Exploring host:virus interactions using pea seed borne mosaic virus (PSbMV) and pea
|Thomas, C.L., Harrison, S., Jones, A.-L.,||
John Innes Centre, Colney Lane
|Revers, F. and Maule, A.J.||
Virologie Vegetale, IBVM-INRA, BP81
Pea seed-borne mosaic virus (PSbMV) is a member of the potyvirus group. The primary host for PSbMV is pea, but the virus also causes disease in lentils and broadbeans. Three pathotypes of PSbMV have been identified, designated P-1, L-1 and P-4. Pathotype P-1 is the most widely distributed and economically significant strain affecting pea. We are interested in both engineered and natural resistance to PSbMV, and have investigated both these areas.
Previous studies have shown that some pea germplasm carries genes for resistance to PSbMV. The resistance gene sbm1 belongs to one of two clusters of recessive genes in pea all conferring resistance to viruses of the potyvirus group. This resistance gene is thought to be different from the other recessive resistance gene that has been characterised (mlo in barley (1)), because it does not involve a hypersensitive response and shows strain specificity. The avirulence factor from PSbMV involved in sbm1 function is the virus genome-linked protein or VPg (3). This protein is believed to be involved in virus replication. It is therefore possible that VPg interacts with the Sbm1 gene product and that this interaction is required for successful replication of the virus.
We are attempting to isolate and characterize the sbm1 resistance gene by focusing on two major methodologies. Sbm1 is on linkage group VI, in a region where there are few chromosomal markers; the nearest marker is located 4 cM from sbm1. Towards a map-based approach, cDNA-AFLP analysis has been used in an attempt to increase the number of markers around sbm1. For this, near isogenic pea lines, one resistant and one susceptible to PSbMV (pathotype P-1) have been compared. An initial screen using 50 primer combinations has identified ten polymorphic bands. These bands have been cloned and are currently being assessed using mapping techniques. In a second approach, which is more direct, VPg (the avirulence determinant for the PSbMV P-1/Sbm1 interaction) is being used as the bait in a yeast two-hybrid screen of cDNA libraries constructed from susceptible and resistant lines of pea. To avoid missing potential interactors as a result of interference from the fused GAL4 DNA binding domain, both N- and C-terminal fusion constructs were made. Screening 6x106 clones with each construct has revealed a number of positive interactors. Verification of the interaction and sequence based identification grouped the 143 identified interactors into 10 classes. The interactor clones were used as probes on mapping filters for pea. None of the interactors mapped to linkage group VI and therefore do not represent the Sbm1 gene product, but they do have the potential to provide information about the functional properties of VPg. The two strongest interactors are being analyzed further to determine their relevance in an in vivo context. Further screening of both libraries is in progress. By using these combined approaches we hope to shed light on the identity of the Sbm1 gene product.
To develop engineered resistance to PSbMV, the viral replicase gene (NIb) has been expressed in transgenic pea lines. Inducible resistance, seen as recovery from infection, was observed in some transgenic peas (2). Resistance was observed in plants in either the homozygous or hemizygous condition and resulted in no overall yield loss despite the initial infection. Resistance was associated with a loss of both viral and transgene RNA, which is indicative of a mechanism based upon induced co-suppression or post transcriptional gene silencing (PTGS).