Expression of maize OXS2a in Arabidopsis stunts plant growth but enhances heat tolerance.

As plants encounter various environmental stresses, judicial allocation of resources to stress response is crucial for plant fitness. The plant OXS2 (OXIDATIVE STRESS 2) family has been reported to play important roles in growth regulation and stress response. Here, we report that the maize OXS2 family member ZmOXS2a when expressed in Arabidopsis retards growth including delayed flowering, but improves heat tolerance.

Enterohemorrhagic Escherichia coli O157:H7 antigens produced in transgenic lettuce effective as an oral vaccine in mice.

Transgene with recombination sites to address biosafety concerns engineered into lettuce to produce EspB and γ-intimin C280 for oral vaccination against EHEC O157:H7. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food-borne pathogen where ruminant farm animals, mainly bovine, serve as reservoirs. Bovine vaccination has been used to prevent disease outbreaks, and the current method relies on vaccines subcutaneously injected three times per year.

Target Lines for Gene Stacking in Rice.

The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the rice genome.

Split-Cre mediated deletion of DNA no longer needed after site-specific integration in rice.

N-cre and C-cre added in separate lines reassemble functional Cre in F1 progeny to excise unnecessary DNA, including cre DNA, thereby eliminating generations needed to cross in and out cre. Crop improvement via transgenesis can benefit through efficient DNA integration strategies. As new traits are developed, new transgenes can be stacked by in planta site-specific integration near previous transgenes, thereby facilitating their introgression to field cultivars as a single segregation locus.

Root microbiome changes associated with cadmium exposure and/or overexpression of a transgene that reduces Cd content in rice.

Cadmium (Cd) is a toxic heavy metal that can accumulate in crop plants. We reported previously the engineering of a low cadmium-accumulating line (2B) of rice through overexpression of a truncated OsO3L2 gene. As expression of this transgene was highest in plant roots, amplicon and metatranscriptome sequencing were used to investigate the possibility that its expression affects root associated microbes.

Target lines for recombinase-mediated gene stacking in soybean.

Five soybean target lines with recombinase sites at suitable genomic positions were obtained and tested for site-specific gene stacking. For introgression of new transgenic traits to field cultivars, adding new DNA to an existing transgene locus would reduce the number of segregating loci to reassemble back into a breeding line. We described previously an in planta transgene stacking system using the Bxb1 integrase to direct new DNA into a genomic target, but for this system to operate, the target locus must have a preexisting recombination site for Bxb1-mediated integration.

Site-Specific Sequence Exchange Between Homologous and Non-homologous Chromosomes.

Transgene integration typically takes place in an easy-to-transform laboratory variety before the transformation event is introgressed through backcrosses to elite cultivars. As new traits are added to existing transgenic lines, site-specific integration can stack new transgenes into a previously created transgenic locus. site-specific integration minimizes the number of segregating loci to assemble into a breeding line, but cannot break genetic linkage between the transgenic locus and nearby undesirable traits.

Recombinase-mediated gene stacking in cotton.

Site-specific gene stacking could reduce the number of segregating loci and expedite the introgression of transgenes from experimental lines to field lines. Recombinase-mediated site-specific gene stacking provides a flexible and efficient solution, but this approach requires a recombinase recognition site in the genome. Here, we describe several cotton (Gossypium hirsutum cv.

Arabidopsis OXIDATIVE STRESS 3 enhances stress tolerance in Schizosaccharomyces pombe by promoting histone subunit replacement that upregulates drug-resistant genes.

Histone replacement in chromatin-remodeling plays an important role in eukaryotic gene expression. New histone variants replacing their canonical counterparts often lead to a change in transcription, including responses to stresses caused by temperature, drought, salinity, and heavy metals. In this study, we describe a chromatin-remodeling process triggered by eviction of Rad3/Tel1-phosphorylated H2Aα, in which a heterologous plant protein AtOXS3 can subsequently bind fission yeast HA2.

Arabidopsis OXS3 family proteins repress ABA signaling through interactions with AFP1 in the regulation of ABI4 expression.

Abscisic acid (ABA) and the AP2/ERF (APETALA2/ETHYLENE-RESPONSIVE FACTOR)-type transcription factor called ABA INSENSITIVE 4 (ABI4) play pivotal roles in plant growth responses to environmental stress. An analysis of seedling development in Arabidopsis ABA hypersensitive mutants suggested that OXS3 (OXIDATIVE STRESS 3), OXS3b, O3L3 (OXS3 LIKE 3), O3L4, and O3L6 were negative regulators of ABI4 expression. We therefore characterized the roles of the OXS3 family members in ABA signaling.

A C-terminal fragment of Arabidopsis OXIDATIVE STRESS 2 can play a positive role in salt tolerance.

The zinc finger transcription factor OXIDATIVE STRESS 2 (OXS2) was previously reported to be involved in oxidative stress tolerance and stress escape. Here we report that an Arabidopsis oxs2-1 mutant is also more sensitive to salt stress. Conversely, the overproduction of a C-terminal fragment of OXS2, the 'AT3' fragment, can enhance salt tolerance in Arabidopsis by upregulating the transcription of at least six salt-induced genes: COR15A, COR47, RD29B, KIN1, ACS2 and ACS6.

SnRK1 regulates chromatin-associated OXS3 family proteins localization through phosphorylation in Arabidopsis thaliana.

In plants, SNF1-related protein kinase 1 (SnRK1) senses nutrient and energy status and transduces this information into appropriate responses. Oxidative Stress 3 (OXS3) and family members share a highly conserved putative N-acetyltransferase catalytic domain (ACD). Here, we describe that the ACD contains two candidate SnRK1 recognition motifs and that SnRK1 can interact with most of the OXS3 family proteins.

Overproduction of plant nuclear export signals enhances diamide tolerance in Schizosaccharomyces pombe.

The nuclear export signal (NES) endows a protein nuclear export ability. Surprisingly, our previous study shows that just the NES peptide of Schizosaccharomyces pombe Oxs1 (SpOxs1) can confer diamide tolerance by competing with transcription factor Pap1 for nuclear transport. This finding intrigued us to test the function of NESs from heterologous organisms.

Spontaneous reactivation of a site-specifically placed transgene independent of copy number or DNA methylation.

As millions of seeds are produced from a breeding line, the long-term stability of transgene expression is vital for commercial-scale production of seeds with transgenic traits. Transgenes can be silenced by epigenetic mechanisms, but reactivation of expression can occur as a result of treatment with chromatin modification inhibitors such as 5-azacytidine, from stress such as heat or UV-B, or in mutants that have acquired a defect in gene silencing. Previously, we targeted a gfp reporter gene into the tobacco (Nicotiana tabacum) genome by site-specific recombination but still found some silenced lines among independent integration events.

OXIDATIVE STRESS 3 regulates drought-induced flowering through APETALA 1.

Stress-induced regulation of flowering time insures evolutionary fitness. Stress-induced late flowering is thought to result from a plant evoking tolerance mechanism to wait out the stress before initiating reproduction. Stress-induced early flowering, on the other hand, is thought to be a stress-escape response.

Nucleocytoplasmic OXIDATIVE STRESS 2 can relocate FLOWERING LOCUS T.

Survival of a species depends on reproductive fitness and a plant's floral transition is controlled by developmental and environmental signals. In Arabidopsis, the floral integrators SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) and FT (FLOWERING LOCUS T) sense various pathway signals to activate floral meristem identity genes. At high stress intensity, greater nuclear accumulation of the zinc-finger transcription factor OXS2 (OXIDATIVE STRESS 2) activates an early-flowering stress-escape response.

Protection from Disulfide Stress by Inhibition of Pap1 Nuclear Export in .

Appropriate subcellular localization of regulatory factors is critical for cellular function. Pap1, a nucleocytoplasmic shuttling transcription factor of , is redox regulated for localization and antistress function. In this study, we find that overproduction of a peptide conjugate containing the nuclear export signal of Oxs1, a conserved eukaryotic protein that, along with Pap1, regulates certain diamide responsive genes, can retain Pap1 in the nucleus before stress by competing for nuclear export.

Engineering low-cadmium rice through stress-inducible expression of OXS3-family member genes.

Cadmium (Cd) as a carcinogen poses a great threat to food security and public health through plant-derived foods such as rice, the staple for nearly half of the world's population. We have previously reported that overexpression of truncated gene fragments derived from the rice genes OsO3L2 and OsO3L3 could reduce Cd accumulation in transgenic rice. However, we did not test the full length genes due to prior work in Arabidopsis where overexpression of these genes caused seedling lethality.

Transfer RNA-derived small RNAs in plants.

Rather than random degradation products, the 18 to 40 nucleotides (nt) transfer RNA-derived small RNAs (tsRNAs) are RNA species generated specifically from pre-RNAs or mature tRNAs in archaea, bacteria and eukaryotes. Recent studies from animal systems have shown that tsRNAs are important non-coding RNAs that regulate gene expression at the transcriptional and/or post-transcriptional levels. They are involved in various biological processes, such as cell proliferation, tumor genesis, stress response and intergenerational epigenetic inheritance.

A new location to split Cre recombinase for protein fragment complementation.

We have previously described a recombinase-mediated gene stacking system in which the Cre recombinase is used to remove lox-site flanked DNA no longer needed after each round of Bxb1 integrase-mediated site-specific integration. The Cre recombinase can be conveniently introduced by hybridization with a cre-expressing plant. However, maintaining an efficient cre-expressing line over many generations can be a problem, as high production of this DNA-binding protein might interfere with normal chromosome activities.

Precise, flexible and affordable gene stacking for crop improvement.

The genetic engineering of plants offers a revolutionary advance for crop improvement, and the incorporation of transgenes into crop species can impart new traits that would otherwise be difficult to obtain through conventional breeding. Transgenes introduced into plants, however, can only be useful when bred out to field cultivars. As new traits are continually added to further improve transgenic cultivars, clustering new DNA near previously introduced transgenes keep from inflating the number of segregating units that breeders must assemble back into a breeding line.

A Pap1-Oxs1 signaling pathway for disulfide stress in Schizosaccharomyces pombe.

We describe a Pap1-Oxs1 pathway for diamide-induced disulfide stress in Schizosaccharomyces pombe, where the nucleocytoplasmic HMG protein Oxs1 acts cooperatively with Pap1 to regulate transcription. Oxs1 and Pap1 form a complex when cells are exposed to diamide or Cd that causes disulfide stress. When examined for promoters up-regulated by diamide, effective Pap1 binding to these targets requires Oxs1, and vice versa.

Protocol for In Vitro Stacked Molecules Compatible with In Vivo Recombinase-Mediated Gene Stacking.

Previously, we described a method for a recombinase-directed stacking of new DNA to an existing transgenic locus. Here, we describe how we can similarly stack DNA molecules in vitro and that the in vitro derived gene stack can be incorporated into an Agrobacterium transformation vector by in vitro recombination. After transfer to the chromosome by Agroinfection, the transgenic locus harbors a new target site that can be used for the subsequent in vivo stacking of new DNA.

Method for Biolistic Site-Specific Integration in Plants Catalyzed by Bxb1 Integrase.

Crop improvement is a never ending process. With a transgenesis approach, it is not inconceivable to envision a continuous addition of new transgenes to existing cultivars. Previously, we described a recombinase-directed gene stacking method in tobacco (Hou et al.