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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 Brassica yellows virus genotype A P0 protein interacts with Rubisco assembly factor 2 in tobacco, affecting its nuclear accumulation and promoting viral infection., In interactions between poleroviruses and their hosts, few cellular proteins have been identified that directly interact with the multifunctional virus P0 protein. To help explore the functions of P0, we identified a Brassica yellows virus genotype A (BrYV-A) P0BrA-interacting protein from Nicotiana benthamiana, Rubisco assembly factor 2 (NbRAF2), which localizes in the nucleus, cell periphery, chloroplasts, and stromules. We found that its C-terminal domain (amino acids 183–211) is required for self-interaction. A split ubiquitin membrane-bound yeast two-hybrid system and co-immunoprecipitation assays showed that NbRAF2 interacted with P0BrA, and co-localized in the nucleus and at the cell periphery. Interestingly, the nuclear pool of NbRAF2 decreased in the presence of P0BrA and during BrYV-A infection, and the P0BrA-mediated reduction of nuclear NbRAF2 required dual localization of NbRAF2 in the chloroplasts and nucleus. Tobacco rattle virus-based virus-induced gene silencing of NbRAF2 promoted BrYV-A infection in N. benthamiana, and the overexpression of nuclear NbRAF2 inhibited BrYV-A accumulation. Potato leafroll virus P0PL also interacted with NbRAF2 and decreased its nuclear accumulation, indicating that NbRAF2 may be a common target of poleroviruses. These results suggest that nuclear NbRAF2 possesses antiviral activity against BrYV-A infection, and that BrYV-A P0BrA interacts with NbRAF2 and alters its localization pattern to facilitate virus infection.

The yellow dwarf viruses (YDVs) of the Luteoviridae family represent the most widespread group of cereal viruses worldwide. They include the Barley yellow dwarf viruses (BYDVs) of genus Luteovirus, the Cereal yellow dwarf viruses (CYDVs) and Wheat yellow dwarf virus (WYDV) of genus Polerovirus. All of these viruses are obligately aphid transmitted and phloem-limited. The first described YDVs (initially all called BYDV) were classified by their most efficient vector. One of these viruses, BYDV-RMV, is transmitted most efficiently by the corn leaf aphid, Rhopalosiphum maidis. Here we report the complete 5612 nucleotide sequence of the genomic RNA of a Montana isolate of BYDV-RMV (isolate RMV MTFE87, Genbank accession no. KC921392). The sequence revealed that BYDV-RMV is a polerovirus, but it is quite distantly related to the CYDVs or WYDV, which are very closely related to each other. Nor is BYDV-RMV closely related to any other particular polerovirus. Depending on the gene that is compared, different poleroviruses (none of them a YDV) share the most sequence similarity to BYDV-RMV. Because of its distant relationship to other YDVs, and because it commonly infects maize via its vector, R. maidis, we propose that BYDV-RMV be renamed Maize yellow dwarf virus-RMV (MYDV-RMV).