Screening of secreted proteins of Sporisorium reilianum f. sp. zeae for cell death suppression in Nicotiana benthamiana

Sporisorium reilianum f. sp. zeae (SRZ) is a biotrophic fungus causing head smut in maize. Maize infection with SRZ leads to very little cell death suggesting the presence of cell-death suppressinpg effectors. Several hundred effector proteins have been predicted based on genome annotation, genome comparison, and bioinformatic analysis. For only very few of these effectors, an involvement in virulence has been shown. In this work, we started to test a considerable subset of these predicted effector proteins for a possible function in suppressing cell death. We generated an expression library of 62 proteins of SRZ under the control of a strong constitutive plant promoter for delivery into plant cells via Agrobacterium tumefaciens -mediated transient transformation. Potential apoplastic effectors with high cysteine content were cloned with signal peptide while potential intracellular effectors were also cloned without signal peptide to ensure proper localization after expression in plant cells. After infiltration of Nicotiana benthamiana leaves, infiltration sites were evaluated for apparent signs of hypersensitive cell death in absence or presence of the elicitin INF1 of Phytophthora infestans . None of the tested candidates was able to induce cell death, and most were unable to suppress INF1-induced cell death. However, the screen revealed one predicted cytoplasmic effector (sr16441) of SRZ that was able to reliably suppress INF1-induced cell death when transiently expressed in N. benthamiana lacking its predicted secretion signal peptide. This way, we discovered a putative function for one new effector of SRZ

Fumigação com voláteis de Muscodor albus para controle de doenças pós-colheita

Postharvest diseases cause considerable losses of fruits and vegetables, from the harvest to their consumption, especially in developing countries. The biological control, particularly by biofumigation, has been pointed as an alternative to reduce postharvest losses. However, for using in the plant disease control, the previous knowledge regarding etiology is essential. A systematic etiological survey was, therefore, conducted in the local market of Viçosa and CEASAMINAS to generate information about the postharvest diseases in Minas Gerais state. The fungi associated with postharvest diseases were isolated, identified and subsequently submitted to in vitro e in vivo tests to verify the potential of Muscodor albus, an endophytic fungus that produces antimicrobials volatiles, in controlling postharvest diseases of fruits and vegetables by biological fumigation. In vitro test was conducted using bipartite Petri dishes with PDA medium, in which M. albus was previously grown for six days in one of the compartments. The mycelial plugs of each pathogen were placed in the other compartment of each plate and cultivated for sixty hours. The M. albus antimicrobial volatiles presented fungistatic or fungicide action depending on the phytopathogenic fungi tested. For the in vivo postharvest micofumigation test, the pathosystem Botrytis cinerea – strawberry was selected because of the in vitro results and the economic importance of gray mold for the crop. Strawberries produced with no pesticides use were wound-inoculated and placed in 11L plastic boxes containing M. albus cultures. The M. albus cultures were grown in Petri dishes containing PDA and the inoculated strawberries were placed in packaging in a way that had no direct contact between fungi and fruits. The micofumigation effectively controlled gray mold and the total decay in postharvest strawberries.Doenças pós-colheita causam perdas expressivas de frutas e hortaliças desde o momento da colheita até o consumo, especialmente em países em desenvolvimento. O controle biológico, particularmente por meio de biofumigação, tem sido apontado como alternativa para reduzir esse problema. Entretanto, para o uso no controle de doenças de plantas, o conhecimento prévio sobre a etiologia é essencial. Um levantamento sistemático foi, portanto, conduzido no mercado local de Viçosa e na CEASAMINAS, a fim de gerar informações sobre doenças pós-colheita no Estado de Minas Gerais. Os fungos associados às doenças pós-colheita foram isolados, identificados e posteriormente submetidos a testes in vitro e in vivo visando verificar o potencial de Muscodor albus, fungo endofítico produtor de voláteis antimicrobianos, em controlar doenças pós-colheita de frutas e hortaliças por fumigação biológica. O teste in vitro foi conduzido utilizando-se placas bipartidas contendo meio BDA, nas quais M. albus foi previamente cultivado por seis dias em um dos compartimentos. Os discos contendo micélio de cada patógeno foram dispostos no outro compartimento de cada placa e cultivados por sessenta horas. Os voláteis antimicrobianos de M. albus apresentaram ação fungistática ou fungicida a depender do fungo fitopatogênico testado. Para o teste in vivo de micofumigação em pós-colheita foi selecionado o patossistema Botrytis cinerea - morango em virtude dos resultados in vitro e da importância econômica do mofo cinzento para a cultura. Morangos adquiridos sem a aplicação de agrotóxicos foram inoculados por ferimentos e dispostos em caixas plásticas de 11L contendo culturas de M. albus. As culturas de M. albus foram crescidas em placas de Petri, contendo BDA e os morangos inoculados foram dispostos em bandejas, de forma a não haver contato direto entre o fungo e os frutos. A micofumigação proporcionou um controle efetivo do mofo cinzento e de podridões totais em morango em pós-colheita

Screening of Secreted Proteins of Sporisorium reilianum f. sp. zeae for Cell Death Suppression in Nicotiana benthamiana

Sporisorium reilianum f. sp. zeae (SRZ) is a biotrophic fungus causing head smut in maize. Maize infection with SRZ leads to very little cell death suggesting the presence of cell-death suppressinpg effectors. Several hundred effector proteins have been predicted based on genome annotation, genome comparison, and bioinformatic analysis. For only very few of these effectors, an involvement in virulence has been shown. In this work, we started to test a considerable subset of these predicted effector proteins for a possible function in suppressing cell death. We generated an expression library of 62 proteins of SRZ under the control of a strong constitutive plant promoter for delivery into plant cells via Agrobacterium tumefaciens -mediated transient transformation. Potential apoplastic effectors with high cysteine content were cloned with signal peptide while potential intracellular effectors were also cloned without signal peptide to ensure proper localization after expression in plant cells. After infiltration of Nicotiana benthamiana leaves, infiltration sites were evaluated for apparent signs of hypersensitive cell death in absence or presence of the elicitin INF1 of Phytophthora infestans . None of the tested candidates was able to induce cell death, and most were unable to suppress INF1-induced cell death. However, the screen revealed one predicted cytoplasmic effector (sr16441) of SRZ that was able to reliably suppress INF1-induced cell death when transiently expressed in N. benthamiana lacking its predicted secretion signal peptide. This way, we discovered a putative function for one new effector of SRZ