The bZIP transcription factor ELONGATED HYPOCOTYL5 (HY5) represents a major hub in the light-signaling cascade both under visible and UV-B light. The mode of transcriptional regulation of HY5, especially under UV-B light, is not well characterized. B-BOX (BBX) transcription factors regulate HY5 transcription and also posttranscriptionally modulate HY5 to control photomorphogenesis under white light. Here, we identify BBX31 as a key signaling intermediate in visible and UV-B light signal transduction in Arabidopsis (Arabidopsis thaliana). BBX31 expression is induced by UV-B radiation in a fluencedependent manner. HY5 directly binds to the promoter of BBX31 and regulates its transcript levels. Loss-and gain-of-function mutants of BBX31 indicate that it acts as a negative regulator of photomorphogenesis under white light but is a positive regulator of UV-B signaling. Genetic interaction studies suggest that BBX31 regulates photomorphogenesis independent of HY5. We found no evidence for a direct BBX31-HY5 interaction, and they primarily regulate different sets of genes in white light. Under high doses of UV-B radiation, BBX31 promotes the accumulation of UV-protective flavonoids and phenolic compounds. It enhances tolerance to UV-B radiation by regulating genes involved in photoprotection and DNA repair in a HY5-dependent manner. Under UV-B radiation, overexpression of BBX31 enhances HY5 transcriptional levels in a UV RESISTANCE LOCUS8-dependent manner, suggesting that BBX31 might regulate HY5 transcription.

An improved method of Agrobacterium-mediated transformation of cowpea was developed employing both sonication and vacuum infiltration treatments. 4 day-old cotyledonary nodes were used as explants for co-cultivation with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pSouv-cry1Ac. Among the different injury treatments, vacuum infiltration and their combination treatments tested, sonication for 20 s followed by vacuum infiltration for 5 min with A. tumefaciens resulted in highest transient GUS expression efficiency (93% explants expressing GUS at regenerating sites). After 3 days of co-cultivation, the explants were cultured in 150 mg/l kanamycin-containing selection medium and putative transformed plants were recovered. The presence, integration and expression of nptII and cry1Ac genes in T0 transgenic plants were confirmed by polymerase chain reaction (PCR), genomic Southern and qualitative reverse transcription (RT)-PCR analysis. Western blot hybridization and enzyme-linked immunosorbent assay (ELISA) detected and demonstrated the accumulation of Cry1Ac protein in transgenic plants. The cry1Ac gene transmitted in a Mendelian fashion. The stable transformation efficiency increased by 88.4% using both sonication-assisted Agrobacterium-mediated transformation (SAAT) and vacuum infiltration than simple Agrobacterium-mediated transformation in cowpea.

Cowpea is one of the important grain legumes. Storage pests, Callosobruchus maculatus and C. chinensis cause severe damage to the cowpea seeds during storage. We employ a highly efficient Agrobacterium-mediated cowpea transformation method for introduction of the bean (Phaseolus vulgaris) alpha-amylase inhibitor-1 (alphaAI-1) gene into a commercially important Indian cowpea cultivar, Pusa Komal and generated fertile transgenic plants. The use of constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404, thiol compounds during cocultivation and a geneticin based selection system resulted in twofold increase in stable transformation frequency. Expression of alphaAI-1 gene under bean phytohemagglutinin promoter results in accumulation of alphaAI-1 in transgenic seeds. The transgenic protein was active as an inhibitor of porcine alpha-amylase in vitro. Transgenic cowpeas expressing alphaAI-1 strongly inhibited the development of C. maculatus and C. chinensis in insect bioassays.

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Agrobacterium-mediated transformation of indica rice has been established in only a limited number of cultivars because the regeneration of plants from transformed embryogenic calli is highly cultivar-specific. Establishment of a highly efficient plant regeneration system from shoot apex explants applicable to many cultivars of indica rice will accelerate the application of transformation technology in breeding programs and functional genomics study. We established an efficient shoot multiplication and plant regeneration system from shoot apices of indica rice using thidiazuron (TDZ) as a plant growth regulator. Shoot apices cultured on MS basal medium devoid of plant growth regulators formed solitary shoots in 90% of cultures. Addition of TDZ or benzylaminopurine to regeneration medium significantly influenced formation of multiple shoots directly from shoot apex explants without an intervening callus stage. Best shoot proliferation response (10.3 shoots per explant) was recorded when shoot apices were cultured on media supplemented with 4 mg/l TDZ. No synergistic effect on shoot proliferation was observed when indole-3-acetic acid and indole-3-butyric acid were supplemented to media containing 4 mg/l TDZ. The regeneration system was efficient in evoking multiple shoot proliferation in eight different cultivars of indica rice. Shoots were rooted in MS basal medium and plantlets were acclimatized with 100% survival rate. The shoot apex explants of all the eight cultivars of indica rice were found competent to Agrobacterium-mediated transformation while explants from IR-64 showed highest transient GUS expression. This variety-independent transformation amenable regeneration system from shoot apices may widely be applicable for genetic transformation of indica varieties.

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.

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