverse genetics had been recently adopted to generate attenuated mutants that have potential in cross-protection. But studies on the variability of the progeny viruses of the attenuated mutants are scarce. Sugarcane mosaic virus (SCMV; genus Potyvirus , family Potyviridae) is the prevalent virus inducing maize dwarf mosaic disease in China. Here, we showed that the substitution of arginine with isoleucine in the FRNK motif at position 184 of helper component-proteinase (HC-Pro) abolished its RNA silencing suppression (RSS) activity, drastically reduced the virulence and accumulation level of SCMV, and impaired the synergism between SCMV and maize chlorotic mottle virus. The attenuated mutant could protect maize plants from a severe infection of SCMV. However, a spontaneous mutation of glycine at position 440 to arginine in HC-Pro rescued the virulence and synergism with maize chlorotic mottle virus of SCMV and the RSS activity of HC-Pro. Similar results were obtained with tobacco vein banding mosaic virus and watermelon mosaic virus. These results provide novel evidence for the complementary mutation of potyviruses in maintaining the HC-Pro RSS activity and potyviral virulence and remind us of evaluating the potential risk of attenuated mutants thoroughly before applying for the control of plant viral diseases via cross-protection. [ABSTRACT FROM AUTHOR]

The frequent mutations recovered recently from the pollen of select maize lines resulted from the meiotic mobilization of specific low-copy number long-terminal repeat (LTR) retrotransposons, which differ among lines. Mutations that arise at male meiosis produce kernels with concordant mutant phenotypes in both endosperm and embryo because the two sperms that participate in double fertilization are genetically identical. Those are in a majority. However, a small minority of kernels with a mutant endosperm carry a nonconcordant normal embryo, pointing to a postmeiotic or microgametophytic origin. In this study, we have identified the basis for those nonconcordant mutations. We find that all are produced by transposition of a defective LTR retrotransposon that we have termed dRemp (defective retroelement mobile in pollen). This element has several unique properties. Unlike the mutagenic LTR retrotransposons identified previously, dRemp is present in hundreds of copies in all sequenced lines. It seems to transpose only at the second pollen mitosis because all dRemp insertion mutants are nonconcordant yet recoverable in either the endosperm or the embryo. Although it does not move in most lines, dRemp is highly mobile in the Corn Belt inbred M14, identified earlier by breeders as being highly unstable. Lastly, it can be recovered in an array of structures, ranging from solo LTRs to tandem dRemp repeats containing several internal LTRs, suggestive of extensive recombination during retrotransposition. These results shed further light on the spontaneous mutation process and on the possible basis for inbred instability in maize. [ABSTRACT FROM AUTHOR]

The Russells began their studies of the hereditary effects of radiation in the late 1940s, and their experiments contributed much to what is known about the induction of gene mutations in mice. I had a close association with them for about 26 years, and they relied on me considerably for database management and statistical support. In 1994, I was shocked to discover that, in experiments on males, they had failed to report numerous spontaneous mutations that arose during the perigametic interval and were detected as clusters of mutations. I realized that their nondisclosure of this information meant that the decades-long application of their data to estimate hereditary risks of radiation to humans using the doubling-dose approach had resulted in a several-fold overestimation of risk. I accordingly reported the situation to funding agencies. The resulting complicated situation is referred to here as the Selby-Russell Dispute. Highlights of the resulting investigation, as well as what occurred afterward, are described, and reasons will be provided to show why, in my opinion, the hereditary risk from radiation in humans was likely overestimated by at least 10-fold because the Russells decided not to report critical information from their massive experiments. [ABSTRACT FROM AUTHOR]

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For more than two decades, weedy rice (Oryza sativa L.) has been controlled in rice fields by using imidazolinone (IMI) herbicide-resistant rice technology (Clearfield®). Outcrossing in weedy rice populations and spontaneous mutations are potential problems with herbicide-resistant crop management technologies, such as the IMI-resistant rice. The aim of this study was to characterize the mechanism of IMI herbicide resistance in weedy rice through dose-response bioassay study and evaluating amino acid substitutions in acetolactate synthase (ALS) protein. A total of 118 suspected IMI-resistant weedy rice samples, which survived in the field after an IMI herbicide application, were collected at harvest time from Türkiye in 2020 and 2021. Single-dose imazamox application experiment revealed that 38 plants survived herbicide treatment. The imazamox resistance of the surviving plants was confirmed by dose-response experiment. ALS gene region underwent a sanger DNA partial sequencing. No substitution was found in 10 samples, however, amino acid substitutions were found in 26 samples with S563N, one sample with S653T, and one sample with E630D. The S653N point is the same substitution point that serves as the origin of resistance for the Clearfield® rice varieties that are commonly cultivated in the region. It has been hypothesized that the gene flow from IMI-resistant rice may be the cause of resistance in the IMI resistant weedy rice samples with S653N. The other substitution, S653T, were considered spontaneous mutation to IMI resistance. Interestingly, the S653T mutation was detected for the first time in weedy rice. The mechanism of resistance of 10 resistant weedy rice was not confirmed in this study, however, it may be a non-target resistance or another mutation point in target site, but evidently, they did not acquire resistance by gene flow from IMI-resistant rice. It has been concluded that the effectiveness of IMI-resistant rice technology in controlling weedy rice has drastically decreased due to possible gene flow, spontaneous mutation and non-target resistance. In addition to cultural controls like clean seed, clean machinery and crop rotation, other herbicide-tolerant rice systems such as Provisia® and Roxy-RPS® rice are needed to create a diverse weedy rice management ensemble available for rice production and move towards sustainable rice farming. [Display omitted] • Gene flow is the major factor for IMI resistance in weedy rice. • The multiple significant amino acid substitution was determined in the ALS region. • The substitution S653T was identified for the first time in the weedy rice biotypes. • The target site resistance mechanism in the associated IMI-resistant weedy rice. [ABSTRACT FROM AUTHOR]

BackgroundA single measles vaccination provides lifelong protection. No antigenic variants that escape immunity have been observed. By contrast, influenza continually evolves new antigenic variants, and the vaccine has to be updated frequently with new strains. Both measles and influenza are RNA viruses with high mutation rates, so the mutation rate alone cannot explain the differences in antigenic variability.ResultsWe develop a new hypothesis to explain antigenic stasis versus change. We first note that the antigenically static viruses tend to have high reproductive rates and to concentrate infection in children, whereas antigenically variable viruses such as influenza tend to spread more widely across age classes. We argue that, for pathogens in a naive host population that spread more rapidly in younger individuals than in older individuals, natural selection weights more heavily a rise in reproductive rate. By contrast, pathogens that spread more readily among older individuals gain more by antigenic escape, so natural selection weights more heavily antigenic mutability.ConclusionThese divergent selective pressures on reproductive rate and antigenic mutability may explain some of the observed differences between pathogens in age-class bias, reproductive rate, and antigenic variation.

While studying spontaneous mutations at the maize bronze (bz) locus, we made the unexpected discovery that specific low-copy number retrotransposons are mobile in the pollen of some maize lines, but not of others. We conducted large-scale genetic experiments to isolate new bz mutations from several Bz stocks and recovered spontaneous stable mutations only in the pollen parent in reciprocal crosses. Most of the new stable bz mutations resulted from either insertions of low-copy number long terminal repeat (LTR) retrotransposons or deletions, the same two classes of mutations that predominated in a collection of spontaneous wx mutations [Wessler S (1997) The Mutants of Maize, pp 385-386]. Similar mutations were recovered at the closely linked sh locus. These events occurred with a frequency of 2-4 × 10-5 in two lines derived fromW22 and in 4Co63, but not at all in B73 or Mo17, two inbreds widely represented in Corn Belt hybrids. Surprisingly, the mutagenic LTR retrotransposons differed in the active lines, suggesting differences in the autonomous element make-up of the lines studied. Some active retrotransposons, like Hopscotch, Magellan, and Bs2, a Bs1 variant, were described previously; others, like Foto and Focou in 4Co63, were not. By high-throughput sequencing of retrotransposon junctions, we established that retrotranposition of Hopscotch, Magellan, and Bs2 occurs genome-wide in the pollen of active lines, but not in the female germline or in somatic tissues. We discuss here the implications of these results, which shed light on the source, frequency, and nature of spontaneous mutations in maize. [ABSTRACT FROM AUTHOR]

Bacteriophage contamination and cell lysis have been recurring issues with some actinomycetes used in the pharmaceutical fermentation industry since the commercialization of streptomycin in the 1940s. In the early years, spontaneous phage-resistant mutants or lysogens were isolated to address the problem. In some cases, multiple phages were isolated from different contaminated fermentors, so strains resistant to multiple phages were isolated to stabilize the fermentation processes. With the advent of recombinant DNA technology, the early scaleup of the Escherichia coli fermentation process for the production of human insulin A and B chains encountered contamination with multiple coliphages. A genetic engineering solution was to clone and express a potent restriction/modification system in the production strains. Very recently, an E. coli fermentation of 1,3-propanediol was contaminated by a coliphage related to T1. CRISPR/Cas9 technology was applied to block future contamination by targeting seven different phage genes for double-strand cleavage. These approaches employing spontaneous mutation, genetic engineering, and synthetic biology can be applied to many current and future microorganisms used in the biotechnology industry. [ABSTRACT FROM AUTHOR]

Estimates of the fitness effects of spontaneous mutations are important for understanding the adaptive potential of species. Here, we present the results of mutation accumulation experiments over 265–512 sequential generations in four species of marine unicellular green algae, Ostreococcus tauri RCC4221, Ostreococcus mediterraneus RCC2590, Micromonas pusilla RCC299, and Bathycoccus prasinos RCC1105. Cell division rates, taken as a proxy for fitness, systematically decline over the course of the experiment in O. tauri, but not in the three other species where the MA experiments were carried out over a smaller number of generations. However, evidence of mutation accumulation in 24 MA lines arises when they are exposed to stressful conditions, such as changes in osmolarity or exposure to herbicides. The selection coefficients, estimated from the number of cell divisions/day, varies significantly between the different environmental conditions tested in MA lines, providing evidence for advantageous and deleterious effects of spontaneous mutations. This suggests a common environmental dependence of the fitness effects of mutations and allows the minimum mutation/genome/generation rates to be inferred at 0.0037 in these species.

The mutation rate of an organism is influenced by the interaction of evolutionary forces such as natural selection and genetic drift. However, the mutation spectrum (i.e. the frequency distribution of different types of mutations) can be heavily influenced by DNA repair. Using mutation-accumulation lines of the extremophile bacterium Deinococcus radiodurans Δ mutS1 and the model soil bacterium Pseudomonas fluorescens wild-type and MMR− (Methyl-dependent Mismatch Repair-deficient) strains, we report the mutational features of these two important bacteria. We find that P. fluorescens has one of the highest MMR repair efficiencies among tested bacteria. We also discover that MMR of D. radiodurans preferentially repairs deletions, contrary to all other bacteria examined. We then, for the first time, quantify genome-wide efficiency and specificity of MMR in repairing different genomic regions and mutation types, by evaluating the P. fluorescens and D. radiodurans mutation data sets, along with previously reported ones of Bacillus subtilis subsp. subtilis, Escherichia coli, Vibrio cholerae, and V. fischeri. MMR in all six bacteria shares two general features: 1) repair efficiency is influenced by the neighboring base composition for both transitions and transversions, not limited to transversions as previously reported; and 2) MMR only recognizes indels <4 bp in length. This study demonstrates the power of mutation accumulation lines in quantifying DNA repair and mutagenesis patterns. [ABSTRACT FROM AUTHOR]