The barley stripe mosaic virus expression system reveals the wheat C2H2 zinc finger protein TaZFP1B as a key regulator of drought tolerance.

BACKGROUND: Drought stress is one of the major factors limiting wheat production globally. Improving drought tolerance is important for agriculture sustainability. Although various morphological, physiological and biochemical responses associated with drought tolerance have been documented, the molecular mechanisms and regulatory genes that are needed to improve drought tolerance in crops require further investigation. We have used a novel 4-component version (for overexpression) and a 3-component version (for underexpression) of a barley stripe mosaic virus-based (BSMV) system for functional characterization of the C2H2-type zinc finger protein TaZFP1B in wheat. These expression systems avoid the need to produce transgenic plant lines and greatly speed up functional gene characterization. RESULTS: We show that overexpression of TaZFP1B stimulates plant growth and up-regulates different oxidative stress-responsive genes under well-watered conditions. Plants that overexpress TaZFP1B are more drought tolerant at critical periods of the plant's life cycle. Furthermore, RNA-Seq analysis revealed that plants overexpressing TaZFP1B reprogram their transcriptome, resulting in physiological and physical modifications that help wheat to grow and survive under drought stress. In contrast, plants transformed to underexpress TaZFP1B are significantly less tolerant to drought and growth is negatively affected. CONCLUSIONS: This study clearly shows that the two versions of the BSMV system can be used for fast and efficient functional characterization of genes in crops. The extent of transcriptome reprogramming in plants that overexpress TaZFP1B indicates that the encoded transcription factor is a key regulator of drought tolerance in wheat.

A New Barley Stripe Mosaic Virus Allows Large Protein Overexpression for Rapid Function Analysis.

Understanding the genetic and molecular bases of gene function is of increasing importance to harness their potential to produce plants with novel traits. One important objective is the improvement of plant productivity to meet future demands in food crop production. Gene function is mostly characterized through overexpression or silencing in transgenic plants. This approach is a lengthy procedure, especially in cereals. Plant viral expression systems can be used for rapid expression of proteins. However, current systems have a small cargo capacity and have mostly been used for gene silencing. Here, a four-component barley stripe mosaic virus-based system with high cargo capacity was constructed for the rapid and stable expression of recombinant proteins in different plant species, allowing function analyses at different stages of development. Fluorescent marker proteins are expressed at high levels within 1 week, and a proof of efficient function analysis is shown using the aluminum malate transporter1 gene. In addition to the ability of gene cotransformation, this work demonstrates that the four-component barley stripe mosaic virus-based system allows the overexpression of cDNAs of up to 2,100 nucleotides (encoding a protein of ∼78 kD), thereby providing an invaluable tool to accelerate functional genomics and proteomic research in monocot and dicot species.

A rapid and efficient method for uniform gene expression using the barley stripe mosaic virus.

BACKGROUND: The barley stripe mosaic virus (BSMV) has become a popular vector to study gene function in cereals. However, studies have been limited to gene silencing in leaves of barley or wheat. In addition, the method produces high variability between different leaves and plants. To overcome these limitations, we explored the potential of modifying the inoculation protocol for BSMV gene overexpression. An improved light, oxygen or voltage-sensing (iLOV) domain-based fluorescent protein was used as a reporter of gene expression to monitor the infection and spread of BSMV. Tobacco (Nicotiana benthamiana) leaves were infected via agroinfiltration and the leaves were homogenized to extract the BSMV particles and inoculate wheat tissues using the traditional leaf abrasion method or by incubation during seed imbibition in a Petri dish. RESULTS: Compared to the leaf abrasion method, the seed imbibition method resulted in a high and uniform detection of iLOV in both roots and leaves of different wheat cultivars and other monocot and dicot species within 7 days after germination. The progression of viral infection via the imbibition method as measured by the expression of iLOV was more stable in different organs and tissues and is transmissible to the next generation. CONCLUSION: Our results show that BSMV can be used as a vector for the expression of small genes such as iLOV in wheat roots and leaves. The inoculation by seed imbibition allows genes to be expressed rapidly and uniformly in wheat and different monocot and dicot species compared to the traditional leaf abrasion method. It also produces high successful transformation as early as 7 days post infection allowing gene function studies during the first generation of infected plants. Furthermore, the method is simple, rapid, and inexpensive compared to the production of transgenic plants.

Genome wide identification of C1-2i zinc finger proteins and their response to abiotic stress in hexaploid wheat.

The C1-2i wheat Q-type C2H2 zinc finger protein (ZFP) transcription factor subclass has been reported to play important roles in plant stress responses. This subclass of ZFPs has not been studied in hexaploid wheat (Triticum aestivum) and we aimed to identify all members of this subclass and evaluate their responses to different abiotic stresses causing oxidative stress. Exploiting the recently published wheat draft genome sequence, we identified 53 members (including homoeologs from A, B and D genomes) of the C1-2i wheat Q-type C2H2 ZFPs (TaZFPs) representing 21 genes. Evolution analysis revealed that 9 TaZFPs members are directly inherited from the parents Triticum urartu and Aegilops tauschii, while 15 diverged through neoploidization events. This TaZFP subclass is responsive to the oxidative stress generator H2O2 and to high light, drought stress and flooding. Most TaZFPs are responsive to H2O2 (37/53), high light (44/53), flooding (31/53) or drought (37/53); 32 TaZFPs were up-regulated by at least 3 stresses and 16 were responsive to all stresses tested. A large number of these TaZFPs were physically mapped on different wheat draft genome sequences with known markers useful for QTL mapping. Our results show that the C1-2i subclass of TaZFPs is associated with responses to different abiotic stresses and that most TaZFPs (30/53 or 57 %) are located on group 5 chromosomes known to be involved in environment adaptation. Detailed characterization of these novel wheat TaZFPs and their association to QTL or eQTL may help to design wheat cultivars with improved tolerance to abiotic stress.