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The wheat fungal pathogens Puccinia graminis f. sp. tritici (Pgt), P. striiformis f. sp. tritici, and P. triticina, causing stem, stripe, and leaf rust, respectively, pose a threat to global wheat production. Genetic resistance in the form of resistance (R) genes provides the best protection, but rust fungal populations change frequently by mutating avirulence (AVR) effectors matching specific R genes, thereby defeating resistance. Hence, characterization of AVR effectors is needed to understand the evolution of pathogen populations, provide insights for extending the effectiveness of R genes, and yield tools for the identification and isolation of R genes. Functional characterization of Avr genes in rust fungi is challenging in these biotrophic pathogens that lack a reliable and efficient transformation system. Studies indicate that the recently engineered foxtail mosaic virus (FoMV) shows promise as an expression system in cereals. In this study, we utilized two confirmed AVR effectors from Pgt, AVRSr35 and AVRSr50, to assess the applicability of FoMV for investigating rust fungus Avr genes. We showed that vector FoMV PV101 carrying PgtAvrSr35 induced a hypersensitive response (HR) in wheat having the corresponding Sr35 resistance gene. However, when carrying PgtAvrSr50, no HR or even mild viral symptoms were seen in a wheat line having Sr50, as this particular wheat line was not susceptible to FoMV. Several wheat cultivars did not support FoMV replication. The results here show that FoMV PV101 is effective as an expression vector for studying rust fungi AVR effectors, but its applicability relies on the susceptibility of wheat cultivars to FoMV. [Figure: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)