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[ES] El silenciamiento por RNA es un mecanismo fundamental en la defensa de las plantas frente a virus. Los RNA de doble cadena víricos disparan este mecanismo al ser reconocidos por unas enzimas con actividad RNasa III, conocidas como DCL (Dicer-like), que median su procesamiento en pequeños RNA (small RNAs, sRNA) de entre 20 y 24 nucleótidos. A continuación, una de las cadenas de los sRNA se incorpora al complejo RISC (RNA-induced silencing complex), cuya molécula efectora es una endonucleasa de la familia de los Argonautas (AGO). Esta unión induce la activación del complejo y conduce a la degradación de RNA de secuencia complementaria al sRNA. Los virus, para luchar contra esta respuesta defensiva del huésped, producen proteínas supresoras del silenciamiento por RNA, abreviadas como VSR (viral RNA silencing suppressors), que son capaces de interferir con una o varias etapas de la ruta del silenciamiento. El virus de las venas amarillas del pimiento 5 (Pepper vein yellows virus 5, PeVYV-5; género Polerovirus, familia Solemoviridae), es un patógeno detectado en España en los últimos años que forma parte de un grupo de virus emergentes que amenazan el cultivo del pimiento. Este agente infeccioso codifica una proteína conocida como P0, cuya actividad VSR ha sido constatada recientemente en el laboratorio y se ha propuesto que está basada en la degradación de proteínas AGO. Para continuar con el estudio de este supresor, en el presente trabajo se ha continuado con la caracterización molecular de una serie de variantes artificiales/naturales del mismo, lo que ha permitido establecer una correlación entre la actividad VSR de P0 y su capacidad para inducir la degradación de AGO. Estos resultados apoyan que el principal modo de acción de P0 es la desestabilización de los efectores de RISC. Además, los resultados han permitido comprobar que, pese a que distintas partes de la molécula P0 parecen estar involucradas en su actividad VSR, esta proteína es capaz de tolerar heterogeneidad de secuencia considerable manteniendo su función. Por último, se ha evaluado, mediante silenciamiento génico inducido por virus (VIGS), la posible participación de dos factores centrales en autofagia, ATG8f y/o ATG8c1, en la degradación de AGO promovida por P0. Los resultados no han permitido corroborar su participación, aunque serán necesarios experimentos adicionales para obtener datos concluyentes a este respecto., [EN] Gene silencing is a fundamental mechanism in the defense against viruses. Viral double-stranded RNAs trigger this mechanism when they are recognized by enzymes with RNase III activity, known as DCL (Dicer-like), which mediate their processing into small RNAs (sRNA) between 20 and 24 nucleotides. Next, one of the sRNA chains is incorporated into the RISC complex (RNA-induced silencing complex), whose effector molecule is an endonuclease of the Argonaut family (AGO). This binding induces the activation of the complex and leads to the degradation of RNA complementary sequences to sRNA. Viruses, to fight against this host defensive response, generate RNA suppressor proteins, known as VSR (viral RNA silencing suppressors), which are capable of interfering with one or more steps of the silencing pathway. Pepper vein yellows virus 5 (PeVYV-5; genus Polerovirus, family Solemoviridae) is a pathogen detected in Spain during the last years which belongs to an emerging group of viruses that threaten pepper culture. This infectious agent encodes a protein known as P0, whose VSR activity has been recently verified in the laboratory and it has been proposed that it is based on the AGO proteins degradation. To continue with the study of this suppressor, in the present work we have continued with the molecular characterization of a series of artificial/natural variants of the suppressor, which has made it possible to establish a connection between the VSR activity of P0 and its capacity to induce the AGO degradation. These results support that the destabilization of RISC effectors is the main mode of action of P0. In addition, the results have confirmed that, even though different parts of P0 molecule seem to be involved in its VSR activity, this protein can tolerate considerable sequence heterogeneity while maintaining its function. Finally, the possible participation of two central factors in autophagy, ATG8f and/or ATG8c1, in the degradation of AGO lead by P0 has been evaluated by viral induced gene silencing (VIGS). The results have not corroborated their participation, so additional experiments will be necessary to obtain conclusive data in this regard.

Simple Summary Polerovirus P0 proteins are multifunctional proteins. Besides their viral suppressor of RNA silencing (VSR) functions, several P0 proteins can induce a cell death phenotype within the infiltrated region of Nicotiana benthamiana or Nicotiana glutinosa. Recently, the Brassica yellows virus (BrYV) genotype A P0 protein (P0BrA) was identified as a strong viral suppressor of RNAi. In this study, we compared the features of the P0 proteins encoded by different genotypes of BrYV and revealed their difference in inducing cell death in N. benthamiana. Key residues in P0BrA for inducing cell death were also identified. We also showed that all three BrYV genotypes had synergistic interaction with PEMV 2 in N. benthamiana. This study provides theoretical guidance for controlling the viral disease caused by poleroviruses in the future. Abstract Brassica yellows virus (BrYV) is a tentative species of the genus Polerovirus, which has at least three genotypes (A, B, and C) in China. The P0 protein of BrYV-A (P0BrA) has been identified as a viral suppressor of RNA silencing (VSR), which can also induce cell death in infiltrated Nicotiana benthamiana leaves. In this study, we demonstrated that the cell death induced by P0BrA was accompanied by the accumulation of reactive oxygen species (ROS) and increased Pathogenesis-related protein genes-1 (PR1) expression. Meanwhile, this cell death phenotype was delayed by salicylic acid (SA) pretreatment. Biological function comparison of the three P0 proteins showed that transiently expressed P0BrB or P0BrC induced a significantly delayed and milder cell death response compared with P0BrA. However, like P0BrA, they also suppressed local and systemic RNA silencing. Six residues of P0BrA essential for inducing cell death were identified by comparative analysis and amino acid substitution assay. We also show that all three BrYV genotypes have synergistic interactions with pea enation mosaic virus 2 (PEMV 2) in N. benthamiana. This study provides theoretical guidance for controlling the viral disease caused by poleroviruses in the future.

The Sugarcane yellow leaf virus (SCYLV) P0, a member of the highly heterologous proteins of poleroviruses, is a suppressor of posttranscriptional gene silencing (PTGS) and has additional activities not seen in other P0 proteins. The P0 protein in previously tested poleroviruses (Beet western yellows virus and Cucurbit aphid-borne yellows virus), suppresses local, but not systemic, PTGS induced by both sense GFP and inverted repeat GF using its F-box-like domain to mediate destabilization of the Argonaute1 protein. We now report that the SCYLV P0 protein not only suppressed local PTGS induced by sense GFP and inverted repeat GF in Nicotiana benthamiana, but also triggered a dosage dependent cell death phenotype in infiltrated leaves and suppressed systemic sense GFP-PTGS. Deletion of the first 15 N-terminal amino acid residues of SCYLV P0 abolished suppression of both local and systemic PTGS and the induction of cell death. In contrast, only systemic PTGS and cell death were lost when the 15 C-terminal amino acid residues were deleted. We conclude that the 15 C-terminal amino acid residue region of SCYLV P0 is necessary for suppressing systemic PTGS and inducing cell death, but is not required for suppression of local PTGS.