Reevaluation of the Reliability and Usefulness of the Somatic Homologous Recombination Reporter Lines

Ülker, Bekir; Hommelsheim, Carl Maximilian; Berson, Tobias; Thomas, Stefan; Chandrasekar, Balakumaran; Olcay, Ahmet Can; Berendzen, Kenneth W.; Frantzeskakis, Lamprinos

doi:10.1105/tpc.112.100404

Cited by 6 publications

(3 citation statements)

References 77 publications

(110 reference statements)

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“…Nonetheless, the rates due to BRCA2B mutants (1.1-fold) were not significantly lower compared to ours (6.3-fold). This discrepancy may arise because Wang et al ( 2010 ) utilized a different GUS mutant line, and the chromosomal position effect on the GUS recombination substrate might influence its sensitivity (Ülker et al, 2012 ). Nonetheless, akin to AtBRCA2A (Wang et al, 2010 ), our results indicate that AtBRCA2B is requisite for maintaining spontaneous SHR rates.…”

Section: Discussionmentioning

confidence: 99%

Essential role of the BRCA2B gene in somatic homologous recombination in Arabidopsis thaliana

Amritha,

Shah

2023

bta

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Constant exposure to various environmental and endogenous stresses can cause structural DNA damage, resulting in genome instability. Higher eukaryotic cells deploy conserved DNA repair systems, which include various DNA repair pathways, to maintain genome stability. Homologous recombination (HR), one of these repair pathways, involves multiple proteins. BRCA2, one of the proteins in the HR pathway, is of substantial research interest in humans because it is an oncogene. However, the study of this gene is limited due to the lack of availability of homozygous BRCA2 -knockout mutants in mammals, which results in embryonic lethality. Arabidopsis thaliana has two copies of the BRCA2 homologs: BRCA2A and BRCA2B . Therefore, the single mutants remain nonlethal and fertile in Arabidopsis . The BRCA2A homolog, which plays a significant role in the HR pathway of germline cells and during the defense response, is well-studied in Arabidopsis . Our study focuses on the functional characterization of the BRCA2B homolog in the somatic cells of Arabidopsis , using the homozygous ΔBRCA2B mutant line. The phenotypic differences of ΔBRCA2B mutants were characterized and compared with wild Arabidopsis plants. The role of BRCA2B in spontaneous somatic HR (SHR) was studied using the ΔBRCA2B -gus detector line. ΔBRCA2B plants have a 6.3-fold lower SHR frequency than the control detector plants. Expression of four other HR pathway genes, including BRE , BRCC36A , RAD50 , and RAD54 , was significantly reduced in ΔBRCA2B mutants. Thus, our findings convey that the BRCA2B homolog plays an important role in maintaining spontaneous SHR rates and has a direct or indirect regulatory effect on the expression of other HR-related genes.

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Section: Discussionmentioning

confidence: 99%

Essential role of the BRCA2B gene in somatic homologous recombination in Arabidopsis thaliana

Amritha,

Shah

2023

bta

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“…By leaving the disinfected plants to grow for a further two weeks our mutation counts were more reliable: instead of needing to detect single blue cells (a consequence of the modified GUS gene) the mutant foci were readily recognisable blue spots. Ulker et al (2012) [ 46 ] did raise a concern over the reliability of mutation reporter lines and sequence of reporter lines after GUS restoration, which was addressed by Puchta and Hohn (2012) [ 47 ]. There are several papers published using these lines to score somatic mutations in different circumstances [ 48 , 49 ].…”

Section: Discussionmentioning

confidence: 99%

Suppression of different classes of somatic mutations in Arabidopsis by vir gene-expressing Agrobacterium strains

et al. 2015

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BackgroundAgrobacterium infection, which is widely used to generate transgenic plants, is often accompanied by T-DNA-linked mutations and transpositions in flowering plants. It is not known if Agrobacterium infection also affects the rates of point mutations, somatic homologous recombinations (SHR) and frame-shift mutations (FSM). We examined the effects of Agrobacterium infection on five types of somatic mutations using a set of mutation detector lines of Arabidopsis thaliana. To verify the effect of secreted factors, we exposed the plants to different Agrobacterium strains, including wild type (Ach5), its derivatives lacking vir genes, oncogenes or T-DNA, and the heat-killed form for 48 h post-infection; also, for a smaller set of strains, we examined the rates of three types of mutations at multiple time-points. The mutation detector lines carried a non-functional β-glucuronidase gene (GUS) and a reversion of mutated GUS to its functional form resulted in blue spots. Based on the number of blue spots visible in plants grown for a further two weeks, we estimated the mutation frequencies.ResultsFor plants co-cultivated for 48 h with Agrobacterium, if the strain contained vir genes, then the rates of transversions, SHRs and FSMs (measured 2 weeks later) were lower than those of uninfected controls. In contrast, co-cultivation for 48 h with any of the Agrobacterium strains raised the transposition rates above control levels. The multiple time-point study showed that in seedlings co-cultivated with wild type Ach5, the reduced rates of transversions and SHRs after 48 h co-cultivation represent an apparent suppression of an earlier short-lived increase in mutation rates (peaking for plants co-cultivated for 3 h). An increase after 3 h co-cultivation was also seen for rates of transversions (but not SHR) in seedlings exposed to the strain lacking vir genes, oncogenes and T-DNA. However, the mutation rates in plants co-cultivated for longer times with this strain subsequently dropped below levels seen in uninfected controls, consistent with the results of the single time-point study.ConclusionsThe rates of various classes of mutations that result from Agrobacterium infection depend upon the duration of infection and the type of pathogen derived factors (such as Vir proteins, oncoproteins or T-DNA) possessed by the strain. Strains with vir genes, including the type used for plant transformation, suppressed selected classes of somatic mutations. Our study also provides evidence of a pathogen that can at least partly counter the induction of mutations in an infected plant.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0595-1) contains supplementary material, which is available to authorized users.

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“…Reports indicate that somatic homologous recombination rates in offspring can be affected by parental environmental exposures (Molinier et al, 2006;Yao and Kovalchuk, 2011;Puchta and Hohn, 2012), but this may not be a highly reproducible response (Pecinka et al, 2009;Ülker et al, 2012). Several recent articles provide evidence for heritable phenotypic changes or chromatin changes during development or in the offspring of plants exposed to biotic stress (Boyko and Kovalchuk, 2011;Dowen et al, 2012;Luna et al, 2012;Rasmann et al, 2012;Slaughter et al, 2012).…”

Section: Epigenetics and Transmission Of Environmental Effects To Offmentioning

confidence: 99%

Epigenetics: Beyond Chromatin Modifications and Complex Genetic Regulation

Schmitz²,

2014

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Reevaluation of the Reliability and Usefulness of the Somatic Homologous Recombination Reporter Lines

Cited by 6 publications

References 77 publications

Essential role of the BRCA2B gene in somatic homologous recombination in Arabidopsis thaliana

Essential role of the BRCA2B gene in somatic homologous recombination in Arabidopsis thaliana

Suppression of different classes of somatic mutations in Arabidopsis by vir gene-expressing Agrobacterium strains

Epigenetics: Beyond Chromatin Modifications and Complex Genetic Regulation

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