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

Mikhail Oliveira Leastro,Vicente Pallás,Jesús Ángel Sánchez-Navarro,Elliot Watanabe Kitajima,Deibis Yorlenis Ortega Castro,Juliana Freitas-Astúa

doi:10.3389/fmicb.2020.01231

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 trans-expression 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.

Highlights

Organisms have a primary cellular defense mechanism known as RNA silencing
Double-stranded RNAs are processed by dicerlike RNases (DCLs) in small RNAs of 20–24 nt in size (Hamilton and Baulcombe, 1999; Matranga and Zamore, 2007; Borges and Martienssen, 2015) the small RNAs are loaded onto Argonaute (AGO) proteins to guide the silencing of DNA or RNA elements by a specific recognition of sequence complementarity (Hammond et al, 2000; Ding and Voinnet, 2007; Pumplin and Voinnet, 2013; Nakanishi, 2016; Pisacane and Halic, 2017)
To test if any of the Citrus leprosis virus C (CiLV-C) proteins have the capacity to suppress local RNA silencing, N. benthamiana 16c leaves were simultaneously co-infiltrated with individual A. tumefaciens cultures containing the binary vector pMOGGFP, as gene silencing inducer, and pMOG800 constructs containing the viral factors tested for potential RNA silencing proteins (RSS). pMOGHCPro and pMOG-empty constructs were used as positive and negative controls, respectively

Summary

Introduction

RNA silencing has a fundamental “sequence specific gene regulatory feature” (Kakumani et al, 2013) and plays an important role in defense against invading microorganisms (pathogens), especially viruses (Pumplin and Voinnet, 2013). This defense mechanism is activated by double-stranded RNA from high genome amplification of invasive microorganisms, transposons or ectopic expressed genes. Functional complementation of defective viral mutants (Chiba et al, 2006; Powers et al, 2008) or viral vectors in which the RSS is correlated with symptoms appearance (Guilley et al, 2009) have been used to identify RSS in the last decade

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Conclusion