Carina Andrea Reyes

Resistance to Citrus psorosis virus in transgenic sweet orange plants is triggered by coat protein–RNA silencing

The lack of naturally occurring resistance to Citrus psorosis virus (CPsV) has demanded exploitation of a transgenic approach for the development of CPsV-resistant sweet orange plants. Transgenic sweet orange plants producing intron-hairpin RNA transcripts (ihpRNA) corresponding to viral cp, 54K or 24K genes were generated and analyzed at the molecular and phenotypic levels. Two independent CPsV challenge assays demonstrated that expression of ihpRNA derived from the cp gene (ihpCP) provided a high level of virus resistance, while those derived from 54K and 24K genes (ihp54K and ihp24K) provided partial or no resistance. The presence of small interfering RNA molecules (siRNAs) in the ihpCP transgenic sweet orange plants prior to virus challenge, indicated that CPsV resistance was due to pre-activated RNA silencing, but siRNAs accumulation level was not directly correlated to the degree of the triggered virus resistance among the different lines. However, pre-activation of the RNA-silencing machinery and a certain minimum accumulation level of siRNA molecules targeting the viral genome are key factors for creating virus-resistant plants. This is the first report of resistance in citrus plants against a negative-strand RNA virus as CPsV.

Citrus psorosis virus 24K protein interacts with citrus miRNA precursors, affects their processing and subsequent miRNA accumulation and target expression

Sweet orange (Citrus sinensis), one of the most important fruit crops worldwide, may suffer from disease symptoms induced by virus infections, thus resulting in dramatic economic losses. Here, we show that the infection of sweet orange plants with two isolates of Citrus psorosis virus (CPsV) expressing different symptomatology alters the accumulation of a set of endogenous microRNAs (miRNAs). Within these miRNAs, miR156, miR167 and miR171 were the most down-regulated, with almost a three-fold reduction in infected samples. This down-regulation led to a concomitant up-regulation of some of their targets, such as Squamosa promoter-binding protein-like 9 and 13, as well as Scarecrow-like 6. The processing of miRNA precursors, pre-miR156 and pre-miR171, in sweet orange seems to be affected by the virus. For instance, virus infection increases the level of unprocessed precursors, which is accompanied by a concomitant decrease in mature species accumulation. miR156a primary transcript accumulation remained unaltered, thus strongly suggesting a processing deregulation for this transcript. The co-immunoprecipitation of viral 24K protein with pre-miR156a or pre-miR171a suggests that the alteration in the processing of these precursors might be caused by a direct or indirect interaction with this particular viral protein. This result is also consistent with the nuclear localization of both miRNA precursors and the CPsV 24K protein. This study contributes to the understanding of the manner in which a virus can alter host regulatory mechanisms, particularly miRNA biogenesis and target expression.

Differential resistance to Citrus psorosis virus in transgenic Nicotiana benthamiana plants expressing hairpin RNA derived from the coat protein and 54K protein genes

Citrus psorosis virus (CPsV), genus Ophiovirus, family Ophioviridae, is the causal agent of a serious disease affecting citrus trees in many countries. The viral genome consists of three ssRNAs of negative polarity. Post-transcriptional gene silencing (PTGS), a mechanism of plant defence against viruses, can be induced by transgenic expression of virus-derived sequences encoding hairpin RNAs. Since the production of transgenic citrus lines and their evaluation would take years, a herbaceous model plant, Nicotiana benthamiana, was used to test hairpin constructs. The expression of self-complementary hairpin RNA fragments from the coat protein (cp) and 54K genes of the Argentine CPsV 90-1-1 isolate conferred resistance on N. benthamiana plants, indicating that these constructs are good candidates for the transformation of citrus plants. The degree of resistance obtained varied depending on the viral sequence chosen. The analysis of the levels of small interfering RNA accumulation and viral RNAs indicated that the construct derived from cp gene was better at inducing PTGS than that originating from the 54K gene. The dependence of PTGS induction on the degree of identity between the target and the inducer transgene sequences was tested using sequences derived from CPV4, a more distant isolate of CPsV, as PTGS targets. Efficient silencing induction was also obtained to this isolate through the expression of the cp-derived hairpin. This is the first report of transgenic-resistant plants within the context of this serious citrus disease.

Citrus psorosis and Mirafiori lettuce big-vein ophiovirus coat proteins localize to the cytoplasm and self interact in vivo.

Citrus psorosis (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV) belong to the family Ophioviridae, plant viruses with filamentous nucleocapsids and segmented genomes of negative polarity, causing the worldwide distributed citrus psorosis and lettuce big-vein diseases, respectively. To gain insight into the replication cycle of these viruses, the subcellular localization of the viral coat proteins (CP) was studied. Immunoblot analysis of fractionated extracts derived from natural and experimental infected hosts indicated that the CP of CPsV occurs in the soluble cytoplasmic fraction. The cytoplasmic localization of this protein was confirmed by confocal microscopy of fluorescent protein (FP)-tagged CP following its expression in either CPsV-infected and healthy Citrus sinensis plants or in Nicotiana benthamiana plants. The same localization was observed for FP-tagged CP of MiLBVV. The CPs of CPsV and MiLBBV can undergo homologous and heterologous interactions as revealed by fluorescent lifetime imaging microscopy and co-immunoprecipitation analysis. A putative leucine zipper motif that is conserved among ophiovirus CP sequences may account for these interactions.

Identification and characterization of two RNA silencing suppressors encoded by ophioviruses

Citrus psorosis virus and Mirafiori lettuce big-vein virus are two members of the genus Ophiovirus, family Ophioviridae. So far, how these viruses can interfere in the antiviral RNA silencing pathway is not known. In this study, using a local GFP silencing assay on Nicotiana benthamiana, the 24K-25K and the movement protein (MP) of both viruses were identified as RNA silencing suppressor proteins. Upon their co-expression with GFP in N. benthamiana 16c plants, the proteins also showed to suppress systemic RNA (GFP) silencing. The MPCPsV and 24KCPsV proteins bind long (114 nucleotides) but not short-interfering (21 nt) dsRNA, and upon transgenic expression, plants showed developmental abnormalities that coincided with an altered miRNA accumulation pattern. Furthermore, both proteins were able to suppress miRNA-induced silencing of a GFP-sensor construct and the co-expression of MPCPsV and 24KCPsV exhibited a stronger effect, suggesting they act at different stages of the RNAi pathway.

RNA-binding protein immunoprecipitation as a tool to investigate plant miRNA processing interference by regulatory proteins of diverse origin

BackgroundDue to the nature of viral RNA genomes, RNA viruses depend on many RNA-binding proteins (RBP) of viral and host origin for replication, dissemination and evasion of host RNA degradation pathways. Some viruses interfere with the microRNA (miRNA) pathway to generate better fitness. The development of an adjusted, reliable and sensitive ribonucleoprotein immunoprecipitation (RIP) assay is needed to study the interaction between RBP of different origin (including viral origin) and miRNA precursors. The method could be further applied to transiently expressed heterologous proteins in different plant species.ResultsHere we describe a modified RIP assay applied to nuclear epitope-tagged proteins of heterologous origin and transiently expressed in Nicotiana benthamiana. The assay includes a combination of optimized steps as well as the careful selection of control samples and rigorous data analysis. It has proven efficient to detect and quantify miRNA processing intermediates associated with regulatory proteins.ConclusionsThe RIP method described here provides a reliable tool to study the interaction of RBPs, such as transiently expressed regulatory proteins with lowly represented host RNA, as is the case of miRNA precursors. This modified method was efficiently adjusted to recover nuclear proteins and reduce unspecific background. The purification scheme optimized here for GFP-tagged proteins can be applied to a wide array of RBPs. The subsequent application of next-generation sequencing technologies will permit to sequence and characterize all RNA species bound in vivo by a given RBP.