The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

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.

UhAVR1, an HR-triggering avirulence effector of Ustilago hordei, is secreted via the ER-Golgi pathway to the cytosol of barley coleoptile cells and contributes to virulence early in infection

The basidiomycete Ustilago hordei (Uh) causes covered smut disease of barley and oats. Virulence effectors that aid the infection process and support the pathogen’s 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 smut pathogens to date that triggers complete immunity in barley cultivars carrying the 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, both in cell culture away from its host, and during barley interaction. Transient expression of this effector in barley and a heterologous host, Nicotiana benthamiana (Nb), supports a cytosolic localization. Delivery of UhAVR1 via foxtail mosaic virus, Pseudomonas species or Agrobacterium-mediated suppression of cell inducers in barley and Nb support a role in the suppression of a common component(s) of ETI and PTI which is conserved in both plant systems.

Identification of sources of winter barley resistance to smut (Ustilago hordei)

The current paper presents the 3-year assessment results of domestic and foreign winter barley varieties and lines for smut resistance. The smut pathogen in the Rostov region causes one of the most harmful diseases, which, in addition to direct crop losses (head destruction), causes the so-called hidden losses, which can several times exceed direct ones. An effective way to counteract the disease is to cultivate resistant varieties, and farmers need the reliable sources of resistance to the pathogen. The immunological properties of winter barley varieties were studied in an infectious nursery (2016–2018). Seed infection with spores was carried out by S. Dimitrov’s method (1968) using a nutrient medium. Inoculated seeds were sown at the end of the optimal time. There was estimated a number of infected stems. The variety “Dobrynya 3” was used as a susceptible test-variety, which percentage of infection throughout the years ranged from 20.6 to 71.8%. There were studied 115 domestic and foreign varieties and lines. Among 69 domestic commercial varieties and breeding lines, 11 ones (15.9%) showed good resistance, 21 ones (30.4%) showed weak susceptibility, 30 ones (43.5%) possessed average susceptibility and seven varieties (10.1%) were severely affected by the pathogen. Among foreign varieties, resistance to barley smut has been established in seven German varieties, in three French varieties, in two Swiss varieties, one Czech variety and one Ukrainian variety. The varieties “Rocca”, “Nixe” (Germany), “Metelitsa” (Ukraine) are classified as the best ones in resistance to the pathogen during the artificial infection. The French variety “18513 ЕН11” had no affected plants during the study and was identified as an immune variety. The identified sources of winter barley resistance to smut are recommended for use in breeding programs for immunity.

TECHNOLOGY OF BARLEY BREEDING FOR RESISTANCE TO SMUT FUNGI

The technology of creating varieties of barley resistant to the smut diseases has been developed. Their cultivation will help to reduce crop losses, increase the profitability of seed production, improve the quality of seeds, and reduce the environmental risk from the use of chemical plant protection products. This technology was first implemented in the Kemerovo Region in 1988-1999. The wide distribution of species Ustilago nuda (Jens.) Kell. et Sw. (65 %) and U. nigra Tapke (35 %) was revealed. The covered smut ( Ustilago hordei (Pers.) Kell. Et Sw.) was registered in individual collection varieties. The first and tenth races of a loose smut and the sixth race of a black loose smut were identified. The sources of individual and group immunity to the smut fungi from the world gene pool of barley are distinguished: Moskovsky 3/125, Suzdalets, Ramos (Moscow Region); Kazer (Rostov Region); line 3 KM-1192 (Belarus); Kumir Odessky (Ukraine); Guardian (Canada). The donor properties of resistance to a loose smut in the Bagan variety, line 53 HVS 91/76, line 1899 were studied. They are recommended for use in breeding programs. With their participation a new hybrid fund was created. Variability and inheritance of quantitative traits of productivity of hybrids was studied. To select the recombinants immune to the loose smut in combination with high productivity, the most promising combinations of hybrids have been identified: Bagan × Viner; Bagan × KM-16; l. 53 HVS 91/76 × KM-7; KM-7 × l. 1899; l. 1899 × KM-7. Among them, prospective breeding lines have been selected. The results of the implementation of technology elements in the selection of barley for resistance to smut fungi is the creation of immune highly productive varieties of a new generation: Simon and Luka (Kemerovo Region). They are included in the State register of breeding achievements since 2004.