Virus-Mediated Protein Overexpression (VOX) in Monocots to Identify and Functionally Characterize Fungal Effectors.

One of the important armories that pathogens utilize to successfully colonize the plants is small secreted effector proteins, which could perform a variety of functions from suppression of plant innate immunity to manipulation of plant physiology in favor of the disease. Plants, on the other hand, evolved disease resistance genes that recognize some of the effectors or avirulence (Avr) proteins. Both, identification of the Avr proteins and understanding of the mechanisms of action of other effectors, are important areas of research in the molecular plant-pathogen interactions field as this knowledge is critical for the development of new effective pathogen control measures. To enable functional analysis of the effectors, it is desirable to be able to overexpress them readily in the host plants. Here we describe detailed experimental protocols for transient effector overexpression in wheat and other monocots using binary Barley stripe mosaic virus (BSMV)- and Foxtail mosaic virus (FoMV)-derived vectors. This functional genomics tool, better known as VOX (Virus-mediated protein OvereXpression), is rapid and relatively simple and inexpensive.

Foxtail mosaic virus-induced gene silencing (VIGS) in switchgrass (Panicum virgatum L.).

BACKGROUND: Although the genome for the allotetraploid bioenergy crop switchgrass (Panicum virgatum) has been established, limitations in mutant resources have hampered in planta gene function studies toward crop optimization. Virus-induced gene silencing (VIGS) is a versatile technique for transient genetic studies. Here we report the implementation of foxtail mosaic virus (FoMV)-mediated gene silencing in switchgrass in above- and below-ground tissues and at different developmental stages. RESULTS: The study demonstrated that leaf rub-inoculation is a suitable method for systemic gene silencing in switchgrass. For all three visual marker genes, Magnesium chelatase subunit D (ChlD) and I (ChlI) as well as phytoene desaturase (PDS), phenotypic changes were observed in leaves, albeit at different intensities. Gene silencing efficiency was verified by RT-PCR for all tested genes. Notably, systemic gene silencing was also observed in roots, although silencing efficiency was stronger in leaves (~ 63-94%) as compared to roots (~ 48-78%). Plants at a later developmental stage were moderately less amenable to VIGS than younger plants, but also less perturbed by the viral infection. CONCLUSIONS: Using FoMV-mediated VIGS could be achieved in switchgrass leaves and roots, providing an alternative approach for studying gene functions and physiological traits in this important bioenergy crop.

UhAVR1, an HR-Triggering Avirulence Effector of Ustilago hordei, Is Secreted via the ER-Golgi Pathway, Localizes to the Cytosol of Barley Cells during in Planta-Expression, and Contributes to Virulence Early in Infection.

The basidiomycete Ustilago hordei causes covered smut disease of barley and oats. Virulence effectors promoting infection and supporting pathogen lifestyle have been described for this fungus. Genetically, six avirulence genes are known and one codes for UhAVR1, the only proven avirulence effector identified in smuts to date that triggers complete immunity in barley cultivars carrying resistance gene Ruh1. A prerequisite for resistance breeding is understanding the host targets and molecular function of UhAVR1. Analysis of this effector upon natural infection of barley coleoptiles using teliospores showed that UhAVR1 is expressed during the early stages of fungal infection where it leads to HR triggering in resistant cultivars or performs its virulence function in susceptible cultivars. Fungal secretion of UhAVR1 is directed by its signal peptide and occurs via the BrefeldinA-sensitive ER-Golgi pathway in cell culture away from its host. Transient in planta expression of UhAVR1 in barley and a nonhost, Nicotiana benthamiana, supports a cytosolic localization. Delivery of UhAVR1 via foxtail mosaic virus or Pseudomonas species in both barley and N. benthamiana reveals a role in suppressing components common to both plant systems of Effector- and Pattern-Triggered Immunity, including necrosis triggered by Agrobacterium-delivered cell death inducers.

Virus-Induced Gene Silencing in Poaceae Using a Foxtail Mosaic Virus Vector.

Virus-induced gene silencing (VIGS) is a powerful tool for rapidly knocking down the expression of plant genes to elucidate functional genomics. We have established a VIGS vector for monocot plants derived from Foxtail mosaic virus (FoMV), a positive-sense single-stranded RNA virus. For silencing a targeted gene, plant gene fragment was inserted into the vector between open reading frame 4 (ORF4) and ORF5 under the control of a duplicated coat protein promoter. Plants of different monocot species were infected by mechanical inoculation with sap from FoMV derivative-infected Chenopodium quinoa leaves. Gene silencing was typically observed within 2-3 weeks after inoculation. In this chapter, we describe the detailed protocol for silencing a target gene in various Poaceae plants by using FoMV-based vectors.

Foxtail mosaic virus-induced flowering assays in monocot crops.

Virus-induced flowering (VIF) exploits RNA or DNA viruses to express flowering time genes to induce flowering in plants. Such plant virus-based tools have recently attracted widespread attention for their fundamental and applied uses in flowering physiology and in accelerating breeding in dicotyledonous crops and woody fruit-trees. We now extend this technology to a monocot grass and a cereal crop. Using a Foxtail mosaic virus (FoMV)-based VIF system, dubbed FoMViF, we showed that expression of florigenic Flowering Locus T (FT) genes can promote early flowering and spikelet development in proso millet, a C4 grass species with potential as a nutritional food and biofuel resource, and in non-vernalized C3 wheat, a major food crop worldwide. Floral and spikelet/grain induction in the two monocot plants was caused by the virally expressed untagged or FLAG-tagged FT orthologs, and the florigenic activity of rice Hd3a was more pronounced than its dicotyledonous counterparts in proso millet. The FoMViF system is easy to use and its efficacy to induce flowering and early spikelet/grain production is high. In addition to proso millet and wheat, we envisage that FoMViF will be also applicable to many economically important monocotyledonous food and biofuel crops.

An RNAi suppressor activates in planta virus-mediated gene editing.

RNA-guided CRISPR/Cas9 technology has been developed for gene/genome editing (GE) in organisms across kingdoms. However, in planta delivery of the two core GE components, Cas9 and small guide RNA (sgRNA), often involves time-consuming and labor-intensive production of transgenic plants. Here we show that Foxtail mosaic virus, a monocot- and dicot-infecting potexvirus, can simultaneously express Cas9, sgRNA, and an RNAi suppressor to efficiently induce GE in Nicotiana benthamiana through a transgenic plant-free manner.