1.11.11
USE OF INFECTIOUS COMPLEMENTARY DNA, RECOMBINANT OR TAGGED WITH THE GFP TO STUDY THE PATHOGENICITY OF LETTUCE MOSAIC POTYVIRUS

T CANDRESSE1, SJ YANG1, S GERMAN-RETANA1, E REDONDO1, O LE GALL1, F REVERS1, H LOT2, S SOUCHE2 and J DUNEZ1

INRA, Station de Pathologie Végétale, 1BP 81, F-33883 Villenave d'Ornon Cedex, France; 2BP 94, F-84143 Montfavet Cedex, France

Background and objectives
Lettuce mosaic potyvirus (LMV) causes severe losses on lettuce crops worldwide. Four genes controlling resistance to LMV have been identified in Lactuca sp., of which the two recessive genes mo11 and mo12 are used commercially to protect lettuce cultivars. These two genes afford either resistance (no virus multiplication) or tolerance (symptomless virus multiplication) depending on the LMV isolate, and in addition control the ability of the virus to be seed transmitted. LMV isolates able to overcome these two genes have been observed in Europe and elsewhere, and in some instances caused local epidemics. Our aim is to understand the basis of resistance in lettuce and of resistance-breaking in LMV. For this purpose we have obtained infectious full-length LMV cDNA clones. We have constructed recombinants between two isolates differing in their ability to overcome mo1 (LMV-0 and LMV-E, about 97% sequence identity [1]), in order to map the determinants of resistance-breaking and of symptomatology. The gfp gene of Aequorea victoria was inserted in LMV in order to understand the virus spread in the plant.

Materials and methods
The improved gfp mutant mgfp5 [2] was inserted as an amino-terminal fusion with the HC-Pro domain of the viral polyprotein. Primary infections of lettuce seedlings with LMV cDNA under the control of the cauliflower mosaic virus 35S promoter were performed either by rubbing in the presence of carborundum, or by particle bombardment (Helios Gene-Gun, BioRad). Plants infected with recombinant viruses were controlled visually for symptoms and by ELISA or immunocapture-RT-PCR for the presence of progeny virus. Plants infected with LMV-GFP were observed visually under natural or UV light, or in fluorescence microscopy under blue light.

Results and conclusions
Comparisons of the characteristics of infection derived from virus, or from parental or recombinant cDNA on lettuce cultivars either susceptible (Trocadéro), or carrying the mo11 (Mantilia) or mo12 gene (Vanguard75), allows us to rule out the role of certain regions of the LMV genome in mo1-breaking. Studies are in progress to sharpen this analysis to the level of single amino-acid substitutions between the two LMV isolates.

Intense GFP fluorescence was observed in some lettuce cultivars and in pea, but in other hosts only faint or undetectable fluorescence is observed. However, serial or long-term passaging has shown the gfp insert to be quite stable over time in hosts displaying fluorescence or not. Analysis of the infection pattern of lettuce indicates that young leaves and leaves produced later in the infection process probably differ in their mode of secondary infection.

References
1. Revers F, Yang SJ, Walter J et al., 1997. Virus Research 47, 167-177.
2. Siemering KR, Golbik R, Sever R, Haseloff J, 1996. Current Biology 6, 1653-1663.