Engineering Plant Virus "Vaccines" Using Pepino Mosaic Virus as a Model (2013)

Cross protection refers to the practice of mitigating crop yield losses by preinoculating plants with a mild variant of a plant virus to protect the plants from secondary infection by a severe variant of the same virus.Given its economically important applications in agriculture, cross protection remains a viable approach to mitigate crop losses resulting from infections by emerging and rapidly spreading viruses.However, the absence of a rapid and reliable procedure for developing attenuated plant virus variants is a serious barrier to the effective use of cross protection in managing plant viruses.The goal of my thesis research has been to develop a knowledge-based approach for producing stably attenuated plant viruses for use in cross protection, and to evaluate the effectiveness of the attenuated viruses to protect plants against secondary infections with wild type viruses.Pepino mosaic virus (PepMV) was used as a model plant virus and Nicotiana benthamiana as model experimental plant.PepMV is a single-stranded, positive sense (+) RNA virus that belongs to the genus Potexvirus, in the Flexiviridae family.PepMV causes serious losses in greenhouse tomato productions.Nicotiana benthamiana is a close relative of tomato, and a common model plant with a rapid growth rate.Importantly, it shows easily recognizable symptoms upon PepMV infections.Tomato, the major economic crop host of PepMV, was used to confirm some of the results obtained in N. benthamiana.Two approaches were used to engineer attenuated iii PepMV for use in cross protection.In the first approach, non-conserved amino acid (AA) residues within the capsid protein (CP) of PepMV were identified by multiple alignments that compared PepMV CP with five other potexviruses, and subjected to replacements with their counterparts in Potato virus X, the type member of the genus Potexvirus.This approach, referred to as alignment guided replacement (AGR), permitted the identification of one PepMV mutant with significantly attenuated infection symptoms.This mutant, in which the threonine (T) and alanine (A) residues at AA positions 66 and 67 of PepMV CP were replaced with lysine (K) and aspartic acid (D) residues, respectively, maintained its ability to move systemically but caused very mild symptoms in the systemically infected N. benthamiana and tomato plants.More importantly, plants pre-infected with the KD mutant became resistant to secondary infections with wild-type PepMV, hence identifying it as a robust inducer of cross protection.In the second approach, the focus was on the RNA-dependent RNA polymerase (RdRp) gene of PepMV and, while maintaining its AA sequence, more frequently used codons were replaced with their least used counterparts for every AA residue through a process known as codon de-optimization.PepMV mutants with more than half of the RdRp gene codonde-optimized retained significant levels of pathogenicity in infected plants, suggesting that codon de-optimization alone might not lead to substantial attenuation of PepMV.In summary, my thesis research used two different approaches to engineer attenuated PepMV variants and tested the resultant mutants for both symptom attenuation and cross protection induction.The results demonstrate AGR as a novel, simple, rapid and reliable procedure for generating attenuated, cross protective viral variants.