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A novel CBL-interacting protein kinase (CIPK) gene, ZmCIPK16, was isolated from maize (Zea mays), which has been certified to have two copies in the genome. The ZmCIPK16 is strongly induced in maize seedlings by PEG, NaCl, ABA, dehydration, heat and drought, but not by cold. A yeast two-hybrid assay demonstrated that ZmCIPK16 interacted with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8. Bimolecular fluorescence complementation (BiFC) assays prove that ZmCIPK16 can interact with ZmCBL3, ZmCBL4, ZmCBL5, and ZmCBL8 in vivo. Subcellular localization showed that ZmCIPK16 is distributed in the nucleus, plasma membrane and cytoplasm; this is different from the specific localization of ZmCBL3, ZmCBL4, and ZmCBL5, which are found in the plasma membrane. The results also showed that overexpression of ZmCIPK16 in the Arabidopsis sos2 mutant induced the expression of the SOS1 gene and enhanced salt tolerance. These findings indicate that ZmCIPK16 may be involved in the CBL-CIPK signaling network in maize responses to salt stress.

Potato virus Y (PVY) infections often lead to altered numbers of host plant chloroplasts, as well as changes in morphology and inhibited photosynthesis. The multifunctional protein helper component-proteinase, HC-Pro, has been identified in PVY-infected leaf chloroplasts. We used yeast two-hybrid and bimolecular fluorescence complementation assays to demonstrate that HC-Pro can interact with the chloroplast division-related factor NtMinD in yeast and tobacco cells, respectively. In addition, we confirmed that residues 271 to 314 in NtMinD are necessary for its interaction with PVY HC-Pro in a yeast two-hybrid analysis using four NtMinD deletion mutants. These residues are necessary for the dimerization of NtMinD, which plays a vital role in chloroplast division. Thus, PVY HC-Pro may affect NtMinD activity by inhibiting the formation of NtMinD homodimers, and this may interfere with chloroplast division and contribute to changes in the numbers of chloroplast per cell observed in PVY-infected plants.

A critical factor in edible plant-derived vaccine development is adequate expression of the exogenous antigens in transgenic plants. We synthesized a codon-optimized gene (sVP6) encoding the VP6 protein of human group A rotavirus and inserted it into the alfalfa genome using agrobacterium-mediated transformation. As much as 0.28% of the total soluble protein of the pBsVP6-transgenic alfalfa was sVP6. Female BALB/c mice were gavaged weekly with 10 mg of transgenic alfalfa extract containing 24 microg of sVP6 protein and 10 microg of CpG-rich oligodeoxynucleotides as mucosal adjuvant. Immunized mice developed high titers of anti-VP6 serum IgG and mucosal IgA. Offspring of immunized dams developed less severe diarrhea after challenge with simian rotavirus SA-11, indicating that antibodies generated in the dams provided passive heterotypic protection to the pups. These results suggest that oral immunization with pBsVP6-transgenic alfalfa provides a potential means of protecting children and young animals from severe acute rotavirus-induced diarrhea.

BackgroundThe use of transgenes to improve complex traits in crops has challenged current genetic transformation technology for multigene transfer. Therefore, a multigene transformation strategy for use in plant molecular biology and plant genetic breeding is thus needed.Methodology/Principal FindingsHere we describe a versatile, ready-to-use multigene genetic transformation method, named the Recombination-assisted Multifunctional DNA Assembly Platform (RMDAP), which combines many of the useful features of existing plant transformation systems. This platform incorporates three widely-used recombination systems, namely, Gateway technology, in vivo Cre/loxP and recombineering into a highly efficient and reliable approach for gene assembly. RMDAP proposes a strategy for gene stacking and contains a wide range of flexible, modular vectors offering a series of functionally validated genetic elements to manipulate transgene overexpression or gene silencing involved in a metabolic pathway. In particular, the ability to construct a multigene marker-free vector is another attractive feature. The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system. A proof-of-principle experiment was confirmed by successfully transferring several heterologous genes into the plant genome.Conclusions/SignificanceThis platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call “one-stop breeding.”