Study of the avirulence effector of Ustilago hordei, UhAVR1, during susceptible and resistant host interactions

Plant pathogens secrete virulence factors known as effectors to aid in infection and host colonization. Plants possess a multi-tier defense system to detect and halt pathogens. The basidiomycete fungus Ustilago hordei causes covered smut disease of barley. The previously identified effector UhAVR1 from U. hordei causes avirulence in barley cultivars carrying the resistance gene, Ruh1, whereas Ruh1 absence leads to virulence. To date, UhAVR1 is the only proven avirulence effector of smuts. This study aimed to functionally characterize UhAVR1 and to understand the underlying molecular mechanism leading to susceptibility or resistance in barley. Using fungal strains expressing UhAVR1+SP:mCherry, the secretion of UhAVR1 via the Brefeldin A-sensitive ER-Golgi pathway and by the action of its signal peptide was shown. UhAvr1 transcripts were detected only early during infection of barley seedlings confirming in planta induction upon host sensing. This infection-specific induction contributes to susceptibility in cultivar Odessa (ruh1) or to complete immunity in cultivar Hannchen (Ruh1). Transient expression of UhAVR1 in barley, Nicotiana benthamiana, and Arabidopsis thaliana showed it localizes to the cytosol, the site where it likely performs its functions. The delivery of UhAVR1 via foxtail mosaic virus, Pseudomonas bacteria, and Agrobacterium-mediated suppression of cell death inducers in N. benthamiana and barley support a role in the suppression of a conserved component(s) of plant immunity. RNA-seq analysis at 48 hpi of cultivar Odessa infected with U. hordei revealed that UhAvr1 induces plant fatty acids and suppresses plant defense. Whereas in cultivar Hannchen, UhAvr1 induces ETI and down-regulates PTI. Co-immunoprecipitation assays coupled with mass spectrometry from cultivar Odessa and N. benthamiana transiently expressing UhAVR1-SP:GFP revealed candidate host interactors with a chloroplast role. Previous work genetically located Ruh1 on barley chromosome 7H. This information was used to localize Ruh1 to a 10 million base pair region where 21 predicted resistance genes reside. Although identification of Ruh1 among the candidates was unsuccessful, this works paves the way for further studies on host resistance in this pathosystem. Ultimately, generating knowledge on how pathogens cause disease and how plants defend themselves is pivotal in generating control strategies against plant pathogens.Science, Faculty ofBotany, Department ofGraduat

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.Science, Faculty ofNon UBCBotany, Department ofReviewedFacult

Combate biológico de la mustia hilachosa (Thanatephorus cucumeris) en el frijol en Panamá

The objectiveof this work was to evaluate the capacity of Trichodermasp., Metarrhizium anisopliae and endophytic bacteria ofT. cucumeris (Frank) Donk under fi eld conditions, for thebiological control of web blight (T. cucumeris) in beans.Applications of Bacillus subtilis, Trichoderma sp. (Strain2-Villa Margarita), Trichoderma sp. (Strain 1-Colombia) andM. anisopliae (Colombia) at concentration of 2 x 107 conidia/ml of water), B. subtilis at rates of 0.335, 0.670, 1.005 and1.340 g ai / ml of water + 36 g ai Chlorothalonil /ha, benomyl50 WP with doses of 250 g ai / ha and a control treatment wereconducted. Results revealed high differences (P ≤ 0.0003)between treatments; the treatment with lower severity was M.anisopliae, followed by B. subtilis with 1,340 g ai /ml of water+ Chlorothalonil at 36 g ai/ha with 11.87% and B. subtilisto 0,335 g ai /l of water + Chlorothalonil at 36 g ai/ha, with15.0%. The treatments affected to the greatest degree were B.subtilis with 0,670 g ai/l of water + Chlorothalonil with 36g ai/ha, Trichoderma sp. (Strain 2-Villa Margarita) and thecontrol treatment with 26.2%, 22.25% and 21.85% of severity,respectively. The regression equation for the experiment Y =2502.91-13.021* Sev, was signifi cant and indicated lossesof 13.02 kg for each percent increase of the disease. Themarginal analysis revealed that the treatment with highest gainwas M. anisopliae, recording a net profi t of 28.34%.El objetivo del presente trabajo fue determinar la capacidadbiocontroladora de hongos y bacteria endofíticos en T.cucumeris (Frank) Donk en condiciones de campo. Seevaluó la aplicación de Trichoderma sp. (Cepa 2-FincaMargarita), Trichoderma sp. (Cepa 1-Colombia) y M.anisopliae (Colombia) en concentración de 2 x 107 conidias/ml de agua); B. subtilis a razón de 0,335; 0,670; 1,005 y1,340 g i.a./l agua + Clorotalonil 36 g i.a. /ha; Benomil 50WP en dosis de 250 g i.a./ha y el testigo absoluto. Hubosignificancia (P≤0,0003) entre los tratamientos, la de menorseveridad de T. cucumeris se dio con la adición de M.anisopliae, seguido de B. subtilis con 1340 g i.a./l agua +Clorotalonil 36 g i.a/ha con 11,87% y Bacillus subtilis a0,335 g i.a./l agua + Clorotalonil 36 g i.a/ha con 15,0%. Lostratamientos donde hubo mayor severidad fueron B. subtilis0,670 g i.a./l agua + Clorotalonil 36 g i.a/ha, Trichodermasp. (Cepa 2-Finca Margarita) y el testigo absoluto con26,2; 22,25% y 21,85% de severidad, respectivamente. Laecuación de regresión para el experimento Y=2502,91-13,021*Sev, fue signifi cativa e indicó pérdidas de 13,02 kgpor cada uno por ciento de incremento de la enfermedad. Enel análisis marginal el tratamiento con mayor ganancia fueM. anisopliae, con un beneficio neto de 28,34%

Combate biológico de la mustia hilachosa (Thanatephorus cucumeris) en el frijol en Panamá

The objectiveof this work was to evaluate the capacity of Trichodermasp., Metarrhizium anisopliae and endophytic bacteria ofT. cucumeris (Frank) Donk under fi eld conditions, for thebiological control of web blight (T. cucumeris) in beans.Applications of Bacillus subtilis, Trichoderma sp. (Strain2-Villa Margarita), Trichoderma sp. (Strain 1-Colombia) andM. anisopliae (Colombia) at concentration of 2 x 107 conidia/ml of water), B. subtilis at rates of 0.335, 0.670, 1.005 and1.340 g ai / ml of water + 36 g ai Chlorothalonil /ha, benomyl50 WP with doses of 250 g ai / ha and a control treatment wereconducted. Results revealed high differences (P ≤ 0.0003)between treatments; the treatment with lower severity was M.anisopliae, followed by B. subtilis with 1,340 g ai /ml of water+ Chlorothalonil at 36 g ai/ha with 11.87% and B. subtilisto 0,335 g ai /l of water + Chlorothalonil at 36 g ai/ha, with15.0%. The treatments affected to the greatest degree were B.subtilis with 0,670 g ai/l of water + Chlorothalonil with 36g ai/ha, Trichoderma sp. (Strain 2-Villa Margarita) and thecontrol treatment with 26.2%, 22.25% and 21.85% of severity,respectively. The regression equation for the experiment Y =2502.91-13.021* Sev, was signifi cant and indicated lossesof 13.02 kg for each percent increase of the disease. Themarginal analysis revealed that the treatment with highest gainwas M. anisopliae, recording a net profi t of 28.34%.El objetivo del presente trabajo fue determinar la capacidadbiocontroladora de hongos y bacteria endofíticos en T.cucumeris (Frank) Donk en condiciones de campo. Seevaluó la aplicación de Trichoderma sp. (Cepa 2-FincaMargarita), Trichoderma sp. (Cepa 1-Colombia) y M.anisopliae (Colombia) en concentración de 2 x 107 conidias/ml de agua); B. subtilis a razón de 0,335; 0,670; 1,005 y1,340 g i.a./l agua + Clorotalonil 36 g i.a. /ha; Benomil 50WP en dosis de 250 g i.a./ha y el testigo absoluto. Hubosignificancia (P≤0,0003) entre los tratamientos, la de menorseveridad de T. cucumeris se dio con la adición de M.anisopliae, seguido de B. subtilis con 1340 g i.a./l agua +Clorotalonil 36 g i.a/ha con 11,87% y Bacillus subtilis a0,335 g i.a./l agua + Clorotalonil 36 g i.a/ha con 15,0%. Lostratamientos donde hubo mayor severidad fueron B. subtilis0,670 g i.a./l agua + Clorotalonil 36 g i.a/ha, Trichodermasp. (Cepa 2-Finca Margarita) y el testigo absoluto con26,2; 22,25% y 21,85% de severidad, respectivamente. Laecuación de regresión para el experimento Y=2502,91-13,021*Sev, fue signifi cativa e indicó pérdidas de 13,02 kgpor cada uno por ciento de incremento de la enfermedad. Enel análisis marginal el tratamiento con mayor ganancia fueM. anisopliae, con un beneficio neto de 28,34%