The ascomycete fungus
causes early blight, one of economically the most important tomato diseases. Due to frequent use of fungicides,
has developed resistance with negative economic and environmental ...consequences. Research of new ways to control fungal pathogens has turned its eye to environmentally friendly chemicals with low toxicity such as boronic acids. The aim of our study was therefore to test the antifungal effects of phenylboronic and boric acid
on
.
. We isolated the pathogen from a symptomatic tomato plant and determined the minimum inhibitory concentration of phenylboronic and boric acid on
mycelial growth using the poisoned food technique. The antifungal effect was tested on a wide range of phenylboronic and boric acid concentrations (from 0.04 % to 0.3 %) applied separately to agar with mycelial disc of the pathogen. After five days of incubation, phenylboronic acid at low concentration (0.05 %) completely inhibited mycelial growth. Boric acid, in turn, did not significantly slow down mycelial growth but did reduce sporulation and confirmed its fungistatic effect. Our findings point to the potential use of phenylboronic acid to control phytopathogenic fungi. This is, to our knowledge, the first report on its antifungal effect on an agriculturally important pathogen
. Moreover, since
is also a human pathogen, these results may have clinical ramifications.
Finding a suitable alternative to the small pool of existing antifungal agents is a vital task in contemporary agriculture. Therefore, intensive research has been conducted globally to uncover ...environmentally friendly and efficient agents that can suppress pathogens resistant to the currently used antimycotics. Here, we tested the activity of boric acid (BA) and its derivative phenylboronic acid (PBA) in controlling the early blight symptoms in tomato plants infected with pathogenic fungus
. By following the appearance and intensity of the lesions on leaves of the tested plants, as well as by measuring four selected physiological factors that reflect plant health, we have shown that both BA and PBA act prophylactically on fungal infection. They did it by reducing the amount and severity of early blight symptoms, as well as by preventing deterioration of the physiological traits, occurring upon fungal inoculation. Phenylboronic acid was more efficient in suppressing the impact of
infection. Therefore, we conclude that BA, and even more so PBA, may be used as agents for controlling early blight on tomato plants, as they are both quite effective and environmentally friendly.
Bacterial SSB proteins, as well as their eukaryotic RPA analogues, are essential and ubiquitous. They avidly bind single-stranded DNA and regulate/coordinate its metabolism, hence enabling essential ...DNA processes such as replication, transcription, and repair. The prototypic
SSB protein is encoded by an
gene. Although the
gene promoters harbor an SOS box, multiple studies over several decades failed to elucidate whether
gene expression is inducible and SOS dependent. The SOS regulon is comprised of about 50 genes, whose transcription is coordinately induced under stress conditions. Using quantitative real-time PCR, we determined the
gene expression kinetics in UV- and γ-irradiated
and revealed that
gene expression is elevated in irradiated cells in an SOS-dependent manner. Additionally, the expression of the
gene was determined to indicate the extent of SOS induction. In a mutant with a constitutively induced SOS regulon, the
gene was overexpressed in the absence of DNA damage. Furthermore, we measured
gene expression by droplet digital PCR during unaffected bacterial growth and revealed that
gene expression was equal in wild-type and SOS
bacteria, whereas
expression was higher in the former. This study thus reveals a complex pattern of
gene expression, which under stress conditions depends on the SOS regulon, whereas during normal bacterial growth it is unlinked to SOS induction. The
gene is SOS regulated in such a way that its basal expression is relatively high and can be increased only through stronger SOS induction. The remarkable SOS induction observed in undisturbed wild-type cells may challenge our notion of the physiological role of the SOS response in bacteria.
The procedure illustrated in this paper represents a new method for transcriptome analysis by PCR (Polymerase Chain Reaction), which circumvents the need for elimination of potential DNA ...contamination. Compared to the existing methodologies, our method is more precise, simpler and more reproducible because it preserves the RNA's integrity, does not require materials and/or reagents that are used for elimination of DNA and it also reduces the number of samples that should be set up as negative controls. This novel procedure involves the use of a specifically modified primer during reverse transcription step, which contains mismatched bases, thus producing cDNA molecules that differ from genomic DNA. By using the same modified primer in PCR amplification, only cDNA template is amplified since genomic DNA template is partially heterologous to the primer. In this way, amplification by PCR is unaffected by any potential DNA contamination since it is specific only for the cDNA template. Furthermore, it accurately reflects the initial RNA concentration of the sample, which is prone to changes due to various physical or enzymatic treatments commonly used by the current methodologies for DNA elimination. The method is particularly suitable for quantification of highly repetitive DNA transcripts, such as satellite DNA.
Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In
, DSB repair is initiated by the RecBCD enzyme that ...resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of
after three different DNA-damaging treatments: (i) expression of I-
I endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the
, and
mutants. After I-
I expression and γ-irradiation, this phenotype was efficiently suppressed by the
mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the
mutation abolished cytological defects of
mutants and also partially suppressed the cytological defects of
mutants. However, neither
nor
mutation alone could suppress the cytological defects of UV-irradiated
mutants. The suppression was achieved only by simultaneous inactivation of the
and
genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated
mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in
.
Background and purpose: Homologous recombination in Escherichia coli proceeds via two pathways, RecBCD and RecFOR, which use different enzymes for DNA end resection and loading of RecA recombinase. ...The postsynaptic reactions following RecA-mediated homologous pairing have mostly been studied within the RecBCD pathway. They involve RuvABC helicase/resolvase complex, RecG and RadA helicases that process recombination intermediates to produce recombinant DNA molecules. Also, RecG functionally interacts with the PriA protein in initiation of recombination-dependent replication. Here, we studied the individual and combined effects of ruvABC, recG and radA null mutations on transductional recombination in both pathways. The effect of the priA300 mutation, which acts as a suppressor of the recG mutation, was also tested. The goal was to characterize the postsynaptic stage of transductional recombination in more details, especially in the RecFOR pathway, which is less well-studied.
Materials and methods: Phage P1vir-mediated transduction was used to measure recombination efficiency in a series of recombination mutants. The proA+ marker was used for selection in transductional crosses with various ΔproA recipients.
Results: The ruvABC mutation moderately decreased recombination in both recombination pathways, while radA had no effect. The recG mutation reduced recombination in the RecBCD pathway but not in the RecFOR pathway. The strong recombination defect of recG radA double mutants in both pathways was completely suppressed by the priA300 mutation, and this suppression depended on the functional RuvABC complex.
Conclusions: RecG and RadA proteins have a redundant role in transductional recombination via RecFOR pathway. In both recombination pathways, RecG and RadA functionally interact with PriA, probably during initiation of recombination-dependent replication.
Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in ...Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3' ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD's RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3' tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3' ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis.
Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in
To study DSB processing
, we induced DSBs into the
chromosome by γ-irradiation and measured ...chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with single-stranded DNA (ssDNA). RecA decreased DNA degradation in wild-type,
, and
strains, indicating that it is a general phenomenon in
On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the
mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3' tail (ssExos). 3'-5' ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding the 3' tail from degradation by 3'-5' ssExos. Since 3' tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3' tails emanating from DSBs, along with 3' tail's massive length, or prevention of their degradation by inactivation of 3'-5' ssExos. Thus, we conclude that 3' overhangs are crucial in controlling the extent of DSB processing in
This study suggests a regulatory mechanism for DSB processing in
, wherein 3' tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends.
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