Qi-Wen Niu

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method

Collective efforts of several laboratories in the past two decades have resulted in the development of various methods for Agrobacterium tumefaciens–mediated transformation of Arabidopsis thaliana. Among these, the floral dip method is the most facile protocol and widely used for producing transgenic Arabidopsis plants. In this method, transformation of female gametes is accomplished by simply dipping developing Arabidopsis inflorescences for a few seconds into a 5% sucrose solution containing 0.01–0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying the genes to be transferred. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. A transformation frequency of at least 1% can be routinely obtained and a minimum of several hundred independent transgenic lines generated from just two pots of infiltrated plants (20–30 plants per pot) within 2–3 months. Here, we describe the protocol routinely used in our laboratory for the floral dip method for Arabidopsis transformation. Transgenic Arabidopsis plants can be obtained in approximately 3 months.

Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69159 and amiR-HC-Pro159 are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP159 targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69159 and amiR-HC-Pro159 from a dimeric pre-amiR-P69159/amiR-HC-Pro159 transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 °C, a temperature that compromises small interfering RNA–mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.

Chemical-regulated, site-specific DNA excision in transgenic plants

We have developed a chemical-inducible, site-specific DNA excision system in transgenic Arabidopsis plants mediated by the Cre/loxP DNA recombination system. Expression of the Cre recombinase was tightly controlled by an estrogen receptor-based fusion transactivator XVE. Upon induction by β-estradiol, sequences encoding the selectable marker, Cre, and XVE sandwiched by two loxP sites were excised from the Arabidopsis genome, leading to activation of the downstream GFP (green fluorescent protein) reporter gene. Genetic and molecular analyses indicated that the system is tightly controlled, showing high-efficiency inducible DNA excision in all 19 transgenic events tested with either single or multiple T-DNA insertions. The system provides a highly reliable method to generate marker-free transgenic plants after transformation through either organogenesis or somatic embryogenesis.

KORRIGAN, an Arabidopsis Endo-1,4-β-Glucanase, Localizes to the Cell Plate by Polarized Targeting and Is Essential for Cytokinesis

The formation of the cell plate, a unique structure in dividing plant cells, is pivotal for cytokinesis. A mutation in the Arabidopsis KORRIGAN (KOR) gene causes the formation of aberrant cell plates, incomplete cell walls, and multinucleated cells, leading to severely abnormal seedling morphology. The mutant, designed kor1-2, was identified as a stronger allele than the previously identified kor1-1, which appears to be defective only in cell elongation. KOR1 encodes an endo-1,4-β-d-glucanase with a transmembrane domain and two putative polarized targeting signals in the cytosolic tail. When expressed in tobacco BY2 cells, a KOR1-GFP (green fluorescence protein) fusion protein was localized to growing cell plates. Substitution mutations in the polarized targeting motifs of KOR1 caused the fusion proteins to localize to the plasma membrane as well. Expression of these mutant genes in kor1-2 plants complemented only the cell elongation defect but not the cytokinesis defect, indicating that polarized targeting of KOR1 to forming cell plates is essential for cytokinesis. Our results suggest that KOR1 plays a critical role during cytokinesis.

Arabidopsis DCP2, DCP1, and VARICOSE Form a Decapping Complex Required for Postembryonic Development

mRNA turnover in eukaryotes involves the removal of m7GDP from the 5′ end. This decapping reaction is mediated by a protein complex well characterized in yeast and human but not in plants. The function of the decapping complex in the development of multicellular organisms is also poorly understood. Here, we show that Arabidopsis thaliana DCP2 can generate from capped mRNAs, m7GDP, and 5′-phosphorylated mRNAs in vitro and that this decapping activity requires an active Nudix domain. DCP2 interacts in vitro and in vivo with DCP1 and VARICOSE (VCS), an Arabidopsis homolog of human Hedls/Ge-1. Moreover, the interacting proteins stimulate DCP2 activity, suggesting that the three proteins operate as a decapping complex. Consistent with their role in mRNA decay, DCP1, DCP2, and VCS colocalize in cytoplasmic foci, which are putative Arabidopsis processing bodies. Compared with the wild type, null mutants of DCP1, DCP2, and VCS accumulate capped mRNAs with a reduced degradation rate. These mutants also share a similar lethal phenotype at the seedling cotyledon stage, with disorganized veins, swollen root hairs, and altered epidermal cell morphology. We conclude that mRNA turnover mediated by the decapping complex is required for postembryonic development in Arabidopsis.

Inducible isopentenyl transferase as a high-efficiency marker for plant transformation

Overexpression of the isopentenyltransferase gene (ipt) from the Ti-plasmid of Agrobacterium tumefaciens increases cytokinin levels, leading to generation of shoots from transformed plant cells. When combined with a dexamethasone-inducible system for controlling expression, ipt expression can be used to select for transgenic regenerants without using an antibiotic-resistance marker. The combined system allows efficient cointroduction of multiple genes (in addition to ipt) and produces transgenic plants without morphological or developmental defects.

Molecular Evolution of a Viral Non-Coding Sequence under the Selective Pressure of amiRNA-Mediated Silencing

Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.

Overexpression of PGA37/MYB118 and MYB115 promotes vegetative-to-embryonic transition in Arabidopsis

Formation of somatic embryos from non-germline cells is unique to higher plants and can be manipulated in a variety of species. Previous studies revealed that overexpression of several Arabidopsis genes, including WUSCHEL (WUS)/PLANT GROWTH ACTIVATOR6 (PGA6), BABY BOOM, LEAFY COTYLEDON1 (LEC1), and LEC2, is able to cause vegetative-to-embryonic transition or the formation of somatic embryos. Here, we report that a gain-of-function mutation in the Arabidopsis PGA37 gene, encoding the MYB118 transcription factor, induced vegetative-to-embryonic transition, the formation of somatic embryos from root explants, and an elevated LEC1 expression level. Double mutant analysis showed that WUS was not required for induction of somatic embryos by PGA37/MYB118. In addition, overexpression of MYB115, a homolog of PGA37/MYB118, caused a pga37-like phenotype. A myb118 myb115 double mutant did not show apparent developmental abnormalities. Collectively, these results suggest that PGA37/MYB118 and MYB115 promote vegetative-to-embryonic transition, through a signaling pathway independent of WUS.

The role of miR156/SPLs modules in Arabidopsis lateral root development

miR156 is an evolutionarily highly conserved miRNA in plants that defines an age-dependent flowering pathway. The investigations thus far have largely, if not exclusively, confined to plant aerial organs. Root branching architecture is a major determinant of water and nutrients uptake for plants. We show here that MIR156 genes are differentially expressed in specific cells/tissues of lateral roots. Plants overexpressing miR156 produce more lateral roots whereas reducing miR156 levels leads to fewer lateral roots. We demonstrate that at least one representative from the three groups of miR156 targets SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes: SPL3, SPL9 and SPL10 are involved in the repression of lateral root growth, with SPL10 playing a dominant role. In addition, both MIR156 and SPLs are responsive to auxin signaling suggesting that miR156/SPL modules might be involved in the proper timing of the lateral root developmental progression. Collectively, these results unravel a role for miR156/SPLs modules in lateral root development in Arabidopsis.

Tempo and Mode of Plant RNA Virus Escape from RNA Interference-Mediated Resistance

ABSTRACT A biotechnological application of artificial microRNAs (amiRs) is the generation of plants that are resistant to virus infection. This resistance has proven to be highly effective and sequence specific. However, before these transgenic plants can be deployed in the field, it is important to evaluate the likelihood of the emergence of resistance-breaking mutants. Two issues are of particular interest: (i) whether such mutants can arise in nontransgenic plants that may act as reservoirs and (ii) whether a suboptimal expression level of the transgene, resulting in subinhibitory concentrations of the amiR, would favor the emergence of escape mutants. To address the first issue, we experimentally evolved independent lineages of Turnip mosaic virus (TuMV) (family Potyviridae) in fully susceptible wild-type Arabidopsis thaliana plants and then simulated the spillover of the evolving virus to fully resistant A. thaliana transgenic plants. To address the second issue, the evolution phase took place with transgenic plants that expressed the amiR at subinhibitory concentrations. Our results show that TuMV populations replicating in susceptible hosts accumulated resistance-breaking alleles that resulted in the overcoming of the resistance of fully resistant plants. The rate at which resistance was broken was 7 times higher for TuMV populations that experienced subinhibitory concentrations of the antiviral amiR. A molecular characterization of escape alleles showed that they all contained at least one nucleotide substitution in the target sequence, generally a transition of the G-to-A and C-to-U types, with many instances of convergent molecular evolution. To better understand the viral population dynamics taking place within each host, as well as to evaluate relevant population genetic parameters, we performed in silico simulations of the experiments. Together, our results contribute to the rational management of amiR-based antiviral resistance in plants.