RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA

Abstract

Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus-host-vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA.

The Torradovirus‐specific RNA2‐ORF1 protein is necessary for plant systemic infection

“… Constructs used in this study. Schematic representation of pJL89‐M‐R2 (R2‐WT, A), pJL89‐M‐R2‐GFP (R2‐GFP, B) (Ferriol et al ., ) and the series of RNA2 mutant constructs used in this study (A and B). The RNA genome is depicted with horizontal lines.…”

“…Moreover, the N‐terminal fragment of the ToTV RNA1‐encoded polyprotein induces a hypersensitive response (HR)‐like in Nicotiana benthamiana plants (Wieczorek and Obrepalska‐Steplowska, ). Infectious clones have also been generated for ToANV, previously named tomato marchitez virus isolate M (Ferriol et al ., , b), and have been shown to have the ability to initiate infection via agroinfiltration. This resulted in symptoms indistinguishable from those of wild‐type (WT) infection, and the progeny were whitefly transmissible (Ferriol et al ., ).…”

“…Infectious clones have also been generated for ToANV, previously named tomato marchitez virus isolate M (Ferriol et al ., , b), and have been shown to have the ability to initiate infection via agroinfiltration. This resulted in symptoms indistinguishable from those of wild‐type (WT) infection, and the progeny were whitefly transmissible (Ferriol et al ., ). A ToANV infectious construct expressing the green fluorescent protein (GFP) was also derived (Ferriol et al ., ) and was used to monitor ToANV movement in the inoculated leaf.…”

“…This predicted protein has homologues only in the torradoviruses, and the percentage identity of aligned proteins varies from 61.5% to 76.5% amongst the tomato-infecting torradoviruses, and 21.8% to 54.7% amongst the other torradoviruses; nevertheless, a number of core domains based on identical residues are shared amongst all the sequenced torradoviruses (van der Vlugt et al, 2015). RNA2-ORF2 encodes a polyprotein which is processed by virus-encoded proteases, yielding a predicted movement protein (MP) and the three distinct CPs (Ferriol et al, 2016a). So far, there is no experimental evidence of the translational strategy to express RNA2-ORF2 from the bicistronic torradovirus RNA2.…”

“…This resulted in symptoms indistinguishable from those of wildtype (WT) infection, and the progeny were whitefly transmissible (Ferriol et al, 2016a). A ToANV infectious construct expressing the green fluorescent protein (GFP) was also derived (Ferriol et al, 2016a) and was used to monitor ToANV movement in the inoculated leaf. Furthermore, the ToANV cDNA infectious clones were used to locate the cleavage sites of the ToANV RNA2encoded polyprotein (Ferriol et al, 2016b).…”