Background

Potato virus Y (PVY) is the most economically important virus that infects cultivated potato and causes major crop losses worldwide (Valkonen, 2007). Besides potato, PVY can infect a broad range of other plant species, including mainly other cultivated Solanacea species such as tomato, pepper, or tobacco, as well as weeds and ornamentals (Kerlan, 2006). PVY is naturally transmitted by aphids in a non-persistent manner. Up to 65 aphid species have been reported to be potential vectors for PVY, which makes it difficult to implement efficient viral control and management measures (Lacomme and Jacquot, 2017). Typical for the Potyviridae family, the PVY virion is structured as a flexuous filamentous particle that encapsidates a 9.7 kb positive-sense single-stranded RNA genome. It encodes 10 proteins synthesised from a single open reading frame and one protein, P3N-PIPO, synthesised from an overlapping reading frame (Chung et al., 2008).

Understanding the complex interaction network established between PVY and its host plant is indispensable for designing more efficient and long-lasting virus control measures, such as targeted crop-breeding strategies (Lacomme and Jacquot, 2017). During the last decade, technological advancements have improved our knowledge of potato-PVY interactions (Križnik et al., 2017; Lukan et al., 2018; Stare et al., 2019) and viral mechanisms of the infection cycle (Mäkinen and Hafrén, 2014; Mäkinen et al., 2017). The potential of such technical developments has been demonstrated in our recent study (Kežar et al., 2019). By combining knowledge of the PVY virion structure (based on high-resolution cryo-electron microscopy) with in planta coat protein (CP) functional analysis (using a PVY cDNA clone), we provided insights into the crucial role of CP and its N- and C- terminal regions in PVY infectivity.

The study reveals the value of an easy-to-manipulate PVY cDNA infectious clone for in planta functional analysis of the virus. The production and use of PVY infectious clones has been more challenging than that for other potyviruses due to the expression of cryptic prokaryotic promoters in the PVY genome, which results in products that are cytotoxic to E. coli (Fakhfakh et al., 1996; Jakab et al., 1997). Thus, only a few PVY cDNA clones have been used to date to investigate the viral determinants involved in the vein necrosis symptom (Tribodet et al., 2005), the rate of PVY cell-to-cell movement via inclusion of a GFP reporter gene into the polyprotein (Rupar et al., 2015), and the role of temperature in Ry_sto-mediated extreme resistance (Grech-Baran et al., 2020).

However, the use of PVY infectious clones as a reverse genetics tool for in planta functional analysis of PVY is still scarce because of the low inoculation yields and complex manipulation of some PVY clones. Therefore, we here describe a protocol to functionally characterise the role of the viral CP by using the PVY-N605(123) cDNA clone (Bukovinszki et al., 2007), a full-length infectious clone that was previously developed to avoid the cytotoxic effects to E. coli through intron insertion. We present a detailed description of the cloning strategy to introduce desired CP deletions into the PVY-N605(123) cDNA clone followed by gene gun bombardment of Nicotiana clevelandii plants. The protocol includes characterisation of the resulting virus amplification and spread using reverse transcription quantitative PCR to quantify viral genome and transmission electron microscopy to confirm virion formation. We recently used this protocol to study the role of viral CP N- and C- terminal regions in virus infectivity (Kežar et al., 2019). The same procedure, with an appropriate modification of the mutagenesis strategy, can also be used to introduce any mutation into PVY cDNA or alternatively to insert fluorescent protein into cDNA and tag specific PVY proteins for purification or imaging purposes. Therefore, the protocol described here offers an indispensable tool for in planta analysis of the viral cycle and functional studies of viral proteins.