elF4E - susceptibility factor for potyvirus infection with potential predictive resistance strategies

In this project we want to understand a special protein that helps some viruses to infect plants. This protein, called eukaryotic translation initiation factor 4E, or elF4E, exists in all cells of higher organisms and plays a central role in producing other proteins by reading messenger RNA (mRNA). We showed that plants without this protein can be resistant to infection with some viruses. Although specific viruses (Potyviruses) that depend upon this protein have an RNA with a slightly different structure from that of mRNAs, we believe that elF4E plays a very similar role in helping the viral RNA to produce other proteins. From studies of different crop plants that are resistant to potyviruses, we know of the existence of different forms of elF4E which cannot assist potyvirus infection. It appears that the inability of these resistant plants to produce viral proteins is associated with a failure of elF4E to bind to the starting-end of the viral RNA. In our studies of peas resistant to pea potyvirus (called Pea seed-borne mosaic virus: PSbMV), we have also identified another very surprising property of elF4E. It also has the ability to assist PSbMV to move from one cell to another, to spread the infection through the plant. Hence, altered elF4E prevents the virus from both replicating and spreading in resistant pea plants. Cell-to-cell movement of large molecules is also important for plant growth and development although the mechanisms involved are generally very poorly understood. Therefore, the study of elF4E can tell us about several very important areas of biology: how viruses infect plants and the potential for resistance in plants, the translation of RNAs to produce proteins, and cell-to-cell communication. We will make new changes in the elF4E protein and relate these to the potential of plants to carry out mRNA translation, virus replication, and virus movement from cell to cell. We will also determine the atomic structure of the elF4E and virus proteins that join together in a complex. In this way we will understand how the proteins join together and what we need to do to stop it happening. From this information we will identify mutant pea plants where the complex cannot form and therefore plants with potential to exhibit new resistance to PSbMV and other potyviruses. We will also identify mutants for the experimental plant Arabidopsis, which will establish a range of genetic and biological tools for further dissection of cell-to-cell communications.

In a previous BBSRC-funded programme we showed that the sbm1 recessive resistance gene in pea effective against Pea seed borne mosaic virus (PSbMV) encodes a mutant allele of eukaryotic translation initiation factor 4E (elF4E). This gene is one of a cluster of resistances on linkage group VI in pea that collectively confer resistance to a range of different potyviruses. Based upon related observations for pepper and lettuce, it is most probably that these resistances are multiple functions of the same resistance allele. We have shown that a second resistance cluster on linkage group II is tightly linked to the paralogous gene, elF(iso)4E. Since potyvirus avirulence for most of these resistances is determined by the virus-genome linked protein, VPg, which is covalently attached to the 5¿end of the viral RNA, the most likely scenario is that VPg binds to elF4E for translation and that this interaction is disrupted in resistant plants. We have shown that elF4E performs an additional role during infection in assisting the virus to move from cell to cell. In this project we will carry out a mutagenesis-based dissection of the various functions of elF4E and elF(iso)4E and relate these to the determined crystal structures and models of pea elF4E and elF(iso)4E. By co-crystallising eLF4E with VPG or its interacting peptide, we aim to provide a structure for the functional interaction between the two proteins against which all potyvirus-host resistance interactions can be modelled. This will allow us to predict the nature of resistance-conferring mutations in eLF4E and to identify such mutations using TILLING of pea and Arabidopsis. These and the known elF4E alleles will be tested against a range of potyviruses to identify potential sources of novel resistance and to verify the prediction strategies. The potential for elF4E/elF(iso)4E to aid virus macromolecular trafficking between cells is novel and important since macromolecular trafficking is now known to underlie diverse regulatory roles in growth and development, in addition to pathogen defence. To dissect this process with respect to virus infection, the nature of the viral component (VPg, VPg precursor, viral RNA or virion) interacting with elF4E will be determined, and the importance of the interaction confirmed using the mutants defined above. To further analyse how elF4E/elF(iso)4E might assist cell-to-cell trafficking, Arabidopsis mutants will be identified (by TILLING) that are specifically defective in movement but not translation functions.