Temporal and spatial spread of Soybean mosaic virus in soybeans transformed with the coat protein gene of SMV

Soybean mosaic virus (SMV) causes a serious disease of soybeans, and is found in all major growing regions worldwide. The virus is vectored by over 30 species of aphids in a non-persistent manner. Six soybean lines were generated by Agrobacterium-mediated transformation with the coat-protein gene of SMV strain N. Field plots of each line were established with point sources of aphid-transmissible SMV strain AL-5 in 1999 and 2000. Plots were divided into quadrants, and plant samples within quadrants were bulked together for testing by biotin-avidin ELISA. The Gompertz model was the most appropriate for quantifying and comparing the temporal spread of SMV in the six soybean lines over both years. Two lines (3-24 and 7B-11) had lower infection rates and lower final pathogen incidences, compared to the untransformed control. Ordinary runs analysis revealed clustering of infected quadrants in lines with the highest rates of pathogen progress for both years. Harvested soybeans showed significantly less seed-coat mottling in 3-24 and 7B-11, compared to the untransformed control

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847

DNA double-strand break resection occurs during non-homologous end joining in G1 but is distinct from resection during homologous recombination

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847

Temporal and spatial spread of Soybean mosaic virus in soybeans transformed with the coat protein gene of SMV

Soybean mosaic virus (SMV) causes a serious disease of soybeans, and is found in all major growing regions worldwide. The virus is vectored by over 30 species of aphids in a non-persistent manner. Six soybean lines were generated by Agrobacterium-mediated transformation with the coat-protein gene of SMV strain N. Field plots of each line were established with point sources of aphid-transmissible SMV strain AL-5 in 1999 and 2000. Plots were divided into quadrants, and plant samples within quadrants were bulked together for testing by biotin-avidin ELISA. The Gompertz model was the most appropriate for quantifying and comparing the temporal spread of SMV in the six soybean lines over both years. Two lines (3-24 and 7B-11) had lower infection rates and lower final pathogen incidences, compared to the untransformed control. Ordinary runs analysis revealed clustering of infected quadrants in lines with the highest rates of pathogen progress for both years. Harvested soybeans showed significantly less seed-coat mottling in 3-24 and 7B-11, compared to the untransformed control.</p

Characterization of the slow DNA double-strand break repair component in G1 phase

DNA double-strand breaks (DSBs) represent the most deleterious type of DNA damage as they pose a serious threat to genome integrity. Two major pathways are available for the repair of DSBs: canonical non-homologous end joining (c-NHEJ) and homologous recombination (HR). During c-NHEJ, the DSB ends are re-ligated after minimal end processing steps. The HR pathway is more complex and is initiated by CtIP-dependent DSB end resection to form 3’ ssDNA overhangs for subsequent homology search in the sister chromatid. In wild type human G1-phase cells only c-NHEJ is available for DSB repair, as in this cell cycle phase the homologous sister chromatid required for HR is missing. DSB repair in G1, as well as in G2, shows biphasic kinetics consisting of a fast component that repairs the majority of breaks within the first few hours after damage induction, followed by a slow component that repairs the remaining breaks. The fast component in both G1 and G2 phase is well characterized and represents c-NHEJ, while the slow component in G2 represents repair by HR. Previous work has suggested that the slow repair component in G1 represents a sub-pathway of NHEJ that requires the activities of Artemis and ATM. However, the mechanism underlying the slow repair component in G1 is not fully understood and its characterization was the focus of this work. To specifically study slow repair in G1, high LET α-particle radiation was used to induce complex DNA damages that are repaired with slow kinetics. RPA rapidly binds ssDNA in the cell to protect it from nucleolytic degradation and is phosphorylated in response to DNA damage. Exploiting the qualities of α–particle radiation, an assay was developed to monitor pRPA-foci formation in G1 and used as a tool to measure DSB end resection in this cell-cycle phase. Another approach to study the slow repair component was the quantification of γH2AX foci, a histone modification in response to DSBs, at late time points post IR. Collectively, it was shown that slowly repairing DSBs in G1 undergo resection and subsequent repair by c-NHEJ. This pathway is regulated by Plk3, which after DNA damage phosphorylates CtIP on amino acids Ser327 and Thr847 in G1. Using the pRPA assay, it was demonstrated that Plk3 phosphorylates CtIP on these amino acid residues to promote resection. CtIP phosphorylation on Ser327 also mediates its interaction with Brca1 in G1, which antagonizes 53BP1 to allow resection. The results indicate that the interaction of CtIP and Brca1 is required to promote resection in G1, while depletion of 53BP1 causes hyper-resection of DSBs in G1. The primary function of Brca1 in G1 appears to be the displacement of 53BP1, similar to the mechanism in G2. Furthermore, a number of nucleases required for G1 resection were identified. Similar to the process in G2, G1 resection requires the exonuclease activities of Exo1, EXD2 and Mre11. Contrary to G2, the endonuclease activity of Mre11 is dispensable in G1, as are the activities of BLM/DNA2. Thus, it is proposed that resection in G1 might be initiated from the break end and therefore differs from the mechanism in G2 where Mre11 endonuclease function initiates bi-directional resection several hundred nucleotides away from the break end. γH2AX studies indicated that Artemis, an endonuclease which is specifically required for DBS repair during the slow component, functions downstream of the aforementioned factors. Thus, it is proposed that once resection is initiated in G1, resection intermediates have to be resolved by Artemis to complete repair. Finally, the results indicate that break ends are rejoined via a c-NHEJ process, therefore it was hypothesized that the Ku70/80 heterodimer stays bound to the DSB ends throughout the entire repair time and translocates inwards to expose DNA ends for resection while at the same time limiting the process. Immunofluorescence data support this notion by providing evidence that Ku80 co-localizes with pRPA in G1. Compared to resection in G2, which is always followed up by error-free repair via HR, resection in G1 needs to be much more limited in length. Future work will focus on the elucidation of the mechanisms restricting the extent of resection in G1