Citrus Leprosis Virus C Encodes Three Proteins With Gene Silencing Suppression Activity

Abstract

Citrus leprosis virus C (CiLV-C) belongs to the genus Cilevirus, family Kitaviridae, and is considered the most devastating virus infecting citrus in Brazil, being the main viral pathogen responsible for citrus leprosis (CL), a severe disease that affects citrus orchards in Latin America. Here, proteins encoded by CiLV-C genomic RNA 1 and 2 were screened for potential RNA silencing suppressor (RSS) activity by five methods. Using the GFP-based reporter agroinfiltration assay, we have not found potential local suppressor activity for the five CiLV-C encoded proteins. However, when RSS activity was evaluated using the alfalfa mosaic virus (AMV) system, we found that the p29, p15, and p61 CiLV-C proteins triggered necrosis response and increased the AMV RNA 3 accumulation, suggesting a suppressive functionality. From the analysis of small interfering RNAs (siRNAs) accumulation, we observed that the ectopic expression of the p29, p15, and p61 reduced significantly the accumulation of GFP derived siRNAs. The use of the RSS defective turnip crinkle virus (TCV) system revealed that only the transexpression of the p15 protein restored the cell-to-cell viral movement. Finally, the potato virus X (PVX) system revealed that the expression of p29, p15, and p61 increased the PVX RNA accumulation; in addition, the p29 and p15 enhanced the pathogenicity of PVX resulting in the death of tobacco plants. Furthermore, PVX-p61 infection resulted in a hypersensitive response (HR), suggesting that p61 could also activate a plant defense response mechanism. This is the first report describing the RSS activity for CiLV-C proteins and, moreover, for a member of the family Kitaviridae.

Dichorhaviruses Movement Protein and Nucleoprotein Form a Protein Complex That May Be Required for Virus Spread and Interacts in vivo With Viral Movement-Related Cilevirus Proteins

“…The chimeric AMV RNA 3 expressing the AMV MP wild type (wt) was obtained from Leastro et al (2017b) . As reviewed by Leastro et al (2020) , three independent experiments were performed, each one included the infiltration of three leaves of P12 plants per construct. The foci images in P12 plants were taken with the aid of a Leica MZ16F fluorescence stereomicroscope.…”

“…The trans-complementation assay was performed as previously described by Martinez-Perez et al (2019) . In short, three leaves of N. benthamiana per plant were infiltrated with A. tumefaciens cultures containing the empty pMOG 800 or expressing the LEP protein (negative controls), MP of CiLV-C (positive control) (both obtained from Leastro et al, 2020 ) or the pMOG 800 constructs expressing the CiLV-N or OFV-citrus MPs at OD 600 1. Next, the pTCVΔ92-sGFP plasmid was linearized with Xba I and transcripts were inoculated at 1 day post infiltration.…”

Sequence Relationships of RNA Helicases and Other Proteins Encoded by Blunervirus RNAs Highlight Recombinant Evolutionary Origin of Kitaviral Genomes

“…Instead, VLRA 4 protein p68 ( Figure 1B) contains a sequence motif quite similar to the nuclear localization signal (NLS) of cucumoviral 2b proteins known to have silencing suppressor functions (Du et al, 2014;Bujarski, in press) ( Supplementary Table 2). Thus it can be speculated that VLRAs discovered by in silico analyses of Paulownia tomoentosa transcriptome represent a genome of a putative kitavirus that encodes silencing suppressors (Leastro et al, 2020).…”

IIdentification, characterization, and evolutionary features of >i<Brevipalpus>/i<-transmitted viruses causing citrus leprosis disease

“…Based on the lack of similarity of the sequences and structure of the proteins with all the others available in the public domain, the proteins P15, P61, and P24 of cileviruses have unknown functions , although both P61 and P15 are suggested as possible RNA silencing suppressors (Leastro et al, 2020). Sub-cellular localization assays have shown that these proteins associate with the endoplasmic reticulum (ER) (Leastro et al, 2018).…”

“…In CiLV-C, the RNA2 also contains an intergenic region (IR) of ~1 kb located between the p15 and p61 cistrons. P15, P61, and P24 are proteins without definitive associated functions, although the first two seem to be involved in silencing suppression mechanisms (Leastro et al, 2020) and the latter is conserved among cileviruses, higreviruses, and members of the insect-specific negevirus group . The mp encodes a putative movement protein (MP) of the 30K…”