Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein

Abstract

Mild variants of many viruses are able to protect infected plants from subsequent invasion by more severe variants of the same viruses through a process known as cross-protection. In the past, the cross-protective viral variants were commonly derived from mild field isolates that were sometimes genetically heterogeneous, providing variable levels of cross-protection. Here, we report a novel approach to rapidly generate cross-protective variants of the tomato-infecting Pepino mosaic virus (PepMV) independently of the availability of mild field isolates. Our approach sought to attenuate PepMV by mutating less conserved amino acid residues of the abundantly produced capsid protein (CP). These less-conserved amino acid residues were identified through multiple alignments of CPs of six potexviruses including PepMV, and were altered systematically to yield six PepMV mutants. These mutants were subsequently inoculated onto the model plant Nicotiana benthamiana, as well as tomato, to evaluate their accumulation levels, symptom severities, and cross-protection potentials. The mutant KD, in which the threonine (T) and alanine (A) residues at CP positions 66 and 67 were replaced with lysine (K) and aspartic acid (D), respectively, were found to accumulate to low levels in infected plants, cause very mild symptoms, and effectively protect both N. benthamiana and tomato against secondary infections by wild-type PepMV. These data suggest that our approach represents a simple, fast, and reliable way of generating attenuated viral variants capable of cross-protection.

Random Plant Viral Variants Attain Temporal Advantages During Systemic Infections and in Turn Resist other Variants of the Same Virus

“…It was also suggested that cross-protection might play an important role in shaping population structures of RNA plant viruses 3 . Cross-protection refers to the specific protection against a virus in plants pre-inoculated with a mild isolate of the same virus 10 11 . Mechanistically, cross-protection was once thought to be caused by homology-based RNA silencing, although this notion has been challenged by several more recent studies 9 12 13 14 15 .…”

Genetic diversity and population structure of Pepino mosaic virus in tomato crops of Spain and Morocco

“…In general, our results support the finding of low genetic diversity, but geographical differentiation was counteracted by moderate gene flow. This knowledge could provide insights into fundamental eco‐evolutionary processes and valuable information for the design and implementation of stable and effective control strategies, such as cross‐protection by attenuated PepMV isolates (Agüero et al, ; Chewachong et al, ; Hanssen et al, ), which appears to be a good solution in the short term for managing this viral disease.…”

A New Mechanistic Model for Viral Cross Protection and Superinfection Exclusion

“…That complete establishment of the whole plant “cross protection” requires a substantial head start by the primary virus is not inconsistent with SIE operating at the cellular level. Rather, it suggests that most of the susceptible cells must be pre-occupied by the primary virus in order to block the invasion of the superinfector ( Chewachong et al, 2015 ; Zhang et al, 2015 ). Furthermore, once the cross protection is established, the superinfector genome could not be detected despite repeated attempts over many weeks ( Chewachong et al, 2015 ).…”

“…Rather, it suggests that most of the susceptible cells must be pre-occupied by the primary virus in order to block the invasion of the superinfector ( Chewachong et al, 2015 ; Zhang et al, 2015 ). Furthermore, once the cross protection is established, the superinfector genome could not be detected despite repeated attempts over many weeks ( Chewachong et al, 2015 ). Together these studies suggest that the non-cell-autonomous SIE plays a minimal role in SIE manifestation.…”

“…This highly specific self-discriminatory phenomenon, referred to as cross protection, was first documented almost 90 years ago, and has since been used to successfully manage a number of virus diseases of crop plants ( Sherwood and Fulton, 1982 ; Gonsalves, 1998 ; Goregaoker et al, 2000 ; reviewed by Ziebell and Carr, 2010 ; Folimonova, 2013 ). At the molecular level, the genomes of superinfecting viruses are all but undetectable in most of the cross-protected plants, suggesting a failure of cellular entry or multiplication by the superinfectors (e.g., Chewachong et al, 2015 ). While a unified mechanistic understanding of cross protection remains elusive, it is important to note that similar superinfection exclusion (SIE) effect can be induced by symptomatic – in place of mild – virus isolates as well, as long as the primary and superinfecting viruses are genetically closely related.…”