Viral infection causes detrimental diseases in vegetable and field crops. In addition to direct yield losses and reduced quality, the presence of viruses in plant material harms our export position. The aim of this proposal is to identify susceptibility factors of the host plant that are exploited for proliferation by potexviruses, such as Potato virus X (PVX, which is the type species of the potexviruses) and Pepino mosaic virus (PepMV). The latter virus currently causes severe yield losses in tomato production facilities and resistance traits are not available yet. Hijacking host susceptibility factors by viruses is a general mechanism by which they are able to infect plants. Therefore, the identification of critical host susceptibility factors required for completing the infection cycle of potexviruses in general provides leads for generating durable resistance to this type of viruses.
To find these host susceptibility factors, we will focus on PVX and will follow an integrated approach, consisting of a combination of a virus-induced gene silencing (VIGS) screen, yeast two-hybrid analyses and immuno-precipitations. The VIGS screen involves silencing (gene knock-down) by using a cDNA library of the host itself in the model plant Nicotiana benthamiana, which is host to many different potexviruses, including PepMV and PVX. Silenced plants are inoculated with PVX. When there is knock-down of the expression of a gene encoding an essential host susceptibility factor for potexviruses, viral proliferation will be suppressed. The yeast two-hybrid technique, which is aimed to find new protein-protein interactions, will be used to find proteins from the plant that interact with viral proteins as they could be potential host susceptibility factors. For this, a yeast two-hybrid cDNA library from N. benthamiana will be screened against the five viral proteins that are encoded by the genome of PVX: replicase, triple gene block proteins 1, -2 and -3 and the coat protein. The immuno-precipitations have the same aim. However, in this case the proteins coded by PVX are fused to an affinity tag and these fusions are transiently expressed in N. benthamiana. Subsequently, immuno-precipitation will be performed with an affinity matrix and co-purifying proteins will be identified by mass spectrometry. As mentioned above, proteins that interact with any of the viral proteins are potential host susceptibility factors. To test this, knock-down of the gene encoding such a factor in N. benthamiana by VIGS will be performed to study whether indeed PVX proliferation is compromised after inoculation with this virus. The identified, and presumably generic-acting, host susceptibility factors can subsequently be exploited to generate durable resistance to potexviruses in many plant species. This is achieved by generating, or selecting for, plant lines carrying divergent alleles coding for incompatible host susceptibility factors. Such factors do not support proliferation of the potexvirus anymore, but have retained their generic host function. Alternatively, their intrinsic role for the plant (but not for the virus) can be taken over by redundant, non-divergent factors. Subsequently, this virus-resistant starting material is used in regular breeding programs to obtain virus-resistant commercial crop plants.